WO2020123884A1

WO2020123884A1 - Neuron-derived exosomes and their biomarkers for the diagnosis, prognosis, and treatment of traumatic brain injury and alzheimer's disease

Info

Publication number: WO2020123884A1
Application number: PCT/US2019/066110
Authority: WO
Inventors: Edward J. GOETZL
Original assignee: Goetzl Edward J
Priority date: 2018-12-13
Filing date: 2019-12-13
Publication date: 2020-06-18

Abstract

The present invention relates to plasma neuron-derived exosomal protein biomarkers and diagnostic and prognostic methods for traum atic brain injury (TBI) and neuropathogenicafly related neurodegenerative diseases, such as AD. The invention also provides compositions for detecting neuron-derived exosomes and neuron-derived exosomal protein biomarkers as well as compositions and methods useful for treating acute TBI, chronic repetitive TBI by preventing progression to chronic traumatic encephalopathy (CTE), and Alzheimer' s disease (AD).

Description

NEURON-DERIVED EXOSOMES AND THEIR BIOMARKERS FOR TEE DIAGNOSIS, PROGNOSIS, AND TREATMENT OF TRAUMATIC BRAIN INJURY AND ALZHEIMER’S

DISEASE

RELATED APPLICATIONS

[0001] This application claims priority to the U.S. Provisional Patent Application Serial No. 62/779,427, filed on December 13, 2018, and U.S. Provisional Patent Application Serial No. 62/864,442, filed on lime 20, 2019, which are hereby incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to neuron-derived exosomes and neuron-derived exosoma! protein biomarkers and diagnostic and prognostic methods for traumatic brain injury' (TBI) and Alzheimer’s Disease (AD). The invention also provides compositions for detecting neuron-derived exosomes and neuron-derived exosoma! protein biomarkers as well as compositions and methods useful for treating TBI, preventing and/or treating chronic traumatic encephalopathy (CTE), and preventing and/or treating early and advancing AD.

BACKGROUND OF THE INVENTION

[0003] Mechanical injury to the parenchymal tissues and meninges of the brain, and the subsequent series of further damaging proteolytic, oxidati ve, inflammatory and proteinopathic responses are collectively termed traumatic brain injury (TBI) (Langlois, J. A. et al. (2006) J Head Trauma Rehab il 21, 375-378; Roozenbeek,

B. et al. (2013) Nat Rev Neurol 9, 231-236: and Quillinan, N. et al. (2016) Anesthesiol Clin 34, 453-464). Our understanding of the injurious components and restorative mechanisms of both acute TBI resulting from a single concussi ve blow and the mechanism s of chronic traumatic encephalopathy (CTE) following repeated episodes of acute TBI is incomplete and based largely on results of studies of isolated cellular and rodent models.

[0004] Insidious onset of the persistent features of CTE may appear after a single episode of moderately severe acute TBI or be imposed on a repetitive series of episodes of mild acute TBI. These include some of the elements of proteinopathic neurodegeneration typical of Alzheimer disease (AD) and other senile dem entias, although an increased incidence of late-onset AD has been seen only following single-incident severe TBI (DeKosky, S. T. et al. (2013) Nat Rev Neurol 9, 192-200). Recent data from analyses of outcome in over 300,000 deployed military combatants showed a very' significant incidence of AD dementia after mild TBI as well (Bames DE et al. (2018) JAMA Neurol. 75(9): 1055-61). Although the bridges linking acute TBI to CTE are poorly understood, a few' mechanisms have been recognized. Within 24 hr of acute TBI in rats, hippocampal and cortical brain tissues showed increases in Abeta peptide-generating BACE-1 inRNA and protein, but these returned to normal levels within 7 days (Blasko, I. et al. (2004) J Neural Transm (Vienna) 111, 523-536). In humans suffering single-incident severe TBI, increases in brain tissue levels of amyloid precursor protein and Ab peptides as well as diffuse Ab plaques are found within hours (DeKosky, S. T. et al. (2013) Nat Rev Neurol 9, 192-200). In contrast, the predominant early neuropathology in repetitive mild TBI is patchy distribution of neurofibrillary tangles of tau and neuropil threads of tau throughout the neocortex (DeKosky, S. T. et al. (2013) Nat Rev Neurol 9, 192-200). Plasma levels of tau-positive exosomes were abnormally elevated in former professional football players and these increases correlated with decreases in memory and psychomotor speed (Stem, R. A. et al. (2016) J Alzheimers Dis 51, 1099-1109). Although many abnormalities of cognition and affect or mood are similar in subjects suffering from CTE after acute severe TBI or repetitive mild TBI, relationships between these neurocognitive abnormalities and differing proteinopathic etiologic mechanisms have not been delineated (Prince, C. et al. (2017) Brain Sci 7; Roy, D., et al. (2017) J Neuropsychiatry Clin Neurosci 29, 334-342; and Dailey, N. S. et al. (2018) Front Behav Neurosci 12, 118).

[0005] Investigations of a wide range of neurally-derived proteins in CSF and plasma have not yielded biomarkers that reliably assess the severity of TBI, predict progression of TBI to CTE, or offer useful targets for therapy (DeKosky', S. T. et al. (2013) Nat Rev Neurol 9, 192-200; Agoston, D. V. et al. (2017) Brain Inj 31, 1195-1203; Werhane, M. L. et al. (2017) Concussion 2, CNC30; Kim, H. J. et al. (2018) JCI Insight 3). Thus, there is a need in the art for biomarkers and methods useful for diagnosing and prognosing traumatic brain injury. Methods for detecting neuron-derived exosomal biomarkers associated with pathogenesis of neurological diseases, such as, for example chronic traumatic encephalopathy (CTE) are needed. Additionally, there is a need in the art for compositions for detecting biomarkers as well as compositions and methods useful for treating TBI or preventing CTE and other neurological diseases, such as AD that share neuropathological features with CTE. The present invention meets this need by providing accurate, noninvasive methods for detecting biomarkers that are diagnostic and prognostic for traumatic brain injury and AD. The present invention further provides novel methods, assays, kits, and compositions for diagnosing, prognosing, predicting, preventing and treating traumatic brain injury, CTE and pathogenically related neurodegenerative diseases such as AD.

[0006] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

[0007] The present invention is based on the discovery of altered levels of neuron-derived exosomes and their biomarkers that can be used in the diagnosis, prognosis, and treatment of traumatic brain injury, CTE and AD. These biomarkers can be used alone or in combination with one or more additional biomarkers or relevant clinical parameters in prognosis, diagnosis, or monitoring treatment of traumatic brain injury, chronic traumatic encephalopathy, and Alzheimer’s disease.

[0008] Biomarkers that can be used in the practice of the invention include, but are not limited to, plasma levels of neuron-derived exosomes and their cargo proteins ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium- potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NIL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and

phosphorylated tau (e.g., P-T181-tau, P-S199-tau and P-S396-tau).

[0009] In some embodiments, the present invention provides a method comprising: a) providing a biological sample comprising neuron-derived exosomes from a subject; and b) detecting the presence of one or more biomarkers in the sample, wherein the one or more biomarkers are selected from the group consisting of ras- related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NIL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau. In some embodiments the phosphorylated tau is P-T181-tau and/or P-S396-tau.

[0010] In other embodiments, the present invention provides a method comprising: a) providing a biological sample comprising neuron-derived exosomes from a subject; b) enriching the sample for neuron-derived exosomes; and c) detecting the presence of one or more biomarkers in the sample, wherein the one or more biomarkers are selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium -potassium- chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau. In some embodiments the phosphorylated tau is P-T181-tau and/or P-S396-tau. In some embodiments, the methods of the present invention further comprise determining the level or concentration of one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium- chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau in tire sample. In some embodiments the phosphorylated tau is P-T181-tau and/or P-S396-tau. In some embodiments, the subject has traumatic brain injury (TBI). In some embodiments, the traumatic brain injury is acute TBI or chronic repetitive TBI. In some embodiments, tire TBI is mild, moderate or severe. In some embodiments the subject has Alzheimer’s disease (AD).

[0011] In some embodiments, tire present invention provides a method of detecting markers in a biological sample, the method comprising: a) providing a biological sample comprising neuron-derived exosomes from a subject; b) isolating neuron-derived exosomes from the biological sample; and c) detecting the presence of one or more biomarkers selected from die group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium- chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau from the exosomes. In some embodiments, the subject has traumatic brain injury (TBI). In some embodiments, the traumatic brain injury is acute TBI or chronic repetitive TBI. In some embedments the subject has Alzheimer’s disease (AD).

[0012] In other embodiments, the present invention provides a method of detecting markers in a biological sample, the method comprising: a) providing; i) a biological sample comprising neuron-derived exosomes from a subject and ii) immunoassay reagents for the detection of one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter-1 (NKCC- 1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL- 6, prion cellular protein (PrPc), tau, Ab42, and phosphorylated tau; and b) detecting the presence of one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium- chloride co-transporter-1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau in die sample using said reagents.

[0013] In other embodiments, the present invention provides a method of detecting markers in a biological sample, the method comprising: a) providing; i) a biological sample comprising neuron-derived exosomes from a subject and ii) immunoassay reagents for detection of one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH- Ll), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau; b) isolating neuron-derived exosomes from tiie biological sample and c) detecting the presence of one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH- Ll), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau in the exosomes using said reagents. In some embodiments, the subject has traumatic brain injury (TBI). In some embedments, the traumatic brain injury is acute TBI or chronic repetitive TBI. In some embodiments the subject has Alzheimer’s disease (AD). [0014] In some embodiments, the reagents comprise antibodies for performing an immunoassay. In some embodiments, the immunoassay is selected from the group consisting of an ELISA, radio-immunoassay, automated immunoassay, cytometric bead assay, and immunoprecipitation assay. In other embodiments, the biological sample can be any bodily fluid comprising neuron-derived exosomes, including, but not limited to, whole blood, plasma, serum, lymph, amniotic fluid, urine, saliva, and umbilical cord blood. In some embodiments, the marker is a full-size marker. In other embodiments said marker is a fragment of the full-size marker. In other embodiments, the detecting the presence of the marker in the biological sample comprises detecting the amount of the marker in the biological sample. In some embodiments, the method further comprises the step of determining a treatment course of action based on the detection of the marker or the diagnosis of a traumatic brain disease, CTE, or Alzheimer’s disease. In some embodiments, the traumatic brain injury is acute TBI or chronic repetitive TBI.

[0015] In some embodiments, isolating neuron-derived exosomes from the biological sample comprises: contacting the biological sample with an agent under conditions wherein a neuron-derived exosome present in the biological sample binds to the agent to form a neuron-derived exosome-agent complex; and isolating the neuron-derived exosome from the neuron-derived exosome-agent complex to obtain a sample containing the neuron-derived exosome, wherein the purity of the neuron-derived exosomes present in said sample is greater than the purity of the neuron-derived exosomes present in said biological sample. The agent may be an antibody that specifically binds to a neuron-derived exosome surface marker. In some aspects of the present embedment, the contacting comprises incubating or reacting. Example 1 describes isolation of neuron- derived exosomes from a biological sample, for example, by immunoabsorption using an anti-human CD171 biotinylated antibody specific for this surface protein. In certain embedments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid.

[0016] Biomarker proteins can be measured, for example, by performing immunohistochemistiy, im munocytochemi stry , immunofluorescence, immunoprecipitation, Western blotting, or an enzyme-linked immunosorbent assay (ELISA). In certain embodiments, the level of a biomarker is measured with an immunoassay. For example, the level of the biomarker can be measured by contacting an antibody with the biomarker, wherein the antibody specifically binds to the biomarker, or a fragment thereof containing an antigenic determinant of the biomarker. Antibodies that can be used in tire practice of the invention include, but are not limited to, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, recombinant fragments of antibodies, Fab fragments, Fab' fragments, F(ab')₂ fragments, F_v fragments, or scF_v fragments. In one embedment, the method comprises measuring amounts of an in vitro complex comprising a labeled antibody bound to a neuron-derived exosome biomarker. In one aspect, the neuron-derived exosome biomarker is selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium- chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphoiylated tau. In some embodiments, abnormal levels of the biomarker of r as-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium- potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and/or phosphoiylated tau compared to reference value ranges of the biomarkers for a control subject indicate that the subject has a traumatic brain injury or is at-risk of developing a traumatic brain injury. In some aspects, the control subject is a subject without a current or past brain injury. In some embodiments, decreased plasma levels of neuron-derived exosomes acutely and of the biomarkers ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium -chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neuro filament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and/or phosphoiylated tau compared to reference value ranges of the biomarkers for a control subject indicate that tire subject has a traumatic brain injury or is at-risk of developing chronic traumatic encephalopathy. In some aspects, the control subject is a subject without a traumatic brain injury. In some embodiments, tire one or more biomarkers comprises of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co- transporter-1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and/or phosphoiylated tau. In some embedments, abnormal levels of the biomarker of ras-related small GTPase 10 (RABIO), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium- chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and/or phosphoiylated tau compared to reference value ranges of tire biomarkers for a control subject indicate that the subject has a Alzheimer’s disease or is at-risk of developing Alzheimer’s disease.

[0017] The levels of the biomarkers from neuron-derived exosomes from a subject can be compared to reference value ranges for the biomarkers found in one or more samples of neuron-derived exosomes from one or more subjects without a traumatic brain injury (e.g., control sample, healthy subject without a traumatic brain injury). Alternatively, the levels of the biomarkers from neuron-derived exosomes from a subject can be compared to reference value ranges for the biomarkers found in one or more samples of neuron-derived exosomes from one or more subjects with a traumatic brain injury. In some embodiments, the subject has chronic traumatic encephalopathy or Alzheimer’s disease. In other embodiments, the traumatic brain injury is acute TBI or chronic repetitive TBI. [0018] In some embodiments, the invention provides a method for monitoring the efficacy of a therapy for treating a traumatic brain injury in a patient, the method comprising: a) providing a first biological sample comprising neuron-derived exosomes from the patient before the patient undergoes the therapy and a second biological sample comprising neuron-derived exosomes after the patient undergoes the therapy; b) isolating neuron-derived exosomes from the first biological sample and the second biological sample; and c) detecting one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Ann exin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium- potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and

phosphorylated tau from the neuron-derived exosomes from the first biological sample and the second biological sample; and d) comparing the levels of the one or more biomarkers for the neuron-derived exosomes from the first biological sample to tire levels of the one or more biomarkers for the neuron-derived exosomes from the second biological sample, wherein normalized levels of the one or more biomarkers for the neuron-derived exosomes from the second biological sample compared to the levels of the one or more biomarkers for the neuron-derived exosomes from the first biological sample indicate that tire patient is improving. In some embodiments, the one or more biomarkers comprises ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau. In some embodiments, the traumatic brain injury is acute TBI or chronic repetitive TBI. In certain embodiments, the biological sample is selected from tire group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid.

[0019] In some embodiments, tire invention provides a method for monitoring the efficacy of a therapy for treating Alzheimer’s disease in a patient, the method comprising: a) providing a first biological sample comprising neuron-derived exosomes from the patient before the patient undergoes the therapy and a second biological sample comprising neuron-derived exosomes after the patient undergoes the therapy; b) isolating neuron-derived exosomes from the first biological sample and the second biological sample; and c) detecting one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium- potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and

phosphorylated tau from the neuron-derived exosomes from the first biological sample and the second biological sample; and d) comparing the levels of the one or more biomarkers for the neuron-derived exosomes from the first biological sample to tire levels of the one or more biomarkers for the neuron-derived exosomes from the second biological sample, wherein normalized levels of the one or more biomaricers for the neuron-derived exosomes from the second biological sample compared to the levels of the one or more biomarkers for the neuron-derived exosomes from the first biological sample indicate that the patient is improving. In some embodiments, the one or more biomarkers comprises ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau. In certain embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid.

[0020] In some embodiments, the invention provides a method for monitoring the efficacy of a therapy for treating a traumatic brain injury in a patient, tire method comprising: a) providing a first biological sample comprising neuron-derived exosomes from the patient before the patient undergoes the therapy and a second biological sample comprising neuron-derived exosomes after the patient undergoes the therapy; b) isolating neuron-derived exosomes from the first biological sample and tire second biological sample; and c) detecting one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium- potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and

phosphorylated tau from the neuron-derived exosomes from the first biological sample and the second biological sample; and d) comparing the levels of the one or more biomarkers for the neuron-derived exosomes from the first biological sample to the levels of the one or more biomarkers for the neuron-derived exosomes from the second biological sample, wherein normalized levels of the one or more biomarkers for the neuron-derived exosomes from the second biological sample compared to the levels of the one or more biomarkers for the neuron-derived exosomes from the first biological sample indicate that die patient is improving. In some embodiments, the traumatic brain injury' is acute TBI or chronic repetitive TBI. In certain embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid.

[0021] In some embedments, the invention provides a method for monitoring the efficacy of a therapy for treating Alzheimer’s disease in a patient, the method comprising: a) providing a first biological sample comprising neuron-derived exosomes from the patient before the patient undergoes the therapy and a second biological sample comprising neuron-derived exosomes after the patient undergoes the therapy; b) isolating neuron-derived exosomes from the first biological sample and the second biological sample; and c) detecting one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium- potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NIL), 1L-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and

phosphorylated tau from the neuron-derived exosomes from the first biological sample and the second biological sample; and d) comparing the levels of the one or more biomarkers for the neuron-derived exosomes from the first biological sample to the levels of the one or more biomarkers for the neuron-derived exosomes from the second biological sample, wherein normalized levels of the one or more biomarkers for the neuron-derived exosomes from the second biological sample compared to the levels of the one or more biomarkers for the neuron-derived exosomes from the first biological sample indicate that the patient is improving. In certain embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid.

[0022] In yet other embodiments, the invention provides a method of treating a patient suspected of having a traumatic brain injury, the method comprising: a) detecting neuron-exosomal biomarker levels in the patient or receiving information regarding the neuron-exosomal biomarker levels of the patient, as determined according to a method described herein; and b) administering a therapeutically effective amount of at least one drug that alters neuron-exosomal biomarker levels in the subject. After treatment, the method may further comprise monitoring the response of the patient to treatment.

[0023] In yet other embodiments, the invention provides a method of treating a patient suspected of having Alzheimer’s disease, the method comprising: a) detecting neuron-exosomal biomaiker levels in the patient or receiving information regarding the neuron-exosomal biomarker levels of the patient, as determined according to a method described herein; and b) administering a therapeutically effective amount of at least one drug that alters neuron-exosomal biomarker levels in the subject. After treatment, the method may further comprise monitoring the response of the patient to treatment.

[0024] In other embodiments, the invention provides a method comprising: providing a biological sample from a subject suspected of having a traumatic brain injury or Alzheimer’s disease; detecting the presence or level of at least one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium -potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau; and administering a treatment to the subject. In one embodiment, the method further comprises administering a therapeutically effective amount of at least one drug that treats a traumatic brain injury or Alzheimer’s disease to the subject if increased levels of the one or more biomarkers are detected in the subject. In one embodiment, the method further comprises administering a therapeutically effective amount of at least one drug that treats a traumatic brain injury or Alzheimer’s disease to the subject if decreased levels of the one or more biomarkers are detected in the subject. After treatment, the method may further comprise monitoring the response of the subject to treatment. In some embodiments, the one or more biomarkers comprises r as-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C -terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC- 1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL- 6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau. In some embodiments, the traumatic brain injury is acute TBI or chronic repetitive TBI.

[0025] In other embodiments, the present invention provides a method of treating a subject with a traumatic brain injury or Alzheimer’s disease, comprising: providing a biological sample from the subject; determining the level of at least one or more biomarkers selected from die list consisting ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau using at least one reagent that specifically binds to said biomarkers; and prescribing a treatment regimen based on the level of the one or more biomarkers. In some embodiments, the method further comprises isolating neuron-derived exosomes from the biological sample. In some embodiments, the traumatic brain injury is acute TBI or chronic repetitive TBI. In other embodiments, the traumatic brain injury is mild, moderate, or severe.

[0026] In some embedments, the invention provides a set of biomarkers for assessing traumatic brain injury or Alzheimer’s disease status of a subject, the set comprising one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH- Ll), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter-1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau, wherein neuron-derived exosome levels of the biomarkers in the set are assayed; and wherein the biomarker levels of die set of biomarkers determine the traumatic brain injury status of the subject with at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% specificity. In some aspects, the set of biomarkers determine the neurological disease status of the subject with at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sensitivity. In yet other aspects, the set of biomarkers determine the traumatic brain injury status of the subject with at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% accuracy. In some embedments, the one or more biomarkers comprises ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium- chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau. [0027] In other embodiments, the invention provides a composition comprising at least one in vitro complex comprising a labeled antibody bound to a biomarker protein selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), AP spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphoiylated tau, wherein said biomarker protein is extracted from neuron- derived exosomes of a subject who has been diagnosed with a traumatic brain injury or Alzheimer’s disease, suspected of having a traumatic brain injury or Alzheimer’s disease, or at risk of developing a traumatic brain injury or Alzheimer’s disease. The antibody may be detectably labeled with any type of label, including, but not limited to, a fluorescent label, an enzyme label, a chemiluminescent label, or an isotopic label. In some embodiments, the composition is in a detection device (i.e., device capable of detecting labeled antibody). In some embodiments, the one or more biomarkers comprises ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium- potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and

phosphorylated tau. In some embodiments, the traumatic brain injury is acute TBI or chronic repetitive TBI. In other embodiments, the traumatic brain injury is mild, moderate, or severe.

[0028] In other embodiments, the invention provides a kit for detecting or monitoring a traumatic brain injury or Alzheimer’s disease in a subject. In some embodiments, the kit may include a container for holding a biological sample isolated from a subject who has been diagnosed or suspected of having a traumatic brain injury or Alzheimer’s disease or at risk of developing a traumatic brain injury- or Alzheimer’s disease, at least one agent that specifically detects a biomarker of the present invention; and printed instructions for reacting the agent with neuron-derived exosomes from the biological sample or a portion of the biological sample to detect the presence or amount of at least one biomarker. In other embedments, the kit may also comprise one or more agents that specifically bind neuron-derived exosomes for use in isolating neuron-derived exosomes from a biological sample. In yet other embodiments, the kit may further comprise one or more control reference samples and reagents for performing an immunoassay. In certain embodiments, the agents may be packaged in separate containers. In some embodiments, the kit comprises agents for measuring the levels of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and/or phosphorylated tau. In yet other embedments, the kit further comprises an antibody that binds to a neuron-derived exosome marker, such as, for example, CD 171 (LI CAM neural adhesion protein). [0029] In other embodiments, the invention provides a method for treating a traumatic brain injury, the method comprising the steps of: providing a biological sample from a subject suspected of having a traumatic brain injury, wherein the sample comprises neuron-derived exosomes; measuring the level of one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium- chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), 1L-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau from the biological sample, wherein an altered level of the one or more biomarkers in the sample relative to the level in a control sample is indicative of a need for treatment; and administering an effective amount of an agent to the subject thereby treating the traumatic brain injury in the subject. In some embodiments, the agent is a diuretic agent, an anti-anxiety agent, an anticoagulant agent, an anticonvulsant agent, and antidepressant agent, a muscle relaxant agent, a stimulant agent, an anti-seizure agent, or a coma-inducing agent.

[0030] In other embodiments, the invention provides methods for identifying agents, devices, or protocols for treating a traumatic brain injury or preventing progression of a traumatic brain injury (TBI) to chronic traumatic encephalopathy (CTE), tire method comprising: a) providing a first biological sample comprising neuron-derived exosomes from a subject before the subject undergoes treatment with the agent, device, or protocol and a second biological sample from the subject comprising neuron-derived exosomes after the subject undergoes treatment with the agent, device, or protocol; b) isolating neuron-derived exosomes from the first biological sample and the second biological sample; and c) detecting one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co- transporter-1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau from the neuron-derived exosomes from the first biological sample and the second biological sample; and d) comparing the levels of tire one or more biomarkers from the neuron-derived exosomes from the first biological sample to the levels of the one or more biomarkers for the neuron-derived exosomes from the second biological sample, wherein normalized levels of the one or more biomarkers form the neuron-derived exosomes from the second biological sample compared to the levels of the one or more biomarkers from the neuron-derived exosomes from tire first biological sample indicate that the agent, device, or protocol is effective for treating traumatic brain injury or preventing progression of a traumatic brain injury (TBI) to chronic traumatic encephalopathy (CTE), and abnormal levels of the one or more biomarkers from the neuron-derived exosomes from the second biological sample compared to the levels in a control sample indicate that the agent, device, or protocol is not effective at treating a traumatic brain injury or preventing the progression of a traumatic brain injury to chronic traumatic encephalopathy. In some embodiments, tire device is a helmet, padded headgear, a collar, a faceguard, a brace, padding, or a mouth guard. In other embodiments, the agent is acetaminophen, ibuprofen, aspirin, a neuro-steroid, or an antidepressant.

[0031] In other embodiments, the invention provides methods for identifying agents, devices, or protocols for treating Alzheimer’s disease or preventing progression of Alzheimer’s disease, the method comprising: a) providing a first biological sample comprising neuron-derived exosomes from a subject before the subject undergoes treatment with the agent, device, or protocol and a second biological sample from the subject comprising neuron-derived exosomes after the subject undergoes treatment with the agent, device, or protocol; b) isolating neuron-derived exosomes from the first biological sample and the second biological sample; and c) detecting one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C -terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium -potassium-chloride co-transporter-1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau from the neuron-derived exosomes from the first biological sample and the second biological sample; and d) comparing the levels of the one or more biomarkers from the neuron-derived exosomes from die first biological sample to die levels of the one or more biomarkers for the neuron-derived exosomes from the second biological sample, wherein normalized levels of the one or more biomarkers form the neuron-derived exosomes from the second biological sample compared to the levels of the one or more biomarkers from the neuron-derived exosomes from the first biological sample indicate that the agent, device, or protocol is effective for treating Alzheimer’s disease or preventing Alzheimer’s disease, and abnormal levels of the one or more biomarkers from the neuron-derived exosomes from die second biological sample compared to the levels in a control sample indicate that the agent, device, or protocol is not effective at treating Alzheimer’s disease or preventing Alzheimer’s disease.

[0032] In other embodiments, the invention provides methods for identifying agents, devices, or protocols for treating a traumatic brain injury or preventing progression of a traumatic brain injury (TBI) to chronic traumatic encephalopathy (CTE), die method comprising: a) providing a first biological sample comprising neuron-derived exosomes from a subject before the subject suffers a traumatic brain injury and a second biological sample from the subject comprising neuron-derived exosomes after the subject suffers a traumatic brain injury and undergoes treatment with the agent, device, or protocol; b) isolating neuron-derived exosomes from the first biological sample and the second biological sample; and c) detecting one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C- terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride cotransporter-1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau from the neuron-derived exosomes from the first biological sample and the second biological sample; and d) comparing the levels of the one or more biomarkers from the neuron-derived exosomes from the first biological sample to the levels of the one or more biomarkers for die neuron-derived exosomes from the second biological sample, wherein normalized levels of the one or more biomarkers form the neuron-derived exosomes from the second biological sample compared to the levels of the one or more biomarkers from the neuron-derived exosomes from the first biological sample indicate that the agent, device, or protocol is effective for treating traumatic brain injury or preventing progression of a traumatic brain injury (TBI) to chronic traumatic encephalopathy (CTE), and abnormal levels of the one or more biomarkers from the neuron-derived exosomes from the second biological sample compared to the levels in the first biological sample indicate that the agent, device, or protocol is not effective at treating a traumatic brain injury or preventing the progression of a traumatic brain injury to chronic traumatic encephalopathy. In some embedments, the device is a helmet, padded headgear, a collar, a faceguard, a brace, padding, or a mouth guard. In other embedments, the agent is acetaminophen, ibuprofen, aspirin, a neuro-steroid, or an antidepressant.

[0033] In other embodiments, the invention provides methods for treating a subject having a traumatic brain injury, comprising, administering an effective amount of a PrPc inhibitor to the subject, thereby treating the traumatic brain injury in the subject. In some embodiments, the PrPc inhibitor is an antibody. In other embodiments, the antibody is a monoclonal antibody. In still other embodiments, more than one PrPc inhibitor is administered to the subject. In certain embedments, two or more anti-PrPc antibodies are administered to the subject.

[0034] In other embodiments, the invention provides methods for treating a subject having Alzheimer’s disease, comprising, administering an effective amount of a PrPc inhibitor to the subject, thereby treating the Alzheimer’s disease in the subject. In some embodiments, the PrPc inhibitor is an antibody. In other embedments, the antibody is a monoclonal antibody. In still other embedments, more than one PrPc inhibitor is administered to the subject. In certain embedments, two or more anti-PrPc antibodies are administered to the subject.

[0035] In other embodiments, the invention provides methods for treating a subject having a traumatic brain injury, comprising, administering an effective amount of a synaptogyrin-3 inhibitor to the subject, thereby treating the traumatic brain injury in the subject. In some embodiments, the synaptogyrin-3 inhibitor is an antibody. In other embodiments, the antibody is a monoclonal antibody. In still other embodiments, more than one synaptogyrin-3 inhibitor is administered to the subject. In certain embodiments, two or more anti- synaptogyrin-3 antibodies are administered to the subject.

[0036] In other embodiments, the invention provides methods for treating a subject having Alzheimer’s disease, comprising, administering an effective amount of a synaptogyrin-3 inhibitor to the subject, thereby treating the Alzheimer’s disease in the subject. In some embodiments, the synaptogyrin-3 inhibitor is an antibody. In other embodiments, the antibody is a monoclonal antibody. In still other embodiments, more than one synaptogyrin-3 inhibitor is administered to the subject. In certain embodiments, two or more anti- synaptogyrin-3 antibodies are administered to the subject. [0037] In other embodiments, the invention provides methods for treating a subject having a traumatic brain injury, comprising administering an effective amount of a composition that downregulates the expression of PrPc and/or synaptogyrin-3 in the subject, thereby treating the traumatic brain injury in the subject.

[0038] In other embodiments, the invention provides methods for treating a subject having Alzheimer’s disease, comprising administering an effective amount of a composition that downregulates the expression of PrPc and/or synaptogyrin-3 in the subject, thereby treating the Alzheimer’s disease in the subject.

[0039] In some embodiments, the invention provides methods for prognosing traumatic brain injury or Alzheimer’s disease in a subject, the method comprising the steps of: obtaining the level of at least one biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium- chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), 1L-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau from a biological sample comprising neuron-derived exosomes; and prognosing the traumatic brain injury or Alzheimer’s disease based on the levels of the at least one biomarkers in the sample.

[0040] In other embodiments, tire invention provides methods of screening to determine whether a test compound can inhibit or reduce Abeta42 and/or P-tau binding activity of neuronal prion protein (PrPc) and/or synaptogyrin-3, said method comprising: providing a reaction mixture that comprises a preparation comprising Abeta42 and/or P-tau, neuronal prion protein (PrPc) and/or synaptogyrin-3, and a plurality of test compounds; incubating said reaction mixture under conditions which, in the absence of an inhibitor of said binding activity, allow said Abeta42 and/or P-tau to bind to neuronal prion protein (PrPc) and/or synaptogyrin- 3; and determining whether said binding activity is inhibited or reduced.

[0041] These and other embedments of the present invention will readily occur to those of skill in the art in light of the disclosure herein, and all such embodiments are specifically contemplated.

INCORPORATION BY REFERENCE

[0042] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figures 1 A-l J set forth data showing levels of NDE physiological cargo proteins in cross-sectional control, acute TBI and chronic TBI groups. Each point represents the value for a control or TBI participant and the horizontal line in point clusters is the mean level for that group. Mean+S.E.M. control, acute TBI and chronic TBI participant values, respectively, are 2994+237 pg/ml, 1654+166 pg/ml and 2634+225 for C81 (A), 2012±159 pg/ml, 539±126 pg/ml and 2380±244 pg/ml for RAB10 (B), 7824±1374 pg/ml, 69,133±7,691 pg/ml and 13,167+2,828 pg/ml for annexin VII (C), 71,740+4,755 pg/ml, 176,892+12,764 pg/ml and

79,628+9,894 pg/ml for UCH-L1 (D), 21,194+2214 pg/ml, 40,794+4281 pg/ml and 17,783±2641 pg/ml for all spectrin breakdown products (E), 1255+135 pg/ml, 3328+341 pg/ml and 1087+102 pg/ml for claudin-5 (F), 322+51.3 pg/ml, 689+151 pg/ml and 405+95.4 pg/ml for occludin (G), 2319+204 pg/ml, 6428+529 pg/ml and 3040+422 pg/ml for NKCC-1 (H), 281+35.3 pg/ml, 2507+230 pg/ml and 1000+217 pg/ml for aquaporin 4 (I), and 399+23.2 pg/ml, 1250+183 pg/ml, and 530+67.5 pg/ml for synaptogyrin-3 (J). The significance of differences between values for controls and AD patients was calculated by an unpaired Student’s t test;

[0044] Figures 2A-2F set forth data showing levels of NDE proteinopathic cargo proteins in cross-sectional control, acute mTBI and chronic mTBI groups. Each point represents the value for a control or mTBI participant and the horizontal line in point clusters is the mean level for that group. Mean+S.E.M. control, acute mTBI and chronic mTBI participant values, respectively, are 3561+378 pg/ml, 5260+708 pg/ml and 57354952 for total tau (A), 39.4+2.93 pg/ml, 61.1+7.10 pg/ml and 64.2+4.87 for Ab42 (B), 150+11.3 pg/ml, 257+25.4 pg/ml and 327+25.1 for P-T181-tau (C), 77.4+7.95 pg/ml, 91.5+7.25 pg/ml and 124+13.4 for P- S396-tau (D), 8.43+1.93 pg/ml, 763+168 pg/ml and 132+40.6 pg/ml for IL-6 (E), and 409+31.2 pg/ml, 2364+187 pg/ml and 2081+165 pg/ml for PRPc (F). The significance of differences shown between values for controls and acute mTBI subjects and between controls and chronic mTBI subjects were calculated by an unpaired

[0045] Figures 3 A-3E set forth data showing NDE levels of normal functional cargo proteins in cross- sectional control, cognitive impairment (Cl) with no history of TB1, acute mTBI without Cl and acute mTBI with Cl groups. Each point represents the value for one participant and the horizontal line in point clusters is the mean level for that group. Mean+S.E.M. control, Cl w/o TBI, mTBI w/o Cl and mTBI with Cl, respectively, are 1711+101 pg/ml, 1474+154 pg/ml, 1595+139 pg/ml and 1505+145 pg/ml for claudin-5 (A), 47256+3249 pg/ml, 45274+3882 pg/ml, 35831+3503 pg/ml and 47087+3486 pg/ml for annexin VII (B), 4787+397 pg/ml, 3029±251 pg/ml, 3623+408 pg/ml and 3522+241 pg/ml for aquaporin 4 (C), 79864922 pg/ml, 17149+1207 pg/ml, 6949±702 pg/ml and 17730+995 pg/ml for PrPc (D), and 81.7+11.0 pg/ml, 145+19.7 pg/ml, 64.3+9.14 pg/ml, and 142+13.3 pg/ml for synaptogyrin-3 (E). The significance of differences shown between values for controls and Cl w/o TBI subjects and between values for mTBI w/o Cl and mTBI with Cl subjects, respectively, were calculated by an unpaired Student’s t test; *=p<0.01, **=p<0.001.

[0046] Figures 4A-4D set forth data showing NDE levels of putatively proteinopathic cargo proteins in cross- sectional control, cognitive impairment (Cl) with no history of TBI, acute mTBI without Cl and acute mTBI with Cl groups. Each point represents the value for one participant and the horizontal line in point clusters is the mean level for that group. Mean+S.E.M. control, Cl wVo TBI, mTBI w/o Cl and mTBI with Cl, respectively, are 85.2+6.67 pg/ml, 156+17.3 pg/ml, 63.5+6.70 pg/ml and 173+14.6 pg/ml for P-T181-tau (A), 93.5±10.0 pg/ml, 97.7±12.4 pg/ml, 44.7±3.92 pg/ml and 151±15.4 pg/ml for P-S396-tau (B), 7.49±1.16 pg/ml, 31.5+6.64 pg/ml, 11.3+1.83 pg/ml and 57.5+12.1pg/ml for Ab42 (C), and 44.2+8.54 pg/ml, 59.7+8.21 pg/ml, 43.2+11.2 pg/ml and 117+22.2 pg/ml for IL-6 (D). The significance of differences shown between values for controls and Cl w/o TBI subjects and between values for mTBI w/o Cl and mTBI with Cl subjects, respectively, were calculated by an unpaired Student’s t test; *=p<0.01, **=p<0.001.

[0047] Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of“including,”“comprising,” or“having,”“containing”,“involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. It must be noted that as used herein and in the appended claims, the singular forms“a, ?9 <6 an,” and“the” include plural references unless context clearly dictates otherwise. Thus, for example, a reference to“a fragment” includes a plurality of such fragments, a reference to an“antibody” is a reference to one or more antibodies and to equivalents thereof known to those skilled in the art, and so forth.

DESCRIPTION OF THE INVENTION

[0048] It is to be understood that the invention is not limited to the particular methodologies, protocols, cell lines, assays, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is intended to describe particular embodiments of the present invention, and is in no way intended to limit die scope of the present invention as set forth in the appended claims.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0050] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology-, cell biology, genetics, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Gennaro, A.R, ed. (1990) Remington’s Phamaceutical Sciences, 18th ed., Mack Publishing Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell Scientific Publications); Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag).

[0051] The present invention relates, in part, to the discovery that neuron-derived exosomal biomarkers can be used for the diagnosis and prognosis of traumatic brain injury. The inventor has demonstrated that neuron- derived exosome (NDE) levels were significantly reduced in subjects having acute TBI, but not chronic TBI, compared to those in control subjects. NDE levels of functional neural proteins were abnormal relative to those of control subjects in acute but not chronic TBI, including r as-related small GTPase 10 (RAB10), annexin VII, ubiquitin C-lerminal hydrolase LI (UCH-L1), AP spectrin fragments, claudin-5, sodium- potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, and prion cellular protein (PrPc). In chronic TBI, there are elevated NDE levels of Ab40, Ab42, phosphorylated tau, including P-T181-tau and P-S396-tau. Abnormalities in NDE levels of neuron-derived exosomal proteins in acute TBI, but not chronic TBI, and greater elevations of some neuropathological proteins in chronic TBI than acute TBI delineate phase-specific mechanisms.

[0052] The inventor has identified various NDE proteins that act as progression factors of neurological disease which may contribute to the initiation and progression of chronic traumatic encephalopathy (CTE). As disclosed herein, abnormal levels of progression factor NDE proteins are found in acute and chronic TBI, as they also had been in early AD. The progression factor proteins generally have the capacity to bind, concentrate and deliver to neurons one or more primary neurotoxic proteins, such as Ab40, Ab42 or phosphorylated tau. Progression factor proteins include prion cellular protein (PrPc) binds Ab40, Ab42, synaptogyrin-3 binds all tau forms, IL-6 for inflammation, and aquaporin 1 and 4 for edema.

[0053] The present invention also provides compositions for use in the methods described herein. Such compositions may include small molecule compounds; peptides and proteins including antibodies or functionally active fragments thereof.

[0054] The present invention further provides kits for identifying a subject having a traumatic brain injury or prescribing a therapeutic regimen or predicting benefit from therapy in a subject having a traumatic brain injury or at risk of developing a neurological disease following a traumatic brain injury. In these embodiments, the kits comprise one or more antibodies which specifically bind neuron-derived exosomes, one or more antibodies which specifically bind a neuron-derived exosomal biomarker of the present invention, one or more containers for collecting and or holding the biological sample, and instructions for the kits use. [0055] The section headings are used herein for organizational purposes only, and are not to be construed as in any way limiting the subject matter described herein.

Biological Sample

[0056] The present invention provides biomarkers and diagnostic and prognostic methods for traumatic brain injury and the neurodegeneration of AD. Biomarkers are detected from neuron-derived exosomes from a biological sample obtained from a subject. A biological sample comprising exosomes may be obtained from a subject. The biological sample obtained from the subject is typically blood, but can be any sample from bodily fluids, tissue or cells comprising the vesicles to be analyzed. The biological sample may include, but is not limited to, whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cerebrospinal fluid, a cervical swab, tears, saliva, a buccal swab, skin, organs, and biopsies. Alternatively, exosomes can be obtained from cultured cells by collection of secreted exosomes from the surrounding culture media.

[0057] In some embedments, the biological sample of the invention is obtained from blood. In some embodiments, about 1-10 mL of blood is drawn from a subject. In other embedments, about 10 -50 mL of blood is drawn from a subject. Blood can be drawn from any suitable area of the body, including an arm, a leg, or blood accessible through a central venous catheter. In some embodiments, blood is collected following a treatment or activity. For example, blood can be collected following a medical exam. The timing of collection can also be coordinated to increase the number and/or composition of exosomes present in the sample. For example, blood can be collected following exercise or a treatment that induces vascular dilation.

[0058] Blood may be combined with various components following collection to preserve or prepare samples for subsequent techniques. For example, in some embodiments, blood is treated with an anticoagulant, a cell fixative, a protease inhibitor, a phosphatase inhibitor, or preservative(s) for protein or DNA or RNA following collection. In some embedments, blood is collected via venipuncture using a needle and a syringe that is emptied into collection tubes containing an anticoagulant such as EDTA, heparin, or acid citrate dextrose (ACD). Blood can also be collected using a heparin-coated syringe and hypodermic needle. Blood can also be combined with components that will be useful for cell culture. For example, in some embodiments, blood is combined with cell culture media or supplemented cell culture media (e.g., cytokines).

Enrichment or Isolation of Neuron-derived Exosomes

[0059] Samples can be enriched for neuron-derived exosomes through positive selection, negative selection, or a combination of positive and negative selection. In some embodiments, exosomes are directly captured. In other embodiments, blood cells are captured and exosomes are collected from the remaining biological sample.

[0060] Samples can also be enriched for exosomes based on the biochemical properties of exosomes. The first step is physical isolation entailing polymer precipitation with centrifugation in one or two cycles. Then, for example, samples can be enriched for exosomes based on differences in antigens. In some of the embodiments, antibody-conjugated magnetic or paramagnetic beads in magnetic field gradients or fluorescently labeled antibodies with flow cytometry are used. In some of the embodiments based on metabolic differences, dye uptake/exclusion measured by flow cytometry or another sorting technology is used. Samples can also be enriched for exosomes based on other biochemical properties known in the art. For example, samples can be enriched for exosomes using ligands or soluble receptors.

[0061] In some embodiments, surface markers are used to positively enrich neuron-derived exosomes in the sample. In other embodiments, cell surface markers that are not found on exosomes are used to negatively enrich exosomes by depleting cell populations. Modified versions of flow cytometry sorting may also be used to further enrich for neuron-derived exosomes using surface markers or intracellular or extracellular markers conjugated to fluorescent labels. Intracellular and extracellular markers may include nuclear stains or antibodies against intracellular or extracellular proteins preferentially expressed in exosomes. Cell surface markers may include cell surface antigens that are preferentially expressed on neuron-derived exosomes. In some embodiments, the cell surface marker is a neuron-derived exosome surface marker, including, for example, CD171 (LI CAM neural adhesion protein). In some embodiments, a monoclonal antibody that specifically binds to CD171 (e.g., mouse anti-human CD171 antibody) is used to enrich or isolate neuron- derived exosomes from the sample. In certain aspects, the antibody against CD 171 is biotinylated. In this embodiment, the biotinylated antibody can form an antibody-exosome complex that can be subsequently isolated using strep tavidin-agarose resin or beads. In other embodiments, the antibody is a monoclonal antihuman CD171 antibody. Cell surface markers may include antibodies against cell surface antigens that are preferentially expressed on exosomes (e.g., NCAM). In some embodiments, the cell surface marker is a neuron-derived exosome surface marker, including, for example, NCAM or CD 171. In some embodiments, a monoclonal NCAM, CD9, CD63, CD81, neuron-specific enolase or CD171 antibody is used to enrich or isolate exosomes from the sample. In certain aspects, the NCAM, CD9, CD63, CD81, neuron-specific enolase or CD171 antibody is biotinylated. In this embodiment, biotinylated NCAM or CD171 antibody can form an antibody-exosome complex that can be subsequently isolated using streptavidin-agarose resin or beads. In other embodiments, the NCAM, CD9, CD63, CD81, neuron-specific enolase or CD171 antibody is a monoclonal anti-human NCAM, CD9, CD63, CD81, neuron-specific enolase or CD171 antibody.

[0062] In other embodiments, neuron-derived exosomes are isolated or enriched from a biological sample comprising: contacting a biological sample with an agent under conditions wherein a neuron-derived exosome present in said biological sample binds to said agent to form a neuron-agent complex; and isolating said exosome from said exosome-agent complex to obtain a sample containing said exosome, wherein the purity of the exosomes present in the sample is greater than the purity of exosomes present in the biological sample. In certain embodiments, the contacting is incubating or reacting. In certain embodiments, the exosomes are neuron-derived exosomes. In certain embodiments, the agent is an antibody or a lectin. Lectins useful for forming an exosome-lectin complex are described in U.S. Patent Application Publication No. 2012/0077263. In some embodiments, multiple isolating or enriching steps are performed. In certain aspects of the present embodiment, a first isolating step is performed to isolate exosomes from a blood sample freed of plasma membrane-derived membrane vesicles and a second isolating step is performed to isolate neuron-derived exosomes from other exosomes. In other embodiments, the exosome portion of the exosome-agent complex is lysed using a lysis reagent and the protein levels of the lysed exosome are assayed. In some embodiments, the anti body -exosome complex is created on a solid phase. In yet other embodiments, the methods further comprise releasing the exosome from the antibody-exosome complex. In certain embedments, the solid phase is non-magnetic beads, magnetic beads, agarose, or sepharose. In other embodiments, the vesicle is released by exposing the antibody-exosome complex to low pH between 3.5 and 1.5. In yet other embedments, the released exosome is neutralized by adding a high pH solution. In other embodiments, the released exosomes are lysed by incubating the released exosomes with a lysis solution. In still other embodiments, tire lysis solution contains inhibitors for proteases and phosphatases.

Traumatic Brain Injury

[0063] Traumatic brain injury (TBI), also known as intracranial injury, occurs when an external force injures the brain. TBI can be classified based on severity (mild, moderate, or severe), mechanism (closed or penetrating head injury), or other features (e.g., occurring in a specific location or over a widespread area).

TBI can result in physical, cognitive, social, emotional, and behavioral symptoms, and outcome can range from complete recovery to permanent disability or death.

[0064] The present invention provides methods for detecting diagnosing or prognosing a traumatic brain injury in a subject and/or identifying a subject at risk of developing a neurological disease following a traumatic brain injury, or prescribing a preventative and/or therapeutic regimen or predicting benefit from therapy. Abnormal levels of neuron-derived exosomes and neuron-derived exosomal proteins were found in TBI patients compared to matched control subjects. Hence, reduced levels of neuron-derived exosomes acutely and abnormal levels of neuron-derived exosomal biomarkers acutely or chronically are indicative of development or worsening of effects of traumatic brain injury. Accordingly, detection of neuron-derived exosomal biomarker abnormalities can be used to identify' individuals who will benefit from therapy.

Abnormalities in NDE levels of neuro-functional proteins in acute TBI, but not chronic TBI, and greater elevations of some neuropathological proteins in chronic TBI than acute TBI delineate phase-specific mechanisms.

[0065] Subject suffering from a traumatic brain injury can progress to chronic traumatic encephalopathy (CTE). CTE is a degenerative brain disease found in subjects with a history of repetitive brain trauma or head injuries. CTE is associated with the development of other neurological diseases, including for example, dementia. Features of CTE may appear after a single episode of moderately severe acute TBI or be imposed on a repetitive series of episodes of mild acute TBI. These include some of the elements of proteinopathic neurodegeneration typical of Alzheimer disease (AD) and other senile dementias, although an increased incidence of late-onset AD has been established only following single-incident TBI of any degree of severity (Barnes DE et al. (2018) JAMA Neurol. 75(9): 1055-61).

[0066] Neuron-derived exosomal biomarkers of the present invention can be used in methods for the prognosis and diagnosis of traumatic brain injury and other neurological diseases, including AD. As shown in Example 1, neuron-derived exosome (NDE) levels were significantly reduced in subjects with acute TBI, but not chronic TBI, compared to those in control subjects. NDE levels of functional neural proteins were abnormal relative to those of control subjects in acute but not chronic TBI, including ras-related small GTPase 10 (RAB10), annexin VII, ubiquitin C -terminal hydrolase LI (UCH-L1), AP spectrin fragments, claudin-5, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, and prion cellular protein (PrPc),. In chronic TBI, there are elevated NDE levels of Ab40, Ab42, phosphorylated tau, including P-T181-tau and P-S396-tau. Abnormalities in NDE levels of neuro-functional proteins in acute TBI, but not chronic TBI, and greater elevations of some neuropathological proteins in chronic TBI than acute TBI delineate phase-specific mechanisms.

[0067] Neuron-derived exosomal biomarkers of the present invention can be used to identify progression of neurological disease in subjects having a traumatic brain injury or AD. Various NDE proteins can serve as progression factors of neurological disease. As described herein, abnormal levels of progression factor proteins from NDEs are present in acute and chronic TBI. The progression factor proteins generally have the capacity to bind, concentrate and deliver to neurons one or more primary neurotoxic proteins, such as Ab40, Ab42 or phosphorylated tau. Progression factor proteins include prion cellular protein (PrPc) binds Ab40, Ab42, synaptogyrin-3 binds all tau forms, IL-6 for inflammation, and aquaporin 1 and 4 for edema.

[0068] Traumatic brain injury can increase the risk that a subject will subsequently develop a neurological disease. In some embodiments, the neurological disease is chronic traumatic encephalopathy (CTE). In some embodiments, the neurological disease is selected from the group consisting of: Alzheimer's disease (AD), vascular disease dementia, frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Lewy body dementia, tangle-predominant senile dementia, Pick's disease (PiD), atgyrophilic grain disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Guam parkinsonism-dementia complex, FTDP-17, Lytico-Bodig disease, multiple sclerosis, and Parkinson's disease.

[0069] In certain embodiments, the subject is a mammalian subject, including, e.g., a cat, a dog, a rodent, etc. In preferred embedments, the subject is a human subject.

[0070] In some embedments, the present invention enables a medical practitioner to diagnose or prognose a traumatic brain injury or AD in a subject. In some embedments, the traumatic brain injury is mild, moderate or severe. In yet other embodiments, tire present invention enables a medical practitioner to identify a subject at risk of developing a neurological disease following a traumatic brain injury. In some embodiments, the neurological disease is chronic traumatic encephalopathy. In other embodiments, the present invention enables a medical practitioner to predict whether a subject will later develop a neurological disease following a traumatic brain injury, such as, for example, chronic traumatic encephalopathy. In further embodiments the present invention enables a medical practitioner to prescribe a therapeutic regimen or predict benefit from therapy in a subject having a neurological disease or at risk of developing a neurological disease. For example, the administration of one or more treatments following a traumatic brain injury, guided by protein levels in neuron-derived exosomes of individual patients, could limit progression to CTE preventatively.

Biomarkers

[0071] Neuron-derived exosomal cargo levels of biomarker proteins are assayed for a subject having or at- risk of having a traumatic brain injury' or AD. In some embodiments, one or more biomarkers selected from the group consisting of r as-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter-1 (NKCC- 1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL- 6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau are assayed in order to detect whether or not a subject has a traumatic brain injury or AD. In one embodiment, all of the biomarkers, ras- related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau, are assayed in combination to detect a traumatic brain injury or AD. In some embodiments, the one or more biomarkers are assayed in the preclinical phase. In other embodiments, the traumatic brain injury is mild, moderate, or severe.

[0072] One of ordinary skill in the art has several methods and devices available for the detection and analysis of the biomarkers of the instant invention. With regard to polypeptides or proteins in patient test samples, immunoassay devices and methods are often used. These devices and methods can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of an analyte of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule.

[0073] Preferably the markers are analyzed using an immunoassay, although other methods are well known to those skilled in the art (for example, the measurement of marker RNA levels). The presence or amount of a marker is generally determined using antibodies specific for each marker and detecting specific binding. Any suitable immunoassay may be utilized, for example, enzyme- linked immunoassays (ELISA),

radioimmunoassay (RIAs), competitive binding assays, planar waveguide technology, and the like. Specific immunological binding of the antibody to the marker can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase and the like.

[0074] The use of immobilized antibodies specific for the biomarkers is also contemplated by the present invention. The antibodies could be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay place (such as microtiter wells), pieces of a solid substrate material (such as plastic, nylon, paper), and the like. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.

[0075] The analysis of a plurality of biomarkers may be carried out separately or simultaneously with one test sample. Several biomarkers may be combined into one test for efficient processing of a multiple of samples. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same individual. Such testing of serial samples will allow the identification of changes in marker levels over time. Increases or decreases in biomarker levels, as well as the absence of change in biomarker levels, would provide useful information about disease status that includes, but is not limited to the appropriateness of drug therapies, the effectiveness of various therapies, identification of the severity of a traumatic brain injury, susceptibility to neurological disease, and prognosis of the patient's outcome, including risk of development of a neurological disease, such as, for example, risk of developing CTE.

[0076] An assay consisting of a combination of the biomarkers referenced in the instant invention may be constructed to provide relevant information related to differential diagnosis. Such a panel may be constructed using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more individual markers. The analysis of a single biomarker or subsets of biomarkers comprising a larger panel of biomarkers could be carried out using methods described within die instant invention to optimize clinical sensitivity or specificity in various clinical settings.

[0077] The analysis of markers could be carried out in a variety' of physical formats as well. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings. Particularly useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality' of different analytes. Such formats include protein microarrays, or“protein chips” and capillary devices.

[0078] Biomarkers of the present invention serve an important role in the early detection and monitoring of traumatic brain injury or AD. Biomarkers are typically substances found in a bodily sample that can be detected and/or measured. The measured amount can correlate with underlying disorder or disease pathophysiology and probability of developing a neurological disease (e.g., CTE) in the future. In patients receiving treatment for their condition, the measured amount will also correlate with responsiveness to therapy.

[0079] In some embedments, the biomarker is measured by a method selected from the group consisting of immunohistochemistry, immunocytochemistry, immunofluorescence, immunoprecipitation, western blotting, and ELISA.

Clinical Assay Performance

[0080] The methods of the present invention for detecting neurological disease may be used in clinical assays to diagnose or prognose a neurological disease in a subject, identify a subject at risk of a neurological disease (e.g., CTE), and/or for prescribing a therapeutic regimen or predicting benefit from therapy in a subject having a neurological disease. Clinical assay performance can be assessed by determining the assay’s sensitivity, specificity, area under the ROC curve (AUC), accuracy, positive predictive value (PPV), and negative predictive value (NPV). Disclosed herein are assays for diagnosing or prognosing a traumatic brain injury in a subject, identifying a subject at risk of developing a neurological disease (e.g., CTE), or for prescribing a therapeutic regimen or predicting benefit from therapy in a subject having a traumatic brain injury.

[0081] The clinical performance of the assay may be based on sensitivity. The sensitivity of an assay of the present invention may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. The clinical performance of the assay may be based on specificity. The specificity of an assay of the present invention may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. The clinical performance of the assay may be based on area under the ROC curve (AUC). The AUC of an assay of the present invention may be at least about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. The clinical performance of the assay may be based on accuracy. The accuracy of an assay of the present invention may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.

Compositions

[0082] Compositions useful in the methods of the present invention include compositions that specifically recognize one or more neuron-derived exosomal biomarkers associated with traumatic brain injury, including r as-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NIL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau, or any combination thereof. In some embodiments, the composition enhances the activity' of at least one biomarker. In other embodiments, the composition decreases the activity of at least one biomarker. In some embodiments, the composition increases the levels of at least one biomaricer in the subject. In other embodiments, the composition decreases the levels of at least one biomaricer in the subject. In yet other embodiments, the composition comprises a peptide, a nucleic acid, an antibody, or a small molecule.

[0083] In certain embodiments, the present invention relates to compositions that specifically detect a biomaricer associated with traumatic brain injury. As detailed elsewhere herein, the present invention is based upon the finding that neuron-derived exosomal ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau proteins are specific biomaricers for traumatic brain injury. In one embedment, the compositions of the invention specifically bind to and detect one or more of the biomaricers ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau, or any combination thereof. The composition of the present invention can comprise an antibody, a peptide, a small molecule, a nucleic acid, and the like.

[0084] In some embedments, the composition comprises an antibody, wherein the antibody specifically binds to a biomarker or neuron-derived exosomes. The term“antibody” as used herein and further discussed below is intended to include fragments thereof which are also specifically reactive with a biomarker or vesicle (e.g., exosome). Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)z fragments can be generated by treating antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. Antigen-binding portions may also be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. In certain embedments, the antibody further comprises a label attached thereto and able to be detected (e.g., the label can be a radioisotope, fluorescent compound, enzyme or enzyme co-factor).

[0085] In certain embodiments, an antibody of the invention is a monoclonal antibody, and in certain embedments, the invention makes available methods for generating novel antibodies that specifically bind the biomarker or the exosome of the invention. For example, a method for generating a monoclonal antibody that specifically binds a biomaricer or exosome, may comprise administering to a mouse an amount of an immunogenic composition comprising the biomaricer or exosome, or fragment thereof, effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the biomarker or exosome. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to the biomarker or exosome. The monoclonal antibody may be purified from the cell culture.

[0086] The term“specifically reactive with” or“specifically binds” as used in reference to an antibody is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (e.g., a biomarker or exosome) and other antigens that are not of interest. In certain methods employing the antibody, such as therapeutic applications, a higher degree of specificity in binding may be desirable. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate effectively between the desired antigens and cross-reacting polypeptides. One characteristic that influences the specificity of an antibody:antigen interaction is the affinity of the antibody for the antigen. Although the desired specificity may be reached with a range of different affinities, generally preferred antibodies will have an affinity (a dissociation constant) of about

or less.

[0087] Antibodies can be generated to bind specifically to an epitope of an neuron-derived exosome or a biomarker of the present invention, including, for example, neuron-derived exosome surface markers, such as CD171.

[0088] In addition, tire techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. A variety of different techniques are available for testing interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the Biacore binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Md.), western blots, immunoprecipitation assay s, immunocytochemistry, and

immunohistochemistry.

[0089] In some embedments, tire present invention relates to compositions used for treating traumatic brain injury or preventing progression to a neurological disease (e.g. CTE). As detailed elsewhere herein, abnormal levels of neuron-derived exosomes and neuron-derived exosomal biomarkers are implicated in the pathology of traumatic brain injury. Therefore, in one embodiment, the present invention provides compositions that inhibit or reduce abnormalities in levels of neuron-derived exosomes or neuron-derived exosomal biomarkers. Compositions useful for preventing and/or reducing abnormalities in levels of a neuron-derived exosomes or neuron-derived exosomal biomarkers may include proteins, peptides, nucleic acids, small molecules, and the like.

Methods of Treatment [0090] The present invention provides methods of treating a traumatic brain injury associated with neuron- derived exosomal abnormalities in a subject, comprising administering to the subject an effective amount of a composition, wherein the composition normalizes amounts or inhibits deleterious activities of neuron-derived exosomal proteins

[0091] Furthermore, the methods of the invention can be used for monitoring the efficacy of therapy in a patient. The method comprises: analyzing the levels of one or more biomarkers selected from the group consisting of r as-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C -terminal hydrolase LI (LJCH- Ll), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau from neuron-derived exosomes from biological samples from the patient before and after the patient undergoes the therapy, in conjunction with respective reference levels for the biomarkers. Abnormal levels of neuron-derived exosomal biomarkers including ras-related small GTPase 10 (RABIO), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau correlate with increased traumatic brain injury severity and recency (e.g., acute vs. chronic TBI). Abnormal levels of progression factor proteins, prion cellular protein (PrPc), synaptogyrin-3, IL-6, and aquaporin 1 and 4 correlate with increased risk of progression to CTE. Treatment methods of the present invention help to normalize levels of neuron-derived exosomes and neuron-derived exosomal proteins, ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium -potassium- chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL),v tau, Ab40, Ab42, and phosphorylated tau and correlate with reduced traumatic brain injury severity and indicate that the condition of the patient is improving (e.g., lower risk of progression to CTE or dementia).

[0092] In some embodiments, the methods of the invention provide a method for treating traumatic brain injury or Alzheimer’s disease the method comprising the steps of: obtaining a biological sample from a subject suspected of having a traumatic brain injury or Alzheimer’s disease, wherein the sample comprises neuron-derived exosomes; measuring the level of one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau from die biological sample, wherein an altered level of the one or more biomarkers in the sample relative to the level in a control sample is indicative of a need for treatment; and administering an effective amount of an agent to the subject thereby treating the traumatic brain injury or Alzheimer’s disease in tire subject.

[0093] In other embodiments, the methods of the invention provide a method for treating a subject having a traumatic brain injury or Alzheimer’s disease, comprising, administering an effective amount of a synaptogyrin-3 inhibitor to the subject, thereby treating the traumatic brain injury or Alzheimer’s disease in the subject. In some embodiments, the synaptogyrin-3 inhibitor is an antibody. In other embodiments, the antibody is a monoclonal antibody. In some embodiments, the method for treating a subject having a traumatic brain injury or Alzheimer’s disease comprises administering one or more synaptogyrin-3 inhibitors to the subject, thereby treating the traumatic brain injury or Alzheimer’s disease in the subject. In some embodiments, one anti-synaptogyrin-3 antibody is administered to the subject. In other embodiments, two anti-synaptogyrin-3 antibodies are administered to the subject. In other embodiments, three anti-synaptogyrin-3 antibodies are administered to the subject. In still other embodiments, four or more anti-synaptogyrin-3 antibodies are administered to the subject. The administration of different combinations of anti-synaptogyrin-3 antibodies may be used to treat the traumatic brain injury or Alzheimer’s disease in the subject.

[0094] In other embodiments, the methods of the invention provide a method for treating a subject having a traumatic brain injury or Alzheimer’s disease, comprising, administering an effective amount of a PrPc inhibitor to the subject, thereby treating the traumatic brain injury or Alzheimer’s disease in the subject. In some embodiments, the PrPc inhibitor is an antibody. In other embodiments, the antibody is a monoclonal antibody. The monoclonal anti-PrPc antibodies can be: prion protein monoclonal antibody (3F4); epitope amino acids 109-112, Enzo Life Sciences, cat. # ENZ-ABS-119-0200; anti-prion protein monoclonal antibody (F89/160.1.5); epitope amino acids 146-159, ThermoFisher Scientific, cat. # MAI-750; anti-prion protein monoclonal antibody (8H4); epitope amino acids 145-180, Sigma-Aldrich, cat. # P0110-200 uL; 3) anti-prion protein monoclonal antibody (T16-R); epitope amino acids 33-46, Abeam, cat.# abl36919. In some embedments, the method for treating a subject having a traumatic brain injury comprises administering one or more PrPc inhibitors to tire subject, thereby treating the traumatic brain injury in tire subject. In some embodiments, one anti-PrPC antibody is administered to the subject. In other embodiments, two anti-PrPC antibodies are administered to the subject. In other embodiments, three anti-PrPC antibodies are administered to the subject. In still other embodiments, four or more anti-PrPC antibodies are administered to the subject. The administration of different combinations of anti-PrPc antibodies may be used to treat the traumatic brain injury or Alzheimer’s disease in the subject.

[0095] In other embodiments, the methods of the invention provide a method for treating a subject having a traumatic brain injury or Alzheimer’s disease, comprising, administering an effective amount of a synaptogyrin-3 inhibitor to the subject, thereby treating the traumatic brain injury or Alzheimer’s disease in the subject. In some embodiments, the synaptogyrin-3 inhibitor is an antibody. In other embodiments, the antibody is a monoclonal antibody. The monoclonal anti- synaptogyrin-3 antibodies can be: anti-synaptogyrin-3 monoclonal antibodies from Santa Cruz Biotechnology, clone E-l 1 (IgGl kappa), that is specific for amino acids 160-229 at the C-terminus of synaptogyrin-3. The monoclonal anti- synaptogyrin-3 antibodies can be: clone G-8 (IgG2bkappa), that is specific for amino acids 50-68 at the N-terminus of synaptogyrin-3 and clone G-3 (IgG2bkappa), that also is specific for amino acids 50-68 at the N-terminus of synaptogyrin-3. In some embodiments, the method for treating a subject having a traumatic brain injury comprises administering one or more synaptogyrin-3 inhibitors to the subject, thereby treating the traumatic brain injury in the subject. In some embodiments, one anti-synaplogyrin-3 antibody is administered to the subject. In other embodiments, two anti-synaptogyrin-3 antibodies are administered to the subject. In other embodiments, three anti- synaptogyrin-3 antibodies are administered to the subject. In still other embodiments, four or more anti- synaptogyrin-3 antibodies are administered to the subject. The administration of different combinations of anti-synaptogyrin-3 antibodies may be used to treat the traumatic brain injury or Alzheimer’s disease in the subject.

[0096] In other embodiments, the methods of the invention provide a method for treating a subject having a traumatic brain injury or Alzheimer’s disease, comprising administering an effective amount of a composition that downregulates the expression of PrPc and/or synaptogyrin-3 in the subject, thereby treating the traumatic brain injury' or Alzheimer’s disease in the subject. Such compositions may include small molecule compounds; peptides and proteins including antibodies or functionally active fragments thereof; and polynucleotides including small interfering ribonucleic acids (siRNAs), micro-RNAs (miRNAs), ribozymes, and anti-sense sequences. (See, e.g., Zeng (2003) Proc Natl Acad Sci USA 100:9779-9784; and Kurreck (2003) Eur J Biochem 270: 1628-1644.) CR1SPR gene-editing techniques may be used to prepare compositions useful for downregulating PrPC and/or synaptogyrin-3 in the subject.

Kits

[0097] Another aspect of the invention encompasses kits for detecting or monitoring traumatic brain injury or Alzheimer’s disease in a subject. A variety of kits having different components are contemplated by the current invention. Generally speaking, the kit will include the means for quantifying one or more biomarkers in a subject. In another embodiment, the kit will include means for collecting a biological sample, means for quantifying one or more biomarkers in the biological sample, and instructions for use of the kit contents. In certain embodiments, the kit comprises a means for enriching or isolating neuron-derived exosomes in a biological sample. In further aspects, the means for enriching or isolating neuron-derived exosomes comprises reagents necessary to enrich or isolate neuron-derived exosomes from a biological sample. In certain aspects, the kit comprises a means for quantifying the amount of a biomarker. In further aspects, the means for quantifying the amount of a biomarker comprises reagents necessary to detect the amount of a biomarker. Screening

[0098] In other aspects, the invention provides methods of screening to determine whether a

test compound can inhibit or reduce Abeta42 and/or P-tau binding activity of neuronal prion protein (PrPc) and/or synaptogyrin-3, the method comprising: providing a reaction mixture that comprises a preparation comprising Abeta42 and/or P-tau, neuronal prion protein (PrPc) and/or synaptogyrin-3, and a plurality of test compounds; incubating the reaction mixture under conditions which, in the absence of an inhibitor of the binding activity, allow the Abeta42 and/or P-tau to bind to neuronal prion protein (PrPc) and/or synaptogyrin- 3; and determining whether the binding activity is inhibited or reduced. Such methods are useful for identifying compounds that inhibit or reduce Abeta42 and/or P-tau binding activity of neuronal prion protein (PrPc) and/or synaptogyrin-3. Compounds identified using these methods may be useful for treating TBI.

[0099] These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.

EXAMPLES

[00100] The invention will be further understood by reference to tire following examples, which are intended to be purely exemplary of the invention. These examples are provided solely to illustrate the clamed invention. The present invention is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only. Any methods that are functionally equivalent are within the scope of the invention. Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from tire foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Example 1: Detection of Plasma Neuron-Derived Exosomes and Their Cargo Proteins in Acute and Repetitive Traumatic Brain Injury.

[00101 ] Neuron-derived exosomes and their cargo proteins were detected in plasma samples from subjects with traumatic brain injury (TBI) as follows. Blood samples were obtained from study participants and included ten additional controls of similar ages (see Table 1). Head injuries in study participants were sustained in the course of playing diverse sports, but predominantly hockey or lacrosse. Acute TBI was defined as having a concussion that met standard criteria for mild TBI in the seven days before donation of plasma for the study (Ruff, R. M. et al. (2009) Arch Clin Neuropsychol 24, 3-10). Subjects with acute TBI had suffered 0 (7/18) or one (8/18) or two (3/18) mild TBIs prior to the episode of this study, but all occurred at least eight months before the study occasion (8 to 72 months). Chronic repetitive TBI was defined as having had at least two past concussions that met standard criteria for mild TBI (two for 10/14 and three or more for 4/14), but none for at least three months (range three to 12 months) before donation of plasma for this study (Table 1). Only 2 of 21 controls had had mild TBIs distantly. On entry into tire study, all participants had a neurological examination, cognitive testing and an MRI.

Statistical significance of difference from acute TBI group=^+,p=0.014; **, pO.0001

[00102]Ten milliliters of venous blood were drawn into 0.5 ml saline with EDTA, incubated for 10 minutes at room temperature, and centrifuged for 15 minutes at 2500x g. Plasmas were stored in 0.25-ml aliquots at - 80°C.

[00103] Aliquots of 0.25 ml plasma were incubated with 0.1 ml thromboplastin D (Thermo Fisher Scientific, Waltham, MA, USA), followed by addition of 0.15 ml of calcium- and magnesium-free Dulbecco’s balanced salt solution with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail (Thermo Fisher Scientific), as described (Kapogiannis, D. et al. (2015) Faseb J 29, 589-596). After centrifugation at 3000 g for 30 min at 4°C, NDEs were harvested from resultant supernatants by sequential ExoQuick (System Biosciences, Mountain View, CA, USA) precipitation and immunoabsorption enrichment with mouse anti-human CD171 (LICAM neural adhesion protein) biotinylated antibody (clone 5G3;

eBiosciences, San Diego, CA, USA) as described (Kapogiannis, D. et al. (2015) Faseb J 29, 589-596).

Exosomes were counted, as described (Mustapic, M. et al. (2017) Front Neurosci 11, 278), and lysed in mammalian protein extraction reagent (M-PER; ThermoFisher Scientific) that contained protease and phosphatase inhibitor cocktails prior to storage at -80°C.

[00104]NDE proteins were quantified by ELISA kits for human aquaporin-4, occludin, claudin-5, alpha II spectrin breakdown products (SPDP145) and tetraspanning exosome marker CD81 (Cusabio-American Research Products, Waltham, MA, USA), sodium-potassium-chloride co-transporter 1 (NKCC-1) (Cloud- Clone Corp., American Research Products), synaptogyrin-3 (Abbkine, Wuhan, China, American Research Products), ras -related small GTPase RABIO and annexin VII (Biomalik, Wilmington, DE), UCH-L1 (RayBiotech, Norcross, GA). Ab42 (ultrasensitive ELISA), P-S396-tau and total tau (Invitrogen,

ThermoFisher Scientific, Vienna, Austria), and P-T181-tau (FUJIREBIO, US, Inc., Malvern, PA).

[00105] The mean value for all determinations of CD81 in each assay group was set at 1.00 and relative values of CD81 for each sample were used to normalize their recovery. One investigator (EJG) conducted all ELISAs without knowledge of the clinical data for any patient.

[00106] The Shapiro-Wilks test showed that data in all sets were distributed normally. Statistical significance of differences between the mean value for each TBI group and that of its age- and gender-matched control group was determined with an unpaired Student’s t test, including a Bonferroni correction (Prism 6; GraphPad Software, La Jolla, CA, USA).

[00107] As shown in Figure 1A, the concentrations ofNDEs was significantly lower in plasmas of subjects with acute TBI, who donated blood within 7 days of suffering a concussion, than in plasmas of control subjects with no history of TBI or of those with chronic repetitive TBIs, but none for three or more months. The differences in NDE levels shown by the exosome biomarker protein CD81 were confirmed by counts, that were 213+15.7 x 10⁹/ml (mean+S.E.M.) for controls, 141+12.8 x 10⁹/ml for acute TBI subjects (pcO.0001 compared to controls) and 212+14.2 x 10⁹/ml for chronic TBI subjects.

[00108] The R as -related small GTPase RAB10 is required for formation, intracellular movements and secretion ofNDEs and some other extracellular vesicles (Bucci, C. et al. (2014) Membranes (Basel) 4, 642- 677). Thus, it appears likely that diminished CD81 -normalized levels of RAB10 observed in NDEs of acute TBI subjects relative to those in NDEs of chronic TBI subjects and controls (Fig. IB) are at least in part responsible for concomitant decreases in plasma concentrations ofNDEs in acute TBI subjects. The phospholipid-binding protein annexin-7 also may be involved in intracellular traffic of vesicles (Creutz, C. E. (1992) Science 258, 924-931), but it has other predominant functions including maintenance of neuronal viability and membrane transport functions. The higher levels of annexin VII in NDEs and presumably neurons of acute TBI subjects relative to those of chronic TBI subjects and controls (Fig. 1C) may reflect the precedence of these other activities. To focus on NDE cargo protein processing after TBI, we quantified NDE levels of UCH-L1 that is an abundant neuron-specific facilitator of elimination of damaged proteins through ubiquilinylation targeting for destructive removal by the ATP-dependent proteosomal pathway (Jackson, P. et al. (1981) J Neurol Sci 49, 429-438). Levels of UCH-L1 in CSF and serum were shown to be elevated for several days after acute TBI (Papa, L. et al. (2012) J Trauma Acute Care Surg 72, 1335-1344). Here it is demonstrated that CD81 -normalized NDE levels of UCH-L1 are higher in NDEs and presumably neurons of acute TBI subjects than in those of chronic TBI subjects and controls (Fig. ID). This finding supports upregulation in acute TBI of one ubiquitin-dependent pathway of elimination of damaged neuronal proteins.

[00109] We next quantified effects of TBI on NDE levels of representative proteins required for normal functions of the neuronal cytoskeleton, neuronal water channels and ion carriers, and the blood-brain barrier (BBB). The membrane-associated periodic skeleton in axons of all eukaryotic neurons consists of actin rings separated by spectrin tetramer“spacers”, is necessary for functional organization of membranes and is cleaved by diverse proteases after brain injury (Huh, G. Y. et al. (2001) Neurosci Lett 316, 41-44; Unsain, N. et al. (2018) Front Synaptic Neurosci 10, 10). NDE levels of cleavage fragments of oil (AP) spectrin were significantly elevated after acute TBI relative to those detected in controls and chronic TBI, suggesting breakdown of die human neuronal periodic skeleton by TBI (Fig. IE). Occludin and claudin-5 are the main protein components of the tight junction of the BBB, that increase in concentration in brain tissues and blood after blast injury of rodent brains (Kuriakose, M. et al. (2018) Sci Rep 8, 8681). NDE levels of claudin-5 were significantly higher and of occludin were only marginally higher after acute TBI than for controls and subjects with chronic TBI (Fig. IF, 1G). In neurons, NKCC-1 is a principal co-transporter of ions and aquaporin 4 is the major water channel. CD81 -normalized NDE levels of NKCC-1 and aquaporin 4 both were significantly elevated after acute TBI relative to levels in controls and subjects with chronic TBI (Fig. 1H, II) implying a possible role in the acute development of brain edema following TBI. NDE levels of aquaporin 4, but not NKCC-1, also were significantly elevated after chronic TBI relative to levels in controls (Fig. II).

Synaptogyrin-3 is a protein of presynaptic vesicles, that has the highest affinity for tau of all synaptic proteins examined and whose binding of tau restricts synaptic vesicle mobility with consequent neural dysfunction (Mclnnes, J. et al. (2018) Neuron 97, 823-835 e828). NDE levels of synaptogyrin-3 are significantly higher after acute TBI and marginally higher in chronic TBI than for controls (Fig. 1 J).

[00110] Mean NDE levels of the putatively neuropathogenic proteins Ab42 and P-T181-tau were similarly higher in acute and chronic repetitive TBI titan controls (Fig. 2B, 2C). Mean NDE levels of P-S396-tau in chronic repetitive TBI, but not acute TBI, are significantly elevated above those of controls and also are significantly higher than in acute TBI (P=0.0317) (Fig. 2D). Levels of total tau in NDEs of acute and chronic TBI participants were only marginally higher than in controls (Fig. 2 A). Of the 14 TBI participants with the highest NDE levels of Ab42 and the 16 TBI participants with the highest NDE levels of P-T181-tau, 9 were in both groups.

[00111]Plasma levels of NDEs were decreased significantly relative to those of controls in acute TBI but not chronic repetitive TBI, as assessed by counts and content of the exosome marker protein CD81 (Fig. 1A). Changes in the levels of functional proteins quantified in NDE cargo also correlated with the phase of TBI as mean CD81 -normalized concentrations relative to those of controls were significantly abnormal for acute TBI, but not for chronic repetitive TBI (Fig. 1). These proteins included RAB 10 that regulates formation and activities of synaptic and other neuronal vesicles, UCH-L1 that facilitates removal of damaged cellular proteins, annexin VII and NKCC-1 that constitute and control neuronal membrane ion transporters, as well as neuronal viability, and All spectrin and claudin-5 that maintain neuronal structure and tight-junctions of the BBB (Figs. 1B-1F and 1H). Aquaporin 4 was the only NDE cargo protein examined that was represented at significantly higher mean levels in acute and chronic TBI than in controls, although the acute TBI level was significantly higher than the chronic repetitive TBI level (PO.OOl ) (Fig. II). The minor increase in mean NDE level of occludin in acute TBI relative to that of controls was only marginally significant (Fig. 1G). Similarly, the significant increase in mean NDE levels of synaptogyrin-3 observed in acute TBI was only marginally maintained in chronic TBI (Fig. 1J). The abnormal levels of these many native physiologically functional neuronal proteins may account in part for the decreased concentration of plasma NDEs, reduced removal of damaged proteins, altered BBB functions and brain tissue edema characteristically observed in acute TBI. The NDE functional protein abnormalities of acute TBI appear to be of limited duration, with tire exception of elevated aquaporin 4, as they were not observed in the patients with chronic TBI who had had an acute TBI as recently as 30 days before plasma sampling (Fig. 1).

[00112] In sharp contrast to the NDE cargo of native functional proteins affected by acute TBI, tire most significant changes in chronic TBI were in proteins considered to be neuronally toxic in diverse

neurodegenerative diseases (Fig. 2). The mean NDE levels of Ab42, P-T181-tau and P-S396-tau were significantly higher in chronic TBI participants than in controls, whereas lesser elevations of mean NDE levels of Ab42 and P-T181-tau were observed in acute TBI than chronic TBI, and no significant increase in mean NDE level of P-S396-tau was observed in acute TBI. Mean NDE levels of IL-6 and PRPc were significantly elevated acutely and chronically, but acute increases were greater than those observed chronically. Mean NDE levels of total tau were higher in acute and chronic TBI subjects relative to that of controls. The magnitude of increases in PrPc, synaptogyrin-3, AB42 and P-tau species were similar to those observed in AD.

[00113] These results demonstrated that the methods of the present invention are useful for detecting biomarkers and measuring biomarker protein levels in nueron-derived exosomes in subjects with traumatic brain injury. These results further demonstrated that the methods of the present invention may be used to detect exosomal biomarkers associated with pathogenesis of neurological diseases, including TBI, CTE, and Alzheimer’s disease. These results further showed that methods of tire present invention are useful for prognosis, diagnosis, treating or monitoring treatment of exosomal abnormalities associated with neurological diseases. The results suggested that the methods of the present invention would be useful for treating neurological diseases, including, for example, TBI, CTE and AD.

Example 2: Elevated Levels of Neuropathogenic Proteins and Their Receptors in Neuron-Derived Plasma Exosomes After Traumatic Brain Injury.

[00114] Long-term effects of traumatic brain injury (TBI) on levels of plasma neuron-derived exosome (NDE) biomarker proteins were examined as follows. Plasma was obtained decades after the most recent military service-associated TBI from four groups in a veterans home, who were matched for age and gender; no TBI or cognitive impairment (CI)(n=42), Cl without TBI (n=19), TBI without Cl (n=21) and TBI with Cl (n=26). [00115]TBI was defined for the affected groups three and four as a head injury resulting in any form of medical care. The two groups classified as never having sustained a TBI had no history of any head injury that produced neurological symptoms or required medical care. Confirmation of TBI history was found in VHCY medical records of 52% of the TBI participants and was absent from those of all non-TBI participants. The Ohio State University TBI Identification Method (OSI-TBI ID), a structured clinical interview recommended by the National Institute of Neurological Disorders and Stroke (NINDS) as a Common Data Element (CDE) for the retrospective assessment of lifetime TBI in clinical research, was used to assess TBI history and determine the severity of TBI.

[00116] General cognitive abilities were assessed by the MMSE (range of scores=0-30). The Auditory Verbal Learning Task (AVLT), including Learning Trials (total trials 1-5) and Delayed Recall, were used to determine levels of learning and memory, respectively. Mental processing speed was quantified by the Wechsler Adult Intelligence Scale-Revised (WAIS-R) Digit Symbol Task.

[00117] Impairment was defined using non-conventional standard definitions. A composite Z-score was created from the AVLT learning score, AVLT delay score, MMSE, and WAIS Digit Symbol. The composite score was based on the Alzheimer’s Disease Cooperative Study Preclinical Alzheimer Cognitive Composite score, a measure of early cognitive impairment. First, each participant’s raw cognitive test scores were compared to demographically-corrected normative data for each measure- Mayo’s Older Americans

Normative Studies (MOANS) age-corrected norms for AVLT Learning Trials and Delayed Recall and Alzheimer’s Disease Centers’ Uniform Data Set age-, gender-, and education-corrected norms for MMSE and WAIS-R Digit Symbol. Second, each individual’s raw' test scores were converted into demographically corrected z-scores using these normative data, indicating how much each individual’s performance differs from that of healthy, demographically -similar peers. Third, the individual z-scores for each participant were combined to create a composite cognitive score.

[00118] Cognitive impairment (Cl) was defined as a cognitive composite score below normative values by- more than one standard deviation (SD), medical record dementia diagnosis, or history of taking a dementia prescription medication (donepezil, memantine, or rivastigmine) that was recorded in their medical records.

[00119] The first and second study groups were composed of control subjects who had not experienced any TBI. Subjects of the first group (n=42) had normal cognition, whereas those of the second group (n=19) had developed cognitive impairment (Cl). Subjects of groups three (n=21) and four (n=26) had experienced one to four episodes of TBI during military service 34 to 51 years ago that was not followed by cognitive impairment in those of group three, but was followed by cognitive impairment in those of group four.

[00120] Venous blood was obtained for plasma by the standard phlebotomy technique using EDTA for anticoagulation. Enrichment of plasma neuronal-derived exosomes (NDEs) for extraction and ELISA quantification of proteins

[00121] Aliquots of 0.25 ml plasma were incubated with 0.1 ml thromboplastin D (Thermo Fisher Scientific, Waltham, MA, USA), followed by addition of 0.15 ml of calcium- and magnesium-free Dulbecco’s balanced salt solution with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail (Thermo Fisher Scientific), as described in Goetzl et al. (2016) Faseb J. 12:4141-8. After centrifugation at 3000 g for 30 min at 4°C, NDEs were harvested from resultant supernatants by sequential ExoQuick (System Biosciences, Mountain View, CA, USA) precipitation and immunoabsorption enrichment with mouse antihuman CD171 (LI CAM neural adhesion protein) biotinylated antibody (clone 5G3; eBiosciences, San Diego, CA, USA) as described Goetzl et al. (2016) Faseb J. 12:4141-8. Exosomes were counted and their size ranges determined, as described in Mustapic M et al. (2017) Front Neurosci. 11:278, and then lysed in mammalian protein extraction reagent (M-PER; ThermoFisher Scientific) that contained protease and phosphatase inhibitor cocktails prior to storage at -80°C.

[00122JNDE proteins were quantified by ELISA kits for human claudin-5 and tetraspanning exosome marker CD81 (Cusabio-American Research Products, Waltham, MA, USA), prion cellular protein (PrPc), aquaporin-1 and aquaporin-4 (Cloud-Clone Corp.-American Research Products), synaptogyrin-3 (Abbkine, Wuhan, China- American Research Products), IL-6 (R&D Systems, Minneapolis, MN), annexin VII (Biomatik, Wilmington, DE), Ab42 (ultrasensitive ELISA) and P-S396-tau (Invitrogen, ThermoFisher Scientific, Vienna, Austria), and P-T181-tau (FUJIREBIO, US, Inc., Malvern, PA).

[00123]The mean value for all determinations of CD81 in each assay group was set at 1.00 and relative values of CD81 for each sample were used to normalize their recovery. One investigator (EJG) conducted all ELISAs without knowledge of the clinical data for any subject.

Statistical analyses

[00124] The Shapiro-Wilks test showed whether data in each set were distributed normally. Some data for only two analytes (IL-6 and Ab42) were not distributed normally and the significance of their difference from control values was determined by the Mann-Whitney rank-sum test. For all other sets, statistical significance of differences between die mean value for each control group and that of the corresponding group with Cl was determined with an unpaired Student’s t test, including a Bonferroni correction (Prism 7; GraphPad Software, La Jolla, CA, USA).

Results

[00125] There were no significant differences in ages among subjects of the four study groups, nor were there significant differences between the two groups wo TBI and those wTBI with respect to the percentages of male or minority race participants or the total years devoted to education (Table 2). Levels of cognition, quantified by two distinct testing methods, were significantly higher for group 1 than 2 wo TBI and for group 3 than 4 w TBI (Table 2). In comparing groups 3 and 4 w TBI, the numbers of subjects in each group who sustained one TBI and two or more TBIs were no different and the times since the most recent TBI also were not different (Table 3). Similarly, the percentage of subjects in each group who had moderate or severe TBIs also were not different (Table 3).

[00126] CDS 1 -normalized levels of NDE cargo proteins, such as claudin-5 and annexin VII, that are representative of those elevated only transiently within one week after an impact sport-induced mTBI were the same for cognitively normal controls, subjects with cognitive impairment (Cl) without TBI, and those who had TBI many years before testing without or with subsequent Cl (Figs. 2A, 2B). CDS 1 -normalized levels of NDE aquaporin 4, that were elevated within one week and for at least 3 to 12 months following an impact sport-induced mTBI also were the same for controls, subjects with Cl without TBI, and those who had TBI many years before testing without or with subsequent Cl (Fig. 2C). The CDSl-noimalized NDE levels of PrPc and synaptogyrin-3, that were elevated within one week and for at least 3 to 12 months following an impact sport-induced mTBI, were significantly higher in those with Cl than without Cl many years after TBI and also were significantly higher in subjects with Cl without TBI than cognitively normal controls (Figs. ID, IE).

[00127] CDS 1 -normalized NDE levels of the neuropathogenic proteins P-T181-tau and Ab42, were significantly elevated within one week with persistence for at least 3 to 12 months following an impact sport- induced mTBI and also are elevated up to 10 years prior to clinical presentation of AD. The present results show that these proteins also were significantly higher in those with Cl than without Cl many years after TBI and in subjects with Cl without TBI than cognitively normal controls (Figs. 2A, 2C). CDS 1 -normalized NDE levels of the neuropathogenic proteins P-S396-tau and IL-6 are elevated variably in AD depending on die stage and severity of neurodegeneration. These same NDE proteins are consistently elevated after sport-induced mTBI with significant increases in P-S396-tau only after 3 months and for at least 12 months and in IL-6 within one week with persistence for at least 3 to 12 months. For this set of plasmas from military deployment TBI, both P-S396-tau and IL-6 were significantly higher in those with Cl than without Cl many years after TBI, but were not higher in subjects with Cl without TBI than cognitively normal controls (Figs. 2B, 2D). For most analytes, there is considerable overlap of the values for mTBI subjects without and with CL However, for PrPc, P-T181-tau and P-S396-tau, only 20% or fewer of the values for subjects with Cl overlap into the range for those without CL Thus, in summary, neuronal PrPc and synaptogyrin-3 putative binding proteins along with their respective neuropathogenic ligands Ab42 and the P-tau species P-T181-tau and P-S396-tau all were significantly higher in military TBI subjects with Cl than without Cl many years after TBI sustained in military service as they also were elevated in AD patients evaluated concurrently.

Example 3: Identification of Drugs That Decrease Neuron Surface Expression of PrPc or Synaptogyrin- 3 and Decrease Binding of Ab peptides to PrPc or P-tau Species to Synaptogyrin-3.

[00128] The identification of drugs that decrease neuron surface expression of PrPc of synatogyiin-3 are carried out as follows. Human neuroblastoma cells (SH-SY5Y or NB7 lines) are cultured for 2, 4, 24 or 48 hours without and with different concentrations of a candidate drug before harvesting the cells. Next, cells are labeled without and with fixation by fluorescent antibody to PrPc or synaptogyrin-3 and net fluorescence decrease below control cell levels is quantified to determine effects of the drug on surface levels of the proteins. If effects on surface expression are detected, total cellular proteins are extracted into M-PER detergent for separate ELISA quantification of total neuroblastoma cell levels of PrPc and synaptogyrin-3.

[00129] Studies to identify drugs that decrease binding of Ab peptides to PrPc or P-Tau species to synaptogyrin-3 are carried out as follows. Human neuroblastoma cells (SH-SY5Y or NB7 lines) are cultured overnight as monolayers in 24-well plates. Then at 4°C, cells are incubated for 1 hour without and with levels of anti-PrPc antibody or anti-synaptogyrin-3 antibody known to block binding of aggregated synthetic biotin- Abo (from Ab42, AnaSpec, San Jose, CA) and P-T181-tau-biotin, respectively. The difference in levels of binding of fluorescent-streptavidin to the cell-associated biotinylated ligands without and with antibody treatment is used as a first approximation of specific binding to the neuroblastoma cell receptors. Then different concentrations of drugs are added in tire binding mixture to determine any effects on specific interactions between cellular PrPc and biotin-Abo or between cellular synaptogyrin-3-biotin and P-T181-tau. If neuroblastoma cell levels of PrPc or synaptogyrin-3 are too low for reliable signals, then cells will be transfected with established methods.

Example 4: Treatment with Anti-PrPc Antibody Prevents Neuron Cytotoxicity of Ab42o. [00130] The effect of treatment with anti-PrPc antibody to prevent neuron cytotoxicity of Ab42o was determined as follows. PC12 cells were cultured in PDL-coated Sarstedt plastic T75 tissue culture flasks in DMEM supplemented with 10% horse serum and 5% fetal bovine serum in a 5% C02 humidified atmosphere at 37°C. Differentiation of PC 12 cells was induced in poly-D-lysine-coated, Olympus flat-bottom 12-well plates at a density of 15,000 cells/well in serum-free DMEM by addition of 50 ng/ml of nerve growth factor (NGF). PC12 cells showed signs of differentiation, including neurite sprouting, within 2 to 3 days. Ab42o was introduced into cultures without or one hour after one or more anti-PrPc antibodies, using anti-PrPc antibody alone and no additions as controls. After 1-3 days of incubation, phase contrast images of PC 12 cells were obtained, medium was collected for NDE isolation and cytotoxicity assays, and cellular proteins were extracted.

[00131] Similarly, human neuroblastoma SH-SY5Y cells were maintained in MEM:F12 with 10% FBS and differentiated on rat tail collagen-coated glass cover slips in MEM:F12 + 3% FBS supplemented with 15 nM retinoic acid. Ab42o was introduced into cultures without or one hour after one or more anti-PrPc antibodies, with anti-PrPc antibody alone and no additions as controls followed by 48 hours of incubation. Medium was collected for NDE isolation and cytotoxicity assay's, and cellular proteins were extracted.

[00132] Cytotoxicity was quantified by the percentage release of total neuron cytosolic lactic acid dehydrogenase (LDH) into the medium (Cytoxicity Assay kit, ThennoFisher, Inc.).

[00133] As shown in Table 4 below, the anti-PrPc antibody directed to the amino-terminus of PrPc (T16-R) significantly inhibited cytotoxic effects of both concentrations of Ab42o (p<0.01). In contrast, the anti-PrPc antibody directed to tire carboxyl-terminal domain of PrPc (F89/160.1.5) had no apparent effect on the cytotoxicity of the lower level of Ab42o and only marginally inhibited the cytotoxic effect of the higher level of Ab42o (p<0.05). However, the anti-PrPc antibody directed to the carboxyl-terminal domain of PrPc (F89/160.1.5) significantly enhanced the protective effect of the anti-PrPc antibody directed to the amino- terminus of PrPc (T16-R) against the higher concentration of Ab42o (p<0.01).

[00134] These results demonstrated that the methods of the present invention are useful for preventing neuronal cytotoxicity of Ab42o. These results further suggested that methods of the present invention would be useful for treating neurological diseases, including, for example, traumatic brain injury, CTE, and AD. Example 5: Administration of a Synaptogyrin-3 Inhibitor Prevents Ddeterious Effects of Mild

Traumatic Brain Injury (mTBl)

[00135] A mild TBI mouse model was previously developed to mimic concussive head injury in humans (Hsueh et al. (2019) Cell Transplant 28, 1183-1196 and Lecca et al. (2019) Neurobiol Dis 130, 104528). The model resembles, in severity and time-course, a person falling under their own weight and hitting their head or two humans of equal weight clashing heads at slow running speed. In one series of experiments, each 30 g mouse was anesthetized with isoflurane and then hit on the side of the head between the eye and the ear with a 30 g free weight falling from a height of 80 cm. The anesthetized mice were unaware of the mild TBI procedure and hence do not have anxiety as a confounding factor in later behavioral evaluations. On recovering from anesthetic, mild TBI mice cannot be readily differentiated by inspection or simple observation of behavior from untraumatized but anesthetized control mice. Mice may be treated before mild TBI or subjected to mild TBI, randomized into treatment groups and then treated.

[00136] Prior studies of plasma neuron-derived exosome (NDE) and astrocyte-derived exosome (ADE) contents of relevant proteins in human AD and mTBI have shown significantly increased levels of NDE PrPc receptor for amyloid peptides and synaptogyrin-3 (Sgyr3) receptor for P-tau species and of ADE neurotoxic C components, including C3b and C5b-9, in mTBI ( Goetzl et al. (2018) Faseb J 32, 888-893; Goetzl et al. (2018) Ann Neurol 83, 544-552; Goetzl et al. (2019) Faseb J 33, 5082-5088; and Goetzl et al. (2019)7 Neurotrauma). Quantification of levels of NDE PrPc receptor for amyloid peptides and Sgyr3 receptor for P- tau species as well as ADE neurotoxic C components, including C3b and C5b-9, showed that significant increases were evoked in mice by mTBI described above.

[00137] The effects of mild TBI were evaluated in a broad series of behavioral tests. Y-maze and Novel Object Recognition paradigms demonstrated impairments in spatial and visual memory from four to seven days post- mTBI onward. Anxiety was evaluated, but not found, using an elevated plus maze test.

[00138] Immunohistochemical evaluation of brain tissue in mice with mild TBI was performed. Mice were euthanized at selected times post-TBl, and their brains perfused with PBS to clear the cerebral vasculature before immersion fixation in 4% paraformaldehyde. Coronal sections were cut with special attention to the lesional site and hippocampus, and then sections were subjected to routine staining as well as

immunohistochemistry. Fluoro-Jade C was used to evaluate overall neural cellular loss and NeuN staining allowed for analysis of neuronal loss. Diffuse neuronal cell loss was found throughout regions of the hippocampus and cerebral cortex ipsilateral to the side of injury. Prior studies including silver staining, TUNEL staining, and staining of p53, have confirmed this to be chiefly apoptotic (i.e., programmed) cell death. Neuroinflammation was routinely evident throughout hippocampus and cerebral cortex - both ipsilateral and contralateral to mild and moderate TBI. A variety of immunohistochemical techniques were used to evaluate neuroinflammation, including staining microglia for IBA1 and TNF, as well as astrocytes for IL-6 and TNF. Synaptic integrity was quantified immunohistochemically using staining of synaptophysin for pre-synaptic regions and of post-synaptic density 95 protein (SAP-90) for post-synaptic structures.

[00139] Plasma total extracellular vesicles were precipitated and NDEs and ADEs separately isolated immunochemically as described for human studies and more recently for mice using mouse-specific capture antibodies ( Goetzl et al. (2016) Faseb J 30, 3853-3859, Mustapic et al. (2017) Front Neurosci 11, 278). ELISAs were used to quantify NDE proteins shown to reflect changes relevant to mTBI, such as prion cellular protein, synaptogyrin-3, annexin VII, claudin-5 and NKCC-1, as well as ADE complement proteins beginning with the predominant effectors C3b and C5b-9 ( Goetzl et al. (2018) Ann Neurol 83, 544-552).

[00140] Prevention of mild TBI in mice using an anti-synaptogyrin-3 antibody were performed as follows. An anti-synaptogyrin-3 antibody directed to the extracellular carboxy-terminus of synaptogyrin-3 was administered, 1 mg/mouse, IP 2 to 4 hours prior to induction of mTBI for prevention. At predicted CNS concentrations of this antibody of 0.5 to 1 ug/ml, there were no effects on viability of cultured mouse neurons at 24 or 48 hours.

[00141] Mild TBI elevated NDE levels of the blood-brain barrier component claudin-5, the prion cellular protein receptor for amyloidpl-42 and the synaptogyrin-3 receptor for tau/P-tau significantly by 24 hours and remained increased for up to 7 days after mTBI (see Table 5 below). Similarly, mild TBI elevated ADE levels of the neurotoxic complement effectors C3b and C5b-9 terminal complement complex at 24 hours and for up to 7 days after mTBI. Prior administration of the anti-synaptogyrin-3 antibody at 1 mg/mouse IP substantially reduced all increases in levels of plasma neurotoxic NDE and ADE proteins elicited by mTBI (see Table 6 below).

[00142] Behavioral effects of mTBI were also decreased by prior administration of the anli-synaplogyrin-3 antibody at 1 mg/mouse IP, as demonstrated by results of the Y-maze and Novel Object Recognition tests (see Table 7 below). Performance times for sham control mice were set at 100%, so that higher % values after mTBI showed the consequently delayed performance times and the ameliorating preventative effects of the anti-synaptogyrin-3 antibody.

[00143] These results showed that an anti-synaptogyrin-3 antibody reduced the deleterious effects of mild TBI in mice. These results further showed that methods and compositions of the invention are useful for preventing and/or treating TBI. Moreover, methods and compositions of the invention are useful for improving outcome of a subject that suffers from TBI.

[00144] Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. These results further suggested that methods of the present invention would be useful for treating neurological diseases, including, for example, traumatic brain injury, CTE, and AD.

Example 6: Elevated Levels of Neuropathogenic Proteins and Their Receptors in Neuron-Derived Plasma Exosomes in Subjects with Alzheimer’s Disease. [00145] Neuron-derived exosomes and their cargo proteins were detected in plasma samples from subjects with Alzheimer’s disease (AD) as follows. Ten milliliters of venous blood were drawn into 0.5 ml saline with EDTA, incubated for 10 minutes at room temperature, and centrifuged for 15 minutes at 2500x g. Plasmas were stored in 0.25-ml aliquots at -80°C. Aliquots of 0.25 ml plasma were incubated with 0.1 ml

thromboplastin D (Thermo Fisher Scientific, Waltham, MA, USA), followed by addition of 0.15 ml of calcium- and magnesium-free Dulbecco’s balanced salt solution with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail (Thermo Fisher Scientific), as described

(Kapogiannis, D. et al. (2015) Faseb J 29, 589-596). After centrifugation at 3000 g for 30 min at 4°C, NDEs were harvested from resultant supernatants by sequential ExoQuick (System Biosciences, Mountain View,

CA, USA) precipitation and immunoabsorption enrichment with mouse anti-human CD171 (LI CAM neural adhesion protein) biotinylated antibody (clone 5G3; eBiosciences, San Diego, CA, USA) as described (Kapogiannis, D. et al. (2015) Faseb J 29, 589-596). Exosomes were counted, as described (Mustapic, M. et al. (2017) Front Neurosci 11, 278), and lysed in mammalian protein extraction reagent (M-PER;

ThermoFisher Scientific) that contained protease and phosphatase inhibitor cocktails prior to storage at -80°C.

[00146] NDE proteins were quantified by ELISA kits for human prion cellular protein (PrPc) and

synaptogyrin-3 (Abbkine, Wuhan, China-American Research Products). NDE levels of synatogyrin-3 and PrPc were elevated in preclinical subjects compared to controls (see Table 8 below). Also shown in Table 8, NDE levels of synatogyrin-3 and PrPc were elevated in human subjects with early Alzheimer’s disease compared to healthy controls (see Table 8 below).

[00147] The results showed that plasma NDE levels of neuropathic proteins synaptogyrin-3 and PrPc are elevated in preclinical and early Alzheimer’s disease subjects. These results demonstrated that the methods and compositions of the invention are useful for predicting and/or diagnosing Alzheimer’s disease. These results further suggested that methods of the present invention would be useful for treating Alzheimer’s disease with synaptogyrin-3 and PrPc inhibitors.

[00148] All references cited herein are hereby incorporated by reference herein in their entirety.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method comprising: a) providing a biological sample comprising neuron-derived exosomes from a subject; b) enriching tire sample for neuron-derived exosomes; and c) detecting the presence of one or more biomarkers selected from the group consisting of r as-related small GTPase 10 (RABIO), Annexin VII, ubiquitin C-terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium- chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau in the sample.

2. The methods of claim 1, wherein the biological sample is selected from the list consisting of whole blood, plasma, serum, lymph, amniotic fluid, urine, saliva, and umbilical cord blood.

3. The method of claim 1, wherein the marker is a full-size marker or a fragment of the full-size marker.

4. The method of claim 1, wherein the detecting the presence of the marker in the biological sample comprises detecting the amount of the marker in the biological sample.

5. The method of claim 1 , further comprising the step of determining a treatment course of action based on the detection of the one or more biomarkers.

6. A method comprising: a) providing a biological sample comprising neuron-derived exosomes from a subject having a traumatic brain injury or suspected of having a traumatic brain injury; b) isolating neuron- derived exosomes from tire biological sample; and c) detecting the presence of one or more biomarkers selected from the group consisting of r as-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C- terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride cotransporter-1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau in the exosomes.

7. The method of claim 6, wherein the isolating neuron-derived exosomes from the biological sample comprises: contacting the biological sample with an agent under conditions wherein an neuron-derived exosome present in the biological sample binds to the agent to form an neuron-derived exosome-agent complex; and isolating the neuron-derived exosome from the neuron-derived exosome-agent complex to obtain a sample containing the neuron-derived exosome, wherein the purity of the neuron-derived exosomes present in said sample is greater than the purity of die neuron-derived exosomes present in said biological sample.

8. The method of claim 6, wherein the agent is an antibody.

9. The method of claim 8, wherein the antibody is an anti-CD171 antibody.

10. The methods of claim 6, wherein the biological sample is selected from the list consisting of whole blood, plasma, serum, lymph, amniotic fluid, urine, saliva, and umbilical cord blood.

11. The method of claim 6, wherein the marker is a full-size marker or a fragment of the full-size marker.

12. The method of claim 6, wherein the detecting the presence of the marker in the biological sample comprises detecting the amount of the marker in the biological sample.

13. The method of claim 6, further comprising the step of determining a treatment course of action based on the detection of the one or more biomarkers.

14. The method of clam 6, wherein the traumatic brain injury' is acute TBI or chronic repetitive TBI.

15. The method of clam 6, wherein the subject has or is at-risk of having a neurological disease.

16. The method of clam 15, where in the neurological disease is selected from the group consisting of chronic traumatic encephalopathy (CTE), Alzheimer's disease (AD), vascular disease dementia,

frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Lewy body dementia, tangle-predominant senile dementia, Pick's disease (PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Guam parkinsonism -dementia complex, FTDP-17, Lytico-Bodig disease, multiple sclerosis, and Parkinson's disease.

17. A method for treating a subject having a traumatic brain injury, comprising the steps of: providing a biological sample from a subject suspected of having a traumatic brain injury, wherein the sample comprises neuron-derived exosomes; measuring the level of one or more biomarkers selected from the group consisting of ras-related small GTPase 10 (RAB10), Annexin VII, ubiquitin C -terminal hydrolase LI (UCH-L1), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphorylated tau from the biological sample, wherein an altered level of the one or more biomarkers in the sample relative to the level in a control sample is indicative of a need for treatment; and administering an effective amount of an agent to the subject thereby treating the traumatic brain injury' in the subject.

18. The method of claim 17, wherein the phosphoiylated tau is P-Tl 81-tau and/or P-S396-tau.

19. A method for treating a subject having a traumatic brain injury or Alzheimer’s disease, comprising, administering an effective amount of a PrPc inhibitor and/or synaptogyrin-3 inhibitor to the subject, thereby treating the traumatic brain injury' or Alzheimer’s disease in the subject.

20. The method of claim 19, wherein the inhibitor is an antibody.

21. The method of claim 20, wherein the antibody is a monoclonal antibody.

22. The method of claim 19, wherein more than one PrPc inhibitor and/or synaptogyrin-3 inhibitor is administered to tire subject.

23. The method of claim 22, wherein two or more anti-PrPc antibodies and or anti-synaptogyrin-3 antibodies are administered to the subject.

24. A method for treating a subject having a traumatic brain injury or Alzheimer’s disease, comprising administering an effective amount of a composition that downregulates the expression of PrPc and/or synaptogyrin-3 in the subject, thereby treating the traumatic brain injury or Alzheimer’s disease in the subject.

25. A method of prognosing traumatic brain injury or Alzheimer’s disease in a subject, the method comprising the steps of: obtaining the level of at least one biomaikers selected from the group consisting of ras-related small GTPase 10 (RABIO), Annexin VII, ubiquitin C -terminal hydrolase LI (UCH-Ll), All spectrin fragments, claudin-5, occludin, sodium-potassium-chloride co-transporter- 1 (NKCC-1), aquaporin 1 and 4, synaptogyrin-3, neurofilament heavy chain (NfH), neurofilament light chain (NfL), IL-6, prion cellular protein (PrPc), tau, Ab40, Ab42, and phosphoiylated tau from a biological sample comprising neuron-derived exosomes; and prognosing the traumatic brain injury or Alzheimer’s disease based on tire levels of the at least one biomarkers in the sample.

26. A method of screening to determine whether a test compound can inhibit or reduce Abeta42 and/or P- tau binding activity of neuronal prion protein (PrPc) and/or synaptogyrin-3, said method comprising: providing a reaction mixture that comprises a preparation comprising Abeta42 and/or P-tau, neuronal prion protein (PrPc) and/or synaptogyrin-3, and a plurality of test compounds; incubating said reaction mixture under conditions which, in the absence of an inhibitor of said binding activity, allow said Abeta42 and/or P-tau to bind to neuronal prion protein (PrPc) and/or synaptogyrin-3; and determining whetiier said binding activity is inhibited or reduced.