WO2016205730A1 - Citrullinated proteins and their breakdown products as tbi biomarkers - Google Patents

Citrullinated proteins and their breakdown products as tbi biomarkers Download PDF

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WO2016205730A1
WO2016205730A1 PCT/US2016/038207 US2016038207W WO2016205730A1 WO 2016205730 A1 WO2016205730 A1 WO 2016205730A1 US 2016038207 W US2016038207 W US 2016038207W WO 2016205730 A1 WO2016205730 A1 WO 2016205730A1
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protein
proteins
tbi
determined
injury
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PCT/US2016/038207
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French (fr)
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Gregory P. MUELLER
James G. FREEDY
Rachel C. LAZARUS
Michael N. FLORA
Gay R HOLSTEIN
Giorgio P MARTINELLI
Ramon Diaz ARRASTIA
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The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc.
Icahn School Of Medicine At Mount Sinai
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease

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  • Traumatic brain injury is a major cause of long-term disability. Acute TBI prompts a constellation of dysfunctional processes, collectively known as "secondary injury” mechanisms. A hallmark secondary injury in TBI is a prolonged imbalance in calcium homeostasis, resulting in a dramatic influx of calcium into brain cells. This influx elicits the generation of damaging reactive oxygen species. Protein carbonylation and citrullination are pathological post-translational modifications that can result from intracellular calcium overload. These modifications have been proposed to play a role in neurodegenerative disorders, including Alzheimer's disease, and multiple sclerosis. Both carbonylation and citrullination can contribute to ongoing dysfunction, either through direct loss of protein function or via immune-based mechanisms where proteins specifically modified by citrullination become targeted by the adaptive immune system.
  • the disclosure is directed to methods of diagnosing traumatic brain injury (TBI) in a subject, the method comprising determining the citruUination level(s) of one or more biomarkers in a biological sample taken from the subject, and comparing the determined citruUination level(s) of the one or more biomarkers against a citruUination level(s) of the same one or more biomarkers from a control subject determined not to be suffering from TBI, wherein an increase in the citruUination level(s) of the one or more biomarkers compared to the citruUination level(s) of the one or more biomarkers from the control subject is indicative that the subject may be suffering from TBI.
  • TBI traumatic brain injury
  • the disclosure is also directed to methods of monitoring the progression of traumatic brain injury (TBI) in a subject, the method comprising analyzing at least two biological samples from the subject taken at different time points to determine a citruUination level(s) of one or more biomarkers in each of the at least two biological samples, and comparing the determined citruUination level(s) of the one or more biomarkers over time to determine if the subject's citrullination level(s) of the one or more specific biomarkers is changing over time, wherein an increase in the citrullination level(s) of the one or more biomarkers over time is indicative that the subject's risk of suffering from TBI is increasing over time.
  • TBI traumatic brain injury
  • Figure 1 Mechanisms of protein carbonylation following mitochondrial dysfunction. In the wake of mitochondrial dysfunction driven by calcium overload, oxygen is reduced to hydroxyl radicals through a series of electron reductions catalyzed by enzymes such as
  • ROS reactive oxygen species
  • FIG. 1 Mechanisms of protein citrullination following intracellular calcium overload.
  • Peptidylarginine deiminase (PAD) enzymes are activated by high levels of intracellular calcium. Once activated, PAD enzymes catalyze the conversion of intrapeptidyl arginine residues to intrapeptidyl citrulline residues. Ammonia (NH 3 ) is released as a byproduct of this reaction. ( Figure adapted from Tesija-Kuna & Zirovic (2008)).
  • FIG. 3 Proposed model of proteomic changes following TBI- induced calcium overload.
  • ischemia and mechanical damage tearing, shearing, and hemorrhage
  • ROS reactive oxygen species
  • Citrullination the conversion of intrapeptidyl arginine to intrapeptidyl citrulline residues, is catalyzed by peptidylarginine deiminase, which is a calcium-dependent enzyme (top panel).
  • Carbonylation the conversion of an intrapeptidyl amino acid (Lys, Cys, His) to a carbonyl group, is driven by the effects of ROS (bottom panel). Both citrullination and carbonylation are irreversible proteomic modifications with significant effects on protein structure and function.
  • Figure 4 Effects of CCI on protein carbonylation in rat brain. Five days after
  • extracts were obtained from dissected regions of brain containing the injury penumbra (I) and the corresponding contralateral control region (C).
  • FIG. 5 Identification of rat brain proteins carbonylated after CCI. Proteins present in extracts of naive rat brain (N) and contralateral (C) and ipsilateral (I) regions of CCI-injured rats were fractionated by fluid-phase IEF (pi ranges listed at bottom) and one-dimensional gel electrophoresis. Proteins were transferred to nitrocellulose, derivatized, and probed with anti- DNP antibody (right). Proteins remaining in the gel were visualized by Coomassie staining (left).
  • FIG. 7 The CCI- induced increase in anti-DNP immunofluorescence is specific for protein carbonylation. Brain tissue was collected 5 days after CCI and evaluated by anti-DNP immunofluorescence. Anti-DNP immunofluorescence in the cerebral cortex contralateral to the injury and the CCI-injured cortex are depicted in (A) and (B), respectively (original
  • Brain tissue was collected 5 days after CCI and evaluated by anti-DNP immunofluorescence (A and C, sham control; B and D, CCI). (A and B)
  • FIG. 10 Figure 10. Representative images depicting the carbonylation scoring system. Brain tissue was collected 5 days after CCI and evaluated by anti-DNP immunofluorescence. Images show relative increments in anti-DNP immunofluorescence (0-3) observed in the
  • penumbra/cortex penumbra/cortex, dorsal third ventricle, and median eminence regions.
  • the distinguishing feature differentiating a score of 2 versus a score of 3 for the dorsal third ventricle was the anti- DNP labeling of peri-ependymal cells located in the parenchyma both lateral and ventral to the dorsal third ventricle. This observation reflected a stippled pattern of carbonylation-positive cells expanding approximately 10 to 50 ⁇ into surrounding tissue from the ependymal lining of the ventricle.
  • FIG. 12 Effects of CCI on protein carbonylation in regions of the cerebral cortex of male and female rats. Brain tissue was collected 5 days after CCI and evaluated by anti-DNP immunofluorescence. Sections were scored for protein carbonylation as described under Material and methods. Data indicate means + SEM. A post hoc Tukey HSD analysis indicated a statistically significant difference ( DDD /? ⁇ 0.001) between the scores of the penumbra versus other cortical regions of the injured and sham animals. Graph is representative of three independent experiments.
  • FIG. 14 Injury upregulates the expression of citrullinated proteins in the cerebral cortex. Brain tissue was collected five days after CCI and evaluated for protein citrullination by anti-protein citrulline immunolabeling using mAB 6B3. The upper panels show immunolabeling in sections of control (left) and injured brain (right) (2X magnification). Lower panels show higher magnifications of cerebral cortex adjacent to the lesion site (right) or a comparable site in control cortex (left) (20X magnification). Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed. PB-cit: Protein-bound citrulline.
  • FIG. 15 Specificity of anti-protein citrulline immunolabeling by mAb 6B3.
  • Panels A and B show immunofluorescent signals from mAb 6B3 anti-protein citrulline labeling of the injured cerebral cortex (panel A) and ipsilateral hippocampus (panel B).
  • Panels C and D show immunolableing of equivalent sections with mAb 6B3 that was preadsorbed with citrullinated protein standards.
  • the control and immunoneutralized preparations of mAb 6B3 were treated identically, with the exception of the presence or absence of neutralizing citrullinated proteins. Data are representative of two independent experiments. Scale bar: 200 ⁇ .
  • FIG. 16 Increased protein citruUination in the cerebral cortex, hippocampus and external capsule following CCI.
  • Anti-protein citrulline immunolabeling by mAb 6B3 is shown for the control brain regions (panels A, D, and G), regions ipsilateral to the lesion (panels B, E, H) and regions contralateral to the lesion (panels C, F, and G).
  • Structures representated are the cerebral cortex (A-C), hippocampus (D-F), and external capsule (G-I).
  • Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed. Scale bar: 200 ⁇ .
  • FIG. 17 Localization of CCI- induced protein citruUination to astrocytes. Panels on the left show the colocalization of mAb 6B3 labeling with anti-GFAP labeling in the external capsule. Panels on the right show the colocalization of mAb 6B3 labeling with anti-GFAP labeling in the cerebral cortex. Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed. Scale bar: 200 ⁇ . PB-cit: Protein-bound citrulline.
  • FIG. 1 CCI did not affect the status of protein citrullination in neurons, microglia or oligodendrocytes.
  • FIG. 19 Effects of CCI on protein citrullination in GFAP-positive cells of the ipsilateral and contralateral hippocampus. Colocalization of anti-protein citrulline and anti-GFAP labeling in astrocytes of the ipsilateral hippocampus is depicted in the pairs of panels presented on the left (A-C). Panel D shows the distinctive anti-citrullinated protein immunolabeling of large, rounded cells observed in the contralateral hippocampus of approximately 20% of CCI animals (4 of 21). While these cells lacked the classical stellate morphology of astrocytes, the anti-citrullinated protein labeling colocalized with GFAP immunolabeling (panels E-G). PB-cit: Protein-bound citrulline.
  • FIG. 20 Specificity of anti-protein citrulline mAb 6B3 detection for western blotting. Displayed on the left are two Coomassie-stained profiles showing the protein composition of native fibrinogen (Fib) and citruUinated fibrinogen (C-Fib). The western blot (right) shows three immunoblots in which: citruUinated fibrinogen was probed with active mAb 6B3 (Active/C-Fib; left lane); citruUinated fibrinogen was probed with immunoneutralized mAb 6B3
  • FIG. 21 Identification of proteins that are citruUinated in response to CCI. Extracts of control (C) and injured (I) cerebral cortex were fractionated by fluid-phase isoelectric focusing into defined pH ranges (shown at top) and then further resolved according to molecular weight using one-dimensional gel electrophoresis. Proteins were then transferred to nitrocellulose membranes and probed for protein-bound citrulline (see Methods) (right panel, "Western blot"). Gels run in parallel were visualized with Coomassie (left panel).
  • FIG. 22 Simulated brain injury in normal human astrocytes reveals a spectrum of GFAP breakdown products and the hyper-citrullination of one GFAP species.
  • Normal human astrocytes were treated with ionomycin (10 ⁇ ; 4h) and analyzed for GFAP (left panel) and protein-bound citrulline immunoreactivity (right panel) by western blot.
  • the results of three independent experiments (A, B, and C) are presented, showing the immunoreactivity in extracts prepared from untreated control cells (Control) and cells treated with ionomycin (+ Iono).
  • the blue arrows indicate intact GFAP, while the orange arrows indicate the hyper-citrullinated GFAP breakdown product.
  • FIG. 23 Potential ongoing effects of protein carbonylation and citruUination in TBI pathology. Damage may induce long-term dysfunction through the calcium-mediated proteomic modifications, carbonylation and citruUination. These modifications can elicit a number of pathological outcomes, including the generation of antigenic epitopes (citruUination) and formation of cytotoxic aggregates (carbonylation).
  • FIG. 24 Injury upregulates the expression of citrullinated proteins in the cerebral cortex.
  • Brain tissue was collected five days after CCI and evaluated for protein citruUination by anti-protein citrulline immunolabeling using mAB 6B3.
  • the upper panels show immunolabeling in sections of control (left) and injured brain (right) (2X magnification).
  • Lower panels show higher magnifications of cerebral cortex adjacent to the lesion site (right) or a comparable site in control cortex (left) (20X magnification).
  • Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed.
  • PB-cit Protein-bound citrulline.
  • FIG. 25 Specificity of anti-protein citrulline immunolabeling by mAb 6B3.
  • Panels A and B show immunofluorescent signals from mAb 6B3 anti-protein citrulline labeling of the injured cerebral cortex (panel A) and ipsilateral hippocampus (panel B).
  • Panels C and D shows immunolableing of equivalent sections with mAb 6B3 that was preadsorbed with citrullinated protein standards.
  • the control and immunoneutralized preparations of mAb 6B3 were treated identically, with the exception of the presence or absence of neutralizing citrullinated proteins. Data are representative of two independent experiments. Scale bar: 200 ⁇ .
  • FIG. 26 Increased protein citruUination in the cerebral cortex, hippocampus and external capsule following CCI.
  • Anti-protein citrulline immunolabeling by mAb 6B3 is shown for the control brain regions (panels A, D, and G), regions ipsilateral to the lesion (panels B, E, H) and regions contralateral to the lesion (panels C, F, and G).
  • Structures representated are the cerebral cortex (A-C), hippocampus (D-F), and external capsule (G-I).
  • Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed.
  • Scale bar 200 ⁇ .
  • FIG. 27 Localization of CCI- induced protein citruUination to astrocytes. Panels on the left show the colocalization of mAb 6B3 labeling with anti-GFAP labeling in the external capsule. Panels on the right show the colocalization of mAb 6B3 labeling with anti-GFAP labeling in the cerebral cortex. Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed. Scale bar: 200 ⁇ .
  • PB-cit Protein-bound citrulline.
  • FIG. 28 CCI did not affect the status of protein citruUination in neurons, microglia or oligodendrocytes.
  • FIG. 29 Effects of CCI on protein citruUination in GFAP-positive cells of the ipsilateral and contralateral hippocampus. Colocalization of anti-protein citrulline and anti-GFAP labeling in astrocytes of the ipsilateral hippocampus is depicted in the pairs of panels presented on the left (A-C). Panel D shows the distinctive anti-citrullinated protein immunolabeling of large, rounded cells observed in the contralateral hippocampus of approximately 20% of CCI animals (4 of 21). While these cells lacked the classical stellate morphology of astrocytes, the anti-citrullinated protein labeling colocalized with GFAP immunolabeling (panels E-G).
  • FIG. 30 Specificity of anti-protein citrulline mAb 6B3 detection for western blotting. Displayed on the left are two Coomassie-stained profiles showing the protein composition of native fibrinogen (Fib) and citruUinated fibrinogen (C-Fib). The western blot (right) shows three immunoblots in which: citruUinated fibrinogen was probed with active mAb 6B3 (Active/C-Fib; left lane); citruUinated fibrinogen was probed with immunoneutralized mAb 6B3
  • FIG 31 Identification of proteins that are citruUinated in response to CCI. Extracts of control (C) and injured (I) cerebral cortex were fractionated by fluid-phase isoelectric focusing into defined pH ranges (shown at top) and then further resolved according to molecular weight using one-dimensional gel electrophoresis. Proteins were then transferred to nitrocellulose membranes and probed for protein-bound citrulline (see Methods) (right panel, "Western blot"). Gels run in parallel were visualized with Coomassie (left panel).
  • Simulated brain injury in normal human astrocytes reveals a spectrum of GFAP breakdown products and the hyper-citrullination of one GFAP species.
  • Normal human astrocytes were treated with ionomycin (10 ⁇ ; 4h) and analyzed for GFAP (left panel) and protein-bound citrulline immunoreactivity (right panel) by western blot.
  • the results of three independent experiments (A, B, and C) are presented, showing the immunoreactivity in extracts prepared from untreated control cells (Control) and cells treated with ionomycin (+ Iono).
  • the blue arrows indicate intact GFAP, while the orange arrows indicate the hyper-citrullinated GFAP breakdown product.
  • Traumatic brain injury is a major public health issue in the U.S., with over 1.7 million cases occurring annually. At least 5.3 million Americans are currently living with ongoing disability due to TBI (149). In civilians, these injuries are largely due to motor vehicle accidents (47), as well as falls, sports, and firearms (152). While on deployment, military populations are at disproportionate risk for blast-injury TBIs caused by improvised explosive devices in theater (58). These blast-induced injuries have become the signature injury of operations in Iraq and Afghanistan, with 10% - 20% of returning veterans reported to have suffered a TBI (45).
  • TBI chronic inflammatory state
  • a chronic inflammatory state can persist in the brain for months, and even years, following TBI (101; 108).
  • TBI cognitive deficits in attention, memory, and executive function
  • elucidating the mechanisms that underlie chronic dysfunction following TBI has proven challenging.
  • Primary injury refers to the direct, physical disruption of neural tissue following impact, such as axonal strain and injury due to rotational acceleration-deceleration (129) or focal injuries such as skull fracture (111).
  • Secondary injury refers to the ensuing constellation of dysfunctional cellular and physiological processes following primary mechanical injury (152).
  • Secondary injury damage refers to a delayed series of interdependent pathological processes that follow primary injury (103; 152). Hemorrhage, edema, and raised intracranial pressure are hallmarks for primary injury (50; 152). As a consequence of ischemia and hypoxia (80), these conditions result in complex cascades of cellular processes that involve mitochondrial dysfunction, calcium overload, inflammation, and oxidative stress, which arise over the course of hours to weeks following TBI (149). These secondary injury processes can lead to serious cognitive dysfunction by disrupting synaptic plasticity, axonal structure, and by inducing cell death through apoptosis, necrosis and autophagy (149).
  • One important component of secondary injury is the disturbance of ion homeostasis (95), which can lead to acute and ongoing changes in cellular physiology (51).
  • ROS reactive oxygen species
  • Hydrogen peroxide is a poorly reactive molecule; however, unlike superoxide, it is able to cross cell membranes (14). In contrast, hydroxyl is highly reactive and much more toxic than superoxide (14; 125). These ROS react with neural membrane lipids through peroxidation, generating reactive carbonyl species (RCS) (155) such as acrolein and 4-hydroxynonenal (127; 128) (see Fig. 1).
  • RCS reactive carbonyl species
  • neuropathologies including Alzheimer's disease and Parkinson's disease (25).
  • Elevated intracellular calcium also causes a less-studied protein modification known as citrullination.
  • Protein citrullination is essential for a number of basic physiological functions, including epidermal hydration (120), epigenetic regulation of gene expression (134), hair growth (120), and neural plasticity in stages of early brain development (57).
  • abnormal hyper-citrullination of proteins may occur.
  • the role of abnormal protein citrullination is best understood in the case of rheumatoid arthritis, an autoimmune disorder involving progressive inflammation of synovial joints (55). In this disorder, the citrullination of proteins prompts an autoimmune response due to the formation of antigenic epitopes.
  • protein citrullination is also implicated in several neurodegenerative disorders with altered calcium homeostasis, including Alzheimer's disease (62), temporal lobe epilepsy (8), glaucoma (18), and multiple sclerosis (MS) (6).
  • PADs peptidylarginine deiminases
  • PAD peptidylarginine deiminases
  • PAD enzymes exist as five isoforms, but only two of these are found in neural tissue: PAD2, which is localized to astrocytes, (37; 64; 109) and PAD4, which is exclusively expressed in neurons (2). As depicted in Fig.
  • Citrullination also affects proteins in a number of ways.
  • the addition of a citrulline residue in the place of arginine results in the loss of a positive charge, which can alter tertiary structure, proteolytic susceptibility, and protein-protein interactions (64; 72).
  • MBP myelin basic protein
  • the citrullination of myelin basic protein (MBP) limits the ability of this protein to appropriately associate with lipids (55), which in turn contributes to demyelination by destabilizing sheath structure (89).
  • MBP myelin basic protein
  • citrullinated proteins are also observed within the extracellular plaques seen in post-mortem brains affected by Alzheimer's disease, suggesting a functional role for this modification in neurodegenerative pathology.
  • citrullination has the potential to create "altered-self ' epitopes that may be antigenic, prompting the adaptive immune system to launch autoimmune responses against previously benign proteins (32; 154).
  • MBP citrullination of MBP in MS leads not only to myelin degradation, but also results in the generation of autoantigenic MBP isomers, which are consequently targeted by T-cell lymphocytes (144).
  • T-cell lymphocytes T-cell lymphocytes
  • Intracellular calcium overload is a well-documented pathological effect of TBI. Because protein carbonylation and citruUination are logical and plausible outcomes of oxidative stress and calcium excitotoxicity, it is important to investigate the expression of these damaging post- translational modifications following injury (Figure 3).
  • Protein carbonylation after traumatic brain injury cell specificity, regional susceptibility, and gender differences
  • Protein carbonylation is a well-documented and quantifiable consequence of oxidative stress in several neuropathologies, including multiple sclerosis, Alzheimer's disease, and
  • TBI traumatic brain injury
  • Immunohistochemistry was used to visualize protein carbonylation in the brains of adult male and female Sprague-Dawley rats subjected to controlled cortical impact (CCI) as an injury model of TBI.
  • CCI cortical impact
  • Cell-specific markers were used to colocalize the presence of carbonylated proteins in specific cell types, including astrocytes, neurons, microglia, and oligodendrocytes. Results also indicated that the injury lesion site, ventral portion of the dorsal third ventricle, and ventricular lining above the median eminence showed dramatic increases in protein
  • astrocytes and limited regions of ependymal cells adjacent to the dorsal third ventricle and the median eminence were most susceptible to postinjury protein carbonylation.
  • these patterns of differential susceptibility to protein carbonylation were gender dependent, with males showing significantly greater protein carbonylation at sites distant from the lesion.
  • Proteomic analyses were also conducted and determined that the proteins most affected by carbonylation in response to TBI include glial fibrillary acidic protein, dihydropyrimidase-related protein 2, fructose-bisphosphate aldolase C, and fructose-bisphosphate aldolase A. Many other proteins, however, were not carbonylated by CCI.
  • Traumatic brain injury is a major public health issue affecting over 1.7 million Americans annually, with falls, collision incidents, and motor vehicle accidents being the leading causes of injury (47).
  • TBI Traumatic brain injury
  • the consequences of TBI can be complex and long lasting, resulting in serious disorders that involve progressive cognitive deficits, epilepsy, and profound behavioral alterations.
  • the molecular and cellular disturbances involved in these longer-term responses are not well understood, one common element in the pathology seems to be the establishment of a chronic inflammatory state that can persist in brain for weeks, months, and perhaps even years after TBI (101; 108).
  • a hallmark of inflammation is oxidative stress, which can be caused by metabolic dysfunction with numerous potential causes (43; 124).
  • ROS reactive oxygen species
  • RCS reactive carbonyl species
  • Protein carbonylation involves the introduction of carbonyl groups into protein-bound amino acids. Protein carbonylation may occur either through direct oxidation of amino acid targets by ROS or via interaction with RCS, which are themselves the product of lipid peroxidation (155). By either mechanism, the resulting carbonyl modifications can disrupt protein function and thereby contribute to injury
  • Rats were purchased from Charles River Laboratories (Morrisville, NC, USA). Rats were housed in a barrier facility for animals accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Before experimental procedures, rats were anesthetized with isoflurane (Baxter Healthcare Corp., Deerfield, IL, USA) vaporized in medical-grade oxygen (100%, Roberts Oxygen Co. Inc., Rockville, MD, USA), placed in a digital cranial stereotactic device (Leica Microsystems, Buffalo Grove, IL, USA), and subjected to unilateral CCI injury over the left hemisphere.
  • isoflurane Boxter Healthcare Corp., Deerfield, IL, USA
  • medical-grade oxygen 100%, Roberts Oxygen Co. Inc., Rockville, MD, USA
  • a digital cranial stereotactic device Leica Microsystems, Buffalo Grove, IL, USA
  • the rats underwent a free-hand craniotomy (-3.8 mm from bregma in males, -3.0 mm in females).
  • CCI was administered through the ImpactOne stereotaxic impactor (Leica Microsystems), which delivered a 3-mm flat-tipped impactor at 20° to a depth of 2 mm at 5 m/s with a 500-ms dwell time.
  • the incision was closed and the rats received buprenorphine 0.05 mg/kg for postoperative analgesia.
  • rat body core temperature was maintained at 37+0.3 °C. Sham-operated rats received no anesthesia, incision, or craniotomy.
  • Brains were removed immediately after euthanization, frozen on powdered dry ice, and stored at -80°C until used. Brains were hand-dissected to produce blocks of penumbral structures enriched in carbonylated proteins as identified immunohistochemically (see below). The contralateral brain region and equivalent region from the naive animals were similarly collected.
  • Protein fractions were prepared by homogenization in 5 volumes/tissue weight extraction solution (8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (Chaps), and 50 mM dithiothreitol (DTT; Sigma-Aldrich, St. Louis, MO, USA) containing 0.8% ampholytes (pH 3-10; Invitrogen Life Technologies, Carlsbad, CA, USA) and lx Complete protease inhibitor mix (Roche, Indianapolis, IN, USA)). After homogenization and subsequent centrifugation (20,000g, 10 min, 4°C), the resulting supernatant was stored at -80°C until used. The efficiency of tissue solubilization was > 95%, as judged by protein assay and Coomassie staining of one-dimensional gels.
  • Protein samples were prepared for gel electrophoresis by combining with an equal volume of 2x reducing loading buffer (Novex NuPAGE LDS sample buffer; Invitrogen;
  • Tissue region pools were prepared and 200- ⁇ 1 aliquots of each pool supernatant were further diluted to 2.865 ml having a final composition of IEF denaturant plus ampholytes (150 ⁇ , pH 3-10; Invitrogen), DTT (50 ⁇ , 2 M stock), and bromophenol blue (10 ⁇ , 10 mg/ml stock).
  • the resulting sample was loaded into the IEF fractionator and focused using the following conditions: 100 V, 1.2 mA, 0 W (15 min); 200 V, 2.0 mA, 0 W (1 h); 400 V, 2.0 mA, 1 W (1 h); 600 V, 1.5 mA, 1 W (1 h).
  • IEF-fractionated proteins were further fractionated by size by one-dimensional gel electrophoresis (see One -dimensional gel electrophoresis).
  • Postelectrophoretic detection of carbonylated proteins was performed as described by Conrad et al. (35) with minor modifications. Briefly, nitrocellulose membranes were washed in 20% methanol/80% Tris-buffered saline/Tween 20 (TBS-T), equilibrated in 2 N hydrochloric acid (HC1), and then incubated with 0.5 mM 2,4-dinitrophenylhydrazine (DNPH; Sigma- Aldrich) in 2 N HC1 (10 min, in the dark). The derivatized membranes were then washed three times with 2 N HC1 (10 min per wash) followed by 50% methanol (five times, 10 min per wash).
  • TBS-T Tris-buffered saline/Tween 20
  • DNPH 2,4-dinitrophenylhydrazine
  • Membranes were then equilibrated in TBS-T, blocked with 5% fetal bovine serum/TBS-T, and probed overnight at 4°C with rabbit anti-DNP (Sigma-Aldrich; Catalog No. 9659, 1: 1000 in TBS-T). Membranes were washed three times with TBS-T and probed with horseradish peroxidase-labeled, goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA; Catalog No. 31460, 1:5000 in TBS-T, 3 h).
  • Blots were washed (TBS-T) and visualized by enhanced chemiluminescence (Novex ECL HRP Chemiluminescent Substrate Reagent Kit; Invitrogen) using a FUJI LAS 3000 imager (Fujifilm, Minato, Tokyo, Japan). The images were analyzed using MultiGauge version 3.0 software (Fujifilm) and the intensity of protein bands was quantified with ImageJ software (W.S. Rasband, ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA). Immunoreactive signals were mapped to corresponding protein features of Coomassie- stained gels. These features were excised and processed for identification via peptide mass fingerprinting.
  • Coomassie- stained gel pieces were destained in 100 mM NH 4 CO 3 /50% acetonitrile at 37 °C for 90 min. Slices were dehydrated in 100% acetonitrile (5 min, at room temperature), followed by drying under vacuum. The dehydrated gel pieces were rehydrated in a minimal volume of a solution of 40 mM NH 4 CO 3 /50% acetonitrile containing 20 ng/ ⁇ trypsin (Trypsin Gold, Mass Spec Grade; Promega, Madison, WI, USA) and incubated overnight at 37°C.
  • Peptide fragments were recovered from the gel slices by sequential washes with 75 ⁇ 1% trifluoroacetic acid (TFA) (1 h) and 50 ⁇ 5% TF A/50% acetonitrile (two washes, 1 h per wash). The washes were pooled, dried under vacuum, and then dissolved in 10 ⁇ of 1% TFA.
  • TFA trifluoroacetic acid
  • Tryptic digests were mixed 1: 1 with a-cyanohydroxycinnamic acid matrix (10 mg/ml in 50% acetonitrile/0.1% TFA) containing bradykinin (1060.5692 Da) and adrenocorticotropic hormone fragment 18-29 (2465.1989 Da) (AnaSpec, San Jose, CA, USA) as internal standards and analyzed by matrix- assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, using a Voyager MALDI-TOF DE STR instrument (PE Biosystems). Peptide mass spectra data were analyzed via the Protein Prospector MS-Fit search engine (http://prospector.ucsf.edu/).
  • MALDI-TOF matrix- assisted laser desorption ionization time-of-flight
  • Criteria for a positive identification were a MOWSE score greater than 1.00x10 and more than two times greater than any other identifications for the search, a coverage percentage greater than 25%, and a matched protein identifying with the appropriate molecular weight and p/ for the fractionation procedure.
  • a sample or gel can be reacted with a biotin- or hapten-hydrazide, with the one or more of the proteins of interest being measured by a sandwich assay using at least one antibody against the one or more protein(s) and/or one or more antibody(ies) against the biotin or hapten.
  • a protein-specific antibody may be employed as the capture antibody.
  • a sample or gel can be reacted with-instead of the hydrazide-a carbonylation specific antibody, as disclosed in Xu, et al. "Detecting Protein Carbonylation in Adipose Tissue and in Cultured Adipocytes", Methods Enzymol, 2014: 538: 249-261, which is incorporated herein by reference.
  • detection can be accomplished by Wetern Blot, using instruments and methods as discussed herein and as disclosed in Lazarus, et al. "Protein
  • MRM Reaction Monitoring
  • Euthanized animals were perfused transcardially with 200 ml phosphate-buffered saline (PBS) followed by 200 ml 4% paraformaldehyde for fixation. Brains were stored in 4% paraformaldehyde overnight at 4°C and then equilibrated in a 30% sucrose solution (-48 h, 4°C). Coronal sections (20 ⁇ thick) were collected from 2.5 mm rostral to 2.5 mm caudal to the CCI lesion site and mounted onto Colormark Plus adhesion slides (Thermo Fisher Scientific). Every tenth section was stained with thionine to create a reference library.
  • PBS phosphate-buffered saline
  • ⁇ -casein protein was chemically carbonylated by incubation with ferric chloride (FeCl 3 ) (15 mg in 2 ml 37.5 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes), pH 7.2, containing 37.5 mM ascorbate and 0.15 mM FeCl 3 ; 5 h, 37°C).
  • FeCl 3 ferric chloride
  • Hepes 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
  • the reaction mixture was then dialyzed against 50 mM Hepes and 1 mM ethylenediaminetetraacetic acid (pH 7.2) overnight at room temperature.
  • a control reaction was conducted similarly with the absence of FeCl 3 .
  • Derivatization of protein carbonyl groups was carried out by combining the protein dialysates with an equal volume of 12% sodium dodecyl sulfate and an equal volume of 10 mM DNPH prepared in 1 N HC1. Reactions proceeded in the dark for 15 min at room temperature and were then neutralized with the addition of 3.25 ml 2 M Tris-HCl. The derivatized proteins were dialyzed against H 2 0 overnight. The carbonylation status of the two preparations was confirmed by Western blot using anti-DNP immunodetection.
  • Preadsorption of the primary antibody was carried out by incubating anti-DNP IgG (-2 ⁇ g) with carbonylated, DNPH-derivatized ⁇ -casein (-100 ⁇ g protein) at 4°C, overnight.
  • An additional control condition was established by similarly incubating anti-DNP antibody in the absence of modified ⁇ -casein protein.
  • Carbonylation immunohistochemistry was quantified according to region using a blinded scoring system, with 0 reflecting the lowest amount of carbonylation fluorescence intensity in a region and 3 reflecting the greatest amount across all sections evaluated. Regions scored were the cortical lesion site, dorsal third ventricle, and median eminence. Sixteen brain sections per animal were scored using sections located from bregma -2.12 mm to bregma -3.30 mm
  • CCI increases protein carbonylation in specific regions of the brain
  • FIG. 5 presents a representative analysis in which proteins present in control and TBI extracts were fractionated by fluid-phase IEF, followed by one-dimensional gel electrophoresis. Proteins having enhanced carbonylation after injury were visualized by Western blotting for DNP, mapped to corresponding features on Coomassie-stained gels, and identified as GFAP, dihydropyrimidase-related protein 2 (also known as collapsing response mediator protein 2, or CRMP2), fructose-bisphosphate aldolase C (ALDOC), and fructose-bisphosphate aldolase A (ALDOA).
  • CRMP2 dihydropyrimidase-related protein 2
  • ADOC fructose-bisphosphate aldolase C
  • ADOA fructose-bisphosphate aldolase A
  • Immunolabeling was concentrated in the ependymal cells and processes lining the floor of the third ventricle at the median eminence of CCI animals (Figure 8, panel D). This pattern of intense labeling was not observed in comparable sections prepared from uninjured control animals ( Figure 8, panel C). Together, these immunohistochemical findings indicate that discrete regions of the brain exhibit an upregulation of protein carbonylation after CCI. This response is most pronounced in the cortical area surrounding the area of injury and in the ependymal areas of the dorsal third ventricle/paraventricular region and the median eminence.
  • CCI-induced protein carbonylation is most significantly associated with astrocytes in the region of a CCI lesion.
  • Brain tissue was collected 5 days after CCI and evaluated by coimmunofluorescence.
  • A-C Representatives of the astrocyte and DNP detection at the injury site
  • D-F representatives of neuron and DNP detection at the injury site.
  • Carbonylated proteins within the penumbra region were identified by anti-DNP
  • Gender influences regional patterns of protein carbonylation
  • a number of studies have indicated that gender plays a role in determining the consequences of oxidative injuries of the brain (13; 118). The present investigation sought to determine whether gender affects protein carbonylation after CCI.
  • a scoring system was developed to quantitatively assess the effects of CCI on protein carbonylation and to investigate the influence of gender on the response. The scoring system was based on overall intensity and extent of anti-DNP immunofluorescence detected in each region. Scores were developed on a 0- 3 scale for each region examined. A score of 0 reflected the basal state of carbonylation observed in control animals and a score of 3 was assigned to the greatest signal observed for a given region, across all animals studied. Representative images for scoring of each region of interest are presented in Figure 10.
  • the response in the injury area was six-fold greater than that observed in regions of cerebral cortex distant from the lesion site ( DDD /? ⁇ 0.0001).
  • the carbonylation score for the contralateral cortex of injured animals was not different from the score observed in the sham animal group. There were no gender differences observed across cortical regions compared here.
  • Protein carbonylation is a well-documented consequence and measureable indicator of oxidative stress in multiple neuropathologies, including Alzheimer's disease and Parkinson's disease (25). Recently, interest has focused on the role of oxidative stress in the pathology of TBI (66). To date, research has concentrated on measures of global carbonylation within the central nervous system, whereas little work has explored the neural regions, cell types, and specific proteins affected by this modification. Furthermore, although significant research has identified gender-based differences in TBI prognosis (52; 118), few researchers have examined how gender affects the magnitude or regional distribution of protein carbonylation after TBI.
  • TBI The pathophysiology of TBI is both complex and dynamic, involving physical injury, ischemia/reperfusion, hypoxia, glutamate excitotoxicity, intracellular calcium overload, and elevated intracranial pressure (34; 136; 145; 152).
  • ROS ROS
  • These molecules drive the formation of RCS, such as acrolein and 4-hydroxynonenal, which are by-products of lipid peroxidation (127; 128).
  • RCS catalyze the generation of intra-protein carbonyls, further compromising cellular functions and exacerbating injury pathology.
  • Astrocytes have been proposed to be the first line of defense against oxidative stress after acute TBI (42; 96; 107). Astrocytes play a key role in balancing the oxidative load of the brain after injury, through their ability to scavenge ROS and thereby buffer the spread of oxidative stress (102). Whereas astrocytes have the capacity to combat oxidative stress to the benefit of other adjacent cells, they themselves are still vulnerable to the actions of ROS, which can cause mitochondrial damage, accelerated senescence, and cell death (30; 68).
  • astrocytes are the dominant cell type showing increased protein carbonylation post-CCI. Whereas some neurons exhibited increased protein carbonylation after CCI, this response was minimal in comparison to astrocytes.
  • the carbonylation status of oligodendrocytes and microglia and macrophages in the lesion area was essentially unaffected by CCI.
  • Ependymal cells form a barrier between cerebrospinal fluid (CSF) and neural tissue, acting as both a physical and a metabolic interface (41),
  • CSF cerebrospinal fluid
  • the mechanism by which oxidative damage is transmitted to specific regions of the ependymal system is not fully understood, but may involve: (1) the transport of reactive molecules from the site of injury via the CSF and (2) a selective sensitivity of the ependymal lining of the dorsal third ventricle and median eminence to these mediators.
  • CSF-borne molecules include 4-hydroxynonenal, acrolein, and malondialdehyde, all of which are highly reactive aldehyde products of ROS -catalyzed lipid peroxidation.
  • the ependymal lining at the base of the third ventricle, directly above the median eminence, is well known for its unique structure (106), neurogenic cell populations (74), and neuroendocrine regulation (113). Therefore, we propose that aldehyde by-products of lipid peroxidation are generated by ROS at the site of acute injury, are transported through the cerebroventricular system, and interact with susceptible, CSF-adjacent periventricular ependymal cells to propagate carbonylated protein species.
  • GFAP a major component of astrocytes
  • ALDOA oxidative modification after injury
  • ALDOC a major component of astrocytes
  • GFAP, CRMP2, and ALDOC have all been identified as being carbonylated in Alzheimer's disease (29; 139), indicating a potential role for this modification in long-term neuropathology.
  • Recent work by Oikawa and associates (99) examining the effects of ischemia/reperfusion injury on the carbonylated proteome of the monkey hippocampus also showed enhanced carbonylation of CRMP2 and ALDOC.
  • GFAP is a major target for carbonylation in experimental autoimmune rhinitis
  • encephalitis a murine model of multiple sclerosis (155). All four of the proteins identified in this work are also functionally grouped, as they all contribute to the maintenance of cellular structure (46; 59; 85; 110). In addition, CRMP2 functions in axonal guidance, serving an important role in neuronal regeneration after injury (142). Taken together, these findings suggest that carbonylation may provide a proteomic basis for mechanisms of sustained pathology after TBI. Understanding the mechanisms, nature, localization, and timing of protein modifications after TBI may provide a guide for the development of more effective TBI therapies that target both oxidative stress and its downstream consequences.
  • the disclosure incudes detection of the following carbonylated proteins GFAP, CRMP2, ALDOA and ALDOC.
  • two of any of the above proteins are determined, three of any of the above proteins are determined or four of any of the above proteins are determined.
  • carbonylated proteins can be detected in any suitable biological sample, including but not limited to blood, blood plasma, blood serum, cerebrospinal fluid (CSF), urine, lymph and/or saliva.
  • suitable biological sample including but not limited to blood, blood plasma, blood serum, cerebrospinal fluid (CSF), urine, lymph and/or saliva.
  • CSF cerebrospinal fluid
  • Protein citruUination A proposed mechanism for long-term pathology in traumatic brain injury
  • Protein citruUination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS) and prion disease. CitruUination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citruUination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citruUination and on the specific cell types involved.
  • Traumatic brain injury is a major cause of injury and death in the US, with over 1.7 million TBIs occurring annually and at least 5.3 million Americans currently living with ongoing disability (149). Traumatic brain injuries in civilians are largely due to automobile accidents, as well as falls, sports, and firearms (152). Military populations are at disproportionately elevated risk for blast-related TBI due to the devastating effects of improvised explosive devices (58). While there is a very large body of information on causes and global consequences of TBI, much less is known about the mechanisms underlying long-term pathology.
  • TBI long-term consequences of TBI can be complex, and often result in progressive cognitive and behavioral changes. Studies have indicated that anywhere from 10% - 50% of individuals with TBI suffer from persistent symptoms following injury (11), including attention deficits and short-term memory loss (149). This long-term dysfunction follows in the wake of two main injury phases: (1) the primary injury, caused by the immediate forces of the trauma (111; 152); and (2) the subsequent secondary injury, which presents as a constellation of dysfunctional molecular processes including impaired metabolism, free radical production, inflammation, and glutamate excitotoxicity (149). At present, it is not well understood how these various dysfunctional processes following acute injury can lead to progressive, chronic pathology after TBI.
  • a hallmark mechanism of secondary injury following TBI is prolonged imbalance in cellular calcium homeostasis, resulting in excitotoxic calcium overload (48; 84; 141). Following TBI, ischemia can lead to a dramatic loss of glucose and oxygen delivery to the central nervous system, thereby limiting the production of ATP. In the absence of adequate energy, ionic homeostasis is lost and calcium toxicity ensues.
  • the abnormally large increase in intracellular calcium originates from both the influx from the extracellular space across multiple calcium channels, and from the mobilization of intracellular calcium stores (143). The result is a pathologically prolonged state of elevated intracellular calcium (141).
  • Alzheimer's disease 62
  • temporal lobe epilepsy (8)
  • glaucoma 18
  • rheumatoid arthritis 77
  • multiple sclerosis (6).
  • CCI was conducted as described in Lazarus et al. (2015). Briefly, adult male and female Sprague-Dawley rats (Charles River Laboratories, Morrisville, North Carolina, USA) were anesthetized, then subjected to unilateral CCI over the left hemisphere administered through a ImpactOne stereotaxic impactor (Leica Microsystems, Buffalo Grove, Illinois, USA), which delivered a 3 mm flat-tipped impactor at 20° to a depth of 2 mm at 5 m / s with a 500 ms dwell time at -3.8 mm bregma in males / - 3.0 mm bregma in females. Sham-operated animals received no anesthesia or CCI treatment.
  • ImpactOne stereotaxic impactor Leica Microsystems, Buffalo Grove, Illinois, USA
  • Tissue was collected and prepared as described previously (73). Briefly, euthanized animals underwent transcardial perfusion (phosphate buffered saline (PBS) followed by 4% paraformaldehyde) after which brains were removed for storage overnight at 4°C in 4% paraformaldehyde and then equilibration in a 30% sucrose solution (2 days, 4°C). Brains were sectioned coronally (20 ⁇ ) across the breadth of the lesion site (2.5 mm rostral to 2.5 mm caudal) with a Leica CM1900 cryostat (Leica Microsystems), and sections were then mounted on slides and stored at -80°C.
  • PBS phosphate buffered saline
  • Leica Microsystems Leica Microsystems
  • mAb 6B3, IgG2b mouse monoclonal anti-citrulline antibody
  • the antibody was purified from expression medium by Protein A affinity chromatography (HiTrap Protein A HP column (17-0403-01; GE Healthcare, Buckinghamshire, United Kingdom) on a GE AKTA FPLC fast protein liquid chromatography instrument (FPLC; 18-1900-26; GE Healthcare), aliquoted for single-use and stored at -80°C.
  • the sections were washed five times with 0.2% Triton X-100/PBS and one time with lx PBS (5 m) and then visualized with an Olympus BX61 fluorescent motorized system microscope (Olympus, Shinjuku, Tokyo, Japan) using iVision-Mac software (Bio Vision Technologies, Exton, Pennsylvania, USA).
  • Tris buffer 50 mM Tris HCl, pH 7.4; 5 mM CaCl 2 ; and 0.73 mM dithiothreitol (DTT; Sigma-Aldrich, St. Louis, MO, USA)).
  • control sample was prepared in an identical manner, without the addition of the PAD enzyme cocktail.
  • Brains were collected, snap-frozen with powdered dry ice, and stored at -80°C until use. Brains were thawed on wet ice and then hand-dissected to produce blocks of tissue
  • Tissue blocks were homogenized in 5 volumes / tissue weight extraction solution, consisting of: 7.7 M urea; 2.2 M thiourea; and 4.4% CHAPS; also containing lx Complete protease inhibitor mix (Roche).
  • Proteins in each of the F-IEF fractionations were further resolved by molecular weight fractionation using conventional one-dimensional gel electrophoresis.
  • Samples were combined with an equal volume of 4x reducing loading buffer (Novex NuPAGE LDS sample buffer; 50 mM DTT; Invitrogen), heated at 70°C (20 min) and then fractionated (10 ⁇ per well) using NuPAGE 4 - 12% Bis-Tris gels (Novex, Invitrogen). Proteins were transferred to nitrocellulose blots using an iBlot transfer apparatus & gel transfer stacks (Nitrocellulose Mini; 1B301002, Invitrogen).
  • blots were reprobed with a second anti-protein citrulline antibody (1:500; MABN328EMD; Millipore; detection with horseradish peroxidase-labeled, goat anti- mouse IgM; 1:2500 in TBS-T; 31440, Thermo Scientific), with final overnight washing, to confirm the 6B3 immunoreactive features and increase the sensitivity of detection. No new signals were revealed by this approach.
  • the amount of protein absorbed to the strip was 200 ⁇ 1 / 200 ⁇ g of citruUinated human fibrinogen (400076, Cayman Chemical, Ann Arbor, Michigan, USA), or 200 ⁇ 1 / 200 ⁇ g human fibrinogen (16088, Cayman Chemical) in TBS-T.
  • the duration of the antibody absorption was 16 h at 4°C.
  • Astrocytes were cultured to -70% confluence in T-75 tissue culture flasks according to vendor instructions. Cells were washed with TBS (4 times) and then treated for 4 h (37°C, 95% 0 2 /5% C0 2 ) with either ionomycin (10 ⁇ ; 10 mL TBS) or dimethyl sulfoxide vehicle (DMSO; 10 ⁇ 1/10 mL TBS). Following incubation, protease inhibitors were added to the flasks (final concentration of lx; Complete protease inhibitor mix; 10269700, Roche) and cells were harvested by scraping and centrifugation (800 x g, 4°C, 10 min).
  • Each primary antibody was paired with an appropriate secondary antibody (6B3: horseradish peroxidase-labeled, goat anti-mouse IgG; 1:2500; 31430; Thermo Scientific; GFAP: horseradish peroxidase-labeled, goat anti-rabbit IgG; 1:3000; HAF008; R&D Systems) and blots were visualized by ECL.
  • 6B3 horseradish peroxidase-labeled, goat anti-mouse IgG; 1:2500; 31430; Thermo Scientific
  • GFAP horseradish peroxidase-labeled, goat anti-rabbit IgG; 1:3000; HAF008; R&D Systems
  • Figure 14 presents the effects of CCI on the expression of citruUinated proteins in the rat cerebral cortex.
  • Immunohistochemical analysis demonstrated that the basal level of protein citrullination as detected by labeling with mAB 6B3 was very low under control conditions.
  • CCI induced a marked increase in the immunolabeling of cells in the cortex. This up- regulation of protein citrullination was most pronounced in the vicinity of the lesion. The morphology of the affected cells was remarkably consistent, suggesting that the effects of CCI on protein citrullination were cell- specific.
  • Dual immunofluorescence revealed astrocytes to be the principal cell type in which protein citrullination was affected by CCI.
  • Figure 17 shows that anti-citrulline labeling in the cortex and external capsule was predominantly colocalized with GFAP. Similar observations were made in other affected brain regions.
  • the contralateral hippocampus also displayed intense 6B3 labeling (Figure 19, panel D) that colocalized with GFAP labeling.
  • the co-labeled cells were morphologically distinct from traditional stellate astrocytes, displaying a rounded, branchless appearance consistent with that of a macrophage ( Figure 19, panels E-G).
  • Figure 20 demonstrates the specificity of the 6B3 antibody for detecting citrullinated proteins in a western blot format. Shown on the left is the Coomassie staining for human fibrinogen (Fib) and the same preparation of fibrinogen that was enzymatically citrullinated by reaction with PAD4 (C-Fib). The protein staining shows that the characteristic profile of purified human fibrinogen is modestly affected by reaction with PAD4.
  • cytoskeletal components including dynamin-1, GFAP, and several forms of tubulin
  • those involved in metabolic processes including peroxiredoxin-1, dihydropyrimidinase-related protein 2, and creatine kinase B-type
  • proteins involved in cell-cell signaling and synaptic transmission include synapsin-2, syntaxin-binding protein 1, and amphyiphysin.
  • the disclosure includes determining the citrullination levels of one or more of the following proteins or protein fragments (as listed in FIG. 21) fructose-bisphosphate aldolase A, peroxiredoxin-1, cofilin-1, glutathione S-transferase P, transgelin, dynamin-1, synapsin-2, moesin, septin-9, stress-induced phosphoprotein 1, dihydropyrimidase-related protein 2, alpha- enolase, gamma-enolase, glial fibrillary acidic protein, fructose-bisphosphate aldolase C, neuronal- specific septin-3, transgelin-3, myelin basic protein, heat shock 70 kDa protein 4, GRIP 1- associated protein 1, transitional endoplasmic reticulum ATPase, NADH-ubiquinone oxidoreductase 75 kDa mitochondrial subunit, vesicle-fusing ATPase, 78 k
  • two of any of the above proteins are determined, three of any of the above proteins are determined, four of any of the above proteins are determined, five of any of the above proteins are determined, six of any of the above proteins are determined, seven of any of the above proteins are determined, eight of any of the above proteins are determined, nine of any of the above proteins are determined, ten of any of the above proteins are determined, eleven of any of the above proteins are determined, twelve of any of the above proteins are determined, thirteen of any of the above proteins are determined, fourteen of any of the above proteins are determined, fifteen of any of the above proteins are determined, sixteen of any of the above proteins are determined, seventeen of any of the above proteins are determined, eighteen of any of the above proteins are determined, nineteen of any of the above proteins are
  • citruUinated proteins can be detected in any suitable biological sample, including but not limited to blood, blood plasma, blood serum, cerebrospinal fluid (CSF), urine, lymph, and/or saliva.
  • CSF cerebrospinal fluid
  • FIG. 22 shows the results of 3 separate experiments investigating the effects of ionomycin treatment on the proteolytic processing of GFAP (left panel) and the generation of citruUinated proteins (right panel). The data show that treatment with ionomycin consistently activated the proteolytic processing of intact GFAP (left panel; blue arrows) to produce a distinctive pattern of breakdown products.
  • Protein citruUination is a calcium-dependent protein modification that has been largely studied in the context of autoimmune disorders, particularly rheumatoid arthritis. Abnormal protein citruUination in rheumatoid arthritis results in the generation of antigenic epitopes that become targeted by the adaptive immune system (55; 69). Protein citruUination is also found to be abnormal in several neurodegenerative disorders, including multiple sclerosis (MS) and Alzheimer's disease, suggesting that citruUinated proteins may also serve as antigenic targets in these conditions and thereby contribute to long-term pathogenesis. To date, however, little research has examined the effects of TBI on protein citruUination, where an acute mechanical injury can lead to progressive and sustained neuropathology. Currently, there are no data regarding the identity of specific proteins affected by citruUination following TBI, or information on the susceptibility of different brain regions or cell types to this modification following injury.
  • TBI intracranial pressure
  • a hallmark of acute TBI pathophysiology is intracellular calcium overload (49). This shift in ionic balance is excitotoxic, activating a number of damaging intracellular cascades, including lipid peroxidation, proteolysis, free radical generation (152), and the activation of pro- apoptotic genes (149) with the loss of dendritic spines. Elevated intracellular calcium is an essential condition for the activation of peptidylarginine deiminase (PAD) enzymes, which catalyze the conversion of intrapeptidyl arginine residues to citrulline residues (61).
  • PAD peptidylarginine deiminase
  • PAD enzymes exist as several isoforms, two of which are found in neural tissue: PAD2 (37; 64; 109) and PAD4 (2).
  • PAD2 is largely localized to astrocytes, while PAD4 is exclusively expressed in neurons (2).
  • PAD2 is localized to reactive astrocytes in the brains of mice affected by a prion disorder, scrapie, where its enzymatic activity is nearly double that of healthy mice (64).
  • Research on the etiology of MS has suggested that PAD2 contributes to the pathology of demyelination, where its activity is thought to destabilize myelin sheath structure (89).
  • the selective citrullination of astrocytes in these particular regions may correspond to long-term dysfunctions associated with TBI, including learning and memory deficits associated with progressive hippocampal atrophy (149).
  • citrullinated GFAP is a characteristic feature in MS and Alzheimer's disease (24; 55; 62; 94), and myelin basic protein (MBP), a major component of myelin sheath structure, is profoundly over-citrullinated in MS (24; 144).
  • MBP is the signature citruUinated autoantigen of MS (23; 144).
  • Autoantibodies targeting amphiphysin are associated with several neurological disorders, including sensory neuronopathy and encephalopathy (88). Additionally,
  • dihydropyrimidinase-related protein 2 (CRMP2) is autoantigenic in autoimmune retinopathy (3).
  • alpha enolase has been identified as a citruUinated autoantigen in rheumatoid arthritis (71), while citruUinated 78 kDa glucose-regulated protein is an autoantigen within the pancreatic beta-cells in Type 1 diabetes (115).
  • this report presents the development of an in vitro model for simulating TBI in a controlled and cell-specific manner. Shown here in normal human astrocytes are the effects of calcium excitotoxicity, a hallmark condition of TBI, on the hyper-citrullination of GFAP. Moreover, the findings confirm the proteolytic processing of GFAP to a series of breakdown products that are consistent with those reported by others using in vitro models of TBI (100; 156). Interestingly, our investigation showed that one of these breakdown products is heavily citrullinated, and thus may serve as the antigen for the development of the anti-GFAP autoantibodies recently reported in TBI (153). Accordingly, the application to this and other models for simulated TBI may provide novel insights into consequences and mechanisms of TBI, and also identify informative biomarkers for assessing brain injury.
  • TBI dramatically up-regulates protein citruUination within astrocytes in specific brain regions. Additionally, this modification affects only a subset of the neural proteome, primarily affecting proteins involved in cytoskeletal structure, metabolic processes, and cell-cell signaling. A large proportion of these proteins have been identified as citrullinated in other pathologies, including MS, Alzheimer's disease, and rheumatoid arthritis, indicating a potential role for this protein modification in ongoing pathological processes. Interestingly, gender does not affect the degree or distribution of this modification in neural tissue, in contrast to previous observations involving TBI-induced protein carbonylation. Accordingly, gender differences in the CNS response to TBI may not involve differential responses in protein citruUination. In summary, this research indicates that abnormal protein citruUination is a feature of TBI that could contribute to ongoing pathological mechanisms following acute injury, possibly including aspects of autoimmune dysfunction.
  • ROS reactive oxygen species
  • Reactive oxygen species can catalyze carbonylation through either direct interaction with proteins, or through the reaction of proteins with reactive carbonyl species, which are formed by the reaction of reactive oxygen species with polyunsaturated fatty acids (127; 128; 155).
  • Protein citrullination in contrast, is not dependent on the presence of reactive oxygen species or reactive carbonyl species, but rather on the action of peptidylarginine deiminase (PAD).
  • PAD peptidylarginine deiminase
  • This family of enzymes is calcium-dependent: as calcium binds to PAD, a conformational change in the protein takes places, moving a catalytic residue into the enzyme's active site to act on arginine residues within other proteins (120). The reaction of PAD with arginine residues converts this amino acid to citrulline.
  • Both carbonylation and citruUination can disrupt normal cellular physiology through a number of changes, including enzymatic inactivation (54), protein aggregation (25), and proteasomal inhibition (25).
  • CITRULLINATION Five days following TBI in a rodent model of CCI, protein carbonylation and citrullination were expressed in specific cell types. Protein carbonylation was selectively expressed in astrocytes and ependymal cells, while protein citrullination was enhanced only in astrocytes. Further investigation revealed that neither of these post-translational modifications was significantly associated with neurons, oligodendrocytes, or microglia / macrophages, as indicated by co-localization with antibodies targeting NeuN, MBP and Ibal, respectively. As discussed in Chapter 3, our in vitro model of simulated TBI utilized the calcium ionophore, ionomycin, to induce excitotoxic calcium overload in normal human astrocytes.
  • TBI induces protein carbonylation in ependymal cells of the dorsal third ventricle and median eminence
  • Ependyma are specialized cells forming the barrier between cerebrospinal fluid and neural tissue, acting as both a metabolic and physical interface between these two spaces (41). Interestingly, only certain populations of ependymal cells expressed increased protein carbonylation following TBI. This may reflect the broad heterogeneity of this cell type, as regards structure and function (12; 65), expression of membrane proteins (12), and differential vulnerability to CSF-borne toxins (65). The populations of affected ependyma lined the dorsal third ventricle and above the median eminence, at sites directly adjacent to CSF-filled cavities.
  • Lipid peroxidation occurs as a very early event following TBI, with levels of peroxidation by-products elevated in neural tissue just thirty minutes following injury (7).
  • astrocyte vulnerability involves the complex interaction between these cells and calcium.
  • TBI a decrease in cerebral blood flow limits the amount of oxygen and glucose delivered to the brain, resulting in hypoxia and lack of metabolic support.
  • astrocytes experience large elevations in intracellular calcium (44), to a much greater degree than the calcium influxes observed in neurons (121).
  • ischemia the disruption of cell membranes and mechanical stretching of cellular components leads to a rush of extracellular calcium into astrocytes. Additionally, mechanisms that normally offset the effects of extracellular glutamate are impaired (149).
  • the glutamate-rich extracellular environment causes the activation of NMD A receptors (51), allowing a massive influx of calcium into cells. This influx further exacerbates the dramatically increased intracellular concentration of calcium due to direct injury (111; 149), causing a failure in energy metabolism (51) and the release of sequestered intracellular calcium from internal stores, including the endoplasmic reticulum and mitochondria (109; 143). While the precise response of astrocytes to calcium excitotoxicity is not fully defined, research has shown that astrocytes may exert a protective effect on neuronal viability by insulating these terminally differentiated cells from the effects of excessive extracellular glutamate (49), such that the accumulation of calcium by astrocytes serves to reduce extracellular calcium, and thus its damaging effects on neurons (44).
  • citrullinated proteins may clear from the injury site sooner than carbonylated proteins.
  • This rapid clearance of citrullinated proteins may be due to at least two possible mechanisms.
  • citruUination can alter tertiary structure to a more "open” conformation, and thereby increase protein susceptibility to proteolytic degradation (87).
  • protein citruUination may prompt cellular apoptosis (82), resulting in few citrullinated protein-containing cells remaining by day five after TBI. This possibility assumes that there is a measure of cell specificity in these citruUination and carbonylation responses.
  • these three regions are particularly susceptible to protein citruUination due to their unique activation of these calcium channels, which mediate calcium influx into astrocytes following hypoxia and ischemia (33).
  • Protein carbonylation was not expressed in these regions at five days following injury, suggesting that oxidative stress in these regions is relatively low as compared to the proximal lesion site. This may be due to regional variations in cellular physiology including: variable antioxidant capacities; relative metabolic needs of each region; and differing levels of reactive metal ions, including copper and iron, which can drive metal-catalyzed protein carbonylation.
  • Neurons do not express protein citruUination after TBI
  • ovarian steroids may protect against the generation of reactive oxygen species and reactive carbonyl species.
  • females recover better following TBI as compared to males (16; 52; 118).
  • TBI- induced protein citruUination predicts that males and females are affected equally; however, this is not the case.
  • females are more profoundly affected than males. This suggests that factors in addition to gender contribute to the chronic pathology of these two conditions.
  • the present experimental design may have investigated a time point that simply was not informative for gender differences in long-term pathology caused by abnormal citruUination.
  • proteomic analyses reported here provide the first insights into the carbonylation and citruUination proteomes of TBI.
  • four proteins were identified as having enhanced carbonylation following injury: (1) GFAP; (2) CRMP2; (3) ALDOC; and (4) ALDOA.
  • These proteins have also been identified as carbonylated in other neurodegenerative disorders, including Alzheimer's disease (GFAP, CRMP, ALDOC) (29; 139),
  • ischemic/reperfusion injury CRMP2, ALDOC
  • GFAP multiple sclerosis
  • the modified proteins identified here have essential roles in cellular repair, structure, metabolism, and signaling.
  • CRMP2 functions in axonal guidance, serving an important role in neuronal regeneration after injury (142), while GFAP is a major structural component of astrocytes.
  • GFAP is a major structural component of astrocytes.
  • dynamin-1 and several forms of beta-tubulin.
  • Another functional group of modified proteins are those involved in metabolic processes, including peroxiredoxin-1 and creatine kinase b.
  • citrullination proteins identified have established roles in cell-cell signaling, including synapsin-2, syntaxin-binding protein 1, and amphiphysin.
  • citrullination alters the tertiary structure of myelin basic protein, reshaping this protein into a more open conformation (87). While this alteration is shown to increase the protein's susceptibility to protease degradation (87), it may also allow access of reactive oxygen species and reactive carbonyl species to previously inaccessible amino acids which may now be carbonylated.
  • a second possibility is that the "double" identification of proteins actually reflects two separate pools of modified proteins. For instance, rather than a single pool of CRMP2 that is both carbonylated and citrullinated, it is possible that we have identified two individual groups, one carbonylated and the other citrullinated. If this is the case, then these separate post- translational groups may reflect the distinct regions affected by each modification, as revealed by our immunohistochemical investigations.
  • citrullination is less studied in the context of neurological pathologies, this modification is known to have significant effects on protein antigenicity. Specifically, in addition to changing structure, citrullination also changes the charge and cleavage sites of affected proteins, leading to the presentation of novel, potentially antigenic epitopes (148). This phenomenon is observed in multiple sclerosis, where the citrullination of myelin basic protein prompts myelin degeneration due to a loosened aggregation of lipids in the sheath structure (89).
  • CitruUination of myelin basic protein also increases its susceptibility to degradation by proteinases, including cathespin D, leading to the formation and release of antigenic, citrulline- containing peptides which are presented to peripheral T cells (23).
  • proteinases including cathespin D
  • citrulline- containing peptides which are presented to peripheral T cells (23).
  • a small fraction of total myelin basic protein contains citrullinated residues; however, this amount increases three-fold in multiple sclerosis, inducing a targeted T cell response (5; 144).
  • Citrullinated epitopes are the hallmark of the adaptive immune response in rheumatoid arthritis. CitruUination in this disorder elicits a dramatic production of targeted autoantibodies, to such a significant degree that anti-citrullinated protein antibodies serve as a diagnostic biomarker for this disease (57). Interestingly, two other proteins identified in this investigation are targeted as antigens in separate pathological conditions. Glucose-regulated protein 78 was identified in this research as citrullinated following TBI. Previous work has shown that the citrullinated form of this protein is an autoantigen in Type 1 Diabetes, where modified glucose-regulated protein 78 is secreted by pancreatic beta cells and prompts the production of autoantibodies (115).
  • the chronic demyelination noted in both multiple sclerosis and following TBI could reflect this shared protein modification, which would suggest a mechanistic link between acute, TBI- driven protein modification and chronic structural changes.
  • the carbonylation of CRMP2 following TBI also represents a possible link for shared symptomatology.
  • This protein which is essential for axonal growth and neural regeneration, is carbonylated in the hippocampus in Alzheimer's disease (29), indicating a possible mechanistic role for CRMP2 carbonylation in the memory deficits seen in both this disorder and following TBI.
  • proteomic analyses of specific regions could reveal biological correlates of region-related cognitive deficits, including memory dysfunction and loss of executive function.
  • region-related cognitive deficits including memory dysfunction and loss of executive function.
  • protein carbonylation and citruUination represent critical mechanistic links between acute physical injury and long-term pathology.
  • a rigorous understanding of these proteomic shifts following TBI will lend insight into the biological mechanisms of this injury and may also point to preventative measures to counteract long-term pathogenesis.
  • Protein citruUination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS) and prion disease. CitruUination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citruUination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citruUination and on the specific cell types involved.
  • Traumatic brain injury is a major cause of injury and death in the US, with over 1.7 million TBIs occurring annually and at least 5.3 million Americans currently living with ongoing disability (Walker and Tesco, 2013). Traumatic brain injuries in civilians are largely due to automobile accidents, as well as falls, sports, and firearms (Werner and Engelhard, 2007). Military populations are at disproportionately elevated risk for blast-related TBI due to the devastating effects of improvised explosive devices (Hoge et al., 2008). While there is a very large body of information on causes and global consequences of TBI, much less is known about the mechanisms underlying long-term pathology.
  • TBI long-term consequences of TBI can be complex, and often result in progressive cognitive and behavioral changes. Studies have indicated that anywhere from 10% - 50% of individuals with TBI suffer from persistent symptoms following injury (Bales et al., 2009), including attention deficits and short-term memory loss (Walker and Tesco, 2013). This long- term dysfunction follows in the wake of two main injury phases: (1) the primary injury, caused by the immediate forces of the trauma (Ray et al., 2002;Werner and Engelhard, 2007); and (2) the subsequent secondary injury, which presents as a constellation of dysfunctional molecular processes including impaired metabolism, free radical production, inflammation, and glutamate excitotoxicity (Walker and Tesco, 2013).
  • citruUination In addition to altering both the normal structure and function of proteins, citruUination generates "altered-self ' epitopes that may be antigenic, prompting autoimmune responses against previously benign proteins (Zhao et al., 2010;Chirivi et al., 2013).
  • Altered calcium homeostasis accompanied by protein citruUination has been implicated in several neurodegenerative disorders, including Alzheimer's disease (Ishigami et al., 2005), temporal lobe epilepsy (Asaga and Ishigami, 2001), glaucoma (Bhattacharya et al., 2006), rheumatoid arthritis (Luban and Li, 2010), and multiple sclerosis (Anzilotti et al., 2010).
  • CCI Controlled Cortical Impact
  • Tissue was collected and prepared as described previously (Lazarus et al., 2015).
  • euthanized animals underwent transcardial perfusion (phosphate buffered saline (PBS) followed by 4% paraformaldehyde) after which brains were removed for storage overnight at 4°C in 4% paraformaldehyde and then equilibration in a 30% sucrose solution (2 days, 4°C).
  • Brains were sectioned coronally (20 um) across the breadth of the lesion site (2.5 mm rostral to 2.5 mm caudal) with a Leica CM 1900 cryostat (Leica Microsystems), and sections were then mounted on slides and stored at -80°C.
  • Tris buffer 50 mM Tris HC1, pH 7.4; 5 mM CaCl 2 ; and 0.73 mM dithiothreitol (DTT; Sigma-Aldrich, St. Louis, MO, USA)).
  • control sample was prepared in an identical manner, without the addition of the PAD enzyme cocktail.
  • Brains were collected, snap-frozen with powdered dry ice, and stored at -80°C until use. Brains were thawed on wet ice and then hand-dissected to produce blocks of tissue
  • tissue blocks were homogenized in 5 volumes / tissue weight extraction solution, consisting of: 7.7 M urea; 2.2 M thiourea; and 4.4% CHAPS; also containing lx Complete protease inhibitor mix (Roche). Samples were clarified by centrifugation (20,000 x g, 10 min, 4°C), and resulting supernatants were fractionated by 2-dimensional electrophoresis (2-DE).
  • F-IEF Fluid-phase Isoelectric Focusing
  • Proteins in each of the F-IEF fractionations were further resolved by molecular weight fractionation using conventional one-dimensional gel electrophoresis.
  • Samples were combined with an equal volume of 4x reducing loading buffer (Novex NuPAGE LDS sample buffer; 50 mM DTT; Invitrogen), heated at 70°C (20 min) and then fractionated (10 ⁇ per well) using NuPAGE 4 - 12% Bis-Tris gels (Novex, Invitrogen), using lx MES (2-[N- morpholino]ethanesulfonic acid) running buffer (Novex, Invitrogen). Proteins were transferred to nitrocellulose blots using an iBlot transfer apparatus & gel transfer stacks (Nitrocellulose Mini; 1B301002, Invitrogen).
  • blots were reprobed with a second anti-protein citrulline antibody (1:500; MABN328EMD; Millipore; detection with horseradish peroxidase-labeled, goat anti- mouse IgM; 1:2500 in TBS-T; 31440, Thermo Scientific), with final overnight washing, to confirm the 6B3 immunoreactive features and increase the sensitivity of detection. No new signals were revealed by this approach.
  • the amount of protein absorbed to the strip was 200 ⁇ 1 / 200 ⁇ g of citruUinated human fibrinogen (400076, Cayman Chemical, Ann Arbor, Michigan, USA), or 200 ⁇ 1 / 200 ⁇ g human fibrinogen (16088, Cayman Chemical) in TBS-T.
  • the duration of the antibody absorption was 16 h at 4°C.
  • Immunoreactive signals of interest were mapped to corresponding features in Coomassie- stained gels. These features were excised and processed for identification through the ESMS- Basic Protein ID service using an Elite Orbitrap mass spectrometer (Thermo Scientific) by the W.M. Keck Mass Spectrometry & Proteomics Resource (W.M. Keck Foundation Biotechnology Resource Laboratory, New Haven, Connecticut, USA). Analyses results were received through and tracked within the Yale Protein Expression Database (YPED, Yale / NIDA Neuroproteomics Center, New Haven, Connecticut, USA). Criteria for a positive identification included: an expectation score less than l.OE-65, a percent coverage greater than 35.0%, and a pi and mw that were consistent with the fractionation data.
  • Astrocytes were cultured to -70% confluence in T-75 tissue culture flasks according to vendor instructions. Cells were washed with TBS (4 times) and then treated for 4 h (37°C, 95% 0 2 /5% C0 2 ) with either ionomycin (10 ⁇ ; 10 mL TBS) or dimethyl sulfoxide vehicle (DMSO; 10 ⁇ 1/10 mL TBS). Following incubation, protease inhibitors were added to the flasks (final concentration of lx; Complete protease inhibitor mix; 10269700, Roche) and cells were harvested by scraping and centrifugation (800 x g, 4°C, 10 min).
  • Each primary antibody was paired with an appropriate secondary antibody (6B3: horseradish peroxidase-labeled, goat anti-mouse IgG; 1:2500; 31430; Thermo Scientific; GFAP: horseradish peroxidase-labeled, goat anti-rabbit IgG; 1:3000; HAF008; R&D Systems) and blots were visualized by ECL.
  • 6B3 horseradish peroxidase-labeled, goat anti-mouse IgG; 1:2500; 31430; Thermo Scientific
  • GFAP horseradish peroxidase-labeled, goat anti-rabbit IgG; 1:3000; HAF008; R&D Systems
  • Citrullination immunohistochemistry was quantified in the cortex using a previously developed scoring system (Lazarus et al., 2015), with a score of 0 reflecting the lowest amount of citrullination fluorescence intensity in a region and a score of 3 reflecting the greatest amount of fluorescence intensity (see Supplementary Figure 1).
  • Four brain sections per animal were scored, and comparisons between groups were analyzed by ANOVA with a Tukey HSD post hoc test (IBM SPSS Statistics for Macintosh, Version 22.0, Armonk, NY: IBM Corp.). A value of p ⁇ 0.05 was considered significant.
  • Figure 24 presents the effects of CCI on the expression of citruUinated proteins in the rat cerebral cortex.
  • Immunohistochemical analysis demonstrated that the basal level of protein citrullination as detected by labeling with mAB 6B3 was very low under control conditions.
  • CCI induced a marked increase in the immunolabeling of cells in the cortex. This up- regulation of protein citrullination was most pronounced in the vicinity of the lesion. The morphology of the affected cells was remarkably consistent, suggesting that the effects of CCI on protein citrullination were cell- specific.
  • CCI produced a marked increase in protein citruUination throughout the injured cortex, extending from lateral to the lesion site to regions of the cortex not directly impacted by CCI (Figure 26, panel B). Scoring of this region revealed that male naive rats (mean score: 0.06) and male CCI rats (mean score: 1.43) were significantly different, /? ⁇ 0.001. Female naive rats (mean score: 0.04) and female CCI rats (mean score: 1.35) were also significantly different, /?
  • Figure 27 shows that anti-citrulline labeling in the cortex and external capsule was predominantly colocalized with GFAP. Similar observations were made in other affected brain regions.
  • the findings presented in Figure 28 further confirm that CCI-induced protein citruUination was not significantly associated with neurons (NeuN), microglia / macrophages (Ibal), or oligodendrocytes (MBP) in the cortex. CitruUination was also not significantly associated with these cell types in any other brain regions investigated (not shown).
  • Figure 30 demonstrates the specificity of the 6B3 antibody for detecting citruUinated proteins in a western blot format. Shown on the left is the Coomassie staining for human fibrinogen (Fib) and the same preparation of fibrinogen that was enzymatically citruUinated by reaction with PAD4 (C-Fib). The protein staining shows that the characteristic profile of purified human fibrinogen is modestly affected by reaction with PAD4.
  • cytoskeletal components including dynamin-1, GFAP, and several forms of tubulin; those involved in metabolic processes (including peroxiredoxin-1, dihydropyrimidinase-related protein 2, and creatine kinase B-type); and proteins involved in cell-cell signaling and synaptic transmission (synapsin-2, syntaxin-binding protein 1, and amphyiphysin).
  • these functional groups of affected proteins - cytoskeletal components, metabolic proteins and proteins involved in cell-cell signaling - may have been preferentially identified as citruUinated due to their relatively high abundance. These abundant protein types may be more easily identifiable through proteomic analyses than less abundant proteins, which may also be citruUinated but not easily identified. In this regard, however, only a small proportion of the very high abundance proteins were identified as being citruUinated, indicating a notable degree of specificity in the citrullination response.
  • Sample 4 moesin, sequence ERQEAEEAKEALLQASR (score 21.64), synapsin-2, sequence YDIRVQK (score 19.95), and septin-9, sequence
  • RVETPASKAPEGSAMPVTDAAPK (score 13.24); Sample 9, myelin basic protein, sequence FFSGDRGAPK (score 60.9); Sample 11, transitional endoplasmic reticulum ATPase, sequence ETVVEVPQVTWEDIGGLEDVKR (score 34.49); Sample 12, NADH-ubiqiunone
  • oxidoreductase 75kDa subunit sequences IAS Q VAALDLGYKPGVEAIRK (score 26.71) and VALIGSPVDLTYRYDHLGDSPK (score 24.23); Sample 13, heat shock cognate 71 kDa, sequence VEIIANDQGNRTTPSYVAFTDTER (score 43.26); Sample 15, creatine kinase B- type, sequence LLIEMEQRLEQGQPIDDLMPAQK (score 42.46); Sample 16, drebrin, sequence LKDQS IFGDQRDEEEES QMK (score 45.82) and sequence KALDARLR (score 29.54), and amphiphysin, sequence LQRELR (score 16.38).
  • the LC MS/MS approach did not identify all the proteins that were detected through the use of the 6B3 anti-citrullinated protein antibody as containing citrullinated residues.
  • Some possible explanations for this include, but are not limited to: (1) overlapping of ion peaks due to similarity in mass with deamidation of Arg, (2) low abundance of citrullination modification to below the detection limit of the LC MS/MS technique but not western blot, (3) poor ionization of a citrullinated peptide, (4) poor fragmentation of a citrullinated peptide, and (5) non- specificity detection of the proteins by western blot.
  • FIG. 32 shows the results of 3 separate experiments investigating the effects of ionomycin treatment on the proteolytic processing of GFAP (left panel) and the generation of citrullinated proteins (right panel). The data show that treatment with ionomycin consistently activated the proteolytic processing of intact GFAP (left panel; blue arrows) to produce a distinctive pattern of breakdown products.
  • Protein citrullination is a calcium-dependent protein modification that has been largely studied in the context of autoimmune disorders, particularly rheumatoid arthritis. Abnormal protein citruUination in rheumatoid arthritis results in the generation of antigenic epitopes that become targeted by the adaptive immune system (Gyorgy et al., 2006;Kidd et al., 2008). Protein citruUination is also found to be abnormal in several neurodegenerative disorders, including multiple sclerosis (MS) and Alzheimer's disease, suggesting that citrullinated proteins may also serve as antigenic targets in these conditions and thereby contribute to long-term pathogenesis.
  • MS multiple sclerosis
  • Alzheimer's disease suggesting that citrullinated proteins may also serve as antigenic targets in these conditions and thereby contribute to long-term pathogenesis.
  • TBI TBI pathophysiology
  • a hallmark of acute TBI pathophysiology is intracellular calcium overload (Floyd et al., 2005). This shift in ionic balance is excitotoxic, activating a number of damaging intracellular cascades, including lipid peroxidation, proteolysis, free radical generation (Werner and Engelhard, 2007), and the activation of pro-apoptotic genes (Walker and Tesco, 2013) with the loss of dendritic spines.
  • Elevated intracellular calcium is an essential condition for the activation of peptidylarginine deiminase (PAD) enzymes, which catalyze the conversion of intrapeptidyl arginine residues to citrulline residues (Ishigami and Maruyama, 2010).
  • PAD peptidylarginine deiminase
  • PAD enzymes exist as several isoforms, two of which are found in neural tissue: PAD2 (Curis et al., 2005;Raijmakers et al., 2005;Jang et al., 2008) and PAD4 (Acharya et al., 2012).
  • PAD2 is largely localized to astrocytes, while PAD4 is exclusively expressed in neurons (Acharya et al., 2012).
  • PAD2 is localized to reactive astrocytes in the brains of mice affected by a prion disorder, scrapie, where its enzymatic activity is nearly double that of healthy mice (Jang et al., 2008).
  • Research on the etiology of MS has suggested that PAD2 contributes to the pathology of demyelination, where its activity is thought to destabilize myelin sheath structure (Musse et al., 2008).
  • astrocytes selectively display long-term defects in calcium signaling following TBI (Rzigalinski et al., 1998) and ultimately, astrocytic death precedes that of neurons in rat models of TBI (Floyd et al., 2005).
  • Related findings involving hypoxia confirm that astroglial injury is due to massive calcium influxes and resulting excitotoxicity (Ishigami and Maruyama, 2010).
  • Models of TBI illuminate the biologic importance of astrocytes in brain injury, serving a protective role by shielding neurons from damage due to oxygen deprivation (Vibulsreth et al., 1987), glutamate neurotoxicity, and calcium excitotoxicity (Mattson and Rychlik, 1990).
  • the mechanistic basis for this observation may relate to the up-regulation of voltage-gated, class C L-type Ca 2+ channels that are selectively expressed in astrocytes and are particularly sensitive to activation by injury in the regions reported here (Chung et al., 2001).
  • nimodipine an L-type calcium channel blocker
  • These findings correspond to a model of calcium excitotoxicity- induced citruUination.
  • the selective citruUination of astrocytes in these particular regions may correspond to long-term dysfunctions associated with TBI, including learning and memory deficits associated with progressive hippocampal atrophy (Walker and Tesco, 2013).
  • citruUination may occur in other cell types at different time points following TBI, and future work is needed to address this possibility.
  • Protein carbonylation is a reflection of oxidative stress (Dalle-Donne et al., 2003), whereas citruUination is a marker of calcium influx (Vossenaar et al., 2004;Kinloch et al., 2005).
  • the gender difference observed in carbonylation may be due to the protective antioxidant effects of ovarian steroids (Roof et al., 1997), whereas a similar mechanism does not appear to exist in the case of citruUination.
  • the extent to which these protein modifications contribute to gender differences in TBI complications and mortality (Gosworth et al.,
  • citrullinated GFAP is a characteristic feature in MS and Alzheimer's disease (Nicholas et al., 2004;Ishigami et al., 2005;Gyorgy et al., 2006;Bradford et al., 2014), and myelin basic protein (MBP), a major component of myelin sheath structure, is profoundly over-citrullinated in MS (Tranquill et al., 2000;Bradford et al., 2014).
  • MBP myelin basic protein
  • GFAP tubulin, peroxiredoxin 1, cofilin- 1 and alpha/gamma enolase are selectively citrullinated in prion disease (Jang et al., 2008). Therefore, the link between abnormal protein citruUination and neurological disease appears strong.
  • the 37 proteins identified as targets for TBI-induced citruUination were derived from the analysis of only 16 gel features. Accordingly, not all of the proteins identified in a single feature are necessarily citrullinated, and may have resulted from the analysis due to their co-purification with another citrullinated species. Nevertheless, the high correlation that exists between the specific proteins identified here and their reported
  • citruUination in various disease states is consistent with the proposal that these proteins are indeed targets for citruUination following TBI.
  • a potential mechanism by which abnormal protein citrullination contributes to neurological disease could involve the adaptive immune system.
  • a significant proportion of the proteins identified here are also recognized as autoantigens, in both neurological and
  • MBP is the signature citruUinated autoantigen of MS (Tranquill et al., 2000;Bradford et al., 2002).
  • Autoantibodies targeting amphiphysin are associated with several neurological disorders, including sensory neuronopathy and
  • TBI dramatically up-regulates protein citruUination within astrocytes in specific brain regions. Additionally, this modification affects only a subset of the neural proteome, primarily affecting proteins involved in cytoskeletal structure, metabolic processes, and cell-cell signaling. A large proportion of these proteins have been identified as citrullinated in other pathologies, including MS, Alzheimer's disease, and rheumatoid arthritis, indicating a potential role for this protein modification in ongoing pathological processes. Interestingly, gender does not affect the degree or distribution of this modification in neural tissue, in contrast to previous observations involving TBI-induced protein carbonylation. Accordingly, gender differences in the CNS response to TBI may not involve differential responses in protein citruUination.
  • citrullinated breakdown products of filial fibrillary acidic protein and citrullinated forms of other proteins, both intact and breakdown products, have been discovered as candidate biomarkers for traumatic brain injury (TBI).
  • TBI traumatic brain injury
  • additional proteins include amphiphysin, crafilin 1, dynamin-1, synapsin-2, septin-9, annexin A6, stress-induced- phosphoprotien-1, dihydropyrimidase-related protein 2, neuronal- specific septin-3, transgelin-3, GRIPI- associated protein 1, transitional endoplasmic reticulum ATPase, vesicle-fusing ATPase,
  • the disclosed methods can be conducted with any suitable assay system.
  • the assay systems contemplated by the present invention are used to conduct any type of diagnostic or analytical method known in the art.
  • Such analytical methods include but are not limited to clinical chemistry assays (e.g., measurements of pH, ions, gases and metabolites, including mass spectrometry), hematological measurements, nucleic acid amplification assays (e.g., polymerase chain reaction (PCR) and ligase chain reaction assays), immunoassays (e.g., direct, sandwich and/or competitive immunoassays, including Western blots and ELISAs, and serological assays), oligonucleotide ligation assays, and nucleic acid hybridization assays.
  • clinical chemistry assays e.g., measurements of pH, ions, gases and metabolites, including mass spectrometry
  • nucleic acid amplification assays e.g., polymerase chain reaction (PCR)
  • Any biological reagent that might be used in such analytical methods can be used in such systems, including but not limited to nucleic acids, nucleotides, oligonucleotides, DNA, RNA, PNA, primers, probes, antibodies or fragments thereof, antigens, small molecules, e.g., drugs or prodrugs, streptavidin, avidin, and biotin.
  • These systems can be portable, e.g., hand-held, and/or operated within a fixed laboratory or field setting, alone or in combination with one or more additional components, assay devices or systems. These systems can be used in a variety of applications, from field operations to laboratory settings, in a wide variety of industries, including but not limited to, medical, clinical, forensic, pharmaceutical, environmental, veterinary, biological, chemical, agricultural, waste management, hazardous chemical, drug testing, and in defense applications, e.g., for the detection of biological warfare agents.
  • the assay systems and consumables used in the present invention can detect an analyte of interest by any suitable method, including but not limited to, optical, electromechanical, radiowave, electromagnetic, colorimetric, fluorimetric, chemiluminescent, electrochemiluminescent, radiochemical, nuclear magnetic resonance, enzymatic, fluorescent, particle-count, and cell-count based detection.
  • the assay consumable includes devices in which one or more steps of an assay process are conducted and such devices can include one or more test sites where an assay measurement is conducted.
  • the assay consumable includes at least one assay test site for an assay.
  • a test site can include a plurality of distinct assay domains, at least two of the domains including reagents for measuring different analytes.
  • the consumable can include a plurality of test sites for a plurality of individual assays.
  • the assay consumable can be a component that provides a reagent or other assay component that is used by the system to conduct an assay.
  • the assay consumable can be a container with one or more compartments for holding assay reagents.
  • the assay consumable (or test sites therein) can be single use or it can be reusable.
  • the assay consumable can be configured to conduct one test or multiple tests (sequentially or in parallel).
  • Test sites refer to regions of a consumable that hold, contact and/or interrogate a sample.
  • a test site can include a plurality of distinct assay domains, at least two such domains include reagents for measuring different analytes.
  • Consumables can comprise multiple test sites which can hold, contact or otherwise interrogate distinct volumes (aliquots) of the same sample and/or volumes of different samples.
  • a sector of an assay consumable refers to grouping of two or more test sites of the consumable. Each test site can be used to conduct a single measurement or multiple measurements on a volume of sample (for example, the measurement of multiple different analytes in a multiplexed assay format).
  • a consumable with multiple test sites can be configured to use all of its test sites in parallel, to use its test sites at different times (e.g., assigning unused test sites to be used as new samples are delivered to the assay system), or a combination of both modes of operation can be enabled.
  • the assay consumable can be any structure useful in diagnostic applications and that structure can be dictated by the particular assay format or detection method employed by the device.
  • assay consumables suitable for use with the invention include, but are not limited to, test tubes, cuvettes, flow cells, assay cartridges and cassettes (which can include integrated fluidics for assay processing), multi-well plates, slides, assay chips, lateral flow devices (e.g., strip tests), flow-through devices (e.g., dot blots), pipette tips, solid phase supports for biological reagents and the like.
  • test sites in the assay consumable are defined by compartments in the assay consumable, e.g., wells, chambers, channels, flow cells and the like.
  • the assay consumable and/or test sites can include one or more components used to carry out an assay measurement according to one or more specific detection methodologies.
  • examples of such components can include, but are not limited to, lateral flow matrices, filtration matrices, optical windows, sensors (e.g., electrochemical and optical sensors), solid phase supports for binding reactions (e.g., coated slides, chips, beads, pins, coated filtration or lateral flow matrices, tubes and the like), reagents (dry or in liquid form), electrodes, analyte selective membranes and the like.
  • the assay consumable can be a device that incorporates a conventional lateral flow test strip, e.g., an immunoassay test strip, as an assay medium.
  • the device is molded to include an identifier or the identifier is affixed to the device without any modification to the structure of the device and/or the assay medium.
  • the device is placed within the analytical system, i.e., the assay system, for analysis and before, during or after the performance of the assay, the identifier controller within, affixed to or associated with the assay system reads the data contained on the identifier and uses that data in the assay or after the assay is completed by the system.
  • the assay consumable and accompanying assay system or reader is capable of performing a multiplex assay.
  • a multiplex assay is a type of assay in which multiple measurements are performed on a single sample, e.g., by distributing samples across multiple test sites and/or by carrying out multiple measurements on volumes of samples in individual test sites.
  • the multiple measurements can include, but are not limited to, (i) multiple replicates of a measurement for an analyte; (ii) multiple measurements of a certain analyte (i.e., multiple non-identical measurements for the same analyte, e.g., measurements that differ in format or in the identity of the assay reagents that are employed); and/or (iii) measurements of multiple different analytes.
  • an assay consumable is configured to carry out, in one or more test sites, multiplex measurements that include at least two assays for two different analytes.
  • Multiplex measurements that can be used with the invention include, but are not limited to, multiplex measurements (i) that involve the use of multiple sensors; (ii) that use discrete assay domains on a surface (e.g., an array) that are distinguishable based on location on the surface; (iii) that involve the use of reagents coated on particles that are distinguishable based on a particle property, such as size, shape, color, etc.; (iv) that produce assay signals that are distinguishable based on optical properties (e.g., absorbance or emission spectrum), (v) that are based on temporal properties of an assay signal (e.g., time, frequency or phase of a signal), and/or (vi) that are based on some other assay characteristic.
  • multiplex measurements i) that involve the use of multiple sensors; (ii) that use discrete assay domains on a surface (e.g., an array) that are distinguishable based on location on the surface; (iii) that involve the use of reagent
  • an assay test site comprises a plurality of distinct assay domains and each domain comprises one or more reagents for measuring a different analyte.
  • Multiplexing information including the location, identity, and composition of each assay domain, is used to identify the assay signal generated at each domain and connect it to a determination of the presence or amount of the corresponding analyte (a process which can include the application of additional consumable data such as signal thresholds and/or calibration parameters).
  • Such multiplexing information can be provided as consumable data and/or stored to the consumable identifier.
  • a test site can be configured to carry out a plurality of multiplexed measurements
  • the assay consumable can include a plurality of test sites. Information regarding the exact configuration of the one or more test sites, assay domains, and/or one or more sectors in a consumable can be included in the information saved to the assay consumable identifier and/or provided as consumable data.
  • This information can include the location and identity of the test sites, assay domains, and/or one or more sectors as well as multiplexing information (as described above) including the number, identity and differentiating characteristics of the individual measurements within a test site, assay domain, and/or sector (e.g., the specific locations, identities and/or assay reagents of assay domains within each test site).
  • multiplexing information including the number, identity and differentiating characteristics of the individual measurements within a test site, assay domain, and/or sector (e.g., the specific locations, identities and/or assay reagents of assay domains within each test site).
  • the use of a test site, assay domain, and/or sector in an assay consumable can also be recorded to the identifier to track the use of the consumable in an assay system.
  • the identifier and/or consumable data can also include information concerning the assay format and specific processing steps to be used for an assay consumable or test site, assay domain, and/or sector of an assay consumable.
  • the identifier and/or consumable data can also include information concerning analytical methods that should be applied by the system once an assay is conducted to analyze the output of an assay in a given test site, assay domain, and/or sector and, optionally, to provide results that combine the output from multiple assays in a test site, assay domain, and/or sectors.
  • test sites can be configured in any suitable configuration, depending on the geometry of the consumable and/or the type of assay conducted with the consumable.
  • the test sites are configured as wells and/or chambers in the assay consumable.
  • the assay consumable of the present invention can be a multi-well plate (e.g., a 24-, 96- , 384- or 1536-well plate), and the wells of the plate can further comprise a plurality (e.g., 2 or more, 4 or more, 7 or more, 25 or more, 64 or more, 100 or more, etc.) of distinct assay domains.
  • Multi-domain multi-well plates that are adapted to allow assay measurements to be conducted using electrode induced luminescence measurements (e.g., electrochemiluminescence measurements) are described in U.S. Application Ser. No. 10/238,391, entitled “Methods and Reader for Conducting Multiple Measurements on a Sample", filed on Sep. 10, 2002, issued as U.S. Patent No. 7,858,321 on December 28, 2010, hereby incorporated by reference.
  • the exact configuration of the domains, test sites, and/or sectors in an assay consumable, as well as the specific identity of each domain, test site, and/or sector and the reagents bound to that domain/test site/sector can be included in the information saved to the assay consumable identifier and/or provided as consumable data.
  • the use of a given domain, test site, and/or sector in an assay consumable can also be recorded to the identifier to track the use of the consumable in an assay system.
  • Assay consumables can be used in a plurality of diverse assays and this diversity leads to a variety of suitable configurations of the associated consumable.
  • the same analyte is measured at different assay domains within a test site, the different assay domains being designed to measure a different property or activity of the analyte.
  • Information concerning the assay format that can be used in an assay consumable, test site and/or assay domain can also be saved to the assay consumable identifier and/or provided as consumable data.
  • the identifier and/or consumable data can also include information concerning analytical methods that should be applied by the system once an assay is conducted to analyze the output of an assay in a given test site and/or domain and compare that output to an assay in a separate test site and/or domain.
  • Such assay consumables include one or more, and in one embodiment, a plurality of test sites and/or assay domains for conducting one or more assay measurements simultaneously or sequentially.
  • the test sites can be configured as wells and/or chambers.
  • These test sites and/or assay domains comprise one or more electrodes for inducing luminescence from materials in the test sites and/or assay domains.
  • the assay consumables can further comprise assay reagents in liquid or dry form, e.g., in the test sites, e.g., wells or chambers, of the consumable.
  • an assay consumable or multi-well assay plate can include several additional elements, e.g., a plate top, plate bottom, wells, working electrodes, counter electrodes, reference electrodes, dielectric materials, electrical connections, and assay reagents.
  • the wells of the plate can be defined by holes or openings in the plate top, or as indentations or dimples on a surface of a plate.
  • the plates can have any number of wells of any size or shape, arranged in any pattern or configuration and can be composed of a variety of different materials.
  • Exemplary embodiments of consumables that can be used in the present invention include industry standard formats for the number, size, shape and configuration of the plate and wells, e.g., 96-, 384-, and 1536-well plates, with the wells configured in two- dimensional arrays.
  • Other formats can include single well plates, 2-well plates, 6-well plates, 24-well plates, and 6144-well plates.
  • Multi-well assay plates can be used once or can be used multiple times and are well suited to applications where the plates are disposable.
  • Various configurations for suitable assay plates can be used in the present invention, including but not limited to those depicted in Figs. 11A, 12A, 13A, 13B, 14A, 15, and 16A of U.S. Application Ser. No.
  • Binding assays for measuring biomarker levels may use solid phase or
  • Suitable assay methods include sandwich or competitive binding assays. Examples of sandwich immunoassays are described in U.S. Patent No. 4,168,146 and U.S. Patent No. 4,366,241, both of which are incorporated herein by reference in their entireties. Examples of competitive immunoassays include those disclosed in U.S. Patent No. 4,235,601, U.S. Patent No. 4,442,204 and U.S. Patent No. 5,208,535, each of which are incorporated herein by reference in their entireties.
  • Multiple biomarkers may be measured using a multiplexed assay format, e.g., multiplexing through the use of binding reagent arrays, multiplexing using spectral
  • Suitable multiplexing methods include array based binding assays using patterned arrays of immobilized antibodies directed against the biomarkers of interest.
  • Various approaches for conducting multiplexed assays have been described (See e.g., US 20040022677; US 20050052646; US 20030207290; US 20030113713; US 20050142033; and US 20040189311, each of which is incorporated herein by reference in their entireties.
  • One approach to multiplexing binding assays involves the use of patterned arrays of binding reagents, e.g., U.S. Patent No.
  • Another approach involves the use of binding reagents coated on beads that can be individually identified and interrogated. See e.g., WO 9926067, which describes the use of magnetic particles that vary in size to assay multiple analytes; particles belonging to different distinct size ranges are used to assay different analytes. The particles are designed to be distinguished and individually interrogated by flow cytometry. Vignali has described a multiplex binding assay in which 64 different bead sets of microparticles are employed, each having a uniform and distinct proportion of two dyes (Vignali, D. A A, "Multiplexed Particle-Based Flow Cytometric Assays" J. ImmunoL Meth. (2000) 243: 243-55).
  • a diagnostic test may be conducted in a single assay chamber, such as a single well of an assay plate or an assay chamber that is an assay chamber of a cartridge.
  • the assay modules e.g., assay plates or cartridges or multi-well assay plates
  • methods and apparatuses for conducting assay measurements suitable for the present invention are described for example, in US 20040022677; US 20050052646; US 20050142033; US 20040189311, each of which is incorporated herein by reference in their entireties.
  • Assay plates and plate readers are now commercially available (MULTI-SPOT ® and MULTI- ARRAY ® plates and SECTOR ® instruments, Meso Scale Discovery, a division of Meso Scale Diagnostics, LLC, Gaithersburg, MD.).
  • the various isoforms of ⁇ can be purchased from AnaSpec, Inc. ( w w . m sijcc .
  • Antibodies specific for the various isoforms of ⁇ may be produced by methods known in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual. (Third Ed.) (2000).
  • the assay consumables can be used in a reader that can be used to induce and measure luminescence, e.g., electrode induced luminescence or
  • the accompanying assay system can also induce and/or measure current and/or voltage, for example, at an electrode.
  • the assay system can incorporate, for example, one or more photodetectors; a light tight enclosure; mechanisms to transport the assay plates into and out of the reader (and in particular, into and out of a light tight enclosure); mechanisms to align and orient the assay plates with the photodetector(s) and/or with electrical contacts; additional mechanisms to track and identify plates (e.g. bar code readers); mechanisms to make electrical connections to plates, one or more sources of electrical energy for inducing luminescence, and appropriate devices, electronics and/or software.
  • the assay reader can also include mechanisms to store, stack, move and/or distribute one or more multi-well assay plates (e.g. plate stackers and/or plate conveyors).
  • the assay system can be configured to measure light from multi-well assay plates by measuring light sequentially from a plurality of sectors or regions of the plate (i.e., a grouping of a plurality of adjacent assay domains within a plate) and/or from the entire plate substantially simultaneously or simultaneously.
  • the assay system can also incorporate additional microprocessors and computers to control certain functions within the system and to aid in the storage, analysis and presentation of data.
  • Various configurations for suitable assay systems can be used in the present invention, including but not limited to those depicted in Figs. 17 to 23 of U.S. Application Ser. No. 2004/0022677, each of which are incorporated herein by reference.
  • Glial fibrillary acidic protein The major protein of glial intermediate filaments in differentiated astrocytes. J Neuroimmunol 8:203 - 14
  • citrullinated antigens in mouse models of autoimmune arthritis and demyelination were citrullinated antigens in mouse models of autoimmune arthritis and demyelination.
  • Demyelination A Role for Tumor Necrosis Factor- Induced Peptidylarginine Deiminase 4 Translocation. Neurobiology of Disease 26: 11387 - 96
  • Aldolase directly interacts with ARNO and modulates cell morphology and acidic vesicle distribution. Am J Physiol Cell Physiol. 300:C1442-C55
  • GFAP-BDP as an Acute Diagnostic Marker in Traumatic Brain Injury: Results from the Prospective Transforming Research and Clinical Knowledge in Traumatic Brain Injury Study.
  • Alzheimer's disease Journal of Alzheimer' s Disease 19:341 - 53
  • Citrulline-containing proteins as a novel marker of neurodegeneration.
  • Curis E., Nicolis, I., Moinard, C, Osowska, S., Zerrouk, N., Benazeth, S., and Cynober, L.
  • L-Citrulline immuno staining identifies nitric oxide production sites within neurons. Neuroscience 114, 111 - 122.
  • Rondas, D., Crevecoeur I., D'hertog, W., Ferreira, G., Staes, A., Garg, A., Eizirik, D., Agostinis,
  • Citrullinated glucose-regulated protein 78 is an autoantigen in type 1 diabetes. Diabetes 64, 573 - 586.

Abstract

The disclosure is directed to methods of diagnosing traumatic brain injury (TBI) in a subject, the method comprising determining the citrullination level(s) of one or more biomarkers in a biological sample taken from the subject, and comparing the determined citrullination level(s) of the one or more biomarkers against a citrullination level(s) of the same one or more biomarkers from a control subject determined not to be suffering from TBI, wherein an increase in the citrullination level(s) of the one or more biomarkers compared to the citrullination level(s) of the one or more biomarkers from the control subject is indicative that the subject may be suffering from TBI.

Description

Citrullinated Proteins and Their Breakdown Products as TBI Biomarkers
Background
[0001] Traumatic brain injury (TBI) is a major cause of long-term disability. Acute TBI prompts a constellation of dysfunctional processes, collectively known as "secondary injury" mechanisms. A hallmark secondary injury in TBI is a prolonged imbalance in calcium homeostasis, resulting in a dramatic influx of calcium into brain cells. This influx elicits the generation of damaging reactive oxygen species. Protein carbonylation and citrullination are pathological post-translational modifications that can result from intracellular calcium overload. These modifications have been proposed to play a role in neurodegenerative disorders, including Alzheimer's disease, and multiple sclerosis. Both carbonylation and citrullination can contribute to ongoing dysfunction, either through direct loss of protein function or via immune-based mechanisms where proteins specifically modified by citrullination become targeted by the adaptive immune system.
[0002] This work investigated carbonylation and citrullination in a rodent model of TBI. We have identified specific regions and cell types susceptible to these modifications following TBI. While protein carbonylation was expressed in astrocytes of the lesion site and ependymal cells of specific periventricular regions, citrullination was found in astrocytes throughout the cerebral cortex, external capsule, and hippocampus. Unexpectedly, only a small subset of proteins in the entire brain proteome was affected by TBI. Four proteins were found to be carbonylated in response to injury, and these same proteins, along with 33 others, were found to be citrullinated following TBI. Many of these 37 proteins are known to be modified similarly in other neuropathologies. While the distribution and magnitude of protein citrullination was unaffected by gender, susceptibility to protein carbonylation was gender dependent, with males showing significantly greater protein carbonylation in ependymal cells distant from the injury lesion, as compared to females.
[0003] These findings show that carbonylation and citruUination are specific processes, with selective effects on regional, cellular, and proteomic susceptibility. Gender differences observed in carbonylation indicate that hormonal mechanisms may serve a protective role in TBI- induced oxidative stress. The dramatic expression of protein citruUination following injury may provide a basis for the development of autoimmune dysfunction in TBI pathology. Accordingly, persistent proteomic changes may underlie ongoing disability, either through direct loss-of-function or secondary immune-based pathological mechanisms.
Summary of the Disclosure
[0004] The disclosure is directed to methods of diagnosing traumatic brain injury (TBI) in a subject, the method comprising determining the citruUination level(s) of one or more biomarkers in a biological sample taken from the subject, and comparing the determined citruUination level(s) of the one or more biomarkers against a citruUination level(s) of the same one or more biomarkers from a control subject determined not to be suffering from TBI, wherein an increase in the citruUination level(s) of the one or more biomarkers compared to the citruUination level(s) of the one or more biomarkers from the control subject is indicative that the subject may be suffering from TBI.
[0005] The disclosure is also directed to methods of monitoring the progression of traumatic brain injury (TBI) in a subject, the method comprising analyzing at least two biological samples from the subject taken at different time points to determine a citruUination level(s) of one or more biomarkers in each of the at least two biological samples, and comparing the determined citruUination level(s) of the one or more biomarkers over time to determine if the subject's citrullination level(s) of the one or more specific biomarkers is changing over time, wherein an increase in the citrullination level(s) of the one or more biomarkers over time is indicative that the subject's risk of suffering from TBI is increasing over time.
Brief Description of the Drawings
[0006] Figure 1. Mechanisms of protein carbonylation following mitochondrial dysfunction. In the wake of mitochondrial dysfunction driven by calcium overload, oxygen is reduced to hydroxyl radicals through a series of electron reductions catalyzed by enzymes such as
NAD(P)H oxidase and superoxide dismutase. Hydroxyl radicals, as well as the intermediate molecules superoxide and hydrogen peroxide, are known as reactive oxygen species (ROS). ROS are highly reactive molecules that target polyunsaturated fatty acids, such as those in the lipid membranes of neural cells, forming breakdown products of lipid peroxidation. These products are categorized as reactive carbonyl species (RCS). Both ROS and RCS catalyze irreversible protein carbonylation.
[0007] Figure 2. Mechanisms of protein citrullination following intracellular calcium overload. Peptidylarginine deiminase (PAD) enzymes are activated by high levels of intracellular calcium. Once activated, PAD enzymes catalyze the conversion of intrapeptidyl arginine residues to intrapeptidyl citrulline residues. Ammonia (NH3) is released as a byproduct of this reaction. (Figure adapted from Tesija-Kuna & Zirovic (2008)).
[0008] Figure 3. Proposed model of proteomic changes following TBI- induced calcium overload. After traumatic injury, ischemia and mechanical damage (tearing, shearing, and hemorrhage) lead to increased extracellular glutamate, an excitatory amino acid (1). This causes significant ionic imbalance, prompting a dramatic influx of calcium into cells (2). High intracellular levels of calcium impair mitochondrial function, causing decreased energy metabolism (3). ATP depletion prevents the efflux of calcium to restore ionic homeostasis (4). In addition, calcium-induced mitochondrial dysfunction leads to the production of reactive oxygen species (ROS) (5). Two post-translational modifications, citrullination and carbonylation, are driven by calcium overload. Citrullination, the conversion of intrapeptidyl arginine to intrapeptidyl citrulline residues, is catalyzed by peptidylarginine deiminase, which is a calcium- dependent enzyme (top panel). Carbonylation, the conversion of an intrapeptidyl amino acid (Lys, Cys, His) to a carbonyl group, is driven by the effects of ROS (bottom panel). Both citrullination and carbonylation are irreversible proteomic modifications with significant effects on protein structure and function.
[0009] Figure 4. Effects of CCI on protein carbonylation in rat brain. Five days after
experimentally administered CCI, extracts were obtained from dissected regions of brain containing the injury penumbra (I) and the corresponding contralateral control region (C).
Lysates were analyzed (4.2 μg/lane) for protein carbonylation by one-dimensional gel electrophoresis followed by Western blotting. Coomassie staining of the protein remaining in the transferred gel was used to confirm equal protein loading (left). DNP Western blotting was performed to detect protein carbonylation in CCI-injured brain samples versus uninjured contralateral brain samples (right). Arrows indicate protein bands for which carbonylation was most affected by CCI. Images are representative of three independent experiments. A total of n=5 CCI animals were examined.
[0010] Figure 5. Identification of rat brain proteins carbonylated after CCI. Proteins present in extracts of naive rat brain (N) and contralateral (C) and ipsilateral (I) regions of CCI-injured rats were fractionated by fluid-phase IEF (pi ranges listed at bottom) and one-dimensional gel electrophoresis. Proteins were transferred to nitrocellulose, derivatized, and probed with anti- DNP antibody (right). Proteins remaining in the gel were visualized by Coomassie staining (left). Features showing increased carbonylation in response to TBI (numbered boxes, right) were mapped to the corresponding Coomassie features (left) and identified by peptide mass fingerprinting as: (1) GFAP (49,957 Da; p/ 5.4), (2) CRMP2 (62,278 Da; p/ 6.0), (3) ALDOC (39,284 Da; p/ 6.7), and (4) ALDOA (39,352 Da; p/8.3). Images are representative of three independent experiments. A total of n=S CCI and n=S naive animals were examined.
[0011] Figure 6. Effects of CCI on protein carbonylation immunohistochemistry in rat brain. Brain tissue was collected 5 days after CCI and evaluated by anti-DNP immunofluorescence. A whole-slice image (top, xl original magnification, scale bar indicates 2 mm) and an image of the lesion site (bottom, x2.5 original magnification, scale bar indicates 2 mm) are shown. Images are representative of three independent experiments. A total of n=l6 CCI animals and n=l6 sham animals were examined, with 16 slices per animal from bregma -2.12 mm to bregma -3.30 mm.
[0012] Figure 7. The CCI- induced increase in anti-DNP immunofluorescence is specific for protein carbonylation. Brain tissue was collected 5 days after CCI and evaluated by anti-DNP immunofluorescence. Anti-DNP immunofluorescence in the cerebral cortex contralateral to the injury and the CCI-injured cortex are depicted in (A) and (B), respectively (original
magnification xlO, scale indicates 200 μιη). Immunoneutralization of the anti-DNP antibody with (C) carbonylated and derivatized β-casein but not (D) unmodified β-casein resulted in reduced DNP immunohistochemical staining of the CCI-injured brain (original magnification x20, scale bar, 100 μιη). Images are representative of three independent experiments. A total of n=l6 CCI animals and n=l6 sham animals were examined, with 16 slices per animal from bregma -2.12 mm to bregma -3.30 mm. [0013] Figure 8. Effects of CCI on protein carbonylation in ependymal cells lining the dorsal third ventricle and median eminence. Brain tissue was collected 5 days after CCI and evaluated by anti-DNP immunofluorescence (A and C, sham control; B and D, CCI). (A and B)
Representatives of the dorsal third ventricle. (C and D) Representatives of the ventricle lining above the median eminence. After CCI, ependymal cells lining the dorsal third ventricle became highly immunoreactive, as did distinct cells located in the adjacent parenchyma of the paraventricular thalamic nucleus (B, arrows). Also after CCI, cells lining the ventricle of the median eminence showed an increase in protein carbonylation (D). (A and B) Original magnification x20, scale 50 μιη. (C and D) Original magnification x40, scale 50 μιη. Images are representative of three independent experiments. A total of n=l6 CCI animals and n=l6 sham animals were examined, with 16 slices per animal from bregma -2.12 mm to bregma -3.30 mm.
[0014] Figure 9. Original magnification x20, scale 100 μιη. Images are representative of three independent experiments. A total of n=3 CCI animals and n=3 sham animals were examined, with four slices per animal from bregma -2.12 mm to bregma -3.30 mm.
[0015] Figure 10. Representative images depicting the carbonylation scoring system. Brain tissue was collected 5 days after CCI and evaluated by anti-DNP immunofluorescence. Images show relative increments in anti-DNP immunofluorescence (0-3) observed in the
penumbra/cortex, dorsal third ventricle, and median eminence regions. The distinguishing feature differentiating a score of 2 versus a score of 3 for the dorsal third ventricle was the anti- DNP labeling of peri-ependymal cells located in the parenchyma both lateral and ventral to the dorsal third ventricle. This observation reflected a stippled pattern of carbonylation-positive cells expanding approximately 10 to 50 μιη into surrounding tissue from the ependymal lining of the ventricle. Penumbra/cortex and median eminence images, original magnification xlO; dorsal third ventricle images, original magnification x20. Images are representative of three independent experiments. A total of n=l6 CCI animals and n=l6 sham animals were examined, with 16 slices per animal from bregma -2.12 mm to bregma -3.30 mm.
[0016] Figure 11. Gender did not affect the degree of protein carbonylation in the cerebral cortex of control and CCI-lesioned rats. Brain tissue was collected 5 days after CCI and evaluated by anti-DNP immunofluorescence. As shown qualitatively for a representative (A) male and (B) female, and quantitatively in Figure 13, the degree of protein carbonylation after CCI in the penumbra region was not significantly affected by gender. Original magnification x20, scale 100 μιη. Images are representative of three independent experiments. A total of n=l6 CCI animals (7 female, 9 male) were examined, with 16 slices per animal from bregma -2.12 mm to bregma -3.30 mm.
[0017] Figure 12. Effects of CCI on protein carbonylation in regions of the cerebral cortex of male and female rats. Brain tissue was collected 5 days after CCI and evaluated by anti-DNP immunofluorescence. Sections were scored for protein carbonylation as described under Material and methods. Data indicate means + SEM. A post hoc Tukey HSD analysis indicated a statistically significant difference (DDD/?<0.001) between the scores of the penumbra versus other cortical regions of the injured and sham animals. Graph is representative of three independent experiments. A total of n=l6 CCI animals (7 female and 9 male) and n=l6 sham animals (9 female and 7 male) were examined, with 16 slices per animal from bregma -2.12 mm to bregma -3.30 mm.
[0018] Figure 13. Gender differences in the effects of CCI on protein carbonylation in the median eminence and dorsal third ventricle. Brain tissue was collected 5 days after CCI and evaluated by anti-DNP immunofluorescence. Sections were scored for protein carbonylation as described under Material and methods. Data indicate means + SEM. N.S., not statistically significant; D/?<0.05; DD/?<0.01. Regarding the median eminence, a one-way ANOVA indicated significantly different levels of protein carbonylation between the animal gender and the injury conditions ( (3,27) =13.096, /?=0.000). A post hoc Tukey HSD analysis indicated a statistically significant difference between male naive and male CCI protein carbonylation (D/?<0.05), a statistically significant difference between male naive and female naive protein carbonylation (D/?<0.05), and a statistically significant difference between male CCI and female CCI protein carbonylation (DD/?<0.01). There was not a statistically significant difference between female naive and female CCI protein carbonylation (N.S., /?=0.192). Regarding the dorsal third ventricle, a one-way ANOVA indicated significantly different levels of protein carbonylation between the animal gender and the injury conditions ( (3,28) =11.874, /?=0.000). A post hoc Tukey HSD analysis indicated a statistically significant difference between male naive and male CCI protein carbonylation (D/?<0.05) and a statistically significant difference between male CCI and female CCI protein carbonylation (DD/?<0.01). There was not a statistically significant difference between male naive and female naive protein carbonylation (N.S., p=0.311) or between female naive and female CCI protein carbonylation (N.S., /?=0.996). Graph is representative of three independent experiments. A total of n=l6 CCI animals (7 female and 9 male) and n=l6 sham animals (9 female and 7 male) were examined, with 16 slices per animal from bregma -2.12 mm to bregma -3.30 mm.
[0019] Figure 14. Injury upregulates the expression of citrullinated proteins in the cerebral cortex. Brain tissue was collected five days after CCI and evaluated for protein citrullination by anti-protein citrulline immunolabeling using mAB 6B3. The upper panels show immunolabeling in sections of control (left) and injured brain (right) (2X magnification). Lower panels show higher magnifications of cerebral cortex adjacent to the lesion site (right) or a comparable site in control cortex (left) (20X magnification). Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed. PB-cit: Protein-bound citrulline.
[0020] Figure 15. Specificity of anti-protein citrulline immunolabeling by mAb 6B3. Panels A and B show immunofluorescent signals from mAb 6B3 anti-protein citrulline labeling of the injured cerebral cortex (panel A) and ipsilateral hippocampus (panel B). Panels C and D show immunolableing of equivalent sections with mAb 6B3 that was preadsorbed with citrullinated protein standards. The control and immunoneutralized preparations of mAb 6B3 were treated identically, with the exception of the presence or absence of neutralizing citrullinated proteins. Data are representative of two independent experiments. Scale bar: 200 μιη.
[0021] Figure 16. Increased protein citruUination in the cerebral cortex, hippocampus and external capsule following CCI. Anti-protein citrulline immunolabeling by mAb 6B3 is shown for the control brain regions (panels A, D, and G), regions ipsilateral to the lesion (panels B, E, H) and regions contralateral to the lesion (panels C, F, and G). Structures representated are the cerebral cortex (A-C), hippocampus (D-F), and external capsule (G-I). Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed. Scale bar: 200 μιη.
[0022] Figure 17. Localization of CCI- induced protein citruUination to astrocytes. Panels on the left show the colocalization of mAb 6B3 labeling with anti-GFAP labeling in the external capsule. Panels on the right show the colocalization of mAb 6B3 labeling with anti-GFAP labeling in the cerebral cortex. Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed. Scale bar: 200 μιη. PB-cit: Protein-bound citrulline.
[0023] Figure 18. CCI did not affect the status of protein citrullination in neurons, microglia or oligodendrocytes. Sections of cerebral cortex ipsilateral to CCI were probed with mAb 6B3 to label protein bound citrulline (PB-Cit; upper panels) and either anti-NeuN, Ibal, or MBP to label neurons, microglia, or oligodendrocytes, respectively (middle panels). The merge of the two signals is presented in the lower panels. Data are representative of 8 separate experiments. Scale bar = 200 μη.
[0024] Figure 19. Effects of CCI on protein citrullination in GFAP-positive cells of the ipsilateral and contralateral hippocampus. Colocalization of anti-protein citrulline and anti-GFAP labeling in astrocytes of the ipsilateral hippocampus is depicted in the pairs of panels presented on the left (A-C). Panel D shows the distinctive anti-citrullinated protein immunolabeling of large, rounded cells observed in the contralateral hippocampus of approximately 20% of CCI animals (4 of 21). While these cells lacked the classical stellate morphology of astrocytes, the anti-citrullinated protein labeling colocalized with GFAP immunolabeling (panels E-G). PB-cit: Protein-bound citrulline.
[0025] Figure 20. Specificity of anti-protein citrulline mAb 6B3 detection for western blotting. Displayed on the left are two Coomassie-stained profiles showing the protein composition of native fibrinogen (Fib) and citruUinated fibrinogen (C-Fib). The western blot (right) shows three immunoblots in which: citruUinated fibrinogen was probed with active mAb 6B3 (Active/C-Fib; left lane); citruUinated fibrinogen was probed with immunoneutralized mAb 6B3
(Neutralized/C-Fib; middle lane); and native fibrinogen was probed with active mAb 6B3 (Active/Fib; right lane). [0026] Figure 21. Identification of proteins that are citruUinated in response to CCI. Extracts of control (C) and injured (I) cerebral cortex were fractionated by fluid-phase isoelectric focusing into defined pH ranges (shown at top) and then further resolved according to molecular weight using one-dimensional gel electrophoresis. Proteins were then transferred to nitrocellulose membranes and probed for protein-bound citrulline (see Methods) (right panel, "Western blot"). Gels run in parallel were visualized with Coomassie (left panel). Sixteen features showing increased citruUination in response to CCI (black numbered boxes, right panel) were mapped to corresponding Coomassie features (red numbered boxes, left panel) and identified by peptide mass finger printing and tandem mass spectrometry. Proteins identified are listed in the lower panel. Images are representative of six independent experiments. A total of n = 4 CCI and n = 4 control animals were examined.
[0027] Figure 22. Simulated brain injury in normal human astrocytes reveals a spectrum of GFAP breakdown products and the hyper-citrullination of one GFAP species. Normal human astrocytes were treated with ionomycin (10 μΜ; 4h) and analyzed for GFAP (left panel) and protein-bound citrulline immunoreactivity (right panel) by western blot. The results of three independent experiments (A, B, and C) are presented, showing the immunoreactivity in extracts prepared from untreated control cells (Control) and cells treated with ionomycin (+ Iono). The blue arrows indicate intact GFAP, while the orange arrows indicate the hyper-citrullinated GFAP breakdown product.
[0028] Figure 23. Potential ongoing effects of protein carbonylation and citruUination in TBI pathology. Injury may induce long-term dysfunction through the calcium-mediated proteomic modifications, carbonylation and citruUination. These modifications can elicit a number of pathological outcomes, including the generation of antigenic epitopes (citruUination) and formation of cytotoxic aggregates (carbonylation).
[0029] Figure 24. Injury upregulates the expression of citrullinated proteins in the cerebral cortex. Brain tissue was collected five days after CCI and evaluated for protein citruUination by anti-protein citrulline immunolabeling using mAB 6B3. The upper panels show immunolabeling in sections of control (left) and injured brain (right) (2X magnification). Lower panels show higher magnifications of cerebral cortex adjacent to the lesion site (right) or a comparable site in control cortex (left) (20X magnification). Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed. PB-cit: Protein-bound citrulline.
[0030] Figure 25. Specificity of anti-protein citrulline immunolabeling by mAb 6B3. Panels A and B show immunofluorescent signals from mAb 6B3 anti-protein citrulline labeling of the injured cerebral cortex (panel A) and ipsilateral hippocampus (panel B). Panels C and D shows immunolableing of equivalent sections with mAb 6B3 that was preadsorbed with citrullinated protein standards. The control and immunoneutralized preparations of mAb 6B3 were treated identically, with the exception of the presence or absence of neutralizing citrullinated proteins. Data are representative of two independent experiments. Scale bar: 200 μιη.
[0031] Figure 26. Increased protein citruUination in the cerebral cortex, hippocampus and external capsule following CCI. Anti-protein citrulline immunolabeling by mAb 6B3 is shown for the control brain regions (panels A, D, and G), regions ipsilateral to the lesion (panels B, E, H) and regions contralateral to the lesion (panels C, F, and G). Structures representated are the cerebral cortex (A-C), hippocampus (D-F), and external capsule (G-I). Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed. Scale bar: 200 μιη.
[0032] Figure 27. Localization of CCI- induced protein citruUination to astrocytes. Panels on the left show the colocalization of mAb 6B3 labeling with anti-GFAP labeling in the external capsule. Panels on the right show the colocalization of mAb 6B3 labeling with anti-GFAP labeling in the cerebral cortex. Data are representative of 15 control animals (8 males and 7 females) and 21 CCI animals (11 males and 10 females). No gender-based differences were observed. Scale bar: 200 μιη. PB-cit: Protein-bound citrulline.
[0033] Figure 28. CCI did not affect the status of protein citruUination in neurons, microglia or oligodendrocytes. Sections of cerebral cortex ipsilateral to CCI were probed with mAb 6B3 to label protein bound citrulline (PB-Cit; upper panels) and either anti-NeuN, Ibal, or MBP to label neurons, microglia, or oligodendrocytes, respectively (middle panels). The merge of the two signals is presented in the lower panels. Data are representative of 8 separate experiments. Scale bar = 200 μη.
[0034] Figure 29. Effects of CCI on protein citruUination in GFAP-positive cells of the ipsilateral and contralateral hippocampus. Colocalization of anti-protein citrulline and anti-GFAP labeling in astrocytes of the ipsilateral hippocampus is depicted in the pairs of panels presented on the left (A-C). Panel D shows the distinctive anti-citrullinated protein immunolabeling of large, rounded cells observed in the contralateral hippocampus of approximately 20% of CCI animals (4 of 21). While these cells lacked the classical stellate morphology of astrocytes, the anti-citrullinated protein labeling colocalized with GFAP immunolabeling (panels E-G). PB-cit: Protein-bound citrulline. [0035] Figure 30. Specificity of anti-protein citrulline mAb 6B3 detection for western blotting. Displayed on the left are two Coomassie-stained profiles showing the protein composition of native fibrinogen (Fib) and citruUinated fibrinogen (C-Fib). The western blot (right) shows three immunoblots in which: citruUinated fibrinogen was probed with active mAb 6B3 (Active/C-Fib; left lane); citruUinated fibrinogen was probed with immunoneutralized mAb 6B3
(Neutralized/C-Fib; middle lane); and native fibrinogen was probed with active mAb 6B3 (Active/Fib; right lane).
[0036] Figure 31. Identification of proteins that are citruUinated in response to CCI. Extracts of control (C) and injured (I) cerebral cortex were fractionated by fluid-phase isoelectric focusing into defined pH ranges (shown at top) and then further resolved according to molecular weight using one-dimensional gel electrophoresis. Proteins were then transferred to nitrocellulose membranes and probed for protein-bound citrulline (see Methods) (right panel, "Western blot"). Gels run in parallel were visualized with Coomassie (left panel). Sixteen features showing increased citrullination in response to CCI (black numbered boxes, right panel) were mapped to corresponding Coomassie features (red numbered boxes, left panel) and identified by peptide mass finger printing and tandem mass spectrometry. Proteins identified are listed in the lower panel. Several of these proteins are also citruUinated in other pathologies, including: ALDOA, ALDOC, GFAP, PRDX1, COF1, ENOA, ENOG, MBP and tubulin (beta) in prion disease (Jang et al., 2008;Jang et al., 2010); GFAP and MBP in Alzheimer's disease and multiple sclerosis (Gyorgy et al., 2006); ENOA in rheumatoid arthritis (Kinloch et al., 2005); and GRP78 in Type I diabetes (Rondas et al., 2014). Images are representative of six independent experiments. A total of n = 4 CCI and n = 4 control animals were examined. [0037] Figure 32. Simulated brain injury in normal human astrocytes reveals a spectrum of GFAP breakdown products and the hyper-citrullination of one GFAP species. Normal human astrocytes were treated with ionomycin (10 μΜ; 4h) and analyzed for GFAP (left panel) and protein-bound citrulline immunoreactivity (right panel) by western blot. The results of three independent experiments (A, B, and C) are presented, showing the immunoreactivity in extracts prepared from untreated control cells (Control) and cells treated with ionomycin (+ Iono). The blue arrows indicate intact GFAP, while the orange arrows indicate the hyper-citrullinated GFAP breakdown product.
Detailed Description of the Disclosure
[0038] Traumatic brain injury (TBI) is a major public health issue in the U.S., with over 1.7 million cases occurring annually. At least 5.3 million Americans are currently living with ongoing disability due to TBI (149). In civilians, these injuries are largely due to motor vehicle accidents (47), as well as falls, sports, and firearms (152). While on deployment, military populations are at disproportionate risk for blast-injury TBIs caused by improvised explosive devices in theater (58). These blast-induced injuries have become the signature injury of operations in Iraq and Afghanistan, with 10% - 20% of returning veterans reported to have suffered a TBI (45).
[0039] The long-term consequences of TBI can be complex and progressive, with anywhere from 10% - 50% of individuals diagnosed with acute TBI going on to suffer from persistent symptoms (11). In addition to the development of cognitive deficits in attention, memory, and executive function (11), a chronic inflammatory state can persist in the brain for months, and even years, following TBI (101; 108). However, elucidating the mechanisms that underlie chronic dysfunction following TBI has proven challenging. Currently, it is not well understood how the acute mechanical injury of TBI can develop into long-term dysfunctions that may last for years. Present understanding of post-injury processes falls into two distinct phases: a first stage of "primary injury," and subsequent "secondary injury." Primary injury refers to the direct, physical disruption of neural tissue following impact, such as axonal strain and injury due to rotational acceleration-deceleration (129) or focal injuries such as skull fracture (111). Secondary injury refers to the ensuing constellation of dysfunctional cellular and physiological processes following primary mechanical injury (152).
MECHANISMS OF SECONDARY INJURIES
[0040] Secondary injury damage refers to a delayed series of interdependent pathological processes that follow primary injury (103; 152). Hemorrhage, edema, and raised intracranial pressure are hallmarks for primary injury (50; 152). As a consequence of ischemia and hypoxia (80), these conditions result in complex cascades of cellular processes that involve mitochondrial dysfunction, calcium overload, inflammation, and oxidative stress, which arise over the course of hours to weeks following TBI (149). These secondary injury processes can lead to serious cognitive dysfunction by disrupting synaptic plasticity, axonal structure, and by inducing cell death through apoptosis, necrosis and autophagy (149). One important component of secondary injury is the disturbance of ion homeostasis (95), which can lead to acute and ongoing changes in cellular physiology (51).
IONIC IMBALANCE AND INTRACELLULAR CALCIUM OVERLOAD FOLLOWING TBI
[0041] Primary injury from mechanical forces can lead to ionic imbalance in the brain, largely due to the effects of glutamate excitotoxicity following TBI. The dramatic increase of extracellular glutamate, an excitatory amino acid, is due to a number of post-injury effects, including the physical disruption of cell membranes and the impairment of energy-dependent glutamate uptake mechanisms (149). This glutamate-rich extracellular environment prompts the activation of a number of ionotropic glutamate receptors, including N-methyl-D-aspartate (NMDA) receptors (51). This affects ionic balance in a number of ways, including increased potassium efflux and the concurrent, dramatic influx of sodium, chloride, and calcium, causing neurons and glial cells to pathologically swell (111). Furthermore, intracellular magnesium levels are reduced, unblocking NMDA receptors and leading to an even greater influx of calcium across the cell membrane (149). This shift in ionic balance is harmful, activating a number of damaging intracellular cascades including lipid peroxidation, proteolysis, free radical generation (152), and the activation of pro-apoptotic genes (149) with the loss of dendritic spines.
[0042] Energy metabolism is also significantly affected by post-TBI ionic imbalance. As the sodium-potassium pump attempts to restore membrane potential, increasing amounts of ATP are required. However, ATP availability is limited by decreased oxygen and glucose, due to impaired cerebral blood flow (51). As calcium accumulation increases from extracellular sources, the failure of energy resources to correct ionic shifts initiates the release of calcium from intracellular stores (109; 143).
[0043] This prolonged rise in intracellular calcium is regarded as a hallmark consequence of TBI (48; 49; 84; 141; 151). Calcium accumulation is seen within hours of brain injury, and persists for several days (51). This overload can lead to several pathological processes, including the activation of calcium-dependent proteases (143; 151), disruption of structural elements such as neurofilaments and microtubules, and the activation of apoptotic genetic signals (51). Prolonged elevation of intracellular calcium also has a profound impact on mitochondria, with dramatic effects on structure, function, and most notably, oxidative stress (51; 143; 149; 151).
OXIDATIVE STRESS AND PROTEIN CARBONYLATION [0044] As intracellular calcium concentration rises, it is sequestered into the mitochondrial matrix across the inner mitochondrial membrane (75; 143). In addition to interfering with ATP synthesis, this ionic accumulation causes the generation of reactive oxygen species (ROS) (75; 111). This generation is due to several possible effects of calcium overload on mitochondria, including interference with respiration, uncoupled electron transfer (104), and a reduced number of electron acceptors in the mitochondrial matrix (31). The main ROS generated by mitochondria is superoxide, a short-lived but highly reactive molecule. Superoxide is converted to several other ROS, including hydrogen peroxide and hydroxyl radicals. Hydrogen peroxide is a poorly reactive molecule; however, unlike superoxide, it is able to cross cell membranes (14). In contrast, hydroxyl is highly reactive and much more toxic than superoxide (14; 125). These ROS react with neural membrane lipids through peroxidation, generating reactive carbonyl species (RCS) (155) such as acrolein and 4-hydroxynonenal (127; 128) (see Fig. 1).
[0045] The actions of ROS and RCS catalyze an important consequence of oxidative stress known as protein carbonylation. Carbonylation involves the modification of amino acids, including lysine, cysteine, or histidine, to carbonyl derivatives within proteins. This modification disrupts both protein structure and function, thereby contributing to injury pathology (39). A growing body of evidence indicates that protein carbonylation occurs in several
neuropathologies, including Alzheimer's disease and Parkinson's disease (25).
[0046] A great deal of research has focused on the role of oxidative stress in the ongoing pathology of TBI (66). However, this research has mainly focused on the use of global carbonylation as a measure of overall oxidative stress in pathology, rather than identifying the specific neural regions, cell types, or proteins affected by this modification. Furthermore, while clinical findings have identified gender-based differences in TBI prognosis (52; 118), little work to date has addressed how gender affects the expression of protein carbonylation in neural tissue following TBI. These gaps in TBI research are significant, as protein carbonylation may underlie long-term changes following injury.
CALCIUM OVERLOAD AND PROTEIN CITRULLINATION
[0047] Elevated intracellular calcium also causes a less-studied protein modification known as citrullination. Protein citrullination is essential for a number of basic physiological functions, including epidermal hydration (120), epigenetic regulation of gene expression (134), hair growth (120), and neural plasticity in stages of early brain development (57). However, in states of pathology, abnormal hyper-citrullination of proteins may occur. The role of abnormal protein citrullination is best understood in the case of rheumatoid arthritis, an autoimmune disorder involving progressive inflammation of synovial joints (55). In this disorder, the citrullination of proteins prompts an autoimmune response due to the formation of antigenic epitopes. However, protein citrullination is also implicated in several neurodegenerative disorders with altered calcium homeostasis, including Alzheimer's disease (62), temporal lobe epilepsy (8), glaucoma (18), and multiple sclerosis (MS) (6).
[0048] Dramatically elevated intracellular calcium is an essential condition for the activation of peptidylarginine deiminases (PADs), the family of enzymes which catalyze the conversion of intra-peptide arginine residues to intra-peptide citrulline residues (61) (See Fig. 2). PAD enzymes exist as five isoforms, but only two of these are found in neural tissue: PAD2, which is localized to astrocytes, (37; 64; 109) and PAD4, which is exclusively expressed in neurons (2). As depicted in Fig. 2, increased free calcium associates with PAD to allosterically activate this enzyme's activity, which catalyzes the conversion of intra-peptidyl arginine to intra-peptidyl citrulline within other proteins. Disordered calcium homeostasis results in an abnormal profile of protein citrullination, resulting in the formation of antigenic epitopes.
[0049] While elevated intracellular calcium concentration is a necessary prerequisite for PAD activation, little attention has been given to the possible presence of citrullinated proteins following TBI-induced calcium overload. Citrullination may play a role in the long-term pathology that follows TBI, as this modification can lead to the development of several forms of dysfunction, including abnormal autoimmune responses.
PATHOLOGICAL EFFECTS OF CARBONYLATION AND CITRULLINATION
[0050] We propose that abnormalities in protein carbonylation and citrullination serve as possible mediators of chronic pathology following TBI. As such, these post-translational modifications represent a potential third, enduring phase of dysfunction following primary and secondary phases.
[0051] The most common effect of protein carbonylation is inactivation of modified enzymes (54). However, carbonylation can also destabilize crucial cellular structure components (155) and promote protein aggregation due to tertiary structure modifications, which can reveal hydrophobic residues to aqueous environments and prompt misfolding (25). The relationship between carbonylation and protein degradation is complex: while carbonylation renders the affected protein more susceptible to breakdown, a high degree of environmental oxidative stress can lead to the carbonylation of proteasomal components. This renders the proteasome dysfunctional, and effectively inhibits the degradation of carbonylated proteins (25). Thus, in the environment of extreme oxidative that can follow TBI, it is possible that carbonylated proteins are no longer targeted by the damaged proteasome for breakdown, and may rather accumulate as misfolded aggregates (25; 54). While several of the proteins that are carbonylated as a consequence of TBI have been identified (101), the functional effect of carbonylation in TBI pathology is not fully recognized, leaving a significant gap in the understanding of the long-term proteomic effects of oxidative stress following injury.
[0052] Citrullination also affects proteins in a number of ways. The addition of a citrulline residue in the place of arginine results in the loss of a positive charge, which can alter tertiary structure, proteolytic susceptibility, and protein-protein interactions (64; 72). In MS, the citrullination of myelin basic protein (MBP) limits the ability of this protein to appropriately associate with lipids (55), which in turn contributes to demyelination by destabilizing sheath structure (89). It has been proposed that the dysfunctional effects of citrullination on myelin sheath structure play a major role in the development of MS (55). Furthermore, citrullinated proteins are also observed within the extracellular plaques seen in post-mortem brains affected by Alzheimer's disease, suggesting a functional role for this modification in neurodegenerative pathology.
[0053] Perhaps most importantly, in addition to altering both protein structure and function, citrullination has the potential to create "altered-self ' epitopes that may be antigenic, prompting the adaptive immune system to launch autoimmune responses against previously benign proteins (32; 154). For example, the citrullination of MBP in MS leads not only to myelin degradation, but also results in the generation of autoantigenic MBP isomers, which are consequently targeted by T-cell lymphocytes (144). As noted above, the effects of citrullination are most significantly studied in the context of rheumatoid arthritis, where anti-citrullinated protein antibodies are utilized as diagnostic biomarkers due to the dramatic antigenicity of this modification in inflamed synovial joint spaces (55). Thus, citrullination has the capacity to prompt an adaptive immune response against selectively modified proteins. An investigation into protein citruUination following brain injury is merited, and may indicate an autoimmune component in the chronic pathology of TBI.
[0054] Intracellular calcium overload is a well-documented pathological effect of TBI. Because protein carbonylation and citruUination are logical and plausible outcomes of oxidative stress and calcium excitotoxicity, it is important to investigate the expression of these damaging post- translational modifications following injury (Figure 3).
[0055] This research investigates the expression of carbonylation and citruUination in a rodent model of TBI, controlled cortical impact (CCI), five days following injury. The first manuscript focuses on protein carbonylation, while the second manuscript examines protein citruUination. Both manuscripts identify specific proteins modified following injury, and examine what regions and neural cell types are most susceptible to each modification. Furthermore, the impact of gender on the expression of these modifications is investigated.
Protein carbonylation after traumatic brain injury: cell specificity, regional susceptibility, and gender differences
[0056] Protein carbonylation is a well-documented and quantifiable consequence of oxidative stress in several neuropathologies, including multiple sclerosis, Alzheimer's disease, and
Parkinson's disease. Although oxidative stress is a hallmark of traumatic brain injury (TBI), little work has explored the specific neural regions and cell types in which protein carbonylation occurs. Furthermore, the effect of gender on protein carbonylation after TBI has not been studied. The present investigation was designed to determine the regional and cell specificity of TBI-induced protein carbonylation and how this response to injury is affected by gender.
Immunohistochemistry was used to visualize protein carbonylation in the brains of adult male and female Sprague-Dawley rats subjected to controlled cortical impact (CCI) as an injury model of TBI. Cell-specific markers were used to colocalize the presence of carbonylated proteins in specific cell types, including astrocytes, neurons, microglia, and oligodendrocytes. Results also indicated that the injury lesion site, ventral portion of the dorsal third ventricle, and ventricular lining above the median eminence showed dramatic increases in protein
carbonylation after injury. Specifically, astrocytes and limited regions of ependymal cells adjacent to the dorsal third ventricle and the median eminence were most susceptible to postinjury protein carbonylation. However, these patterns of differential susceptibility to protein carbonylation were gender dependent, with males showing significantly greater protein carbonylation at sites distant from the lesion. Proteomic analyses were also conducted and determined that the proteins most affected by carbonylation in response to TBI include glial fibrillary acidic protein, dihydropyrimidase-related protein 2, fructose-bisphosphate aldolase C, and fructose-bisphosphate aldolase A. Many other proteins, however, were not carbonylated by CCI. These findings indicate that there is both regional and protein specificity in protein carbonylation after TBI. The marked increase in carbonylation seen in ependymal layers distant from the lesion suggests a mechanism involving the transmission of a cerebral spinal fluid-borne factor to these sites. Furthermore, this process is affected by gender, suggesting that hormonal mechanisms may serve a protective role against oxidative stress.
[0057] Traumatic brain injury (TBI) is a major public health issue affecting over 1.7 million Americans annually, with falls, collision incidents, and motor vehicle accidents being the leading causes of injury (47). The consequences of TBI can be complex and long lasting, resulting in serious disorders that involve progressive cognitive deficits, epilepsy, and profound behavioral alterations. Although the molecular and cellular disturbances involved in these longer-term responses are not well understood, one common element in the pathology seems to be the establishment of a chronic inflammatory state that can persist in brain for weeks, months, and perhaps even years after TBI (101; 108).
[0058] A hallmark of inflammation is oxidative stress, which can be caused by metabolic dysfunction with numerous potential causes (43; 124). At the tissue and cellular levels, the pro- oxidative forces of inflammation following an injury can outweigh the capacity of antioxidative, protective mechanisms such as superoxide dismutase and glutathione peroxidase. The resulting state drives the formation of reactive oxygen species (ROS) and reactive carbonyl species (RCS), products of lipid peroxidation by ROS. In TBI, this condition results from injury-induced ischemia/reperfusion, hypoxia, elevated intracranial pressure, glutamate excitotoxicity, and intracellular calcium overload (34; 145), all of which contribute to the breakdown of
mitochondrial bioenergetics due, in part, to abnormally aggregated proteins (101).
[0059] An important modification of oxidative stress is protein carbonylation, which involves the introduction of carbonyl groups into protein-bound amino acids. Protein carbonylation may occur either through direct oxidation of amino acid targets by ROS or via interaction with RCS, which are themselves the product of lipid peroxidation (155). By either mechanism, the resulting carbonyl modifications can disrupt protein function and thereby contribute to injury
pathology (39). Carbonylation has long been utilized as a metric of oxidative stress levels in various neurodegenerative pathologies, including multiple sclerosis (19) and Alzheimer's disease (139). Although several studies in TBI have explored protein carbonylation, this work has been largely limited to the use of this modification as a measure of total oxidative
stress (114). Here, we identify four proteins that are preferentially carbonylated in response to TBI, investigate the susceptibility of various brain regions to carbonylation, identify cell types within these regions that are most affected by this modification, and, furthermore, differentiate gender-based differences in carbonylation after TBI. The results of this work shed light onto into the differential susceptibility of specific brain regions and cell types to TBI-induced oxidative stress, providing insights into the mechanisms of TBI pathology.
MATERIALS AND METHODS
Controlled Cortical Impact (CCI)
[0060] Adult male and adult female Sprague-Dawley rats (8-9 weeks of age) were purchased from Charles River Laboratories (Morrisville, NC, USA). Rats were housed in a barrier facility for animals accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Before experimental procedures, rats were anesthetized with isoflurane (Baxter Healthcare Corp., Deerfield, IL, USA) vaporized in medical-grade oxygen (100%, Roberts Oxygen Co. Inc., Rockville, MD, USA), placed in a digital cranial stereotactic device (Leica Microsystems, Buffalo Grove, IL, USA), and subjected to unilateral CCI injury over the left hemisphere. Briefly, the rats underwent a free-hand craniotomy (-3.8 mm from bregma in males, -3.0 mm in females). CCI was administered through the ImpactOne stereotaxic impactor (Leica Microsystems), which delivered a 3-mm flat-tipped impactor at 20° to a depth of 2 mm at 5 m/s with a 500-ms dwell time. After CCI, the incision was closed and the rats received buprenorphine 0.05 mg/kg for postoperative analgesia. Throughout all procedures, rat body core temperature was maintained at 37+0.3 °C. Sham-operated rats received no anesthesia, incision, or craniotomy. Animals were euthanized 5 days after injury under anesthesia (10% chloral hydrate solution, 0.4 ml/100 g). All animal handling procedures were performed in compliance with guidelines from the National Research Council for the ethical handling of laboratory animals and were approved by the Institutional Animal Care and Use Committee of USUHS (IACUC
Protocol APG 12-827, Bethesda, MD, USA). Immunoblotting: protein carbonylation detection and identification in brain tissue
Tissue collection and preparation
[0061] Brains were removed immediately after euthanization, frozen on powdered dry ice, and stored at -80°C until used. Brains were hand-dissected to produce blocks of penumbral structures enriched in carbonylated proteins as identified immunohistochemically (see below). The contralateral brain region and equivalent region from the naive animals were similarly collected.
One-dimensional gel electrophoresis
[0062] Protein fractions were prepared by homogenization in 5 volumes/tissue weight extraction solution (8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (Chaps), and 50 mM dithiothreitol (DTT; Sigma-Aldrich, St. Louis, MO, USA) containing 0.8% ampholytes (pH 3-10; Invitrogen Life Technologies, Carlsbad, CA, USA) and lx Complete protease inhibitor mix (Roche, Indianapolis, IN, USA)). After homogenization and subsequent centrifugation (20,000g, 10 min, 4°C), the resulting supernatant was stored at -80°C until used. The efficiency of tissue solubilization was > 95%, as judged by protein assay and Coomassie staining of one-dimensional gels.
[0063] Protein samples were prepared for gel electrophoresis by combining with an equal volume of 2x reducing loading buffer (Novex NuPAGE LDS sample buffer; Invitrogen;
containing 50 mM DTT) and heating at 70°C for 10 min. Samples were then fractionated (25 μ1/4.16 μg per lane) using NuPAGE 10% Bis-Tris gels (Novex-Invitrogen) and transferred to nitrocellulose blots using an iBlot transfer apparatus (Invitrogen).
Fluid-phase isoelectric focusing (IEF) [0064] To reduce the complexity of tissue homogenates for subsequent proteomic analyses, samples were prefractionated by fluid-phase IEF before one-dimensional gel electrophoresis. Samples of injured and contralateral hemisphere, and the corresponding regions from control naive animals (n = 8 for each region), were homogenized in 5 volumes (wt/vol) IEF denaturant consisting of 7.7 M urea, 2.2 M thiourea, and 4.4% Chaps containing lx Complete protease inhibitor mix (Roche) and clarified by centrifugation (20,000g, 10 min, 4°C). Tissue region pools were prepared and 200-μ1 aliquots of each pool supernatant were further diluted to 2.865 ml having a final composition of IEF denaturant plus ampholytes (150 μΐ, pH 3-10; Invitrogen), DTT (50 μΐ, 2 M stock), and bromophenol blue (10 μΐ, 10 mg/ml stock). The resulting sample was loaded into the IEF fractionator and focused using the following conditions: 100 V, 1.2 mA, 0 W (15 min); 200 V, 2.0 mA, 0 W (1 h); 400 V, 2.0 mA, 1 W (1 h); 600 V, 1.5 mA, 1 W (1 h). This resulted in fractions of proteins within the following pi ranges: 3.0-4.6, 4.6-5.4, 5.4-6.2, 6.2-7, and 7-9.1. IEF-fractionated proteins were further fractionated by size by one-dimensional gel electrophoresis (see One -dimensional gel electrophoresis).
Postelectrophoretic detection of carbonylated proteins
[0065] Postelectrophoretic detection of carbonylated proteins was performed as described by Conrad et al. (35) with minor modifications. Briefly, nitrocellulose membranes were washed in 20% methanol/80% Tris-buffered saline/Tween 20 (TBS-T), equilibrated in 2 N hydrochloric acid (HC1), and then incubated with 0.5 mM 2,4-dinitrophenylhydrazine (DNPH; Sigma- Aldrich) in 2 N HC1 (10 min, in the dark). The derivatized membranes were then washed three times with 2 N HC1 (10 min per wash) followed by 50% methanol (five times, 10 min per wash). Membranes were then equilibrated in TBS-T, blocked with 5% fetal bovine serum/TBS-T, and probed overnight at 4°C with rabbit anti-DNP (Sigma-Aldrich; Catalog No. 9659, 1: 1000 in TBS-T). Membranes were washed three times with TBS-T and probed with horseradish peroxidase-labeled, goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA; Catalog No. 31460, 1:5000 in TBS-T, 3 h). Blots were washed (TBS-T) and visualized by enhanced chemiluminescence (Novex ECL HRP Chemiluminescent Substrate Reagent Kit; Invitrogen) using a FUJI LAS 3000 imager (Fujifilm, Minato, Tokyo, Japan). The images were analyzed using MultiGauge version 3.0 software (Fujifilm) and the intensity of protein bands was quantified with ImageJ software (W.S. Rasband, ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA). Immunoreactive signals were mapped to corresponding protein features of Coomassie- stained gels. These features were excised and processed for identification via peptide mass fingerprinting.
Protein identification by peptide mass fingerprinting
[0066] Coomassie- stained gel pieces were destained in 100 mM NH4CO3/50% acetonitrile at 37 °C for 90 min. Slices were dehydrated in 100% acetonitrile (5 min, at room temperature), followed by drying under vacuum. The dehydrated gel pieces were rehydrated in a minimal volume of a solution of 40 mM NH4CO3/50% acetonitrile containing 20 ng/μΐ trypsin (Trypsin Gold, Mass Spec Grade; Promega, Madison, WI, USA) and incubated overnight at 37°C. Peptide fragments were recovered from the gel slices by sequential washes with 75 μΐ 1% trifluoroacetic acid (TFA) (1 h) and 50 μΐ 5% TF A/50% acetonitrile (two washes, 1 h per wash). The washes were pooled, dried under vacuum, and then dissolved in 10 μΐ of 1% TFA. Tryptic digests were mixed 1: 1 with a-cyanohydroxycinnamic acid matrix (10 mg/ml in 50% acetonitrile/0.1% TFA) containing bradykinin (1060.5692 Da) and adrenocorticotropic hormone fragment 18-29 (2465.1989 Da) (AnaSpec, San Jose, CA, USA) as internal standards and analyzed by matrix- assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, using a Voyager MALDI-TOF DE STR instrument (PE Biosystems). Peptide mass spectra data were analyzed via the Protein Prospector MS-Fit search engine (http://prospector.ucsf.edu/). Criteria for a positive identification were a MOWSE score greater than 1.00x10 and more than two times greater than any other identifications for the search, a coverage percentage greater than 25%, and a matched protein identifying with the appropriate molecular weight and p/ for the fractionation procedure.
[0067] In one embodiment, for detection of either carbonylated proteins or citruUinated proteins, a sample or gel can be reacted with a biotin- or hapten-hydrazide, with the one or more of the proteins of interest being measured by a sandwich assay using at least one antibody against the one or more protein(s) and/or one or more antibody(ies) against the biotin or hapten. A protein- specific antibody may be employed as the capture antibody.
[0068] In another embodiment, for detection of either carbonylated proteins or citruUinated proteins, a sample or gel can be reacted with-instead of the hydrazide-a carbonylation specific antibody, as disclosed in Xu, et al. "Detecting Protein Carbonylation in Adipose Tissue and in Cultured Adipocytes", Methods Enzymol, 2014: 538: 249-261, which is incorporated herein by reference.
[0069] In either of these two embodiments, detection can be accomplished by Wetern Blot, using instruments and methods as discussed herein and as disclosed in Lazarus, et al. "Protein
Carbonylation after Traumatic Brain Injury: Cell Specificity, Regional Susceptibility, and Gender Differences" Free Radical Biology and Medicine, 78 (2015) 89-100, incorporated herein by reference.
[0070] In another embodiment, for detection of either carbonylated proteins or citruUinated proteins, enzymatic digestion of sample to peptides then detection of carbonylation or citrullination in specific peptides can be determined through Mass Spectrometry. Antibodies against the proteins can be used to clean and concentrate prior to digest. Optionally, Multiple
Reaction Monitoring (MRM) techniques using isotopically labeled versions of expected peptides can be used to improve quantitation.
Immunohistochemistry
Tissue collection and preparation
[0071] Euthanized animals were perfused transcardially with 200 ml phosphate-buffered saline (PBS) followed by 200 ml 4% paraformaldehyde for fixation. Brains were stored in 4% paraformaldehyde overnight at 4°C and then equilibrated in a 30% sucrose solution (-48 h, 4°C). Coronal sections (20 μιη thick) were collected from 2.5 mm rostral to 2.5 mm caudal to the CCI lesion site and mounted onto Colormark Plus adhesion slides (Thermo Fisher Scientific). Every tenth section was stained with thionine to create a reference library.
Detection of carbonylated proteins and cell-specific markers
[0072] Immuno staining for protein carbonyls was performed as described by Zheng and
Bizzozero (155), with minor modifications. Each section was prepared by incubation with 100 μΐ DNPH (1 mg/ml in 2 N HC1) for 30 min at room temperature. Sections were then washed three times with 0.2% Triton X-100/PBS and blocked for 1 h at room temperature with 10% normal donkey serum. After three more washes with 0.2% Triton X-100/PBS, each tissue section was incubated at 4°C overnight with 100 ul of 1: 1000 rabbit anti-DNP (Sigma-Aldrich; Catalog No. D9656) in 0.3% Triton X-100/PBS. Colocalization experiments were performed by co- incubating sections with cell-specific markers: mouse anti-NeuN (detects neurons; EMD
Millipore, Billerica, MA, USA; Catalog No. MAB377; 1: 1000), mouse anti-glial fibrillary acidic protein (GFAP; detects astrocytes; EMD Millipore; Catalog No. MAB360; 1: 1000), goat anti- Ibal (detects microglia and macrophages; Abeam, Cambridge, UK; Catalog No. ab5076; 1: 100), and mouse anti-01ig2 (detects oligodendrocytes; EMD Millipore; Catalog No. MABN50, 1: 100). Slides were washed three times with 0.2% Triton X-100/PBS and then incubated with 100 μΐ secondary antibody solution: 1: 100 donkey anti-rabbit IgG (H+L), conjugated to green- fluorescent Alexa Fluor 488 dye (Invitrogen); 1: 100 donkey anti-mouse or anti-goat IgG (H+L), conjugated to red-fluorescent Alexa Fluor 594 dye (Invitrogen; Catalog Nos. A-21203 and A- 11058, respectively). After 3 h of incubation, this solution was replaced with 4',6-diamidino-2- phenylindole (DAPI; Sigma-Aldrich; Catalog No. D32670, 0.01% in 0.2% Triton X-100/PBS, 5 min at room temperature) for nuclear staining. Sections were washed three times with 0.2% Triton X-100/PBS and one time with lx PBS and then visualized with a Zeiss LSM 5 Pascal confocal microscope (Zeiss, Jena, Germany).
Preadsorption control
[0073] Specificity of the carbonylation immunohistochemistry was confirmed by
immunoneutralizing the primary antibody with carbonylated protein standard. Briefly, β-casein protein was chemically carbonylated by incubation with ferric chloride (FeCl3) (15 mg in 2 ml 37.5 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes), pH 7.2, containing 37.5 mM ascorbate and 0.15 mM FeCl3; 5 h, 37°C). The reaction mixture was then dialyzed against 50 mM Hepes and 1 mM ethylenediaminetetraacetic acid (pH 7.2) overnight at room temperature. A control reaction was conducted similarly with the absence of FeCl3.
Derivatization of protein carbonyl groups was carried out by combining the protein dialysates with an equal volume of 12% sodium dodecyl sulfate and an equal volume of 10 mM DNPH prepared in 1 N HC1. Reactions proceeded in the dark for 15 min at room temperature and were then neutralized with the addition of 3.25 ml 2 M Tris-HCl. The derivatized proteins were dialyzed against H20 overnight. The carbonylation status of the two preparations was confirmed by Western blot using anti-DNP immunodetection. Preadsorption of the primary antibody was carried out by incubating anti-DNP IgG (-2 μg) with carbonylated, DNPH-derivatized β-casein (-100 μg protein) at 4°C, overnight. An additional control condition was established by similarly incubating anti-DNP antibody in the absence of modified β-casein protein.
Statistical analysis
[0074] Carbonylation immunohistochemistry was quantified according to region using a blinded scoring system, with 0 reflecting the lowest amount of carbonylation fluorescence intensity in a region and 3 reflecting the greatest amount across all sections evaluated. Regions scored were the cortical lesion site, dorsal third ventricle, and median eminence. Sixteen brain sections per animal were scored using sections located from bregma -2.12 mm to bregma -3.30 mm
(control/CCI male rats n=l and 9, respectively; control/CCI female rats n=9 and 7, respectively). Comparisons between groups were analyzed by ANOVA with a Tukey HSD post hoc test. /?<0.05 was considered significant (MultiGauge version 3.0 software; Fujifilm).
RESULTS
CCI increases protein carbonylation in specific regions of the brain
[0075] An initial immunoblotting experiment was performed to confirm that the CCI model resulted in a reproducible pattern of protein carbonylation in brain. We investigated the profile of carbonylated proteins in whole-brain lysates. Injured brain lysates were compared to
contralateral uninjured tissue lysates from the same animal using Coomassie staining and Western blotting for DNP in one-dimensional gels (Figure 4). Total carbonylation (based on total lane intensity) was increased approximately 10% in samples from injured brain tissue compared to the signal observed in contralateral, uninjured brain tissue. Interestingly, carbonylation was enhanced for a small group of specific protein features (arrows) in response to CCI. These results confirmed that CCI causes the upregulation of carbonylation of specific proteins.
[0076] Peptide mass fingerprinting was used to identify specific proteins carbonylated in response to CCI. Figure 5 presents a representative analysis in which proteins present in control and TBI extracts were fractionated by fluid-phase IEF, followed by one-dimensional gel electrophoresis. Proteins having enhanced carbonylation after injury were visualized by Western blotting for DNP, mapped to corresponding features on Coomassie-stained gels, and identified as GFAP, dihydropyrimidase-related protein 2 (also known as collapsing response mediator protein 2, or CRMP2), fructose-bisphosphate aldolase C (ALDOC), and fructose-bisphosphate aldolase A (ALDOA).
[0077] To identify specific regions of the brain in which increased protein carbonylation occurred after TBI, we performed anti-DNP immunohistochemistry. Total brain DNP staining is displayed in Figure 6, with the site of CCI indicated in the upper left cortex.
[0078] At higher magnification, the fluorescence intensity of anti-DNP labeling in the vicinity of the lesion was clearly distinguished from that observed in the uninjured contralateral cortex (Figure 7, panels A and B, respectively). The specificity of the immunolabeling was confirmed by immunoneutralization of the anti-DNP primary antibody. Preincubation of the antibody with a carbonylated control protein, β-casein, effectively eliminated anti-DNP labeling in injured brain tissue (Figure 7, panel C). No reduction in signal was observed using anti-DNP antibody preincubated with unmodified β-casein (Figure 7, panel D).
[0079] In addition to the immediate area of the lesion, CCI increased protein carbonylation in the region of the dorsal third ventricle (Figure 8, panels A and B) and the median eminence (Figure 8, panels C and D) compared to these regions in sham, control animals. Staining was especially pronounced in the ependymal cells lining the third ventricle of both regions. In comparison, basal levels of anti-DNP immunofluorescence in the equivalent regions of sham control animals were very low. The most intense anti-DNP immunofluorescence after CCI was also observed in ependymal cells located adjacent to the paraventricular thalamic nuclei in the CCI-injured animals (Figure 8, panel B). Figure 5 also shows anti-DNP immunofluorescence in the median eminence region of control and CCI-lesioned rats (Figure 8, panels C and D, respectively).
Immunolabeling was concentrated in the ependymal cells and processes lining the floor of the third ventricle at the median eminence of CCI animals (Figure 8, panel D). This pattern of intense labeling was not observed in comparable sections prepared from uninjured control animals (Figure 8, panel C). Together, these immunohistochemical findings indicate that discrete regions of the brain exhibit an upregulation of protein carbonylation after CCI. This response is most pronounced in the cortical area surrounding the area of injury and in the ependymal areas of the dorsal third ventricle/paraventricular region and the median eminence.
Cell specificity of CCI-induced protein carbonylation
[0080] Experiments involving dual immunohistochemical staining were conducted to identify the cell types exhibiting increased protein carbonylation after CCI. The increase in carbonylation observed in the brain tissue surrounding the lesion was most significantly associated with astrocytes (Figure 9, panels A-C). Anti-DNP labeling predominantly colocalized with that of anti-GFAP, a marker for astrocytes. Labeling by anti-DNP was observed within both astrocytes (Figure 9, panels A-C, white arrows) and enlarged, GFAP-negative cells undergoing apparent degeneration (Figure 9, panels A-C, red arrows). In some cases, astrocytic foot processes appeared to surround these enlarged cells. The vast majority (>90%) of astrocytes in the injury lesion expressing carbonylated proteins were reactive astrocytes, as judged by enhanced GFAP expression, cellular hypertrophy, and disruption of individual cellular domains (132).
[0081] As shown in Figure 9, CCI-induced protein carbonylation is most significantly associated with astrocytes in the region of a CCI lesion. Brain tissue was collected 5 days after CCI and evaluated by coimmunofluorescence. (A-C) Representatives of the astrocyte and DNP detection at the injury site, (D-F) representatives of neuron and DNP detection at the injury site.
Carbonylated proteins within the penumbra region were identified by anti-DNP
immunohistochemistry (green, A and D). (B) and (E) represent anti-GFAP (astrocytes, red) and anti-NeuN (neurons, red) detection, respectively, in the injury lesion. (B) also illustrates counterstaining with DAPI (blue) for the detection of nuclei. Merged images (C and F) indicate the detection of carbonylated proteins and their relation to astrocytes (C) and neurons (F).
Astrocytes containing carbonylated proteins are shown in yellow (C). Astrocyte bodies and processes (red) contained carbonylated proteins (green, A), resulting in a yellow colocalization signal as indicated by white arrows (C). Astrocyte processes surrounding degenerating cells that contained carbonylated proteins are indicated by pink arrows (C). Neurons were identified using anti-NeuN (red, E), and protein carbonylation was identified using anti-DNP (green, D). The merged image indicates locations where both labels occur (yellow, F). Arrows indicate neurons that contained carbonylated proteins. (A-C) Original magnification x40, scale 50 μιη. (D-F) Original magnification x20, scale 100 μιη. Images are representative of three independent experiments. A total of n=3 CCI animals and n=3 sham animals were examined, with four slices per animal from bregma -2.12 mm to bregma -3.30 mm.
[0082] Immunohistochemical labeling of carbonylated proteins in the penumbra region also appeared in neurons, as indicated by the colocalization of anti-DNP and anti-NeuN labeling (Figure 9, panels D-F, white arrows). However, this colabeling was limited to only a relatively small proportion of the total number of neurons visualized by NeuN, compared to the relatively greater amount of carbonylated proteins within astrocytes as visualized by GFAP. Data also indicated that protein carbonylation was not significantly associated with oligodendrocytes, microglia, or macrophages.
Gender influences regional patterns of protein carbonylation
[0083] A number of studies have indicated that gender plays a role in determining the consequences of oxidative injuries of the brain (13; 118). The present investigation sought to determine whether gender affects protein carbonylation after CCI. A scoring system was developed to quantitatively assess the effects of CCI on protein carbonylation and to investigate the influence of gender on the response. The scoring system was based on overall intensity and extent of anti-DNP immunofluorescence detected in each region. Scores were developed on a 0- 3 scale for each region examined. A score of 0 reflected the basal state of carbonylation observed in control animals and a score of 3 was assigned to the greatest signal observed for a given region, across all animals studied. Representative images for scoring of each region of interest are presented in Figure 10.
[0084] As shown in Figure 11 and Figure 12, immunohistochemical and quantitative analysis indicated that the degree of protein carbonylation observed in the area of acute injury, 5 days after CCI, did not appreciably differ between male and female rats in that both genders had a maximal score of 3.
[0085] The response in the injury area was six-fold greater than that observed in regions of cerebral cortex distant from the lesion site (DDD/?<0.0001). The carbonylation score for the contralateral cortex of injured animals was not different from the score observed in the sham animal group. There were no gender differences observed across cortical regions compared here.
[0086] We next analyzed the effects of gender on CCI-induced protein carbonylation in the dorsal third ventricle and median eminence regions of brain. As shown in Figure 13,
carbonylation scores for median eminence of control animals were higher for males compared to females. This gender difference was further reflected in the response to CCI in that carbonylation scores were significantly elevated over control values in males but only tended to be so in females. A similar pattern was observed in the dorsal third ventricle, in which CCI-induced protein carbonylation was significantly greater in males compared to females. The carbonylation state of the dorsal third ventricle of control males tended to be higher than that of control females; however, this difference did not achieve statistical significance.
[0087] Protein carbonylation is a well-documented consequence and measureable indicator of oxidative stress in multiple neuropathologies, including Alzheimer's disease and Parkinson's disease (25). Recently, interest has focused on the role of oxidative stress in the pathology of TBI (66). To date, research has concentrated on measures of global carbonylation within the central nervous system, whereas little work has explored the neural regions, cell types, and specific proteins affected by this modification. Furthermore, although significant research has identified gender-based differences in TBI prognosis (52; 118), few researchers have examined how gender affects the magnitude or regional distribution of protein carbonylation after TBI.
[0088] The pathophysiology of TBI is both complex and dynamic, involving physical injury, ischemia/reperfusion, hypoxia, glutamate excitotoxicity, intracellular calcium overload, and elevated intracranial pressure (34; 136; 145; 152). Collectively, these conditions establish an environment of mitochondrial dysfunction, marked by the generation of ROS, including superoxide and hydrogen peroxide. These molecules drive the formation of RCS, such as acrolein and 4-hydroxynonenal, which are by-products of lipid peroxidation (127; 128). RCS, in turn, catalyze the generation of intra-protein carbonyls, further compromising cellular functions and exacerbating injury pathology. The data from this investigation show that the astrocytes in the immediate area of the injury lesion site, along with the ependymal cells lining the dorsal third ventricle and the floor of the third ventricle above the median eminence, contain the highest levels of protein carbonylation at 5 days after CCI. A time point of 5 days post-injury was considered to be sufficiently removed from the injury event to gain insights into the longer-term processes involved in brain injury. Whereas both genders showed similar levels of protein carbonylation at the site of injury, males showed a greater increase in carbonylation in ependymal cells distant from the lesion.
[0089] Within the injury region, the vast majority of astrocytes rich in carbonylated proteins were reactive astroglia, as indicated by complex, overlapping processes and hypertrophic cell bodies (132). Astrocytes have been proposed to be the first line of defense against oxidative stress after acute TBI (42; 96; 107). Astrocytes play a key role in balancing the oxidative load of the brain after injury, through their ability to scavenge ROS and thereby buffer the spread of oxidative stress (102). Whereas astrocytes have the capacity to combat oxidative stress to the benefit of other adjacent cells, they themselves are still vulnerable to the actions of ROS, which can cause mitochondrial damage, accelerated senescence, and cell death (30; 68). Our results support these conclusions by demonstrating that at the site of lesion, astrocytes are the dominant cell type showing increased protein carbonylation post-CCI. Whereas some neurons exhibited increased protein carbonylation after CCI, this response was minimal in comparison to astrocytes. The carbonylation status of oligodendrocytes and microglia and macrophages in the lesion area was essentially unaffected by CCI.
[0090] Our data indicate that CCI induces carbonylation in a subpopulation of cells distant from the region of the acute injury, specifically, in the ependymal lining of the dorsal third ventricle and median eminence. This unanticipated response was more pronounced in male rats compared to females. Ependymal cells form a barrier between cerebrospinal fluid (CSF) and neural tissue, acting as both a physical and a metabolic interface (41), The mechanism by which oxidative damage is transmitted to specific regions of the ependymal system is not fully understood, but may involve: (1) the transport of reactive molecules from the site of injury via the CSF and (2) a selective sensitivity of the ependymal lining of the dorsal third ventricle and median eminence to these mediators. Likely CSF-borne molecules include 4-hydroxynonenal, acrolein, and malondialdehyde, all of which are highly reactive aldehyde products of ROS -catalyzed lipid peroxidation. Unlike ROS, however, these compounds are relatively long-lived and can withstand extracellular transport from their site of generation (26; 127). In this regard, elevated levels of 4-hydroxynonenal have been documented in the CSF of patients with neuropathologies, including amyotrophic lateral sclerosis (130) and Parkinson's disease (126). The selective sensitivity of specific ependymal zones lining the dorsal third ventricle and area above the median eminence may reflect unique functional characteristics of the cells involved. In support of this proposal, ependymal cells lining the ventricles of the brain have been shown to display a broad range of heterogeneity with respect to structure and function (12; 65), membrane protein expression (12), and vulnerability to CSF-borne toxins (65). Similarly, the ependymal lining at the base of the third ventricle, directly above the median eminence, is well known for its unique structure (106), neurogenic cell populations (74), and neuroendocrine regulation (113). Therefore, we propose that aldehyde by-products of lipid peroxidation are generated by ROS at the site of acute injury, are transported through the cerebroventricular system, and interact with susceptible, CSF-adjacent periventricular ependymal cells to propagate carbonylated protein species.
[0091] The significance of increased protein carbonylation in the ependymal regions is not yet fully understood. It is known that a subset of ependymal cells of both the median eminence and the dorsal third ventricle extend foot-like processes into the brain parenchyma and onto resident capillaries (97). Moreover, this subset exhibits endocytosis of macromolecules from the CSF (106), which could lead to a local accumulation of RCS, thus propagating protein carbonylation. Our present immunohistochemical findings are also consistent with the proposal that carbonylation-containing ependymal cells may expand into the surrounding parenchyma, possibly serving as a mechanism for repair.
[0092] This investigation is the first to examine the effects of gender on the differential patterns of protein carbonylation after TBI. Basal levels of protein carbonylation were low but detectable in all brain regions investigated here. Interestingly, normal male rats exhibited a significantly greater degree of protein carbonylation in the ependymal zones of the dorsal third ventricle and median eminence compared to normal females. TBI produced dramatic and comparable increases in carbonylation in the acute injury region in both male and female rats. However, injured males showed a significantly greater increase in protein carbonylation than injured females in the ependymal zones of the dorsal third ventricle and median eminence, sites distant from the lesion. Accordingly, whereas male and female rats seem to respond similarly at the site of acute injury, females may be more resistant to secondary processes at sites distant to the injury. In this regard, clinical studies document that female patients exhibit reduced mortality and decreased complications after TBI (16; 52; 118). It has been proposed that the ovarian steroid hormone progesterone serves to regulate metabolic functions in the brain (60) and counteracts the excitotoxic effects of TBI (78; 137; 138), possibly by protecting against lipid peroxidation and the generation of RCS (119).
[0093] Although it is well recognized that oxidative stress drives many processes of secondary injury after brain injury, clinical trials involving antioxidant interventions have had limited success in preventing the sequelae of TBI (9; 67; 112; 122; 146). Our findings indicate that oxidative stress from TBI occurs within 5 days of injury, both locally at the site of acute impact and at sites distant from the site of impact. The lack of efficacy from antioxidants may be due to post-injury delays such that the irreversible effects of protein carbonylation may have occurred well before antioxidants could be applied. TBI-induced protein carbonylation and other forms of oxidative stress occur rapidly and effects appear to persist for extended periods of time after injury (10; 17; 20; 79). Accordingly, timely administration of antioxidant therapy post-injury has been difficult to achieve clinically. Also, multiple mechanisms are involved in the processes of oxidative stress, suggesting that a single treatment strategy may be inadequate in targeting the full spectrum of oxidative processes requiring management.
[0094] The research here has identified the selective carbonylation of GFAP, CRMP2, ALDOA, and ALDOC in response to TBI. The identification of GFAP, a major component of astrocytes, is consistent with our immunohistochemical findings, showing that these cells are particularly susceptible to oxidative modification after injury. GFAP, CRMP2, and ALDOC have all been identified as being carbonylated in Alzheimer's disease (29; 139), indicating a potential role for this modification in long-term neuropathology. Recent work by Oikawa and associates (99) examining the effects of ischemia/reperfusion injury on the carbonylated proteome of the monkey hippocampus also showed enhanced carbonylation of CRMP2 and ALDOC. Additionally, GFAP is a major target for carbonylation in experimental autoimmune
encephalitis, a murine model of multiple sclerosis (155). All four of the proteins identified in this work are also functionally grouped, as they all contribute to the maintenance of cellular structure (46; 59; 85; 110). In addition, CRMP2 functions in axonal guidance, serving an important role in neuronal regeneration after injury (142). Taken together, these findings suggest that carbonylation may provide a proteomic basis for mechanisms of sustained pathology after TBI. Understanding the mechanisms, nature, localization, and timing of protein modifications after TBI may provide a guide for the development of more effective TBI therapies that target both oxidative stress and its downstream consequences.
[0095] Thus, the disclosure incudes detection of the following carbonylated proteins GFAP, CRMP2, ALDOA and ALDOC. In some embodiments, two of any of the above proteins are determined, three of any of the above proteins are determined or four of any of the above proteins are determined.
[0096] These carbonylated proteins can be detected in any suitable biological sample, including but not limited to blood, blood plasma, blood serum, cerebrospinal fluid (CSF), urine, lymph and/or saliva.
[0097] In conclusion, this study demonstrates in rats that TBI induces carbonylation in specific brain regions, cell types, and proteins and that males are more susceptible to TBI-induced protein carbonylation than females. These results suggest that: (1) region- specific protein carbonylation may play an important role in the etiology of TBI pathology and (2) females are less susceptible to the ongoing pathological spread of protein modifications after injury. These gender- specific effects may point to possible approaches for intervention to address the secondary, pathological spread of protein modifications after TBI-induced oxidative stress.
Protein citruUination: A proposed mechanism for long-term pathology in traumatic brain injury
[0098] Protein citruUination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS) and prion disease. CitruUination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citruUination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citruUination and on the specific cell types involved. Immunofluorescence revealed that controlled cortical impact in rats profoundly up-regulated protein citruUination in the cerebral cortex, external capsule, and hippocampus. This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citruUination in neurons, oligodendrocytes or microglia. Further, proteomic analyses demonstrated that the effects of TBI on citruUination were confined to a relatively small subset of neural proteins. Proteins most notably affected were those also reported to be citrullinated in other disorders including prion disease and MS. In vivo findings were extended in an in vitro model of simulated TBI employing normal human astrocytes. Pharmacologically-induced calcium excitotoxicity was shown to activate the citruUination and breakdown of glial fibrillary acidic protein, producing a novel candidate TBI biomarker and potential target for autoimmune recognition. In summary, these findings demonstrate that the effects of TBI on protein citrullination are selective with respect to brain region, cell type and proteins modified, and may contribute to a role for autoimmune dysfunction in chronic pathology following TBI.
[0099] Traumatic brain injury (TBI) is a major cause of injury and death in the US, with over 1.7 million TBIs occurring annually and at least 5.3 million Americans currently living with ongoing disability (149). Traumatic brain injuries in civilians are largely due to automobile accidents, as well as falls, sports, and firearms (152). Military populations are at disproportionately elevated risk for blast-related TBI due to the devastating effects of improvised explosive devices (58). While there is a very large body of information on causes and global consequences of TBI, much less is known about the mechanisms underlying long-term pathology.
[00100] The long-term consequences of TBI can be complex, and often result in progressive cognitive and behavioral changes. Studies have indicated that anywhere from 10% - 50% of individuals with TBI suffer from persistent symptoms following injury (11), including attention deficits and short-term memory loss (149). This long-term dysfunction follows in the wake of two main injury phases: (1) the primary injury, caused by the immediate forces of the trauma (111; 152); and (2) the subsequent secondary injury, which presents as a constellation of dysfunctional molecular processes including impaired metabolism, free radical production, inflammation, and glutamate excitotoxicity (149). At present, it is not well understood how these various dysfunctional processes following acute injury can lead to progressive, chronic pathology after TBI.
[00101] A hallmark mechanism of secondary injury following TBI is prolonged imbalance in cellular calcium homeostasis, resulting in excitotoxic calcium overload (48; 84; 141). Following TBI, ischemia can lead to a dramatic loss of glucose and oxygen delivery to the central nervous system, thereby limiting the production of ATP. In the absence of adequate energy, ionic homeostasis is lost and calcium toxicity ensues. The abnormally large increase in intracellular calcium originates from both the influx from the extracellular space across multiple calcium channels, and from the mobilization of intracellular calcium stores (143). The result is a pathologically prolonged state of elevated intracellular calcium (141).
[00102] The downstream effects of cellular calcium toxicity have been examined closely in regard to mitochondrial dysfunction and oxidative stress. However, little attention has been given to the role of TBI-induced calcium overload in the activation of peptidylarginine deiminase (PAD) enzymes. This family of calcium-dependent enzymes catalyzes the post-translational modification of citruUination, resulting in the conversion of intrapeptidyl arginine residues to citrulline residues. In addition to altering both the normal structure and function of proteins, citruUination generates "altered-self ' epitopes that may be antigenic, prompting autoimmune responses against previously benign proteins (32; 154). Altered calcium homeostasis
accompanied by protein citruUination has been implicated in several neurodegenerative disorders, including Alzheimer's disease (62), temporal lobe epilepsy (8), glaucoma (18), rheumatoid arthritis (77), and multiple sclerosis (6).
[00103] At present, understanding of the specific proteins modified by citruUination is very limited outside the field of immunology, where the antigenic properties of abnormal
citruUination are studied largely within the context of rheumatoid arthritis. Additionally, there is no data available regarding the regional and cellular specificity of protein citruUination in neural tissue following TBI. Here, we identify the regions and cell types most susceptible to protein citruUination following TBI, and also identify a subset of neural proteins that are preferentially citruUinated in response to injury. Furthermore, we present the development of an in vitro model for simulating TBI through astrocytic calcium excitotoxicity, and identify a citrullinated breakdown product of glial fibrillary acidic protein (GFAP). Until now, citrullination has been primarily associated with chronic, progressive autoimmune and neural disorders. The present findings indicate that protein citrullination is a feature of TBI that may contribute to the long- term pathogenic mechanisms following acute injury, including those that involve the adaptive immune system.
MATERIALS AND METHODS
Controlled Cortical Impact (CCI)
[00104] CCI was conducted as described in Lazarus et al. (2015). Briefly, adult male and female Sprague-Dawley rats (Charles River Laboratories, Morrisville, North Carolina, USA) were anesthetized, then subjected to unilateral CCI over the left hemisphere administered through a ImpactOne stereotaxic impactor (Leica Microsystems, Buffalo Grove, Illinois, USA), which delivered a 3 mm flat-tipped impactor at 20° to a depth of 2 mm at 5 m / s with a 500 ms dwell time at -3.8 mm bregma in males / - 3.0 mm bregma in females. Sham-operated animals received no anesthesia or CCI treatment. Animals were euthanized five days following injury. All animal handling procedures were performed in compliance with guidelines from the National Research Council for the ethical handling of laboratory animals, as approved by the Institutional Animal Care and Use Committee of USUHS (IACUC Protocol APG 12-827, Bethesda, Maryland, USA). Immunohistochemistry
Tissue collection and preparation
[00105] Tissue was collected and prepared as described previously (73). Briefly, euthanized animals underwent transcardial perfusion (phosphate buffered saline (PBS) followed by 4% paraformaldehyde) after which brains were removed for storage overnight at 4°C in 4% paraformaldehyde and then equilibration in a 30% sucrose solution (2 days, 4°C). Brains were sectioned coronally (20 μιη) across the breadth of the lesion site (2.5 mm rostral to 2.5 mm caudal) with a Leica CM1900 cryostat (Leica Microsystems), and sections were then mounted on slides and stored at -80°C. The following groups of gender / conditions were prepared for immunohistochemical analysis: n = 11 male rats, CCI; n = 8 male rats, sham-operated control; n = 10 female rats, CCI; n = 7 female rats, sham-operated control.
Detection of cell-specific citrullination
[00106] The region and cell- specific effects of CCI on protein citrullination were determined using a mouse monoclonal anti-citrulline antibody (mAb 6B3, IgG2b) (81). The antibody was purified from expression medium by Protein A affinity chromatography (HiTrap Protein A HP column (17-0403-01; GE Healthcare, Buckinghamshire, United Kingdom) on a GE AKTA FPLC fast protein liquid chromatography instrument (FPLC; 18-1900-26; GE Healthcare), aliquoted for single-use and stored at -80°C.
[00107] Immunodetection of citrullinated proteins was performed as follows. Brain sections were incubated at 4°C overnight with 125 μΐ of 1: 1000 mouse anti-citrulline 6B3 antibody in 0.3% Triton X-100/PBS, with 1: 100 normal donkey serum (NDS) as a blocking agent (16 sections per animal). Co-localization experiments were performed by co-incubating these sections with mAb 6B3 and either: (1) goat anti-GFAP (1: 1000; ab53554; Abeam, Cambridge, England) (astrocytes); (2) goat anti-ionized calcium-binding adapter molecule (Ibal; 1:500; ab5076; Abeam) (microglia); (3) rabbit anti-neuronal nuclei (NeuN; 1:500; abl04225; Abeam) (neurons); or (4) rabbit anti-myelin basic protein (MBP; 1: 1000; ab40390; Abeam)
(oligodendroglia). Slides were washed five times with 0.2% Triton X-100/PBS and then incubated for 30 min with 125 μΐ of secondary antibody solution: 1: 100 donkey anti-mouse IgG (H+L), conjugated to green-fluorescent Alexa Fluor 488 dye (A-21202; Invitrogen, Waltham, Massachusetts, USA); and either 1: 100 donkey anti-goat, conjugated to red-fluorescent Alexa Fluor 594 (A- 11058, Invitrogen), or 1: 100 donkey anti-rabbit, conjugated to red-fluorescent Alexa Fluor 594 (A-21207; Invitrogen). The sections were washed five times with 0.2% Triton X-100/PBS and one time with lx PBS (5 m) and then visualized with an Olympus BX61 fluorescent motorized system microscope (Olympus, Shinjuku, Tokyo, Japan) using iVision-Mac software (Bio Vision Technologies, Exton, Pennsylvania, USA).
Preadsorption control
[00108] Specificity of the mAb 6B3 anti-citrulline antibody in immunofluorescence was confirmed through immunoneutralization using a mixture of citrullinated protein molecular weight standards (trypsinogen, glyceraldehyde 3-phosphate dehydrogenase, bovine albumin, trypsin inhibitor, alpha-lactalbumin, carbonic anhydrase, and egg albumin) prepared in-house via 10 hour incubation at 37°C with active PAD enzyme cocktail (0.5 μg / ul; P312-37C-25;
SignalChem, Richmond, British Columbia, Canada) in Tris buffer (50 mM Tris HCl, pH 7.4); 5 mM CaCl2; and 0.73 mM dithiothreitol (DTT; Sigma-Aldrich, St. Louis, MO, USA)).
Concurrently, a control sample was prepared in an identical manner, without the addition of the PAD enzyme cocktail.
Identification of citrullinated protein species in injured rat brain
Tissue collection and preparation
[00109] Brains were collected, snap-frozen with powdered dry ice, and stored at -80°C until use. Brains were thawed on wet ice and then hand-dissected to produce blocks of tissue
encompassing the lesion site (penumbra region) and the surrounding tissue shown by
immunohistochemistry to have increased protein citrullination following CCI. The equivalent region from naive animals (control tissue) was similarly collected. Tissue blocks were homogenized in 5 volumes / tissue weight extraction solution, consisting of: 7.7 M urea; 2.2 M thiourea; and 4.4% CHAPS; also containing lx Complete protease inhibitor mix (Roche).
Samples were clarified by centrifugation (20,000 x g, 10 min, 4°C), and resulting supernatants were fractionated by 2-dimensional electrophoresis (2-DE).
Fluid-phase Isoelectric Focusing (F-IEF)
[00110] Treatment group pools (control and injury penumbra) were prepared (N = 4/pool) and fractionated by F-IEF. Briefly, 200 μΐ aliquots of each pool was diluted up to 2.865 ml in IEF running solution (7.7 M urea; 2.2 M thiourea; 4.4% CHAPS; ampholytes (150 μΐ, pH 3 - 10; ZM0021, Invitrogen); DTT (50 μΐ, 2 M); and bromophenol blue (10 μΐ, 10 mg/ml). Samples were loaded into the ZOOM IEF Fractionator (ZMF10002; Invitrogen) and focused according to the following conditions: (1) 100 V, 1.2 mA, 0 W (15 min); (2) 200 V, 2.0 mA, 0 W (1 h); (3) 400 V, 2.0 mA, 1 W (1 h); (4) 600 V, 1.5 mA, 1 W (1 h) resulting in fractions of proteins within the following five pi ranges: 3.0 - 4.6; 4.6 - 5.4; 5.4 - 6.2; 6.2 - 7.0; and 7.0 - 9.1.
Molecular weight fractionation
[00111] Proteins in each of the F-IEF fractionations were further resolved by molecular weight fractionation using conventional one-dimensional gel electrophoresis. Samples were combined with an equal volume of 4x reducing loading buffer (Novex NuPAGE LDS sample buffer; 50 mM DTT; Invitrogen), heated at 70°C (20 min) and then fractionated (10 μΐ per well) using NuPAGE 4 - 12% Bis-Tris gels (Novex, Invitrogen). Proteins were transferred to nitrocellulose blots using an iBlot transfer apparatus & gel transfer stacks (Nitrocellulose Mini; 1B301002, Invitrogen).
Immunoblotting [00112] Blots were blocked with 5% instant nonfat dry milk / Tris-buffered saline/Tween 20 (TBS-T) (1 h, room temperature) and then incubated with the 6B3 primary antibody (1:300 in TBS-T; mAb stock = 1.79 mg/mL) for 1 h at room temperature, then 4°C overnight. Following equilibration to room temperature (30 min), membranes were washed in TBS-T (5 times over 60 min), incubated with secondary antibody, horseradish peroxidase-labeled, goat anti-mouse IgG (1:2500 in 5% TBS-T; 31430, Thermo Scientific) for 2 h at room temperature and then visualized by enhanced chemiluminescence (ECL) (Novex ECL HRP Chemiluminescent Substrate Reagent Kit; WP20005, Invitrogen) using the FUJI LAS 3000 Imager (Fujifilm, Minato, Tokyo, Japan). Images collected were analyzed using MultiGauge software (v. 3.0, Fujifilm). In some cases, blots were reprobed with a second anti-protein citrulline antibody (1:500; MABN328EMD; Millipore; detection with horseradish peroxidase-labeled, goat anti- mouse IgM; 1:2500 in TBS-T; 31440, Thermo Scientific), with final overnight washing, to confirm the 6B3 immunoreactive features and increase the sensitivity of detection. No new signals were revealed by this approach.
[00113] The specificity of mAb 6B3 for detecting citruUinated proteins on western blots was confirmed by immunoneutralization, similar to the approach used for immunohistochemistry (see above), but involving solid-phase preadsorption (versus fluid phase) to accurately replicate the conditions of western blotting. In this case, the 6B3 antibody was reacted with a strip of nitrocellulose (Protran® BA85, 0.45 μιη pore size, binding capacity 80 μg/cm ; Sigma- Aldrich; cut into 1.4 cm x 3 cm pieces) to which either the citruUinated or native forms of fibrinogen had been bound. The amount of protein absorbed to the strip was 200μ1 / 200μg of citruUinated human fibrinogen (400076, Cayman Chemical, Ann Arbor, Michigan, USA), or 200μ1 / 200μg human fibrinogen (16088, Cayman Chemical) in TBS-T. The duration of the antibody absorption was 16 h at 4°C.
Protein identifications
[00114] Immunoreactive signals of interest were mapped to corresponding features in
Coomassie- stained gels. These features were excised and processed for identification through the ESMS-Basic Protein ID service using an Elite Orbitrap mass spectrometer (Thermo Scientific) by the W.M. Keck Mass Spectrometry & Proteomics Resource (W.M. Keck Foundation
Biotechnology Resource Laboratory, New Haven, Connecticut, USA). Analyses results were received through and tracked within the Yale Protein Expression Database (YPED, Yale / NIDA Neuroproteomics Center, New Haven, Connecticut, USA). Criteria for a positive identification included: an expectation score less than 1.0E-65, a percent coverage greater than 35.0%, and a pi and mw that were consistent with the fractionation data.
In vitro model of protein citrullination following TBI-induced excitotoxicity in astrocytes
[00115] An in vitro model of simulated TBI was established using normal human astrocytes (NHA CC-2565, Lonza, Allendale, New Jersey, USA) and treatment with the calcium ionophore, ionomycin (Free Acid, Streptomyces conglobatus, 407950, Calbiochem, Billerica,
Massachusetts, USA), to simulate calcium excitotoxicity. Astrocytes were cultured to -70% confluence in T-75 tissue culture flasks according to vendor instructions. Cells were washed with TBS (4 times) and then treated for 4 h (37°C, 95% 02/5% C02) with either ionomycin (10 μΜ; 10 mL TBS) or dimethyl sulfoxide vehicle (DMSO; 10 μ1/10 mL TBS). Following incubation, protease inhibitors were added to the flasks (final concentration of lx; Complete protease inhibitor mix; 10269700, Roche) and cells were harvested by scraping and centrifugation (800 x g, 4°C, 10 min). Cells were extracted by vortexing into IEF denaturant (see section 2.3.2) at a ratio of 1 part pellet to 4 parts buffer (v/v). Samples were clarified by centrifugation (15,000 x g, 4°C, 10 m). The resulting supernatent was processed by one-dimensional gel electrophoresis and immunoblotting as described above. Replicate immunoblots containing samples from three separate experiments each were probed with either anti-citrullinated protein (6B3) or anti-GFAP (1:3000, Z0334, DAKO, Carpinteria, California, USA). Each primary antibody was paired with an appropriate secondary antibody (6B3: horseradish peroxidase-labeled, goat anti-mouse IgG; 1:2500; 31430; Thermo Scientific; GFAP: horseradish peroxidase-labeled, goat anti-rabbit IgG; 1:3000; HAF008; R&D Systems) and blots were visualized by ECL.
[00116] Figure 14 presents the effects of CCI on the expression of citruUinated proteins in the rat cerebral cortex. Immunohistochemical analysis demonstrated that the basal level of protein citrullination as detected by labeling with mAB 6B3 was very low under control conditions. In contrast, CCI induced a marked increase in the immunolabeling of cells in the cortex. This up- regulation of protein citrullination was most pronounced in the vicinity of the lesion. The morphology of the affected cells was remarkably consistent, suggesting that the effects of CCI on protein citrullination were cell- specific.
[00117] The specificity of mAb 6B3 labeling of citruUinated proteins in TBI brain was confirmed by immunoneutralization. As shown in Figure 15, preabsorption of the mAb 6B3 with citruUinated protein standard effectively eliminated the labeling of cells in the cerebral cortex and hippocampus of CCI brain. In this experiment, the active labeling condition utilized mAb 6B3 that had been exposed to the identical absorption procedures as the neutralized mAb 6B3, except that the neutralizing proteins were not citruUinated under the control condition.
Additionally, a secondary-only control was also conducted and further confirmed the specificity of the anti-citrullinated protein labeling (not shown). [00118] The regional effects of CCI on protein citrullination are summarized in Figure 16. CCI produced a marked increase in protein citrullination throughout the injured cortex, extending from lateral to the lesion site to regions of the cortex not directly impacted by CCI (Figure 16, panel B). Similarly, immunolabeling of the injured ipsilateral hippocampal formation (Figure 16, panel E) and external capsule (Figure 16, panel H) revealed a significant degree of protein citrullination in these regions. To a lesser extent, protein citrullination was also observed in fibers extending ventrally from the lesion site towards the midline corpus callosum (not shown). Other brain regions, including the amygdala and caudatoputamen, were completely negative for protein citrullination in these CCI animals. Furthermore, there was no gender difference observed in the regionalization or magnitude of protein citrullination following injury. In general, CCI appeared to have little effect on the status of protein citrullination in contralateral brain structures, with the exception of the dorsal hippocampus, where approximately 20% of injured animals (4 of 21) displayed an intense labeling of unusually large and rounded cells (Figure 16, panel F). Finally, 6B3 immunolabeling was uniformly low across all regions studied in control male and female animals (Figure 16, Panels A, D, and G).
[00119] Dual immunofluorescence revealed astrocytes to be the principal cell type in which protein citrullination was affected by CCI. Figure 17 shows that anti-citrulline labeling in the cortex and external capsule was predominantly colocalized with GFAP. Similar observations were made in other affected brain regions.
[00120] The findings presented in Figure 18 further confirm that CCI-induced protein citrullination was not significantly associated with neurons (NeuN), microglia / macrophages (Ibal), or oligodendrocytes (MBP) in the cortex. Citrullination was also not significantly associated with these cell types in any other brain regions investigated (not shown). [00121] The distinctive profile of CCI-induced protein citrullination in the ipsilateral and contralateral hippocampus is shown in Figure 19. As noted above, increased anti-citrullinated protein labeling was clearly evident in the ipsilateral hippocampus of all animals studied (Figure 19, panel A), and this labeling colocalized with GFAP labeling (Figure 19, panels B and C). In addition, in -20% of injured animals, the contralateral hippocampus also displayed intense 6B3 labeling (Figure 19, panel D) that colocalized with GFAP labeling. The co-labeled cells were morphologically distinct from traditional stellate astrocytes, displaying a rounded, branchless appearance consistent with that of a macrophage (Figure 19, panels E-G).
[00122] Figure 20 demonstrates the specificity of the 6B3 antibody for detecting citrullinated proteins in a western blot format. Shown on the left is the Coomassie staining for human fibrinogen (Fib) and the same preparation of fibrinogen that was enzymatically citrullinated by reaction with PAD4 (C-Fib). The protein staining shows that the characteristic profile of purified human fibrinogen is modestly affected by reaction with PAD4. Presented on the right are immunoblots showing the reactivity of mAb 6B3 with the citrullinated fibrinogen preparation (Active/C-Fib) and the elimination of this reactivity by preabsorption of 6B3 with citrullinated fibrinogen prior to blotting (Neutralized/C-Fib). There was no reactivity of mAb 6B3 with fibrinogen that had not been citrullinated by PAD4 treatment (Active/Fib).
[00123] Proteomic analysis revealed that the effects of CCI on protein citrullination were specific to a discrete subset of proteins making up the entire brain proteome, and further, that the proteins involved are primarily associated with cytoskeletal structure and metabolic processes (Figure 21). Shown in the upper left panel are the proteomes of control and injured cerebral cortex fractionated by fluid phase isoelectric focusing. Each pair of lanes, control (C) and CCI (I), show the proteins present in the four different pi partitions. As visualized by Commassie staining, CCI did not appreciably affect the general pattern of protein staining across the four pi fractions. In contrast, the pattern of protein citrullination was dramatically impacted by CCI (upper right panel). Consistent with immunohistochemistry findings (Figures 15 and 16), little protein citrullination was observed in control cortex (C), whereas CCI (I) resulted in the intense labeling of a distinctive subset of the fractionated proteins. The immunoreactive signals of the western blot were mapped to Commas sie features of the protein gel, and proteins were identified by peptide mass fingerprinting and tandem mass spectrometry. The proteins identified are presented in the lower panel of Figure 21. These proteins are functionally grouped as
cytoskeletal components (including dynamin-1, GFAP, and several forms of tubulin); those involved in metabolic processes (including peroxiredoxin-1, dihydropyrimidinase-related protein 2, and creatine kinase B-type); and proteins involved in cell-cell signaling and synaptic transmission (synapsin-2, syntaxin-binding protein 1, and amphyiphysin).
[00124] The disclosure includes determining the citrullination levels of one or more of the following proteins or protein fragments (as listed in FIG. 21) fructose-bisphosphate aldolase A, peroxiredoxin-1, cofilin-1, glutathione S-transferase P, transgelin, dynamin-1, synapsin-2, moesin, septin-9, stress-induced phosphoprotein 1, dihydropyrimidase-related protein 2, alpha- enolase, gamma-enolase, glial fibrillary acidic protein, fructose-bisphosphate aldolase C, neuronal- specific septin-3, transgelin-3, myelin basic protein, heat shock 70 kDa protein 4, GRIP 1- associated protein 1, transitional endoplasmic reticulum ATPase, NADH-ubiquinone oxidoreductase 75 kDa mitochondrial subunit, vesicle-fusing ATPase, 78 kDa glucose-regulated protein, heat shock cognate 71 kDa protein, annexin A6, syntaxin-binding protein 1, actin, cytoplasmic 1, creatine kinase B-type, tubulin beta-2B chain, tubulin beta-2A chain, tubulin beta- 4B chain, tubulin beta-5 chain, tubulin beta-3 chain, amphiphysin, and drebrin. [00125] In some embodiments, two of any of the above proteins are determined, three of any of the above proteins are determined, four of any of the above proteins are determined, five of any of the above proteins are determined, six of any of the above proteins are determined, seven of any of the above proteins are determined, eight of any of the above proteins are determined, nine of any of the above proteins are determined, ten of any of the above proteins are determined, eleven of any of the above proteins are determined, twelve of any of the above proteins are determined, thirteen of any of the above proteins are determined, fourteen of any of the above proteins are determined, fifteen of any of the above proteins are determined, sixteen of any of the above proteins are determined, seventeen of any of the above proteins are determined, eighteen of any of the above proteins are determined, nineteen of any of the above proteins are
determined, twenty of any of the above proteins are determined, twenty one of any of the above proteins are determined, twenty two of any of the above proteins are determined, twenty three of any of the above proteins are determined, twenty four of any of the above proteins are determined, twenty five of any of the above proteins are determined, twenty six of any of the above proteins are determined, twenty seven of any of the above proteins are determined, twenty eight of any of the above proteins are determined, twenty nine of any of the above proteins are determined, thirty of any of the above proteins are determined, thirty one of any of the above proteins are determined, thirty two of any of the above proteins are determined, thirty three of any of the above proteins are determined, thirty four of any of the above proteins are determined, thirty five of any of the above proteins are determined, thirty six of any of the above proteins are determined or thirty seven of any of the above proteins are determined. [00126] These citruUinated proteins can be detected in any suitable biological sample, including but not limited to blood, blood plasma, blood serum, cerebrospinal fluid (CSF), urine, lymph, and/or saliva.
[00127] To further investigate the cellular mechanisms of injury-induced protein citruUination, a model of simulated TBI was established. In this model, normal human astrocytes (obtained commercially) were incubated in control medium or in medium containing the calcium ionophore, ionomycin, to induce calcium excito toxicity. Figure 22 shows the results of 3 separate experiments investigating the effects of ionomycin treatment on the proteolytic processing of GFAP (left panel) and the generation of citruUinated proteins (right panel). The data show that treatment with ionomycin consistently activated the proteolytic processing of intact GFAP (left panel; blue arrows) to produce a distinctive pattern of breakdown products. Probing with mAb 6B3 indicated that one of the GFAP breakdown products is preferentially citruUinated (right panel; orange arrow) in response to simulated TBI. In addition, several other protein features appeared to be heavily citruUinated in response to ionomycin treatment. The identity of these signals remains to be determined.
[00128] Protein citruUination is a calcium-dependent protein modification that has been largely studied in the context of autoimmune disorders, particularly rheumatoid arthritis. Abnormal protein citruUination in rheumatoid arthritis results in the generation of antigenic epitopes that become targeted by the adaptive immune system (55; 69). Protein citruUination is also found to be abnormal in several neurodegenerative disorders, including multiple sclerosis (MS) and Alzheimer's disease, suggesting that citruUinated proteins may also serve as antigenic targets in these conditions and thereby contribute to long-term pathogenesis. To date, however, little research has examined the effects of TBI on protein citruUination, where an acute mechanical injury can lead to progressive and sustained neuropathology. Currently, there are no data regarding the identity of specific proteins affected by citruUination following TBI, or information on the susceptibility of different brain regions or cell types to this modification following injury.
[00129] The constellation of dysfunctions resulting from TBI is often complex and dynamic, involving pathology caused by acute tissue damage and interactions amongst the ongoing changes caused by ischemia, hypoxia, excitotoxicity, and elevated intracranial pressure (149). A hallmark of acute TBI pathophysiology, however, is intracellular calcium overload (49). This shift in ionic balance is excitotoxic, activating a number of damaging intracellular cascades, including lipid peroxidation, proteolysis, free radical generation (152), and the activation of pro- apoptotic genes (149) with the loss of dendritic spines. Elevated intracellular calcium is an essential condition for the activation of peptidylarginine deiminase (PAD) enzymes, which catalyze the conversion of intrapeptidyl arginine residues to citrulline residues (61).
[00130] PAD enzymes exist as several isoforms, two of which are found in neural tissue: PAD2 (37; 64; 109) and PAD4 (2). In humans, PAD2 is largely localized to astrocytes, while PAD4 is exclusively expressed in neurons (2). Moreover, PAD2 is localized to reactive astrocytes in the brains of mice affected by a prion disorder, scrapie, where its enzymatic activity is nearly double that of healthy mice (64). Research on the etiology of MS has suggested that PAD2 contributes to the pathology of demyelination, where its activity is thought to destabilize myelin sheath structure (89).
[00131] The present investigation in rats shows that TBI-induced protein citruUination is selectively localized to astrocytes. This finding is consistent with the understanding that following ischemia, astrocytes exhibit a dramatic rise in intracellular calcium due to a rapid influx of extracellular calcium through dysfunctional membrane channels, as well as the release of calcium from intracellular stores (49). While influxes in intracellular calcium are also found in neurons following mechanical injury, this rise is far less profound than that observed in astrocytes (121). Furthermore, astrocytes selectively display long-term defects in calcium signaling following TBI (121) and ultimately, astrocytic death precedes that of neurons in rat models of TBI (49). Related findings involving hypoxia confirm that astroglial injury is due to massive calcium influxes and resulting excitotoxicity (61). Models of TBI illuminate the biologic importance of astrocytes in brain injury, serving a protective role by shielding neurons from damage due to oxygen deprivation (147), glutamate neurotoxicity, and calcium excitotoxicity (83). It has been proposed that the intracellular rise of calcium within astrocytes is directly linked to their neuroprotective effects on neurons, most likely due to their ability to sequester large amounts of calcium and thus stabilize the ionic environment for neurons (44). Our finding that protein citrullination is limited to astrocytes is consistent with this proposal.
[00132] Present results indicate that the up-regulation of protein citrullination induced by TBI occurs in astrocytes that are located in the cerebral cortex, external capsule, and hippocampus. In contrast, the status of protein citrullination in other brain regions (such as the amygdala and caudate putamen) and cell types was not appreciably altered by TBI. The mechanistic basis for this observation may relate to the up-regulation of voltage-gated, class C L-type Ca2+ channels that are selectively expressed in astrocytes and are particularly sensitive to activation by injury in the regions reported here (33). The finding that nimodipine, an L-type calcium channel blocker, protects against excitotoxic damage in cultured astrocytes supports this proposal (56). These findings correspond to a model of calcium excitotoxicity-induced citrullination. The selective citrullination of astrocytes in these particular regions may correspond to long-term dysfunctions associated with TBI, including learning and memory deficits associated with progressive hippocampal atrophy (149).
[00133] Our investigation showed that patterns of protein citruUination following TBI were similar between male and female rats. This finding is in contrast to our previous observations concerning the effects of TBI on protein carbonylation. Following injury, male rats showed far greater response in protein carbonylation as compared to female rats (73). Protein carbonylation is a reflection of oxidative stress (40), whereas citruUination is a marker of calcium influx (71; 148). Accordingly, the gender difference observed in carbonylation may be due to the protective antioxidant effects of ovarian steroids (117), whereas a similar mechanism does not appear to exist in the case of citruUination. The extent to which these protein modifications contribute to gender differences in TBI complications and mortality (15; 16; 53) remains to be determined. It should be noted that the present experimental design focused on only one time-point post- TBI (five days), and as such, it is possible that gender differences in post-injury citruUination may be evident at other time-points.
[00134] The present investigation has identified 37 proteins as a very small subset of the entire brain proteome that is citrullinated in response to TBI. This selectivity in protein citruUination indicates that the mechanism involved maintains a high degree of specificity. Furthermore, several of the proteins identified here are reported to be citrullinated in neurodegenerative diseases, and thus may play causative roles in neuropathology. Specifically, citrullinated GFAP is a characteristic feature in MS and Alzheimer's disease (24; 55; 62; 94), and myelin basic protein (MBP), a major component of myelin sheath structure, is profoundly over-citrullinated in MS (24; 144). Similarly, GFAP, tubulin, peroxiredoxin 1, cofilin-1 and alpha/gamma enolase are selectively citrullinated in prion disease (64). Therefore, the link between abnormal protein citruUination and neurological disease appears strong. It should be noted that the 37 proteins identified as targets for TBI-induced citruUination were derived from the analysis of only 16 gel features. Accordingly, not all of the proteins identified in a single feature are necessarily citruUinated, and may have resulted from the analysis due to their co-purification with another citruUinated species. Nevertheless, the high correlation that exists between the specific proteins identified here and their reported citruUination in various disease states is consistent with the proposal that these proteins are indeed targets for citruUination following TBI.
[00135] A potential mechanism by which abnormal protein citruUination contributes to neurological disease could involve the adaptive immune system. A significant proportion of the proteins identified here are also recognized as autoantigens, in both neurological and
autoimmune-related disorders. For example, MBP is the signature citruUinated autoantigen of MS (23; 144). Autoantibodies targeting amphiphysin are associated with several neurological disorders, including sensory neuronopathy and encephalopathy (88). Additionally,
dihydropyrimidinase-related protein 2 (CRMP2) is autoantigenic in autoimmune retinopathy (3). Finally, alpha enolase has been identified as a citruUinated autoantigen in rheumatoid arthritis (71), while citruUinated 78 kDa glucose-regulated protein is an autoantigen within the pancreatic beta-cells in Type 1 diabetes (115). Collectively, these findings support the proposal that injury- induced protein citruUination may generate immunological epitopes that become targets of the adaptive immune system. This process may serve as an underlying basis for chronic and progressive neurological disorders.
[00136] Finally, this report presents the development of an in vitro model for simulating TBI in a controlled and cell-specific manner. Shown here in normal human astrocytes are the effects of calcium excitotoxicity, a hallmark condition of TBI, on the hyper-citrullination of GFAP. Moreover, the findings confirm the proteolytic processing of GFAP to a series of breakdown products that are consistent with those reported by others using in vitro models of TBI (100; 156). Interestingly, our investigation showed that one of these breakdown products is heavily citrullinated, and thus may serve as the antigen for the development of the anti-GFAP autoantibodies recently reported in TBI (153). Accordingly, the application to this and other models for simulated TBI may provide novel insights into consequences and mechanisms of TBI, and also identify informative biomarkers for assessing brain injury.
[00137] In conclusion, the present findings show that TBI dramatically up-regulates protein citruUination within astrocytes in specific brain regions. Additionally, this modification affects only a subset of the neural proteome, primarily affecting proteins involved in cytoskeletal structure, metabolic processes, and cell-cell signaling. A large proportion of these proteins have been identified as citrullinated in other pathologies, including MS, Alzheimer's disease, and rheumatoid arthritis, indicating a potential role for this protein modification in ongoing pathological processes. Interestingly, gender does not affect the degree or distribution of this modification in neural tissue, in contrast to previous observations involving TBI-induced protein carbonylation. Accordingly, gender differences in the CNS response to TBI may not involve differential responses in protein citruUination. In summary, this research indicates that abnormal protein citruUination is a feature of TBI that could contribute to ongoing pathological mechanisms following acute injury, possibly including aspects of autoimmune dysfunction.
[00138] The purpose of this work is to investigate the effects of TBI on two important post- translational protein modifications, carbonylation and citruUination. To date, little research has examined the specific regions, cell types, and proteins affected by these modifications following injury. Furthermore, while significant research has demonstrated gender-based differences in prognosis following TBI (52; 118), little work has investigated how gender impacts the distribution of these protein modifications after injury. Characterization of the selective impacts of carbonylation and citrullination allows insight into pathological pathways and ongoing dysfunction following TBI. Figure 23 presents these proposed pathways of long-term injury caused by TBI-induced carbonylation and citrullination.
[00139] Following TBI, a number of progressive and pathological secondary injury mechanisms take place. One of these injury pathways involves dramatic ionic imbalance, leading to a damaging overload of intracellular calcium. This calcium excitotoxicity is a hallmark effect of TBI (48; 49; 84; 141; 151), and persists for several days following injury (51). Through this cascade, a series of cellular pathways become interrupted. Energy homeostasis is disturbed, as decreased cerebral blood flow limits oxygen and glucose availability (51). As intracellular calcium pathologically increases, the consequences of this accumulation may lead to
dysfunctional protein carbonylation and protein citrullination.
[00140] Calcium overload can prompt carbonylation through the generation of reactive oxygen species (ROS) (75; 111) due to several possible pathways, including damaged respiration pathways, uncoupled electron transfer (104), and a decreased number of electron acceptors in the mitochondrial matrix (31). Reactive oxygen species can catalyze carbonylation through either direct interaction with proteins, or through the reaction of proteins with reactive carbonyl species, which are formed by the reaction of reactive oxygen species with polyunsaturated fatty acids (127; 128; 155).
[00141] Protein citrullination, in contrast, is not dependent on the presence of reactive oxygen species or reactive carbonyl species, but rather on the action of peptidylarginine deiminase (PAD). This family of enzymes is calcium-dependent: as calcium binds to PAD, a conformational change in the protein takes places, moving a catalytic residue into the enzyme's active site to act on arginine residues within other proteins (120). The reaction of PAD with arginine residues converts this amino acid to citrulline. Both carbonylation and citruUination can disrupt normal cellular physiology through a number of changes, including enzymatic inactivation (54), protein aggregation (25), and proteasomal inhibition (25).
[00142] Research presented within these manuscripts shows that carbonylation and citruUination impact separate brain regions five days following injury in a rodent model of TBI. While carbonylation was dramatically up-regulated in lesioned tissue and selective periventricular regions, including the area of the dorsal third ventricle and the median eminence, citruUination was increased in the cortex, external capsule, and hippocampus. Protein carbonylation and citruUination were both localized to astrocytes; however, carbonylation was also found in the ependymal cells of the dorsal third ventricle and median eminence. Furthermore, gender did not impact the magnitude or distribution of citruUination at five days following injury. In contrast, carbonylation was significantly affected by gender, in that male rats showed greater
susceptibility to carbonylation at sites distant from the lesion region. Finally, proteomic analyses identified several proteins carbonylated following TBI, specifically GFAP, ALDOA, ALDOC and CRMP2. All four of these proteins, along with 33 others, were identified as expressing increased citruUination after TBI. The following sections will address the significance of these findings and the implication of this data for understanding the long-term dysfunctions seen after TBI.
REGION AND CELL-SPECIFIC EFFECTS OF TBI ON PROTEIN CARBONYLATION AND
CITRULLINATION [00143] Five days following TBI in a rodent model of CCI, protein carbonylation and citrullination were expressed in specific cell types. Protein carbonylation was selectively expressed in astrocytes and ependymal cells, while protein citrullination was enhanced only in astrocytes. Further investigation revealed that neither of these post-translational modifications was significantly associated with neurons, oligodendrocytes, or microglia / macrophages, as indicated by co-localization with antibodies targeting NeuN, MBP and Ibal, respectively. As discussed in Chapter 3, our in vitro model of simulated TBI utilized the calcium ionophore, ionomycin, to induce excitotoxic calcium overload in normal human astrocytes. This series of experiments revealed that calcium overload induced the proteolytic processing of GFAP to a series of breakdown products, one of which is preferentially hyper-citrullinated. However, when this experimental protocol was repeated with neurons (SH-SY5Y), no citruUinated species were found in these cells prior to or following treatment with ionomycin (data not shown). These data suggest that astrocytes are distinctively susceptible to both protein carbonylation and citrullination at five days following TBI. In contrast, ependymal cells are selectively prone to carbonylation, but not citrullination.
TBI induces protein carbonylation in ependymal cells of the dorsal third ventricle and median eminence
[00144] Ependyma are specialized cells forming the barrier between cerebrospinal fluid and neural tissue, acting as both a metabolic and physical interface between these two spaces (41). Interestingly, only certain populations of ependymal cells expressed increased protein carbonylation following TBI. This may reflect the broad heterogeneity of this cell type, as regards structure and function (12; 65), expression of membrane proteins (12), and differential vulnerability to CSF-borne toxins (65). The populations of affected ependyma lined the dorsal third ventricle and above the median eminence, at sites directly adjacent to CSF-filled cavities. While the ependymal cells near the dorsal third ventricle are not well characterized, the region at the base of the third ventricle just above the median eminence is well-known for its neurogenic cell populations (74) and involvement in neuroendocrine regulation (113).
[00145] It is possible that the unique exposure of ependyma to the CSF, and thus exposure to the reactive molecules transported within its mileu, prompt carbonylation within these cells. While reactive oxygen species have a limited capacity for diffusion across tissue following TBI, reactive carbonyl species are able to travel over longer distances (86). These lipid peroxidation products include the highly reactive aldehyde molecules 4-hydroxynonenal and
malondialdehyde. Lipid peroxidation occurs as a very early event following TBI, with levels of peroxidation by-products elevated in neural tissue just thirty minutes following injury (7).
Furthermore, these reactive molecules are rapidly increased in the CSF following TBI. As little as three hours following head trauma, levels of malondialdehyde are increased in the CSF, as compared to both uninjured controls and non-cerebral injured patients (2). Additionally, increased levels of 4-hydroxynonenal have been found in the CSF of patients with
neurodegenerative disorders, including Parkinson's disease (126) and amyotrophic lateral sclerosis (130), suggesting a role for these aldehydes as indicators of pathology. Evidence has also suggested that reactive carbonyl species are responsible for increased permeability of the blood-brain barrier following injury (1; 131). The specific regions of ependymal cells investigated here may be selectively susceptible to aldehyde-induced permeability, increasing their relative vulnerability to protein carbonylation through interaction with reactive carbonyl species transported through the CSF. Because 4-hydroxynonenal, malondialdehyde, and other reactive carbonyl species are relatively long-lived and can withstand extracellular transport from their site of generation (26; 127), it is conceivable that oxidative damage is transmitted to these selective regions of ependymal cells through the transport of reactive molecules from the site of injury via the ventricular system. The unique sensitivity of these particular ependymal regions may reflect unique functional characteristics of the cells involved, such as membrane
permeability properties or membrane receptor protein heterogeneity. The lack of protein citruUination in ependymal cells is not surprising, given the limited distribution of citrullination- catalyzing PAD enzyme isoforms to mainly astrocytes (PAD2) (61) and neurons (PAD4) (2). Region-specific protein carbonylation and citruUination within astrocytes following TBI
[00146] The greatest increases in protein carbonylation and citruUination following TBI were observed in astrocytes. To our knowledge, the work presented here is the first to show that astrocytes are acutely susceptible to these post-translational modifications following TBI. Five days after TBI, protein carbonylation is expressed in astrocytes of the lesion site (see Chapter 2). In contrast, protein citruUination is expressed only mildly in the lesion site, with the largest increase in this modification seen in astrocytes of the cerebral cortex, external capsule, and hippocampus (see Chapter 3).
[00147] One likely mechanism underlying astrocyte vulnerability to carbonylation and citruUination involves the complex interaction between these cells and calcium. Following TBI, a decrease in cerebral blood flow limits the amount of oxygen and glucose delivered to the brain, resulting in hypoxia and lack of metabolic support. During these post-injury conditions, astrocytes experience large elevations in intracellular calcium (44), to a much greater degree than the calcium influxes observed in neurons (121). Following ischemia, the disruption of cell membranes and mechanical stretching of cellular components leads to a rush of extracellular calcium into astrocytes. Additionally, mechanisms that normally offset the effects of extracellular glutamate are impaired (149). The glutamate-rich extracellular environment causes the activation of NMD A receptors (51), allowing a massive influx of calcium into cells. This influx further exacerbates the dramatically increased intracellular concentration of calcium due to direct injury (111; 149), causing a failure in energy metabolism (51) and the release of sequestered intracellular calcium from internal stores, including the endoplasmic reticulum and mitochondria (109; 143). While the precise response of astrocytes to calcium excitotoxicity is not fully defined, research has shown that astrocytes may exert a protective effect on neuronal viability by insulating these terminally differentiated cells from the effects of excessive extracellular glutamate (49), such that the accumulation of calcium by astrocytes serves to reduce extracellular calcium, and thus its damaging effects on neurons (44).
[00148] It is proposed that mitochondria are central to the effects of calcium overload on the expression of both carbonylated and citrullinated proteins in astrocytes. Large increases in intracellular calcium can lead to mitochondrial dysfunction through interference with calcium sequestration in this organelle and uncoupling normal processes of energy metabolism. Impaired mitochondrial function leads to the enhanced generation of reactive oxygen species that are unchecked due to depleted stores of antioxidants, including glutathione (135). These reactive oxygen species generate carbonylation in two major ways. First, reactive oxygen species directly carbonylate proteins by adding carbonyl groups to amino acids, including histidine, lysine, and cysteine. Second, reactive oxygen species generate aldehyde by-products through lipid peroxidation, and these reactive carbonyl species can introduce carbonyl groups into proteins. Citrullination is also catalyzed by astrocytic calcium overload. While homeostatic intracellular levels of calcium are in the range of the sub-micromolar, the optimal calcium concentration for PAD activation is in the high micromolar to millimolar range (123). Thus, the activation of PAD within astrocytes following TBI confirms proposals of astrocyte susceptibility to calcium overload in citrullination-inducing ischemic conditions.
[00149] Significant protein carbonylation was noted in the astrocytes of the lesion site (Chapter 2). It is probable that this observation reflects the acute, extreme oxidative stress at the most proximal site of tissue damage. Following impact, the lesion site is particularly prone to glutamate excitotoxicity, and thus calcium overload, as the mechanical shearing of cerebral blood vessels in the injured parenchyma leads to the increased release of this excitatory neurotransmitter (90). In contrast to carbonylation, protein citruUination in the lesion site was relatively less affected. This unexpected observation may reflect a function of time. Because our experimental model necessarily examined tissue at only five days following injury, a more rapid time course for protein citruUination at the lesion site might occur. In this manner, citrullinated proteins may clear from the injury site sooner than carbonylated proteins. This rapid clearance of citrullinated proteins may be due to at least two possible mechanisms. First, citruUination can alter tertiary structure to a more "open" conformation, and thereby increase protein susceptibility to proteolytic degradation (87). Alternatively, protein citruUination may prompt cellular apoptosis (82), resulting in few citrullinated protein-containing cells remaining by day five after TBI. This possibility assumes that there is a measure of cell specificity in these citruUination and carbonylation responses.
[00150] Injury most predominantly affected protein citruUination in astrocytes of the cerebral cortex, external capsule, and hippocampus (Chapter 3). The molecular basis for this observation may relate to the up-regulation of a specific calcium channel in these three regions. Voltage- gated, class C L-type Ca2+ channels are uniquely expressed in astrocytes in response to ischemia, and are particularly sensitive to injury-mediated activation in the cortex, external capsule, and hippocampus (33). Nimodipine, an L-type calcium channel blocker, protects against astrocyte damage in conditions of calcium overload (56). It is possible, therefore, that these three regions are particularly susceptible to protein citruUination due to their unique activation of these calcium channels, which mediate calcium influx into astrocytes following hypoxia and ischemia (33). Protein carbonylation was not expressed in these regions at five days following injury, suggesting that oxidative stress in these regions is relatively low as compared to the proximal lesion site. This may be due to regional variations in cellular physiology including: variable antioxidant capacities; relative metabolic needs of each region; and differing levels of reactive metal ions, including copper and iron, which can drive metal-catalyzed protein carbonylation. Neurons do not express protein citruUination after TBI
[00151] While past investigations have documented the presence of PAD2 in neurons, our work in both an in vivo CCI animal model and in vitro model of simulated TBI calcium excitotoxicity failed to show protein citruUination in neurons following injury. Nevertheless, citrullinated proteins are reported to accumulate in neurons in both Alzheimer's pathology (2), and
Parkinson's disease (92). Our findings showed that acute calcium toxicity, as induced in both of our models of injury, prompted astrocyte protein citruUination but not neuronal protein citruUination. This difference may be explained by cell-specific PAD isoforms. As noted earlier, astrocytes and neurons contain two separate isoforms of PAD: while astrocytes express PAD2, neurons express PAD4. Our data suggests that calcium overload may be necessary and sufficient for PAD2 activation, while necessary but insufficient for PAD4 activation. Considering the neurodegenerative nature of Alzheimer's and Parkinson's diseases, it is possible that PAD4 activation reflects a chronic rather than acute mechanism, including prolonged calcium imbalance, or perhaps other signaling pathways that have not yet been elucidated. Further investigation should reveal the precise molecular pathways that catalyze the activation of these specific isozymes, as this will reveal the detailed pathological progression of citrullination in damaged neural tissue.
GENDER MODULATES PROTEIN CARBONYLATION FOLLOWING INJURY
[00152] This investigation demonstrated that protein carbonylation, but not citrullination, is affected by gender. While the magnitude and distribution of protein citrullination following TBI was not observed to be different between males and females following injury, the degree of protein carbonylation was significantly influenced by gender. Specifically, protein carbonylation in uninjured male rats was significantly greater than that observed in uninjured female rats, indicating a gender difference in the status of protein carbonylation in healthy animals. While male and female rats expressed a comparable degree of protein carbonylation in the lesion site, regions distant from the lesion site, specifically the ependymal zones of the dorsal third ventricle and the median eminence, were profoundly affected in males as compared to females.
[00153] As discussed above, it is possible that the carbonylation of proteins in ependymal cells reflects the effects of soluble reactive carbonyl species, in the form of reactive oxygen species- driven lipid peroxidation products. It has been proposed that the ovarian steroid progesterone may protect against the formation of reactive carbonyl species by preventing lipid peroxidation (117). It is therefore possible that female hormones serve a protective role against the formation of CSF-borne reactive carbonyl species. The observation that both male and female rats displayed similar degrees of protein carbonylation in the lesion site itself may be due to the fact that protein carbonylation in the site of direct injury reflects the acute action of transient reactive oxygen species rather than reactive carbonyl species. [00154] Under current conditions, ovarian steroids may protect against the generation of reactive oxygen species and reactive carbonyl species. In support of this proposal is that females recover better following TBI as compared to males (16; 52; 118). The absence of gender effects in TBI- induced protein citruUination predicts that males and females are affected equally; however, this is not the case. Rather, in chronic autoimmune disorders involving abnormal citruUination, most notably multiple sclerosis (36) and rheumatoid arthritis (38), females are more profoundly affected than males. This suggests that factors in addition to gender contribute to the chronic pathology of these two conditions. Alternatively, the present experimental design may have investigated a time point that simply was not informative for gender differences in long-term pathology caused by abnormal citruUination.
SELECTIVE MODIFICATION OF PROTEINS FOLLOWING TBI
[00155] The proteomic analyses reported here provide the first insights into the carbonylation and citruUination proteomes of TBI. As noted in Chapter 2, four proteins were identified as having enhanced carbonylation following injury: (1) GFAP; (2) CRMP2; (3) ALDOC; and (4) ALDOA. These proteins have also been identified as carbonylated in other neurodegenerative disorders, including Alzheimer's disease (GFAP, CRMP, ALDOC) (29; 139),
ischemic/reperfusion injury (CRMP2, ALDOC) (99), and multiple sclerosis (GFAP) (155).
[00156] Our work investigating protein citruUination (Chapter 3) revealed 37 separate protein species citruUinated in response to injury. In addition to identifying several cytoskeletal and metabolically-related proteins, this investigation showed that GFAP, CRMP2, ALDOA and ALDOC are also citruUinated in addition to being carbonylated. Furthermore, a large proportion of these 37 proteins have been identified as being citruUinated in other pathologies, including multiple sclerosis, Alzheimer's disease, prion disease, and rheumatoid arthritis (24; 55; 62; 64; 71; 93; 144).
[00157] The modified proteins identified here have essential roles in cellular repair, structure, metabolism, and signaling. For example, CRMP2 functions in axonal guidance, serving an important role in neuronal regeneration after injury (142), while GFAP is a major structural component of astrocytes. In addition to GFAP, several other structural proteins were found to be citruUinated following injury, including dynamin-1 and several forms of beta-tubulin. Another functional group of modified proteins are those involved in metabolic processes, including peroxiredoxin-1 and creatine kinase b. Furthermore, several of the citrullination proteins identified have established roles in cell-cell signaling, including synapsin-2, syntaxin-binding protein 1, and amphiphysin.
[00158] Interestingly, our findings are concordant with the results of other proteomic investigations on protein citrullination in rheumatoid arthritis and multiple sclerosis. Both diseases are associated with increased citrullination of key structural proteins and enzymes involved in cellular metabolic processes (69). Furthermore, several of the citruUinated proteins identified here, including ALDOA, ALDOC, and myelin basic protein have been shown to be citruUinated in other neural pathologies (22; 63). Proteomic analyses have also indicated that carbonylated proteins identified in this investigation are also found in other neurodegenerative pathologies. For example, carbonylated forms of both CRMP2 and GFAP characterize
Alzheimer's disease (21; 28; 139) and aging rodent brain tissue (105; 153). GFAP has also exhibited increased carbonylation in these pathologies. Table 1 presents modified proteins identified by this investigation, along with a review of literature showing evidence that these proteins are carbonylated and / or citruUinated in other pathologies. [00159] These findings suggest that particular proteins are selectively susceptible to either carbonylation or citrullination across multiple pathologies. Furthermore, four of the proteins identified here are altered by both modifications following TBI. The "double" modification of the proteins CRMP2, ALDOA, ALDOC, and GFAP may be due to at least two possible mechanisms. First, it is possible that an initial proteomic modification may yield a protein that is more susceptible to a subsequent modification. For example, it is known that protein
citrullination alters the tertiary structure of myelin basic protein, reshaping this protein into a more open conformation (87). While this alteration is shown to increase the protein's susceptibility to protease degradation (87), it may also allow access of reactive oxygen species and reactive carbonyl species to previously inaccessible amino acids which may now be carbonylated. A second possibility is that the "double" identification of proteins actually reflects two separate pools of modified proteins. For instance, rather than a single pool of CRMP2 that is both carbonylated and citrullinated, it is possible that we have identified two individual groups, one carbonylated and the other citrullinated. If this is the case, then these separate post- translational groups may reflect the distinct regions affected by each modification, as revealed by our immunohistochemical investigations.
Figure imgf000075_0001
Figure imgf000076_0001
[00160] As shown in Table 1, increased expression of selective protein carbonylation and citrullination in multiple neurodegenerative pathologies. Proteomic analyses in this investigation revealed several proteins carbonylated and citrullinated following TBI (blue column). Many of these proteins are modified in other pathological neural disorders, including Alzheimer's disease, prion disease, multiple sclerosis/EAE (experimental autoimmune encephalomyelitis), and aging brain tissue. Asterisk (*) indicates evidence of modification-induced autoantigenicity.
POTENTIAL LONG-TERM EFFECTS OF PROTEIN CARBONYLATION AND CITRULLINATION ON TBI
PATHOLOGY [00161] Protein susceptibility to carbonylation is selective, but there is no clear explanation as to the underlying mechanism of this specificity. Research has suggested that this vulnerability may be due to the presence of specialized binding sites, unique protein conformation, or the relative frequency of amino acids that are targeted by oxidation, including lysine, histidine, and cysteine (39).
[00162] The effects of protein carbonylation have been extensively investigated in the context of neurodegenerative disorders. Through alteration of protein structure, carbonylation impacts cellular physiology by alteration or inactivation of essential processes. For example, the carbonylation of tubulin in neuroblastoma cells can inhibit neurite outgrowth, which is essential for neural plasticity and regeneration (91). In this manner, oxidative stress can lead to a chronic, functional shift in injury recovery through proteomic modification. Furthermore, excessive carbonylation in environments of extreme oxidative stress can inhibit pathways for protein degradation. While mild levels of carbonylation increase a protein's susceptibility to breakdown, an acute degree of carbonylation can inhibit degradation due to the protein's inability to fit into the catalytic sites of the proeasomal complex (91). As a result, aggregation of carbonylation proteins can occur, forming cytotoxic accumulates that are associated with disorders including Alzheimer's and Parkinson's diseases (98).
[00163] While citrullination is less studied in the context of neurological pathologies, this modification is known to have significant effects on protein antigenicity. Specifically, in addition to changing structure, citrullination also changes the charge and cleavage sites of affected proteins, leading to the presentation of novel, potentially antigenic epitopes (148). This phenomenon is observed in multiple sclerosis, where the citrullination of myelin basic protein prompts myelin degeneration due to a loosened aggregation of lipids in the sheath structure (89). CitruUination of myelin basic protein also increases its susceptibility to degradation by proteinases, including cathespin D, leading to the formation and release of antigenic, citrulline- containing peptides which are presented to peripheral T cells (23). In healthy neural tissue, a small fraction of total myelin basic protein contains citrullinated residues; however, this amount increases three-fold in multiple sclerosis, inducing a targeted T cell response (5; 144).
Citrullinated epitopes are the hallmark of the adaptive immune response in rheumatoid arthritis. CitruUination in this disorder elicits a dramatic production of targeted autoantibodies, to such a significant degree that anti-citrullinated protein antibodies serve as a diagnostic biomarker for this disease (57). Interestingly, two other proteins identified in this investigation are targeted as antigens in separate pathological conditions. Glucose-regulated protein 78 was identified in this research as citrullinated following TBI. Previous work has shown that the citrullinated form of this protein is an autoantigen in Type 1 Diabetes, where modified glucose-regulated protein 78 is secreted by pancreatic beta cells and prompts the production of autoantibodies (115). The current investigation also identified the protein amphiphysin as citrullinated following TBI. To our knowledge, this protein has not been noted as citrullinated in other disorders. However, several neurological disorders, including encephalopathy and myelopathy, show increased expression of autoantibodies targeting amphiphysin. It is possible that this immune response is triggered by citrullinated epitopes as of yet unrevealed. Future research is required to determine whether citruUination is the mechanism underlying the antigenic nature of amphiphysin in neural pathologies.
[00164] The work presented here describes the distribution and selective proteomic effects of carbonylation and citruUination that occur five days following TBI. This investigation has shown that protein carbonylation and citruUination affect distinct neural regions, but are both dramatically expressed in astrocytes following injury. While gender influences the magnitude and regionalization of protein carbonylation, there is no such influence on protein citrullination after TBI. Furthermore, we have identified four proteins that are selectively carbonylated after injury. These four proteins, along with 33 others, are also citruUinated at five days following TBI.
[00165] This work has identified many proteins that are similarly carbonylated or citruUinated in neurodegenerative disorders, including Alzheimer's disease, prion disease, and multiple sclerosis. It is conceivable that shared modifications across disorders are indicative of shared pathological pathways. For examples, our findings indicate that myelin basic protein is citruUinated following TBI, just as this protein is citruUinated in multiple sclerosis. Research has shown that myelin basic protein undergoes proteolysis at specific cleavage sites following controlled cortical impact, reflecting similar processes in multiple sclerosis, as described earlier (76). The chronic demyelination noted in both multiple sclerosis and following TBI could reflect this shared protein modification, which would suggest a mechanistic link between acute, TBI- driven protein modification and chronic structural changes. The carbonylation of CRMP2 following TBI also represents a possible link for shared symptomatology. This protein, which is essential for axonal growth and neural regeneration, is carbonylated in the hippocampus in Alzheimer's disease (29), indicating a possible mechanistic role for CRMP2 carbonylation in the memory deficits seen in both this disorder and following TBI.
[00166] Currently, it is not well understood how the acute, mechanical impact of TBI leads to chronic pathology that can last anywhere from months to years. Protein modifications shared between TBI and neurodegenerative disorders may suggest a mechanistic link between physical injury and sustained dysfunction. Specific changes in protein structure and function, seen as early at five days following TBI, could lead to the pathological symptomatology mirrored in diseases such as Alzheimer's and multiple sclerosis. Revealing the specific proteomic effects of carbonylation and citruUination provides mechanistic insight into how acute injury translates into a chronic neurodegenerative disease. Future work addressing the time-course of these modifications, from days to weeks to years following TBI, will add significant understanding of how aberrant post-translational modifications translate into pathology. Further proteomic analyses of specific regions, including the hippocampus and cerebral cortex, could reveal biological correlates of region-related cognitive deficits, including memory dysfunction and loss of executive function. We propose, therefore, that protein carbonylation and citruUination represent critical mechanistic links between acute physical injury and long-term pathology. A rigorous understanding of these proteomic shifts following TBI will lend insight into the biological mechanisms of this injury and may also point to preventative measures to counteract long-term pathogenesis.
[00167] Protein citruUination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS) and prion disease. CitruUination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citruUination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citruUination and on the specific cell types involved. Immunofluorescence revealed that controlled cortical impact in rats profoundly up-regulated protein citruUination in the cerebral cortex, external capsule, and hippocampus. This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citruUination in neurons, oligodendrocytes or microglia. Further, proteomic analyses demonstrated that the effects of TBI on citruUination were confined to a relatively small subset of neural proteins. Proteins most notably affected were those also reported to be citrullinated in other disorders including prion disease and MS. In vivo findings were extended in an in vitro model of simulated TBI employing normal human astrocytes.
Pharmacologically-induced calcium excitotoxicity was shown to activate the citruUination and breakdown of glial fibrillary acidic protein, producing a novel candidate TBI biomarker and potential target for autoimmune recognition. In summary, these findings demonstrate that the effects of TBI on protein citruUination are selective with respect to brain region, cell type and proteins modified, and may contribute to a role for autoimmune dysfunction in chronic pathology following TBI.
[00168] Traumatic brain injury (TBI) is a major cause of injury and death in the US, with over 1.7 million TBIs occurring annually and at least 5.3 million Americans currently living with ongoing disability (Walker and Tesco, 2013). Traumatic brain injuries in civilians are largely due to automobile accidents, as well as falls, sports, and firearms (Werner and Engelhard, 2007). Military populations are at disproportionately elevated risk for blast-related TBI due to the devastating effects of improvised explosive devices (Hoge et al., 2008). While there is a very large body of information on causes and global consequences of TBI, much less is known about the mechanisms underlying long-term pathology.
[00169] The long-term consequences of TBI can be complex, and often result in progressive cognitive and behavioral changes. Studies have indicated that anywhere from 10% - 50% of individuals with TBI suffer from persistent symptoms following injury (Bales et al., 2009), including attention deficits and short-term memory loss (Walker and Tesco, 2013). This long- term dysfunction follows in the wake of two main injury phases: (1) the primary injury, caused by the immediate forces of the trauma (Ray et al., 2002;Werner and Engelhard, 2007); and (2) the subsequent secondary injury, which presents as a constellation of dysfunctional molecular processes including impaired metabolism, free radical production, inflammation, and glutamate excitotoxicity (Walker and Tesco, 2013). At present, it is not well understood how these various dysfunctional processes following acute injury can lead to progressive, chronic pathology after TBI. This ongoing pathology includes deficits in executive function, attention, processing speed, learning and memory formation, and well as behavioral changes in both emotion and affect (Bales et al., 2009;Walker and Tesco, 2013).
[00170] A hallmark mechanism of secondary injury following TBI is prolonged imbalance in cellular calcium homeostasis, resulting in excitotoxic calcium overload (Fineman et al.,
1993;McIntosh et al., 1997;Sun et al., 2008). Following TBI, ischemia can lead to a dramatic loss of glucose and oxygen delivery to the central nervous system, thereby limiting the production of ATP. In the absence of adequate energy, ionic homeostasis is lost and calcium toxicity ensues. The abnormally large increase in intracellular calcium originates from both the influx from the extracellular space across multiple calcium channels, and from the mobilization of intracellular calcium stores (Szydlowskaa and Tymianskia, 2010). The result is a
pathologically prolonged state of elevated intracellular calcium (Sun et al., 2008).
[00171] The downstream effects of cellular calcium toxicity have been examined closely in regard to mitochondrial dysfunction and oxidative stress. However, little attention has been given to the role of TBI-induced calcium overload in the activation of peptidylarginine deiminase (PAD) enzymes. This family of calcium-dependent enzymes catalyzes the post-translational modification of citruUination, resulting in the conversion of intrapeptidyl arginine residues to citrulline residues. In addition to altering both the normal structure and function of proteins, citruUination generates "altered-self ' epitopes that may be antigenic, prompting autoimmune responses against previously benign proteins (Zhao et al., 2010;Chirivi et al., 2013). Altered calcium homeostasis accompanied by protein citruUination has been implicated in several neurodegenerative disorders, including Alzheimer's disease (Ishigami et al., 2005), temporal lobe epilepsy (Asaga and Ishigami, 2001), glaucoma (Bhattacharya et al., 2006), rheumatoid arthritis (Luban and Li, 2010), and multiple sclerosis (Anzilotti et al., 2010).
[00172] At present, understanding of the specific proteins modified by citruUination is very limited outside the field of immunology, where the antigenic properties of abnormal
citruUination are studied largely within the context of rheumatoid arthritis. Additionally, there is no data available regarding the regional and cellular specificity of protein citruUination in neural tissue following TBI. Here, we identify the regions and cell types most susceptible to protein citruUination following TBI, and also identify a subset of neural proteins that are preferentially citruUinated in response to injury. Furthermore, we present the development of an in vitro model for simulating TBI through astrocytic calcium excitotoxicity, and identify a citruUinated breakdown product of glial fibrillary acidic protein (GFAP). Until now, citruUination has been primarily associated with chronic, progressive autoimmune and neural disorders. The present findings indicate that protein citruUination is a feature of TBI that may contribute to the long- term pathogenic mechanisms following acute injury, including those that involve the adaptive immune system.
1. Materials and Methods
1.1. Controlled Cortical Impact (CCI) [00173] CCI was conducted as described in Lazarus et al. (2015). Briefly, adult male and female Sprague-Dawley rats (Charles River Laboratories, Morrisville, North Carolina, USA) were anesthetized, then subjected to unilateral CCI over the left hemisphere administered through a ImpactOne stereotaxic impactor (Leica Microsystems, Buffalo Grove, Illinois, USA), which delivered a 3 mm flat-tipped impactor at 20° to a depth of 2 mm at 5 m / s with a 500 ms dwell time at -3.8 mm bregma in males / - 3.0 mm bregma in females. Naive animals received no anesthesia or CCI treatment. Animals were euthanized five days following injury. All animal handling procedures were performed in compliance with guidelines from the National Research Council for the ethical handling of laboratory animals, as approved by the Institutional Animal Care and Use Committee of USUHS (IACUC Protocol APG 12-827, Bethesda, Maryland, USA). 1.2. Immunohistochemistry
1.2.1. Tissue collection and preparation
[00174] Tissue was collected and prepared as described previously (Lazarus et al., 2015).
Briefly, euthanized animals underwent transcardial perfusion (phosphate buffered saline (PBS) followed by 4% paraformaldehyde) after which brains were removed for storage overnight at 4°C in 4% paraformaldehyde and then equilibration in a 30% sucrose solution (2 days, 4°C). Brains were sectioned coronally (20 um) across the breadth of the lesion site (2.5 mm rostral to 2.5 mm caudal) with a Leica CM 1900 cryostat (Leica Microsystems), and sections were then mounted on slides and stored at -80°C. The following groups of gender / conditions were prepared for immunohistochemical analysis: n = 11 male rats, CCI; n = 8 male rats, naive control; n = 10 female rats, CCI; n = 7 female rats, naive control.
1.2.2. Detection of cell-specific citrullination [00175] The region and cell- specific effects of CCI on protein citrullination were determined using a mouse monoclonal anti-citrulline antibody (rnAb 6B3, IgG2b) (Martinelli et al., 2002). The antibody was purified from expression medium by Protein A affinity chromatography (HiTrap Protein A HP column (17-0403-01; GE Healthcare, Buckinghamshire, United Kingdom) on a GE AKTA FPLC fast protein liquid chromatography instrument (FPLC; 18-1900-26; GE Healthcare), aliquoted for single-use and stored at -80°C.
[00176] Immunodetection of citrullinated proteins was performed as follows. Brain sections were incubated at 4°C overnight with 125 μΐ of 1: 1000 mouse anti-citrulline 6B3 antibody in 0.3% Triton X-100/PBS, with 1: 100 normal donkey serum (NDS) as a blocking agent (16 sections per animal). Co-localization experiments were performed by co-incubating these sections with mAb 6B3 and either: (1) goat anti-GFAP (1: 1000; ab53554; Abeam, Cambridge, England) (astrocytes); (2) goat anti-ionized calcium-binding adapter molecule (Ibal; 1:500; ab5076; Abeam) (microglia); (3) rabbit anti-neuronal nuclei (NeuN; 1:500; abl04225; Abeam) (neurons); or (4) rabbit anti-myelin basic protein (MBP; 1: 1000; ab40390; Abeam)
(oligodendroglia). Slides were washed five times with 0.2% Triton X-100/PBS and then incubated for 30 min with 125 μΐ of secondary antibody solution: 1: 100 donkey anti-mouse IgG (H+L), conjugated to green-fluorescent Alexa Fluor 488 dye (A-21202; Invitrogen, Waltham, Massachusetts, USA); and either 1: 100 donkey anti-goat, conjugated to red-fluorescent Alexa Fluor 594 (A- 11058, Invitrogen), or 1: 100 donkey anti-rabbit, conjugated to red-fluorescent Alexa Fluor 594 (A-21207; Invitrogen). The sections were washed five times with 0.2% Triton X-100/PBS and one time with lx PBS (5 m) and then visualized with an Olympus BX61 fluorescent motorized system microscope (Olympus, Shinjuku, Tokyo, Japan) using iVision-Mac software (Bio Vision Technologies, Exton, Pennsylvania, USA). 1.2.3. Preadsorption control
[00177] Specificity of the mAb 6B3 anti-citrulline antibody in immunofluorescence was confirmed through immunoneutralization using a mixture of citrullinated protein molecular weight standards (trypsinogen, glyceraldehyde 3-phosphate dehydrogenase, bovine albumin, trypsin inhibitor, alpha-lactalbumin, carbonic anhydrase, and egg albumin) prepared in-house via 10 hour incubation at 37°C with active PAD enzyme cocktail (0.5 μg / ul; P312-37C-25;
SignalChem, Richmond, British Columbia, Canada) in Tris buffer (50 mM Tris HC1, pH 7.4); 5 mM CaCl2; and 0.73 mM dithiothreitol (DTT; Sigma-Aldrich, St. Louis, MO, USA)).
Concurrently, a control sample was prepared in an identical manner, without the addition of the PAD enzyme cocktail.
1.3. Identification of citrullinated protein species in injured rat brain
1.3.1. Tissue collection and preparation
[00178] Brains were collected, snap-frozen with powdered dry ice, and stored at -80°C until use. Brains were thawed on wet ice and then hand-dissected to produce blocks of tissue
encompassing the lesion site and the surrounding tissue shown by immunohistochemistry to have increased protein citruUination following CCI. The equivalent region from naive animals (control tissue) was similarly collected. Tissue blocks were homogenized in 5 volumes / tissue weight extraction solution, consisting of: 7.7 M urea; 2.2 M thiourea; and 4.4% CHAPS; also containing lx Complete protease inhibitor mix (Roche). Samples were clarified by centrifugation (20,000 x g, 10 min, 4°C), and resulting supernatants were fractionated by 2-dimensional electrophoresis (2-DE).
1.3.2. Fluid-phase Isoelectric Focusing (F-IEF) [00179] Treatment group pools (control and injured tissue) were prepared (N = 4/pool) and fractionated by F-IEF. Briefly, 200 μΐ aliquots of each pool was diluted up to 2.865 ml in IEF running solution (7.7 M urea; 2.2 M thiourea; 4.4% CHAPS; ampholytes (150 ul, pH 3 - 10; ZM0021, Invitrogen); DTT (50 μΐ, 2 M); and bromophenol blue (10 μΐ, 10 mg/ml). Samples were loaded into the ZOOM IEF Fractionator (ZMF10002; Invitrogen) and focused according to the following conditions: (1) 100 V, 1.2 mA, 0 W (15 min); (2) 200 V, 2.0 mA, 0 W (1 h); (3) 400 V, 2.0 mA, 1 W (1 h); (4) 600 V, 1.5 mA, 1 W (1 h) resulting in fractions of proteins within the following five pi ranges: 3.0 - 4.6; 4.6 - 5.4; 5.4 - 6.2; 6.2 - 7.0; and 7.0 - 9.1.
1.3.3. Molecular weight fractionation
[00180] Proteins in each of the F-IEF fractionations were further resolved by molecular weight fractionation using conventional one-dimensional gel electrophoresis. Samples were combined with an equal volume of 4x reducing loading buffer (Novex NuPAGE LDS sample buffer; 50 mM DTT; Invitrogen), heated at 70°C (20 min) and then fractionated (10 μΐ per well) using NuPAGE 4 - 12% Bis-Tris gels (Novex, Invitrogen), using lx MES (2-[N- morpholino]ethanesulfonic acid) running buffer (Novex, Invitrogen). Proteins were transferred to nitrocellulose blots using an iBlot transfer apparatus & gel transfer stacks (Nitrocellulose Mini; 1B301002, Invitrogen).
1.3.4. Immunoblotting
[00181] Blots were blocked with 5% instant nonfat dry milk / Tris-buffered saline/Tween 20 (TBS-T) (1 h, room temperature) and then incubated with the 6B3 primary antibody (1:300 in TBS-T; mAb stock = 1.79 mg/mL) for 1 h at room temperature, then 4°C overnight. Following equilibration to room temperature (30 min), membranes were washed in TBS-T (5 times over 60 min), incubated with secondary antibody, horseradish peroxidase-labeled, goat anti-mouse IgG (1:2500 in 5% TBS-T; 31430, Thermo Scientific) for 2 h at room temperature and then visualized by enhanced chemiluminescence (ECL) (Novex ECL HRP Chemiluminescent Substrate Reagent Kit; WP20005, Invitrogen) using the FUJI LAS 3000 Imager (Fujifilm, Minato, Tokyo, Japan). Images collected were analyzed using MultiGauge software (v. 3.0, Fujifilm). In some cases, blots were reprobed with a second anti-protein citrulline antibody (1:500; MABN328EMD; Millipore; detection with horseradish peroxidase-labeled, goat anti- mouse IgM; 1:2500 in TBS-T; 31440, Thermo Scientific), with final overnight washing, to confirm the 6B3 immunoreactive features and increase the sensitivity of detection. No new signals were revealed by this approach.
[00182] The specificity of mAb 6B3 for detecting citruUinated proteins on western blots was confirmed by immunoneutralization, similar to the approach used for immunohistochemistry (see above), but involving solid-phase preadsorption (versus fluid phase) to accurately replicate the conditions of western blotting. In this case, the 6B3 antibody was reacted with a strip of nitrocellulose (Protran® BA85, 0.45 μιη pore size, binding capacity 80 μg/cm ; Sigma- Aldrich; cut into 1.4 cm x 3 cm pieces) to which either the citruUinated or native forms of fibrinogen had been bound. The amount of protein absorbed to the strip was 200μ1 / 200μg of citruUinated human fibrinogen (400076, Cayman Chemical, Ann Arbor, Michigan, USA), or 200μ1 / 200μg human fibrinogen (16088, Cayman Chemical) in TBS-T. The duration of the antibody absorption was 16 h at 4°C.
1.3.5. Protein identifications
Immunoreactive signals of interest were mapped to corresponding features in Coomassie- stained gels. These features were excised and processed for identification through the ESMS- Basic Protein ID service using an Elite Orbitrap mass spectrometer (Thermo Scientific) by the W.M. Keck Mass Spectrometry & Proteomics Resource (W.M. Keck Foundation Biotechnology Resource Laboratory, New Haven, Connecticut, USA). Analyses results were received through and tracked within the Yale Protein Expression Database (YPED, Yale / NIDA Neuroproteomics Center, New Haven, Connecticut, USA). Criteria for a positive identification included: an expectation score less than l.OE-65, a percent coverage greater than 35.0%, and a pi and mw that were consistent with the fractionation data.
1.4. In vitro model of protein citrullination following TBI-induced excitotoxicity in astrocytes
[00183] An in vitro model of simulated TBI was established using normal human astrocytes (NHA CC-2565, Lonza, Allendale, New Jersey, USA) and treatment with the calcium ionophore, ionomycin (Free Acid, Streptomyces conglobatus, 407950, Calbiochem, Billerica,
Massachusetts, USA), to simulate calcium excitotoxicity. Astrocytes were cultured to -70% confluence in T-75 tissue culture flasks according to vendor instructions. Cells were washed with TBS (4 times) and then treated for 4 h (37°C, 95% 02/5% C02) with either ionomycin (10 μΜ; 10 mL TBS) or dimethyl sulfoxide vehicle (DMSO; 10 μ1/10 mL TBS). Following incubation, protease inhibitors were added to the flasks (final concentration of lx; Complete protease inhibitor mix; 10269700, Roche) and cells were harvested by scraping and centrifugation (800 x g, 4°C, 10 min). Cells were extracted by vortexing into IEF denaturant (see section 2.3.2) at a ratio of 1 part pellet to 4 parts buffer (v/v). Samples were clarified by centrifugation (15,000 x g, 4°C, 10 m). The resulting supernatent was processed by one-dimensional gel electrophoresis and immunoblotting as described above. Replicate immunoblots containing samples from three separate experiments each were probed with either anti-citrullinated protein (6B3) or anti-GFAP (1:3000, Z0334, DAKO, Carpinteria, California, USA). Each primary antibody was paired with an appropriate secondary antibody (6B3: horseradish peroxidase-labeled, goat anti-mouse IgG; 1:2500; 31430; Thermo Scientific; GFAP: horseradish peroxidase-labeled, goat anti-rabbit IgG; 1:3000; HAF008; R&D Systems) and blots were visualized by ECL.
1.5. Statistical analysis
[00184] Citrullination immunohistochemistry was quantified in the cortex using a previously developed scoring system (Lazarus et al., 2015), with a score of 0 reflecting the lowest amount of citrullination fluorescence intensity in a region and a score of 3 reflecting the greatest amount of fluorescence intensity (see Supplementary Figure 1). Four brain sections per animal were scored, and comparisons between groups were analyzed by ANOVA with a Tukey HSD post hoc test (IBM SPSS Statistics for Macintosh, Version 22.0, Armonk, NY: IBM Corp.). A value of p < 0.05 was considered significant.
Results
[00185] Figure 24 presents the effects of CCI on the expression of citruUinated proteins in the rat cerebral cortex. Immunohistochemical analysis demonstrated that the basal level of protein citrullination as detected by labeling with mAB 6B3 was very low under control conditions. In contrast, CCI induced a marked increase in the immunolabeling of cells in the cortex. This up- regulation of protein citrullination was most pronounced in the vicinity of the lesion. The morphology of the affected cells was remarkably consistent, suggesting that the effects of CCI on protein citrullination were cell- specific.
[00186] The specificity of mAb 6B3 labeling of citruUinated proteins in TBI brain was confirmed by immunoneutralization. As shown in Figure 25, preabsorption of the mAb 6B3 with citruUinated protein standard effectively eliminated the labeling of cells in the cerebral cortex and hippocampus of CCI brain. In this experiment, the active labeling condition utilized mAb 6B3 that had been exposed to the identical absorption procedures as the neutralized mAb 6B3, except that the neutralizing proteins were not citrullinated under the control condition.
Additionally, a secondary-only control was also conducted and further confirmed the specificity of the anti-citrullinated protein labeling (not shown).
[00187] The regional effects of CCI on protein citruUination are summarized in Figure 26. CCI produced a marked increase in protein citruUination throughout the injured cortex, extending from lateral to the lesion site to regions of the cortex not directly impacted by CCI (Figure 26, panel B). Scoring of this region revealed that male naive rats (mean score: 0.06) and male CCI rats (mean score: 1.43) were significantly different, /? < 0.001. Female naive rats (mean score: 0.04) and female CCI rats (mean score: 1.35) were also significantly different, /? < 0.001 (n = 11 male rats, CCI; n = 8 male rats, naive control; n = 10 female rats, CCI; n = 7 female rats, naive control). Similarly, immunolabeling of the injured ipsilateral hippocampal formation (Figure 26, panel E) and external capsule (Figure 26, panel H) showed similar increases in protein citruUination in these regions. To a lesser extent, protein citruUination was also observed in fibers extending ventrally from the lesion site towards the midline corpus callosum (not shown). Other brain regions, including the amygdala and caudatoputamen, were completely negative for protein citruUination in these CCI animals. Furthermore, scoring of the cortex revealed that there was no gender difference observed in the regionalization or magnitude of protein citruUination following injury (CCI: p = 0.968; naive: p = 0.999). In general, CCI appeared to have little effect on the status of protein citruUination in contralateral brain structures, with the exception of the dorsal hippocampus, where approximately 20% of injured animals (4 of 21) displayed an intense labeling of unusually large and rounded cells (Figure 26, panel F). Finally, 6B3 immunolabeling was uniformly low across all regions studied in control male and female animals (Figure 26, Panels A, D, and G).
[00188] Dual immunofluorescence revealed astrocytes to be the principal cell type in which protein citruUination was affected by CCI. Figure 27 shows that anti-citrulline labeling in the cortex and external capsule was predominantly colocalized with GFAP. Similar observations were made in other affected brain regions. The findings presented in Figure 28 further confirm that CCI-induced protein citruUination was not significantly associated with neurons (NeuN), microglia / macrophages (Ibal), or oligodendrocytes (MBP) in the cortex. CitruUination was also not significantly associated with these cell types in any other brain regions investigated (not shown). It should be noted that the lack of co-localization of protein citruUination with NeuN immunoreactivity might reflect diminished NeuN expression following TBI, rather than a lack of citruUinated proteins within neurons. The distinctive profile of CCI-induced protein citruUination in the ipsilateral and contralateral hippocampus is shown in Figure 29. As noted above, increased anti-citrullinated protein labeling was clearly evident in the ipsilateral hippocampus of all animals studied (Figure 29, panel A), and this labeling colocalized with GFAP labeling (Figure 29, panels B and C). In addition, in -20% of injured animals, the contralateral hippocampus also displayed intense 6B3 labeling (Figure 29, panel D) that colocalized with GFAP labeling. The co-labeled cells were morphologically distinct from traditional stellate astrocytes, displaying a rounded, branchless appearance consistent with that of a macrophage (Figure 29, panels E-G).
[00189] Figure 30 demonstrates the specificity of the 6B3 antibody for detecting citruUinated proteins in a western blot format. Shown on the left is the Coomassie staining for human fibrinogen (Fib) and the same preparation of fibrinogen that was enzymatically citruUinated by reaction with PAD4 (C-Fib). The protein staining shows that the characteristic profile of purified human fibrinogen is modestly affected by reaction with PAD4. Presented on the right are immunoblots showing the reactivity of mAb 6B3 with the citrullinated fibrinogen preparation (Active/C-Fib) and the elimination of this reactivity by preabsorption of 6B3 with citrullinated fibrinogen prior to blotting (Neutralized/C-Fib). There was no reactivity of mAb 6B3 with fibrinogen that had not been citrullinated by PAD4 treatment (Active/Fib).
[00190] Proteomic analysis revealed that the effects of CCI on protein citrullination were specific to a discrete subset of proteins making up the entire brain proteome, and further, that the proteins involved are primarily associated with cytoskeletal structure and metabolic processes (Figure 31). Shown in the upper left panel are the proteomes of control and injured cerebral cortex fractionated by fluid phase isoelectric focusing. Each pair of lanes, control (C) and CCI (I), show the proteins present in the four different pi partitions. As visualized by Commassie staining, CCI did not appreciably affect the general pattern of protein staining across the four pi fractions. In contrast, the pattern of protein citrullination was dramatically impacted by CCI (upper right panel). Consistent with immunohistochemistry findings (Figures 2 and 3), little protein citrullination was observed in control cortex (C), whereas CCI (I) resulted in the intense labeling of a distinctive subset of the fractionated proteins. The immunoreactive signals of the western blot were mapped to Commassie features of the protein gel, and proteins were identified by peptide mass fingerprinting and tandem mass spectrometry. The proteins identified are presented in the lower panel of Figure 31. These proteins are functionally grouped as
cytoskeletal components (including dynamin-1, GFAP, and several forms of tubulin); those involved in metabolic processes (including peroxiredoxin-1, dihydropyrimidinase-related protein 2, and creatine kinase B-type); and proteins involved in cell-cell signaling and synaptic transmission (synapsin-2, syntaxin-binding protein 1, and amphyiphysin). It should be noted that these functional groups of affected proteins - cytoskeletal components, metabolic proteins and proteins involved in cell-cell signaling - may have been preferentially identified as citruUinated due to their relatively high abundance. These abundant protein types may be more easily identifiable through proteomic analyses than less abundant proteins, which may also be citruUinated but not easily identified. In this regard, however, only a small proportion of the very high abundance proteins were identified as being citruUinated, indicating a notable degree of specificity in the citrullination response.
[00191] Analysis of the mass spectra data set with a variable modification search for the citrullination of arginine residues revealed many sites of citrullination on arginine residues with Mascot score corresponding to p<0.05. The following is a list of the various proteins and corresponding sequences where arginines were shown to be citruUinated: Sample 1, fructose- bisphopshate aldolase A, sequences LQSIGTENTEENR (score: 55.87) and RALANSLACQGK (score: 10.24); Sample 3, dynamin-1, sequences GMEDLIPLVNR (score: 66.52),
SGQASPSRPESPRPPFDL (score: 17.9), and ASETEENGSDSFMHSMDPQLER (score:
15.29); Sample 4, moesin, sequence ERQEAEEAKEALLQASR (score 21.64), synapsin-2, sequence YDIRVQK (score 19.95), and septin-9, sequence
RVETPASKAPEGSAMPVTDAAPK (score 13.24); Sample 9, myelin basic protein, sequence FFSGDRGAPK (score 60.9); Sample 11, transitional endoplasmic reticulum ATPase, sequence ETVVEVPQVTWEDIGGLEDVKR (score 34.49); Sample 12, NADH-ubiqiunone
oxidoreductase 75kDa subunit, sequences IAS Q VAALDLGYKPGVEAIRK (score 26.71) and VALIGSPVDLTYRYDHLGDSPK (score 24.23); Sample 13, heat shock cognate 71 kDa, sequence VEIIANDQGNRTTPSYVAFTDTER (score 43.26); Sample 15, creatine kinase B- type, sequence LLIEMEQRLEQGQPIDDLMPAQK (score 42.46); Sample 16, drebrin, sequence LKDQS IFGDQRDEEEES QMK (score 45.82) and sequence KALDARLR (score 29.54), and amphiphysin, sequence LQRELR (score 16.38).
[00192] The LC MS/MS approach did not identify all the proteins that were detected through the use of the 6B3 anti-citrullinated protein antibody as containing citrullinated residues. Some possible explanations for this include, but are not limited to: (1) overlapping of ion peaks due to similarity in mass with deamidation of Arg, (2) low abundance of citrullination modification to below the detection limit of the LC MS/MS technique but not western blot, (3) poor ionization of a citrullinated peptide, (4) poor fragmentation of a citrullinated peptide, and (5) non- specificity detection of the proteins by western blot.
[00193] To further investigate the cellular mechanisms of injury-induced protein citrullination, a model of simulated TBI was established. In this model, normal human astrocytes (obtained commercially) were incubated in control medium or in medium containing the calcium ionophore, ionomycin, to induce calcium excito toxicity. Figure 32 shows the results of 3 separate experiments investigating the effects of ionomycin treatment on the proteolytic processing of GFAP (left panel) and the generation of citrullinated proteins (right panel). The data show that treatment with ionomycin consistently activated the proteolytic processing of intact GFAP (left panel; blue arrows) to produce a distinctive pattern of breakdown products. Probing with mAb 6B3 indicated that one of the GFAP breakdown products is preferentially citrullinated (right panel; orange arrow) in response to simulated TBI. In addition, several other protein features appeared to be heavily citrullinated in response to ionomycin treatment. The identity of these signals remains to be determined.
[00194] Protein citrullination is a calcium-dependent protein modification that has been largely studied in the context of autoimmune disorders, particularly rheumatoid arthritis. Abnormal protein citruUination in rheumatoid arthritis results in the generation of antigenic epitopes that become targeted by the adaptive immune system (Gyorgy et al., 2006;Kidd et al., 2008). Protein citruUination is also found to be abnormal in several neurodegenerative disorders, including multiple sclerosis (MS) and Alzheimer's disease, suggesting that citrullinated proteins may also serve as antigenic targets in these conditions and thereby contribute to long-term pathogenesis. To date, however, little research has examined the effects of TBI on protein citruUination, where an acute mechanical injury can lead to progressive and sustained neuropathology. Currently, there are no data regarding the identity of specific proteins affected by citruUination following TBI, or information on the susceptibility of different brain regions or cell types to this modification following injury.
[00195] The constellation of dysfunctions resulting from TBI is often complex and dynamic, involving pathology caused by acute tissue damage and interactions amongst the ongoing changes caused by ischemia, hypoxia, excitotoxicity, and elevated intracranial pressure (Walker and Tesco, 2013). A hallmark of acute TBI pathophysiology, however, is intracellular calcium overload (Floyd et al., 2005). This shift in ionic balance is excitotoxic, activating a number of damaging intracellular cascades, including lipid peroxidation, proteolysis, free radical generation (Werner and Engelhard, 2007), and the activation of pro-apoptotic genes (Walker and Tesco, 2013) with the loss of dendritic spines. Elevated intracellular calcium is an essential condition for the activation of peptidylarginine deiminase (PAD) enzymes, which catalyze the conversion of intrapeptidyl arginine residues to citrulline residues (Ishigami and Maruyama, 2010).
[00196] PAD enzymes exist as several isoforms, two of which are found in neural tissue: PAD2 (Curis et al., 2005;Raijmakers et al., 2005;Jang et al., 2008) and PAD4 (Acharya et al., 2012). In humans, PAD2 is largely localized to astrocytes, while PAD4 is exclusively expressed in neurons (Acharya et al., 2012). Moreover, PAD2 is localized to reactive astrocytes in the brains of mice affected by a prion disorder, scrapie, where its enzymatic activity is nearly double that of healthy mice (Jang et al., 2008). Research on the etiology of MS has suggested that PAD2 contributes to the pathology of demyelination, where its activity is thought to destabilize myelin sheath structure (Musse et al., 2008).
[00197] The present investigation in rats shows that TBI-induced protein citrullination is selectively localized to astrocytes. This finding is consistent with the understanding that following ischemia, astrocytes exhibit a dramatic rise in intracellular calcium due to a rapid influx of extracellular calcium through dysfunctional membrane channels, as well as the release of calcium from intracellular stores (Floyd et al., 2005). While influxes in intracellular calcium are also found in neurons following mechanical injury, this rise is far less profound than that observed in astrocytes (Rzigalinski et al., 1998). Furthermore, astrocytes selectively display long-term defects in calcium signaling following TBI (Rzigalinski et al., 1998) and ultimately, astrocytic death precedes that of neurons in rat models of TBI (Floyd et al., 2005). Related findings involving hypoxia confirm that astroglial injury is due to massive calcium influxes and resulting excitotoxicity (Ishigami and Maruyama, 2010). Models of TBI illuminate the biologic importance of astrocytes in brain injury, serving a protective role by shielding neurons from damage due to oxygen deprivation (Vibulsreth et al., 1987), glutamate neurotoxicity, and calcium excitotoxicity (Mattson and Rychlik, 1990). It has been proposed that the intracellular rise of calcium within astrocytes is directly linked to their neuroprotective effects on neurons, most likely due to their ability to sequester large amounts of calcium and thus stabilize the ionic environment for neurons (Duffy and Mac Vicar, 1996). Our finding that protein citrullination is limited to astrocytes is consistent with this proposal. [00198] Present results indicate that the up-regulation of protein citruUination induced by TBI occurs in astrocytes that are located in the cerebral cortex, external capsule, and hippocampus. In contrast, the status of protein citruUination in other brain regions (such as the amygdala and caudate putamen) and cell types was not appreciably altered by TBI. The mechanistic basis for this observation may relate to the up-regulation of voltage-gated, class C L-type Ca2+ channels that are selectively expressed in astrocytes and are particularly sensitive to activation by injury in the regions reported here (Chung et al., 2001). The finding that nimodipine, an L-type calcium channel blocker, protects against excitotoxic damage in cultured astrocytes supports this proposal (Haun et al., 1992). These findings correspond to a model of calcium excitotoxicity- induced citruUination. The selective citruUination of astrocytes in these particular regions may correspond to long-term dysfunctions associated with TBI, including learning and memory deficits associated with progressive hippocampal atrophy (Walker and Tesco, 2013). However, it is possible that citruUination may occur in other cell types at different time points following TBI, and future work is needed to address this possibility.
[00199] Our investigation showed that patterns of protein citruUination following TBI were similar between male and female rats. This finding is in contrast to our previous observations concerning the effects of TBI on protein carbonylation. Following injury, male rats showed far greater response in protein carbonylation as compared to female rats (Lazarus et al., 2015).
Protein carbonylation is a reflection of oxidative stress (Dalle-Donne et al., 2003), whereas citruUination is a marker of calcium influx (Vossenaar et al., 2004;Kinloch et al., 2005).
Accordingly, the gender difference observed in carbonylation may be due to the protective antioxidant effects of ovarian steroids (Roof et al., 1997), whereas a similar mechanism does not appear to exist in the case of citruUination. The extent to which these protein modifications contribute to gender differences in TBI complications and mortality (Goswasser et al.,
1998;Berry et al., 2009) remains to be determined. It should be noted that the present experimental design focused on only one time-point post- TBI (five days), and as such, it is possible that gender differences in post-injury citruUination may be evident at other time-points.
[00200] The present investigation has identified 37 proteins as a very small subset of the entire brain proteome that is possibly citrullinated in response to TBI. This selectivity in protein citruUination indicates that the mechanism involved maintains a high degree of specificity. Furthermore, several of the proteins identified here are reported to be citrullinated in
neurodegenerative diseases, and thus may play causative roles in neuropathology. Specifically, citrullinated GFAP is a characteristic feature in MS and Alzheimer's disease (Nicholas et al., 2004;Ishigami et al., 2005;Gyorgy et al., 2006;Bradford et al., 2014), and myelin basic protein (MBP), a major component of myelin sheath structure, is profoundly over-citrullinated in MS (Tranquill et al., 2000;Bradford et al., 2014). Similarly, GFAP, tubulin, peroxiredoxin 1, cofilin- 1 and alpha/gamma enolase are selectively citrullinated in prion disease (Jang et al., 2008). Therefore, the link between abnormal protein citruUination and neurological disease appears strong. It should be noted that the 37 proteins identified as targets for TBI-induced citruUination were derived from the analysis of only 16 gel features. Accordingly, not all of the proteins identified in a single feature are necessarily citrullinated, and may have resulted from the analysis due to their co-purification with another citrullinated species. Nevertheless, the high correlation that exists between the specific proteins identified here and their reported
citruUination in various disease states is consistent with the proposal that these proteins are indeed targets for citruUination following TBI. [00201] A potential mechanism by which abnormal protein citrullination contributes to neurological disease could involve the adaptive immune system. A significant proportion of the proteins identified here are also recognized as autoantigens, in both neurological and
autoimmune-related disorders. For example, MBP is the signature citruUinated autoantigen of MS (Tranquill et al., 2000;Bradford et al., 2002). Autoantibodies targeting amphiphysin are associated with several neurological disorders, including sensory neuronopathy and
encephalopathy (Murinson and Guarnaccia, 2008). Additionally, dihydropyrimidinase-related protein 2 (CRMP2) is autoantigenic in autoimmune retinopathy (Adamus et al., 2013). Finally, alpha enolase has been identified as a citruUinated autoantigen in rheumatoid arthritis (Kinloch et al., 2005), while citruUinated 78 kDa glucose-regulated protein is an autoantigen within the pancreatic beta-cells in Type 1 diabetes (Rondas et al., 2014). Collectively, these findings support the proposal that injury-induced protein citrullination may generate immunological epitopes that become targets of the adaptive immune system. This process may serve as an underlying basis for chronic and progressive neurological disorders.
[00202] Finally, this report presents the development of an in vitro model for simulating TBI in a controlled and cell-specific manner. Shown here in normal human astrocytes are the effects of calcium excitotoxicity, a hallmark condition of TBI, on the hyper-citrullination of GFAP.
Moreover, the findings confirm the proteolytic processing of GFAP to a series of breakdown products that are consistent with those reported by others using in vitro models of TBI
(Zoltewicz et al., 2012;Okonkwo et al., 2013). Interestingly, our investigation showed that one of these breakdown products is heavily citruUinated, and thus may serve as the antigen for the development of the anti-GFAP autoantibodies recently reported in TBI (Zhang et al., 2014). Accordingly, the application to this and other models for simulated TBI may provide novel insights into consequences and mechanisms of TBI, and also identify informative biomarkers for assessing brain injury.
[00203] In conclusion, the present findings show that TBI dramatically up-regulates protein citruUination within astrocytes in specific brain regions. Additionally, this modification affects only a subset of the neural proteome, primarily affecting proteins involved in cytoskeletal structure, metabolic processes, and cell-cell signaling. A large proportion of these proteins have been identified as citrullinated in other pathologies, including MS, Alzheimer's disease, and rheumatoid arthritis, indicating a potential role for this protein modification in ongoing pathological processes. Interestingly, gender does not affect the degree or distribution of this modification in neural tissue, in contrast to previous observations involving TBI-induced protein carbonylation. Accordingly, gender differences in the CNS response to TBI may not involve differential responses in protein citruUination. Finally the work presented here examined protein citruUination only at five days following injury. According future work is needed to address the presence of citruUination following TBI in an extended time course to investigate the long- lasting effects of TBI on the status of protein citruUination in the central nervous system. In summary, this research indicates that abnormal protein citruUination is a feature of TBI that could contribute to ongoing pathological mechanisms following acute injury, possibly including aspects of autoimmune dysfunction.
[00204] Further, citrullinated breakdown products of filial fibrillary acidic protein and citrullinated forms of other proteins, both intact and breakdown products, have been discovered as candidate biomarkers for traumatic brain injury (TBI). These additional proteins include amphiphysin, crafilin 1, dynamin-1, synapsin-2, septin-9, annexin A6, stress-induced- phosphoprotien-1, dihydropyrimidase-related protein 2, neuronal- specific septin-3, transgelin-3, GRIPI- associated protein 1, transitional endoplasmic reticulum ATPase, vesicle-fusing ATPase,
78 kDa glucose-regulated protein, syntaxin-binding protein 1 and drebin.
[00205] Analysis of the mass spectra data set with a variable modification search for the citruUination of arginine residues revealed many sites of citruUinatien on arginine residues with
Mascot score corresponding to pi<0.05. Table 2 lists the various proteins and corresponding sequences where arginines were shown to be citrullinated.
Table 2. Proteins identified to be citrullinated in response to TBI.
Protein Sequence i Mascot Score trudose-bisphopshate aldolase A LQSIGTENTEENR 55 8? faictose-bisphopshate aldolase A RALANSLACQG 10.24 dynamiri-1 MEDLI VN 66.52 dynamln-1 SGQASPSR ESP PPFDL 7,90 dynamin-1 AS E 'E £ N GSDSF WiH S D PQl.E R 15.20 moesin ERQEAEEAKEALLQAS 2.1.84 synapsin -2 YD!RVQK 19.95 septin-9 R VET PAS APEG S A P VT D AA P K 13.24 myelin basic proiej > FFSGDR-GAPK 60.90 transitional' sndoplasmic reticulum ETWEVPQVTWEDIGGLEDVKR 34.4S ATPase
NADH-ubiqiunone oxidoreduciase 75kDa IASQVAALDLGYKPGVEAIRK 26.71 subunif
NADH-ubiqluocne oxidoreductase 75kDa VA.UGSPVDLTYRYDHLGDSP 24.23 subunis
heat shock cognate 71 KDa VE!iA DGGNRTTPSYVAFTDTER 43.26 creatine kinase !-type LUE EQ RLEQGQP IDDLMPAQK .42 46 drebrin ' L DQ Si F G OQR D E E EE SQM 45,82 drebm ALDARIR 29.54 amp iphysin LQRELR 16.38
[00206] The disclosed methods can be conducted with any suitable assay system. The assay systems contemplated by the present invention are used to conduct any type of diagnostic or analytical method known in the art. Such analytical methods include but are not limited to clinical chemistry assays (e.g., measurements of pH, ions, gases and metabolites, including mass spectrometry), hematological measurements, nucleic acid amplification assays (e.g., polymerase chain reaction (PCR) and ligase chain reaction assays), immunoassays (e.g., direct, sandwich and/or competitive immunoassays, including Western blots and ELISAs, and serological assays), oligonucleotide ligation assays, and nucleic acid hybridization assays. Any biological reagent that might be used in such analytical methods can be used in such systems, including but not limited to nucleic acids, nucleotides, oligonucleotides, DNA, RNA, PNA, primers, probes, antibodies or fragments thereof, antigens, small molecules, e.g., drugs or prodrugs, streptavidin, avidin, and biotin.
[00207] These systems can be portable, e.g., hand-held, and/or operated within a fixed laboratory or field setting, alone or in combination with one or more additional components, assay devices or systems. These systems can be used in a variety of applications, from field operations to laboratory settings, in a wide variety of industries, including but not limited to, medical, clinical, forensic, pharmaceutical, environmental, veterinary, biological, chemical, agricultural, waste management, hazardous chemical, drug testing, and in defense applications, e.g., for the detection of biological warfare agents. The assay systems and consumables used in the present invention can detect an analyte of interest by any suitable method, including but not limited to, optical, electromechanical, radiowave, electromagnetic, colorimetric, fluorimetric, chemiluminescent, electrochemiluminescent, radiochemical, nuclear magnetic resonance, enzymatic, fluorescent, particle-count, and cell-count based detection.
[00208] The assay consumable includes devices in which one or more steps of an assay process are conducted and such devices can include one or more test sites where an assay measurement is conducted. In one embodiment, the assay consumable includes at least one assay test site for an assay. A test site can include a plurality of distinct assay domains, at least two of the domains including reagents for measuring different analytes. Still further, the consumable can include a plurality of test sites for a plurality of individual assays. Alternatively, the assay consumable can be a component that provides a reagent or other assay component that is used by the system to conduct an assay. For example, the assay consumable can be a container with one or more compartments for holding assay reagents. The assay consumable (or test sites therein) can be single use or it can be reusable. The assay consumable can be configured to conduct one test or multiple tests (sequentially or in parallel).
[00209] Test sites, as used herein, refer to regions of a consumable that hold, contact and/or interrogate a sample. A test site can include a plurality of distinct assay domains, at least two such domains include reagents for measuring different analytes. Consumables can comprise multiple test sites which can hold, contact or otherwise interrogate distinct volumes (aliquots) of the same sample and/or volumes of different samples. A sector of an assay consumable refers to grouping of two or more test sites of the consumable. Each test site can be used to conduct a single measurement or multiple measurements on a volume of sample (for example, the measurement of multiple different analytes in a multiplexed assay format). Depending on the specific requirements of an application, a consumable with multiple test sites can be configured to use all of its test sites in parallel, to use its test sites at different times (e.g., assigning unused test sites to be used as new samples are delivered to the assay system), or a combination of both modes of operation can be enabled.
[00210] The assay consumable can be any structure useful in diagnostic applications and that structure can be dictated by the particular assay format or detection method employed by the device. Examples of assay consumables suitable for use with the invention include, but are not limited to, test tubes, cuvettes, flow cells, assay cartridges and cassettes (which can include integrated fluidics for assay processing), multi-well plates, slides, assay chips, lateral flow devices (e.g., strip tests), flow-through devices (e.g., dot blots), pipette tips, solid phase supports for biological reagents and the like. In certain embodiments, test sites in the assay consumable are defined by compartments in the assay consumable, e.g., wells, chambers, channels, flow cells and the like. The assay consumable and/or test sites can include one or more components used to carry out an assay measurement according to one or more specific detection methodologies. Depending on the function of the consumable and the detection modalities employed by the assays system, examples of such components can include, but are not limited to, lateral flow matrices, filtration matrices, optical windows, sensors (e.g., electrochemical and optical sensors), solid phase supports for binding reactions (e.g., coated slides, chips, beads, pins, coated filtration or lateral flow matrices, tubes and the like), reagents (dry or in liquid form), electrodes, analyte selective membranes and the like.
[00211] In one embodiment, the assay consumable can be a device that incorporates a conventional lateral flow test strip, e.g., an immunoassay test strip, as an assay medium. In this example, the device is molded to include an identifier or the identifier is affixed to the device without any modification to the structure of the device and/or the assay medium. In one embodiment, the device is placed within the analytical system, i.e., the assay system, for analysis and before, during or after the performance of the assay, the identifier controller within, affixed to or associated with the assay system reads the data contained on the identifier and uses that data in the assay or after the assay is completed by the system.
[00212] In another embodiment, the assay consumable and accompanying assay system or reader is capable of performing a multiplex assay. A multiplex assay is a type of assay in which multiple measurements are performed on a single sample, e.g., by distributing samples across multiple test sites and/or by carrying out multiple measurements on volumes of samples in individual test sites. The multiple measurements can include, but are not limited to, (i) multiple replicates of a measurement for an analyte; (ii) multiple measurements of a certain analyte (i.e., multiple non-identical measurements for the same analyte, e.g., measurements that differ in format or in the identity of the assay reagents that are employed); and/or (iii) measurements of multiple different analytes. In one specific embodiment, an assay consumable is configured to carry out, in one or more test sites, multiplex measurements that include at least two assays for two different analytes.
[00213] The invention is not restricted to specific approaches for conducting multiplex measurements in a test site and can employ any of the numerous techniques that have been developed for carrying out multiplex measurements. Multiplex measurements that can be used with the invention include, but are not limited to, multiplex measurements (i) that involve the use of multiple sensors; (ii) that use discrete assay domains on a surface (e.g., an array) that are distinguishable based on location on the surface; (iii) that involve the use of reagents coated on particles that are distinguishable based on a particle property, such as size, shape, color, etc.; (iv) that produce assay signals that are distinguishable based on optical properties (e.g., absorbance or emission spectrum), (v) that are based on temporal properties of an assay signal (e.g., time, frequency or phase of a signal), and/or (vi) that are based on some other assay characteristic. Accordingly, interpretation of multiplexed assay results can involve the use of multiplexing information, such as the identity of the assays carried out in each test site and, within a test site, any assay characteristics (identity of specific sensors, location and identity of assay domains, etc.) that are used to distinguish assays carried out in a test site and/or that are used to tie a specific assay identity to the corresponding assay signal. [00214] In one embodiment, an assay test site comprises a plurality of distinct assay domains and each domain comprises one or more reagents for measuring a different analyte. Multiplexing information, including the location, identity, and composition of each assay domain, is used to identify the assay signal generated at each domain and connect it to a determination of the presence or amount of the corresponding analyte (a process which can include the application of additional consumable data such as signal thresholds and/or calibration parameters). Such multiplexing information can be provided as consumable data and/or stored to the consumable identifier.
[00215] A test site can be configured to carry out a plurality of multiplexed measurements
(e.g., it can include a plurality of distinct assay domains, wherein each domain comprises reagents for measuring a different analyte). In one embodiment, the assay consumable can include a plurality of test sites. Information regarding the exact configuration of the one or more test sites, assay domains, and/or one or more sectors in a consumable can be included in the information saved to the assay consumable identifier and/or provided as consumable data. This information can include the location and identity of the test sites, assay domains, and/or one or more sectors as well as multiplexing information (as described above) including the number, identity and differentiating characteristics of the individual measurements within a test site, assay domain, and/or sector (e.g., the specific locations, identities and/or assay reagents of assay domains within each test site). In addition, the use of a test site, assay domain, and/or sector in an assay consumable can also be recorded to the identifier to track the use of the consumable in an assay system. The identifier and/or consumable data can also include information concerning the assay format and specific processing steps to be used for an assay consumable or test site, assay domain, and/or sector of an assay consumable. The identifier and/or consumable data can also include information concerning analytical methods that should be applied by the system once an assay is conducted to analyze the output of an assay in a given test site, assay domain, and/or sector and, optionally, to provide results that combine the output from multiple assays in a test site, assay domain, and/or sectors.
[00216] The test sites can be configured in any suitable configuration, depending on the geometry of the consumable and/or the type of assay conducted with the consumable. In one embodiment, the test sites are configured as wells and/or chambers in the assay consumable. For example, the assay consumable of the present invention can be a multi-well plate (e.g., a 24-, 96- , 384- or 1536-well plate), and the wells of the plate can further comprise a plurality (e.g., 2 or more, 4 or more, 7 or more, 25 or more, 64 or more, 100 or more, etc.) of distinct assay domains. Multi-domain multi-well plates that are adapted to allow assay measurements to be conducted using electrode induced luminescence measurements (e.g., electrochemiluminescence measurements) are described in U.S. Application Ser. No. 10/238,391, entitled "Methods and Reader for Conducting Multiple Measurements on a Sample", filed on Sep. 10, 2002, issued as U.S. Patent No. 7,858,321 on December 28, 2010, hereby incorporated by reference. The exact configuration of the domains, test sites, and/or sectors in an assay consumable, as well as the specific identity of each domain, test site, and/or sector and the reagents bound to that domain/test site/sector can be included in the information saved to the assay consumable identifier and/or provided as consumable data. In addition, the use of a given domain, test site, and/or sector in an assay consumable can also be recorded to the identifier to track the use of the consumable in an assay system.
[00217] Assay consumables can be used in a plurality of diverse assays and this diversity leads to a variety of suitable configurations of the associated consumable. In one assay format, the same analyte is measured at different assay domains within a test site, the different assay domains being designed to measure a different property or activity of the analyte. Information concerning the assay format that can be used in an assay consumable, test site and/or assay domain can also be saved to the assay consumable identifier and/or provided as consumable data. The identifier and/or consumable data can also include information concerning analytical methods that should be applied by the system once an assay is conducted to analyze the output of an assay in a given test site and/or domain and compare that output to an assay in a separate test site and/or domain.
[00218] One example of a multiplex assay consumable and reader is described in U.S.
2004/0022677, issued as U.S. Patent No. 7,842,246 on November 30, 2010, the disclosure of which is incorporated herein by reference in its entirety. Such assay consumables include one or more, and in one embodiment, a plurality of test sites and/or assay domains for conducting one or more assay measurements simultaneously or sequentially. For example, the test sites can be configured as wells and/or chambers. These test sites and/or assay domains comprise one or more electrodes for inducing luminescence from materials in the test sites and/or assay domains. The assay consumables can further comprise assay reagents in liquid or dry form, e.g., in the test sites, e.g., wells or chambers, of the consumable.
[00219] In addition to the test sites and assay domains, an assay consumable or multi-well assay plate can include several additional elements, e.g., a plate top, plate bottom, wells, working electrodes, counter electrodes, reference electrodes, dielectric materials, electrical connections, and assay reagents. The wells of the plate can be defined by holes or openings in the plate top, or as indentations or dimples on a surface of a plate. The plates can have any number of wells of any size or shape, arranged in any pattern or configuration and can be composed of a variety of different materials. Exemplary embodiments of consumables that can be used in the present invention include industry standard formats for the number, size, shape and configuration of the plate and wells, e.g., 96-, 384-, and 1536-well plates, with the wells configured in two- dimensional arrays. Other formats can include single well plates, 2-well plates, 6-well plates, 24-well plates, and 6144-well plates. Multi-well assay plates can be used once or can be used multiple times and are well suited to applications where the plates are disposable. Various configurations for suitable assay plates can be used in the present invention, including but not limited to those depicted in Figs. 11A, 12A, 13A, 13B, 14A, 15, and 16A of U.S. Application Ser. No. 2004/0022677, each of which are incorporated herein by reference. As stated above, the specific configuration and identity of assay test sites, domains, and/or sectors of an assay consumable can be included in the information saved to the assay consumable identifier and/or provided as consumable data.
[00220] Binding assays for measuring biomarker levels may use solid phase or
homogenous formats. Suitable assay methods include sandwich or competitive binding assays. Examples of sandwich immunoassays are described in U.S. Patent No. 4,168,146 and U.S. Patent No. 4,366,241, both of which are incorporated herein by reference in their entireties. Examples of competitive immunoassays include those disclosed in U.S. Patent No. 4,235,601, U.S. Patent No. 4,442,204 and U.S. Patent No. 5,208,535, each of which are incorporated herein by reference in their entireties.
[00221] Multiple biomarkers may be measured using a multiplexed assay format, e.g., multiplexing through the use of binding reagent arrays, multiplexing using spectral
discrimination of labels, multiplexing of flow cytometric analysis of binding assays carried out on particles, e.g., using the Luminex® system. Suitable multiplexing methods include array based binding assays using patterned arrays of immobilized antibodies directed against the biomarkers of interest. Various approaches for conducting multiplexed assays have been described (See e.g., US 20040022677; US 20050052646; US 20030207290; US 20030113713; US 20050142033; and US 20040189311, each of which is incorporated herein by reference in their entireties. One approach to multiplexing binding assays involves the use of patterned arrays of binding reagents, e.g., U.S. Patent No. 5,807,522 and 6,110,426; Delehanty J-B., Printing functional protein microarrays using piezoelectric capillaries, Methods Mol. Biol. (2004) 264: 135-43; Lue R Y et al., Site-specific immobilization of biotinylated proteins for protein microarray analysis, Methods Mol. Biol. (2004) 264: 85-100; Lovett, Toxicogenomics: Toxicologists Brace for Genomics Revolution, Science (2000) 289: 536-537; Berns A, Cancer: Gene expression in diagnosis, nature (2000) 403: 491-92; Walt, Molecular Biology: Bead-based Fiber-Optic Arrays, Science (2000) 287: 451-52 for more details). Another approach involves the use of binding reagents coated on beads that can be individually identified and interrogated. See e.g., WO 9926067, which describes the use of magnetic particles that vary in size to assay multiple analytes; particles belonging to different distinct size ranges are used to assay different analytes. The particles are designed to be distinguished and individually interrogated by flow cytometry. Vignali has described a multiplex binding assay in which 64 different bead sets of microparticles are employed, each having a uniform and distinct proportion of two dyes (Vignali, D. A A, "Multiplexed Particle-Based Flow Cytometric Assays" J. ImmunoL Meth. (2000) 243: 243-55). A similar approach involving a set of 15 different beads of differing size and fluorescence has been disclosed as useful for simultaneous typing of multiple pneumococcal serotypes (Park, M.K et al., "A Latex Bead-Based Flow Cytometric Immunoassay Capable of Simultaneous Typing of Multiple Pneumococcal Serotypes (Multibead Assay)" Clin. Diagn. Lab Immunol. (2000) 7: 486-89. Bishop, JE et al. have described a multiplex sandwich assay for simultaneous quantification of six human cytokines (Bishop, LE. et al., "Simultaneous Quantification of Six Human Cytokines in a Single Sample Using Microparticle-based Flow Cytometric Technology," Clin. Chem (1999) 45: 1693-1694. A diagnostic test may be conducted in a single assay chamber, such as a single well of an assay plate or an assay chamber that is an assay chamber of a cartridge.
[00222] The assay modules, e.g., assay plates or cartridges or multi-well assay plates), methods and apparatuses for conducting assay measurements suitable for the present invention are described for example, in US 20040022677; US 20050052646; US 20050142033; US 20040189311, each of which is incorporated herein by reference in their entireties. Assay plates and plate readers are now commercially available (MULTI-SPOT® and MULTI- ARRAY® plates and SECTOR® instruments, Meso Scale Discovery, a division of Meso Scale Diagnostics, LLC, Gaithersburg, MD.). In addition, the various isoforms of Αβ can be purchased from AnaSpec, Inc. ( w w. m sijcc . com , Fremont, California) or rPeptide ( www - pcpi ldo c -vs. Bogart, Georgia). Antibodies specific for the various isoforms of Αβ may be produced by methods known in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual. (Third Ed.) (2000).
[00223] In this embodiment, the assay consumables can be used in a reader that can be used to induce and measure luminescence, e.g., electrode induced luminescence or
electrochemiluminescence, in assays conducted in or on assay consumables, e.g., multi-well assay plates. The accompanying assay system can also induce and/or measure current and/or voltage, for example, at an electrode. The assay system can incorporate, for example, one or more photodetectors; a light tight enclosure; mechanisms to transport the assay plates into and out of the reader (and in particular, into and out of a light tight enclosure); mechanisms to align and orient the assay plates with the photodetector(s) and/or with electrical contacts; additional mechanisms to track and identify plates (e.g. bar code readers); mechanisms to make electrical connections to plates, one or more sources of electrical energy for inducing luminescence, and appropriate devices, electronics and/or software. The assay reader can also include mechanisms to store, stack, move and/or distribute one or more multi-well assay plates (e.g. plate stackers and/or plate conveyors). The assay system can be configured to measure light from multi-well assay plates by measuring light sequentially from a plurality of sectors or regions of the plate (i.e., a grouping of a plurality of adjacent assay domains within a plate) and/or from the entire plate substantially simultaneously or simultaneously. The assay system can also incorporate additional microprocessors and computers to control certain functions within the system and to aid in the storage, analysis and presentation of data. Various configurations for suitable assay systems can be used in the present invention, including but not limited to those depicted in Figs. 17 to 23 of U.S. Application Ser. No. 2004/0022677, each of which are incorporated herein by reference.
[00224] The described embodiments and examples of the present disclosure are intended to be illustrative rather than restrictive, and are not intended to represent every embodiment or example of the present disclosure. While the fundamental novel features of the disclosure as applied to various specific embodiments thereof have been shown, described and pointed out, it will also be understood that various omissions, substitutions and changes in the form and details of the devices illustrated and in their operation, may be made by those skilled in the art without departing from the spirit of the disclosure. For example, it is expressly intended that all combinations of those elements and/or method steps which perform substantially the same function in substantially the same way to achieve the same results are within the scope of the disclosure. Moreover, it should be recognized that structures and/or elements and/or method steps shown and/or described in connection with any disclosed form or embodiment of the disclosure may be incorporated in any other disclosed or described or suggested form or embodiment as a general matter of design choice. Further, various modifications and variations can be made without departing from the spirit or scope of the disclosure as set forth in the following claims both literally and in equivalents recognized in law.
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Claims

What is Claimed is:
1. A method of diagnosing traumatic brain injury (TBI) in a subject, the method comprising a) determining the citruUination level(s) of one or more biomarkers in a biological sample taken from the subject, and b) comparing the determined citruUination level(s) of the one or more biomarkers against a citruUination level(s) of the same one or more biomarkers from a control subject determined not to be suffering from TBI, wherein an increase in the citruUination level(s) of the one or more biomarkers compared to the citruUination level(s) of the one or more biomarkers from the control subject is indicative that the subject may be suffering from TBI.
2. The method of claim 1, wherein the one or more biomarkers is a protein or a protein fragment.
3. The method of claim 2, wherein the one or more proteins or protein fragments is selected from the group consisting of fructose-bisphosphate aldolase A, peroxiredoxin-1, cofilin-1, glutathione S-transferase P, transgelin, dynamin-1, synapsin-2, moesin, septin-9, annexin A6, stress-induced phosphoprotein 1, syntaxin-binding protein 1, dihydropyrimidase-related protein 2, alpha-enolase, gamma-enolase, glial fibrillary acidic protein, fructose-bisphosphate aldolase C, neuronal- specific septin-3, transgelin-3, myelin basic protein, heat shock 70 kDa protein 4, GRIP 1- associated protein 1, transitional endoplasmic reticulum ATPase, NADH-ubiquinone oxidoreductase 75 kDa mitochondrial subunit, vesicle-fusing ATPase, 78 kDa glucose-regulated protein, heat shock cognate 71 kDa protein, annexin A6, syntaxin-binding protein 1, actin, cytoplasmic 1, creatine kinase B-type, tubulin beta-2B chain, tubulin beta-2A chain, tubulin beta- 4B chain, tubulin beta-5 chain, tubulin beta-3 chain, amphiphysin, and drebrin.
4. The method of claim 3, wherein two of any of the proteins are determined, three of any of the proteins are determined, four of any of the proteins are determined, five of any of the proteins are determined, six of any of the proteins are determined, seven of any of the proteins are determined, eight of any of the proteins are determined, nine of any of the proteins are determined, ten of any of the proteins are determined, eleven of any of the proteins are determined, twelve of any of the proteins are determined, thirteen of any of the proteins are determined, fourteen of any of the proteins are determined, fifteen of any of the proteins are determined, sixteen of any of the proteins are determined, seventeen of any of the proteins are determined, eighteen of any of the proteins are determined, nineteen of any of the proteins are determined, twenty of any of the proteins are determined, twenty one of any of the proteins are determined, twenty two of any of the proteins are determined, twenty three of any of the proteins are determined, twenty four of any of the proteins are determined, twenty five of any of the proteins are determined, twenty six of any of the proteins are determined, twenty seven of any of the proteins are determined, twenty eight of any of the proteins are determined, twenty nine of any of the proteins are determined, thirty of any of the proteins are determined, thirty one of any of the proteins are determined, thirty two of any of the proteins are determined, thirty three of any of the proteins are determined, thirty four of any of the proteins are determined, thirty five of any of the proteins are determined, thirty six of any of the proteins are determined or thirty seven of any of the proteins are determined.
5. The method of claim 1, wherein the biological sample is selected from the group consisting of blood, blood plasma, blood serum, cerebrospinal fluid (CSF) urine, lymph, saliva and combinations thereof.
6. The method of claim 3, wherein the one or more proteins or protein fragments is selected from the group consisting of amphiphysin, cofilin-1, dynamin-1, synapsin-2, septin-9, annexin A6, stress-induced-phosphoprotien-1, dihydropyrimidase-related protein 2, neuronal- specific septin-3, transgelin-3, GRIP 1 -associated protein 1, transitional endoplasmic reticulum ATPase, vesicle-fusing ATPase, 78 kDa glucose-regulated protein, syntaxin-binding protein 1, and drebin.
7. The method of claim 1, wherein the one or more biomarkers is glial fibrillary acidic protein (GFAP) or a protein fragment of GFAP.
8. The method of claim 2, wherein the one or more proteins or protein fragments is selected from the group consisting of fructose-bisphopshate aldolase A, dynamin-1, moesin, synapsin-2, septin-9, myelin basic protein, transitional endoplasmic reticulum ATPase, NADH-ubiqiunone oxidoreductase 75kDa subunit, heat shock cognate 71 kDa, creatine kinase B-type, drebrin, amphiphysin and GFAP.
9. The method of claim 8, wherein the one or more proteins or protein fragments comprises the citrulination sites LQSIGTENTEENR and RALANSLACQGK from fructose-bisphopshate aldolase A, GMEDLIPLVNR, SGQASPSRPESPRPPFDL and
ASETEENGSDSFMHSMDPQLER from dynamin-1, ERQEAEEAKEALLQASR from moesin, YDIRVQK from synapsin-2, R VETP AS KAPEGS AMP VTD A APK from septin-9,
FFSGDRGAPK from myelin basic protein, ETVVEVPQVTWEDIGGLEDVKR from transitional endoplasmic reticulum ATPase, IAS Q V A ALDLG YKPG VE AIRK and
VALIGSPVDLTYRYDHLGDSPK from NADH-ubiqiunone oxidoreductase 75kDa subunit, VEIIANDQGNRTTPSYVAFTDTER, from heat shock cognate 71 kDa,
LLIEMEQRLEQGQPIDDLMPAQK from creatine kinase B-type,
LKDQS IFGDQRDEEEES QMK, KALDARLR from drebrin, or LQRELR from amphiphysin, or the equivalent human protein sequences thereof.
10. The method of claim 8, wherein the one or more biomarkers is a protein fragment from GFAP.
11. The method of claim 1, wherein the TBI is mild TBI (mTBI) or severe TBI (sTBI).
12. The method of claim 1, wherein the TBI is a closed head injury (CHI) or a blast-induced traumatic brain injury (bTBI).
13. The method of claim 1, wherein the subject is human.
14. The method of claim 1, wherein the biological sample is a brain sample.
15. The method of claim 1, wherein the biological sample is taken from the subject at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after a suspected traumatic episode.
16. The method of claim 1, wherein the subject is at risk of suffering from TBI.
17. The method of claim 1, wherein the citrullination level(s) of one or more biomarkers are determined by immunodetection.
18. A method of monitoring the progression of traumatic brain injury (TBI) in a subject, the method comprising a) analyzing at least two biological samples from the subject taken at different time points to determine a citruUination level(s) of one or more biomarkers in each of the at least two biological samples, and b) comparing the determined citruUination level(s) of the one or more biomarkers over time to determine if the subject's citruUination level(s) of the one or more specific biomarkers is changing over time, wherein an increase in the citruUination level(s) of the one or more biomarkers over time is indicative that the subject's risk of suffering from TBI is increasing over time.
19. The method of claim 18, wherein the one or more biomarkers is a protein or a protein fragment.
20. The method of claim 19, wherein the one or more proteins or protein fragments is selected from the group consisting of fructose-bisphosphate aldolase A, peroxiredoxin-1, cofilin-
1, glutathione S-transferase P, transgelin, dynamin-1, synapsin-2, moesin, septin-9, annexin A6, stress-induced phosphoprotein 1, syntaxin-binding protein 1, dihydropyrimidase-related protein
2, alpha-enolase, gamma-enolase, glial fibrillary acidic protein, fructose-bisphosphate aldolase C, neuronal- specific septin-3, transgelin-3, myelin basic protein, heat shock 70 kDa protein 4, GRIP 1- associated protein 1, transitional endoplasmic reticulum ATPase, NADH-ubiquinone oxidoreductase 75 kDa mitochondrial subunit, vesicle-fusing ATPase, 78 kDa glucose-regulated protein, heat shock cognate 71 kDa protein, annexin A6, syntaxin-binding protein 1, actin, cytoplasmic 1, creatine kinase B-type, tubulin beta-2B chain, tubulin beta-2A chain, tubulin beta- 4B chain, tubulin beta-5 chain, tubulin beta-3 chain, amphiphysin, and drebrin.
21. The method of claim 20, wherein the one or more proteins or protein fragments is selected from the group consisting of amphiphysin, cofilin-1, dynamin-1, synapsin-2, septin-9, annexin A6, stress-induced-phosphoprotien-1, dihydropyrimidase-related protein 2, neuronal- specific septin-3, transgelin-3, GRIP 1 -associated protein 1, transitional endoplasmic reticulum ATPase, vesicle-fusing ATPase, 78 kDa glucose-regulated protein, syntaxin-binding protein 1, and drebin.
22. The method of claim 18, wherein the one or more biomarkers is glial fibrillary acidic protein (GFAP) or a protein fragment of GFAP.
23. The method of claim 19, wherein the one or more proteins or protein fragments is selected from the group consisting of fructose-bisphopshate aldolase A, dynamin-1, moesin, synapsin-2, septin-9, myelin basic protein, transitional endoplasmic reticulum ATPase, NADH- ubiqiunone oxidoreductase 75kDa subunit, heat shock cognate 71 kDa, creatine kinase B-type, drebrin, amphiphysin and GFAP.
24. The method of claim 23, wherein the one or more proteins or protein fragments comprises citrulination sites in LQS IGTENTEENR and RALANSLACQGK from fructose- bisphopshate aldolase A, GMEDLJPLVNR, SGQASPSRPESPRPPFDL and
ASETEENGSDSFMHSMDPQLER from dynamin-1, ERQEAEEAKEALLQASR from moesin, YDIRVQK from synapsin-2, R VETP AS KAPEGS AMP VTD A APK from septin-9,
FFSGDRGAPK from myelin basic protein, ETVVEVPQVTWEDIGGLEDVKR from transitional endoplasmic reticulum ATPase, IAS Q V A ALDLG YKPG VE AIRK and
VALIGSPVDLTYRYDHLGDSPK from NADH-ubiqiunone oxidoreductase 75kDa subunit, VEIIANDQGNRTTPSYVAFTDTER, from heat shock cognate 71 kDa,
LLIEMEQRLEQGQPIDDLMPAQK from creatine kinase B-type,
LKDQS IFGDQRDEEEES QMK, KALDARLR from drebrin, or LQRELR from amphiphysin, or the equivalent human protein sequences thereof.
25. The method of claim 23, wherein the one or more biomarkers is a protein fragment from GFAP.
26. The method of claim 18, wherein the TBI is mild TBI (mTBI) or severe TBI (sTBI).
27. The method of claim 18, wherein the TBI is a closed head injury (CHI) or a blast-induced traumatic brain injury (bTBI).
28. The method of claim 18, wherein the subject is human.
29. The method of claim 18, wherein the biological samples are brain samples.
30. The method of claim 18, wherein at least one of the biological samples is taken from the subject at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after a suspected traumatic episode.
31. The method of claim 18, wherein the subject is at risk of suffering from TBI.
32. The method of claim 18, wherein the citrullination level(s) of one or more biomarkers are determined by immunodetection.
33. A method of diagnosing traumatic brain injury (TBI) in a subject, the method comprising a) determining the carbonylation level(s) of one or more biomarkers in a biological sample taken from the subject, and b) comparing the determined carbonylation level(s) of the one or more biomarkers against a carbonylation level(s) of the same one or more biomarkers from a control subject determined not to be suffering from TBI, wherein an increase in the carbonnylation level(s) of the one or more biomarkers compared to the carbonylation level(s) of the one or more biomarkers from the control subject is indicative that the subject may be suffering from TBI.
34. The method of claim 33, wherein the one or more biomarkers is a protein or a protein fragment.
35. The method of claim 34, wherein the one or more proteins or protein fragments is selected from the group consisting of GFAP, CRMP2, ALDOA and ALDOC.
36. The method of claim 35, wherein two of any of the proteins are determined, three of any of the proteins are determined or four of any of the proteins are determined.
37. The method of claim 33, wherein the biological sample is selected from the group consisting of blood, blood plasma, blood serum, cerebrospinal fluid (CSF) urine, lymph, saliva and combinations thereof.
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