WO2008042190A2 - Détection de maladies neurodégénératives - Google Patents

Détection de maladies neurodégénératives Download PDF

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WO2008042190A2
WO2008042190A2 PCT/US2007/020795 US2007020795W WO2008042190A2 WO 2008042190 A2 WO2008042190 A2 WO 2008042190A2 US 2007020795 W US2007020795 W US 2007020795W WO 2008042190 A2 WO2008042190 A2 WO 2008042190A2
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tdp
tissue
neurodegenerative disease
ftld
ubiquitin
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PCT/US2007/020795
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WO2008042190A3 (fr
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Virginia M.Y. Lee
John Q. Trojanowski
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The Trustees Of The University Of Pennsylvania
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Publication of WO2008042190A2 publication Critical patent/WO2008042190A2/fr
Publication of WO2008042190A3 publication Critical patent/WO2008042190A3/fr
Priority to US12/412,015 priority Critical patent/US8354236B2/en
Priority to US13/712,085 priority patent/US20130184441A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention pertains to detecting or diagnosing a neurodegenerative disease in a subject, methods for determining the efficacy of a drug against a neurodegenerative disease, and novel antibodies that bind to a protein.
  • FTDs are clinically, genetically, and pathologically heterogeneous, and are the second most common cause of dementia under age 65 (G. M. McKhann et al, Arch. Neurol. 58, 1803 (2001); M. S. Forman et al, Ann. Neurol 59, 952 (2006)).
  • MND motor neuron disease
  • FTD with parkinsonism linked to chromosome 17 is usually associated with neurofibrillary tau pathology caused by pathogenic mutations in the microtubule associated protein tau (MAPT) (M. Hutton et al, Nature 393, 702 (1998); P. Poorkaj et al, Ann. Neurol 43, 815 (1998)), which is designated here as FTDP-17T.
  • M. Hutton et al, Nature 393, 702 (1998); P. Poorkaj et al, Ann. Neurol 43, 815 (1998) which is designated here as FTDP-17T.
  • FTDP- 17 families do not develop tau pathology and lack MAPT gene mutations, but instead develop UBIs (designated as FTDP- 17U).
  • PGRN progranulin
  • Ubiquitin-positive, tau- and ⁇ -synuclein-negative inclusions are hallmark lesions of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS), but the identity of the disease protein specific to either disorder has heretofore remained unknown.
  • MAbs monoclonal antibodies
  • TDP-43 TAR DNA-binding protein
  • kits for assessing the absence or presence of a neurodegenerative disease in a subject comprising characterizing TDP-43 in a tissue of the subject. Also provided are methods for diagnosing a neurodegenerative disease in a subject comprising contacting a tissue of the subject with an antibody that binds to TDP-43, and determining the extent of binding of the antibody to the tissue. The present invention also pertains to methods of determining efficacy of a drug against a neurodegenerative disease comprising determining whether the drug modulates the activity of a modified form of TDP-43. There are also disclosed purified antibodies that bind to TDP-43 or fragments thereof. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 depicts the use of immunohistochemical screening to identify TDP-43 as the major disease protein in UBIs of FTLD-U.
  • FIG. 2 shows the results of studies to identify protein spots for LC -MS/MS analyses.
  • FIG. 3 shows the spectrum of FTLD-U neuropathology detected by anti-TDP- 43 antibodes.
  • FIG. 4 provides experimental results demonstrating that TDP-43 immunoreactivity is detected in UBIs of all FTLD-U cases but not in inclusions of other neurodegenerative diseases.
  • FIG. 5 provides the results of biochemical analyses of TDP-43 in sporadic and familial FTLD-U.
  • FIG. 6 depicts the results of studies demonstrating that pathological TDP-43 is hyperphosphorylated and ubiquitinated.
  • FIG. 7 illustrates the finding that UBIs and ALS are immunolabeled by anti- TDP-43 antibodies.
  • FIG. 8 demonstrates that hyperphosphorylated TDP-43 disease protein is recovered from multiple central nervous system regions of sporadic ALS cases.
  • TDP-43 TAR DNA-binding protein
  • AD Alzheimer's disease
  • TDP-43 is the equivalent in other neurodegenerative diseases, including FTD/ ALS.
  • kits for assessing the absence or presence of a neurodegenerative disease in a subject comprising characterizing TDP-43 in a tissue of said subject.
  • the disclosed methods can further comprise comparing the TDP-43 in the tissue of the subject with data for TDP-43 in a tissue of a subject in which the neurodegenerative disease is known to be absent, or with data for TDP-43 in a tissue of a subject in which the neurodegenerative disease is known to be present, or with both.
  • the methods may comprise the additional step of comparing TDP-43 from a test subject with TDP-43 from a subject or subjects in which the presence or absence of a neurodegenerative disease is known.
  • the neurodegenerative disease can be frontotemporal lobar degeneration.
  • the neurodegenerative disease can be amyotrophic lateral sclerosis. All TDP-43 -affected neurodegenerative diseases are contemplated as being within the scope of the present invention.
  • the characterization of TDP-43 in the tissue of the subject can comprise detecting certain post-translational modifications of TDP-43. As provided in Example 3, infra, it has been discovered that specific biochemical modifications can give rise to pathological forms of TDP-43.
  • the characterizing can comprise determining the phosphorylation state of TDP-43.
  • the characterization can also comprise determining the ubiquitination state of TDP- 43. Exemplary methods for determining the phosphorylation or ubiquitination state of TDP-43 are disclosed in Example 3, below, and alternative methods are readily appreciated by those skilled in the present art.
  • the characterization can also comprise determining the presence or absence of C-terminal breakdown or cleavage fragments of TDP-43.
  • the C-terminal fragments can comprise fragments of from about 24 kD to about 26 kD.
  • the molecular signature of the TDP-43 protein can include the presence of C-terminal breakdown or cleavage products migrating at -25 kD.
  • the characterization can include the detection of similar or other post-translational modifications of TDP-43.
  • TDP-43 is a ubiquitously-expressed, highly conserved nuclear protein (Y. M. Ayala et al, J. MoI. Biol. 348, 575 (2005)).
  • the tissue in which the TDP-43 protein is characterized in accordance with the disclosed methods of assessing the absence or presence of a neurodegenerative disease is preferably a cerebrospinal tissue, i.e., tissue located in or derived from the brain or spinal cord or a combination thereof.
  • the tissue can be of the frontal cortex, temporal cortex, hippocampus, or brain stem, or a combination thereof.
  • a tissue of refers both to tissue that is located in situ and to tissue that has been partially or fully moved within or extracted from the subject; as such, all manners of access to tissue are contemplated as being within the scope of the present invention.
  • kits for diagnosing a neurodegenerative disease in a subject comprising contacting a tissue of the subject with an antibody that binds to TDP-43 or a fragment thereof; and, determining the extent of binding of the antibody to the tissue.
  • the tissue has a lesion.
  • the lesion may be a ubiquitin-positive, tau- and ⁇ -synuclein-negative inclusion ("UBI").
  • UBI ubiquitin-positive, tau- and ⁇ -synuclein-negative inclusion
  • TDP-43 is present in UBIs, for example, in UBIs of subjects having fronto temporal lobar degeneration or amyotrophic lateral sclerosis.
  • the UBI may be cytoplasmic, neuritic, or nuclear.
  • the tissue with which the anti-TDP-43 antibody is contacted is tissue of the central nervous system (i.e., cerebrospinal tissue).
  • tissue of the central nervous system i.e., cerebrospinal tissue.
  • exemplary cerebrospinal tissue includes tissue of the hippocampus, neocortex, brain stem, and spinal cord.
  • Antibodies for use in the disclosed methods for diagnosing a neurodegenerative disease may be purchased from a commercial vendor (e.g. , Mouse Anti-Human TDP-43 Monoclonal Antibody; Abnova Corp., Taipei City, Taiwan), or may be prepared according to established protocols or as described herein (see Example 1).
  • the antibodies are purified antibodies that bind to TDP-43 or fragments thereof in ubiquitin-positive, tau- and ⁇ -synuclein-negative inclusions in subjects having Type 1 ubiquitin-positive frontotemporal lobar degeneration, or in subjects having Type 2 ubiquitin-positive frontotemporal lobar degeneration, which are also disclosed and claimed herein.
  • novel anti-TDP-43 antibodies themselves, including purified antibodies that bind to TDP-43 or fragments thereof in ubiquitin-positive, tau- and ⁇ -synuclein-negative inclusions in subjects having Type 1 ubiquitin-positive frontotemporal lobar degeneration, or in subjects having Type 2 ubiquitin-positive frontotemporal lobar degeneration.
  • Antibodies that bind TDP- 43 in UBIs can be produced according to established protocols or as described herein or using variations thereon.
  • the extent of binding of the anti-TDP-43 antibody to the tissue may be determined by techniques recognized by those skilled in the art. Secondary antibodies that are directed to a species-specific portion of the anti-TDP-43 primary antibody may be bound to a detection label and contacted with the tissue after or contemporaneously with the contacting of the tissue with the anti-TDP-43 antibody. Alternatively, the anti-TDP-43 antibody may be directly conjugated to a detection label.
  • Detection labels or tags are well known in the art and may include fluorophores, gold nanoparticles, biotin, alkaline phosphatase, horseradish peroxidase, and the like, Immunohistochemical techniques are also widely understood by those skilled in the art.
  • Also provided are methods for determining the efficacy of a drug against a neurodegenerative disease comprising determining whether the drug modulates the activity of a modified form of TDP-43.
  • the neurodegenerative disease is frontotemporal lobar degeneration or amyotrophic lateral sclerosis, although other TDP-43 - affected diseases are also contemplated.
  • Neurodegenerative diseases can be defined by the presence of ubiquitinated, misfolded protein aggregates in the cytoplasm and/or nucleus of nerve cells. M. S. Forman, J. Q. Trojanowski, V. M.-Y. Lee, Nat. Med. 10, 1055 (2004). Disease proteins in neurodegenerative diseases are also often pathologically phosphorylated. V. M.-Y.
  • the modified form of TDP-43 in the instant methods may be a hyperphosphorylated form of TDP-43.
  • the modified form of TDP-43 may also be a ubiquitinated form of TDP-43, or may comprise C-terminal breakdown or cleavage fragments of TDP-43.
  • the breakdown or cleavage fragments may comprise fragments of from about 24 kD to about 26 kD.
  • the determination of whether the drug modulates the activity of a modified form of TDP-43 may be made as of the drug's effect in vivo, e.g., in a tissue of a subject, or may be made as of the drug's effect on a modified form of TDP-43 that has been removed from a tissue.
  • Some embodiments of the provided methods comprise determining whether said drug modulates the activity of a modified form of TDP-43 in a cerebrospinal tissue of a subject.
  • the cerebrospinal tissue may have a lesion.
  • the lesion can be a ubiquitin-positive, tau- and ⁇ -synuclein-negative inclusion (UBI), and the UBI can be cytoplasmic, neuritic, or nuclear.
  • UBI ubiquitin-positive, tau- and ⁇ -synuclein-negative inclusion
  • the determination of whether a drug effects the modulation of a modified form of TDP-43 can follow the contacting of the drug with the modified form of TDP-43.
  • the contacting of the drug with the modified form of TDP-43 which may be performed in the context of, inter alia, cell or tissue culture, live animals, human patients, or under a variety of experimental conditions readily recognized by those skilled in the art, can comprise incubation or inoculation with the drug, although other means of contacting the drug with the modified form of TDP-43 are also contemplated.
  • EXAMPLES [0033] The present invention is further defined in the Examples included herein.
  • FIG. 1 depicts the use of immunohistochemical screening to identify TDP-43 as the major disease protein in UBIs of FTLD-U.
  • FIG. IA-D shows that novel MAb 182 specifically labels the ubiquitin-positive long neuritic UBIs predominantly in the upper cortical layers in FTLD-U Type 1 cases (FIG. IA, B), while MAb 406 specifically immunostains numerous UBIs in FTLD-U Type 2 cases (FIG. 1C, D).
  • FIG. IA-D shows that novel MAb 182 specifically labels the ubiquitin-positive long neuritic UBIs predominantly in the upper cortical layers in FTLD-U Type 1 cases (FIG. IA, B), while MAb 406 specifically immunostains numerous UBIs in FTLD-U Type 2 cases (FIG. 1C, D).
  • MAbs 182 and 406 detect disease-specific bands ⁇ 24 kD and 26 kD, respectively from urea fractions of frontal gray matter extracts of FTLD-U Type 1 (case #11) and Type 2 (case #18) in immunoblots, but not from AD or CO.
  • Anti-tau MAbs T14/46 which detected pathological hyperphosphorylated tau from AD brains is included here as disease control.
  • the scale bar shown in FIG. IA corresponds to 25 ⁇ m for FIG. IA-D.
  • Frozen brain tissues and fixed, paraffin-embedded tissue blocks were obtained from following institutions: the Center for Neurodegenerative Disease Research (CNDR) Brain Bank at the University of Pennsylvania, USA; Center for Neuropathology and Prion Research Brain Bank at the University of Kunststoff, Germany; Department of Pathology, University of British Columbia, Canada (source of UBC- 17); Department of Neurosciences, University of California San Diego, USA (source of HDDD2). Consent for autopsy was obtained from legal representative from all subjects in accordance with local Institutional Review Boards. Neuropathological diagnostic assessment of FTLD-U, PiD, ALS, AD, DLB, PD, MSA, PSP, CBD, NIFID and neuropathologically normal controls (CO) was performed in accordance with published guidelines.
  • Antibodies used in this study included: 1) anti-ubiquitin antibodies: mouse MAb 1510 (Chemicon, Temecula, CA), rabbit polyclonal antibody (Dako, Carpinteria, CA), mouse MAb UblB4 (unpublished, CNDR), 2) anti-tau antibodies: mouse MAbs T14 and T46 (CNDR) (1, 2), mouse MAb PHF-I (3) (a gift from Dr. P.
  • anti-TDP-43 antibodies rabbit polyclonal antibody (ProteinTech Group, Chicago, IL); mouse MAb 2E2-D3 (Abnova Corp., Taipei, Taiwan), 4) anti-FTLD-U antibodies: MAbs 182 and 406 (see below for antibody production), 5) anti- ⁇ -synuclein: rat MAb 15G7 (4), and 6) anti- ⁇ -internexin (Zymed Laboratories Inc., San Francisco, CA).
  • Frozen sections (10 ⁇ m) from FTLD-U brains were used for screening of newly generated MAbs. Briefly, frozen sections were air-dried (30 min), fixed in icecold acetone (5 min) and air- dried (30 min) again. Endogenous peroxidase was quenched with 0.3% H 2 O 2 in methanol (15 min) and immunohistochemistry performed as described for paraffin-embedded sections. Double-labeling immunofluorescence was performed as previously described (D. M. Sampathu et al.) using Alexa Fluor 488 and 594 conjugated secondary antibodies (Molecular Probes, Eugene, OR).
  • the SARK insoluble materials were extracted in 0.25 mL/g urea buffer (7M urea, 2M thiourea, 4% 3-[(3- Cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), 30 mM Tris, pH 8.5). Proteins were resolved in Tris-glycine 5-20% gradient SDS-PAGE, transferred to nitrocellulose and probed with primary and secondary antibodies (horseradish peroxidase-conjugated anti- mouse IgG or anti-rabbit IgG (Jackson ImmunoReasearch, West Grove, PA)).
  • Murine MAbs 406 (case # 18) and 182 (case #11) were generated using high Mr (>250 kD) and Mr 20-30 materials, respectively, from urea fractions of FTLD-U frontal cortex as immunogen as previously described (D. M. Sampathu et al). Briefly, urea fractions (100-150 ⁇ g protein/mouse) were separated using 5-20% gradient SDS-PAGE, and the portion of the gel containing proteins with Mr > 250 kD (including the stacking gel) or Mr 20-30 was minced, homogenized in phosphate-buffered saline, emulsified with incomplete Freund's adjuvant, and injected subcutaneously into BALB/c mice.
  • Boost injections 25-50 ⁇ g protein/mouse were made on days 21, 35, and 49, followed by intraperitoneal injection of immunogens without adjuvant on day 63. Fusion was conducted on day 66 using Sp2 myeloma cells as fusion partner. Resulting hybridoma supernatants were screened by immunohistochemistry on paraffin-embedded and frozen sections of FTLD-U cortex known to contain UBIs. All positive MAbs were determined to be of the IgM class using standard light and heavy chain antibody subtype analysis.
  • 2D-PAGE Two-Dimensional (2D)-PAGE.
  • 2D-P AGE was performed with the ZOOM® IPGRunnerTM system (Invitrogen Corp., Carlsbad, CA) using pH 3-lOL or pH 3-10NL strip for the first dimension separation and 4-12% Bis-Tris PAGE for the second dimension according to manufacturer's protocol.
  • Gels were either stained with Colloidal Blue (Invitrogen Corp., Carlsbad, CA) or transferred to nitrocellulose membrane and immunblotted with MAbs 406 or 182.
  • Protein spots corresponding to immuno-positive spots were excised from gels, digested with sequencing grade trypsin and the peptides separated by nano liquid chromatography on a Cl 8 capillary column.
  • Eluted peptides were sequenced on line with a nanospray Qstar-XL mass spectrometer (Applied Biosystems, Foster City, CA). Data were acquired and analyzed with Analyst QS software, and Mascot dll script was used for database search. Protein total score >70 with confidence >95% was accepted as positive identification.
  • Table 1 lists the demographic characteristics of FTLD-U cases used in this study.
  • UBC- 17 and HDDD2 are families with published linkage to chromosome 17 (G. M. McKhann et al, Arch. Neurol. 58, 1803 (2001); M. S. Forman et ah, Ann. Neurol. 59, 952 (2006)).
  • TDP-43 Three peptides corresponding to amino acid residues 252-263, 276-293 and 409-414 of the TAR-DNA-binding protein 43 (TDP-43) were identified (data not shown). Significantly, the 409-414 peptide is at the extreme C terminus of TDP-43 suggesting that both the 24 and 26 kD fragments are truncated in the middle of TDP-43 and extend to its C-terminus.
  • TDP-43 coded for by TARDP on chromosome 1 was initially cloned as a human protein capable of binding to a polypyrimidine-rich motif in the HIV transactive response DNA (S. H. Ou, F. Wu, D. Harrich, L. F. Garcia-Martinez, R. B. Gaynor, J. Virol. 69,3584 (1995)) and later identified independently as part of a complex involved in the splicing of the cystic fibrosis transmembrane conductance regulator gene (E. Buratti et al, EMBO J. 20, 1774 (2001)). It contains two RNA-recognition motifs (RRMs) as well as a glycine-rich C-terminal sequence (H.
  • RRMs RNA-recognition motifs
  • EXAMPLE 2 Demonstrating that TDP-43 is present in Familial and Sporadic FTLD-U and Co-localizes with Ubiquitin
  • FIG. 3 depicts the results of tests designed to reveal the spectrum of FTLD-U neuropathology detected by anti-TDP-43 antibodies.
  • FIG. 2A-2L shows: double-label immunofluorescence demonstrating immunolabeling of long neuritic profiles from Type 1 cases with MAb 182 (A) and anti-TDP-43 (B); cytoplasmic UBIs in Type 2 with MAb 406 (D) and anti-TDP-43 (E); UBIs in Type 3 with anti-ubiquitin (G) and anti-TDP-43 (H); UBIs in HDDD2 with anti-ubiquitin (J) and anti-TDP-43 (K). Overlays demonstrating co-localization of the corresponding immunostainings are shown in FIG.
  • Lewy- body like round inclusions in motor neurons of spinal cord (S), and lentiform (T) as well as round (U) intranuclear UBIs in HDDD2 are labeled by anti-TDP-43.
  • Scale bar in FIG. 3 A corresponds to 50 ⁇ m (A-P, and S), 25 ⁇ m (Q and R) and 20 ⁇ m (T and U).
  • UBIs in the motor neurons of spinal cord and brainstem in FTLD-U cases with and without clinical signs of MND were immunostained by anti-TDP-43 antibodies (FIG. 3 S and data not shown) as were the nuclear UBIs characteristic of FTDP- 17U cases (FIG. 3 T, 3U).
  • TDP-43 is a highly specific and novel disease protein found in neuronal UBIs of all FTLD-U subtypes and FTDP- 17U.
  • EXAMPLE 3 Demonstrating that TDP-43 in Familial and Sporadic FTLD-U is Hyperphosphorylated, Ubiquitinated, and Cleaved to Generate Disease-Specific Insoluble C- terminal Fragments
  • Immunoprecipitated proteins were eluted with SDS sample buffer (10 mM Tris, pH 6.8, 1 mM EDTA, 40 mM DTT, 1% SDS, 10% sucrose), resolved by 5-20% SDS-PAGE and analyzed by immunoblot as described above.
  • FIG. 5 A provides the results of an immunoblot analysis of sequential extracts from frontal cortex of FTLD-U Type 1 and 2 with rabbit anti-TDP-43, and shows pathologic -25 kD bands (*), 45 kD band (**), and high M r smear (***) in the urea fraction.
  • Column 1 is the LS fraction;
  • column 2 is the HS/TX fraction;
  • column 3 is the SARK fraction; and
  • column 4 is the UREA fraction.
  • 5B is an immunoblot analysis of urea fractions from hippocampal/temporal cortex of FTLD-U Types 1-3 and frontal cortex of FTDP- 17U, and shows the distinct pathological profile of TDP-43 which was not detectable in other neurodegenerative diseases and CO brains.
  • TDP-43 protein While full length TDP-43 protein was present in all soluble and insoluble fractions of FTLD-U Type 1, Type 2 as well as AD and CO, a strong labeling of bands -25 kD similar to bands detected by Mab 182 and 406 were only detectable in the urea fractions of FTLD-U Type 1 and 2, respectively (* in FIG. 5A). Further, a higher molecular band -45 kD and a high molecular smear were specifically recognized by TDP-43 antibodies in the urea fractions of the FTLD-U cases compared to AD and CO (** and ***, respectively, in FIG. 5A).
  • the molecular signature of the TDP-43 disease protein includes the presence of C-terminal breakdown/cleavage products migrating at -25 kD, a -45 kD Mr variant and a high Mr TDP-43 -immunoreactive smear, although levels of these pathological species of TDP-43 varied, which may reflect the extent of TDP-43 neuropathology in diverse brain regions of different FTLD-U cases.
  • TDP-43 To determine the specific biochemical modifications giving rise to these pathological forms of TDP-43, the phosphorylation state of TDP-43 in FTLD-U was investigated, since disease proteins in other neurodegenerative disorders (e.g., tau and ⁇ - synuclein) are pathologically phosphorylated (see V. M.-Y. Lee, M. Goedert, J. Q. Trojanowski, Ann. Rev. Neurosci. 24, 1121 (2001); H. Fujiwara et al, Nat. Cell Biol. 4, 160 (2002)) and TDP- 43 contains numerous potential phosphorylation sites (predicted by NetPhos 2.0 server).
  • TDP-43 is the Disease Protein in UBIs of Sporadic ALS
  • FTLD-U and ALS may be parts of a single clinicopathological spectrum, and that they may share similar pathogenic mechanisms which affect different populations of CNS neurons.
  • Classic ALS cases were examined for the presence of TDP-43 positive UBIs (case # 54-72, Table 1, supra).
  • FIG. 7A-L depicts double-label immunofluorescence of ALS UBIs with anti- ubiquitin (A,D,G, J) and anti-TDP-43 (B,E,H,K) showing co-localization in round UBIs in spinal cord motor neuron (A-C), skein-like UBIs in spinal cord motor neuron (D-F), Lewy-body like UBIs in hypoglossal neuron (G-I) and skein-like UBIs in hypoglossal neuron (J-L). Merge images of sections are shown in FIG. 7C, F, I, and L.
  • 7M-S shows DAB-immunostaining with anti- TDP -43 labels Lewy-body like (M), round (N) and skein-like inclusions (O and P) in motor neurons of the spinal cord and medulla.
  • Cytoplasmic UBIs in hippocampal dentate granule neurons (Q) and few UBIs in frontal cortex (R and S) were also stained by TDP-43.
  • Asterisks in (Q) depict normal nuclear staining, arrows point to missing nuclear staining in UBI-bearing neurons (arrowheads).
  • the scale bar in FIG. 7A corresponds to 25 ⁇ m (FIG. 7A-P), and the scale bar in FIG. 7Q corresponds to 50 ⁇ m (FIG. 7Q-S).
  • FIG. 8 A shows immunoblots of urea fractions from frontal cortex (FC), temporal cortex (TC), and spinal cord (SC) of multiple ALS cases probed with anti-TDP-43 antibody, and demonstrates variable presence of the pathologic C-terminal fragments (*), 45 kD band (**), and high M 1 - smear (***).
  • FC frontal cortex
  • TC temporal cortex
  • SC spinal cord
  • 8B depicts immunoblots of dephosphorylated ALS urea extracts with alkaline phosphatase with anti-TDP-43 antibody, and revealed a collapse of the 45 kD band into the 43 kD band as well as an increase in complexity of truncated TDP-43 -immunoreactive bands ⁇ 23-27 kD.
  • FIG. 8A A disease-specific protein signature for TDP-43 in the ALS samples resembling that described above for FTLD-U was detected (FIG. 8A). Similar to FTLD-U, hyperphosphorylated 45 kD TDP-43 and its 25 kD breakdown products as well as the high M 1 - smear of TDP-43 proteins were highly variable from one CNS region to another and from one ALS case to another, while dephosphorylation of the urea fractions demonstrated that the 45 kD band in ALS corresponds to pathologically hyperphosphorylated TDP-43 as in FTLD-U (FIG. 8B). However, since the presence of UBIs in ALS cases is more variable than FTLD-U, not all brain regions examined in all cases exhibited pathological TDP-43.
  • TDP-43 as the major disease protein in UBIs of FTLD-U and ALS that form the signature lesions of these disorders. This was accomplished by generating novel MAbs to insoluble material from UBI-enriched brain regions of FTLD-U brains in conjunction with immunohistochemical and biochemical analyses complemented by parallel studies using two independently generated anti-TDP-43 specific antibodies.
  • TDP-43 is the major disease protein of UBIs in FTLD-U and ALS.
  • TDP-43 is the major disease protein of UBIs in FTLD-U and ALS.

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Abstract

L'invention concerne des procédés d'évaluation de l'absence ou de la présence d'une maladie neurodégénérative chez un sujet, ce procédé consistant à caractériser TDP-43 dans un échantillon de tissu prélevé chez le sujet. L'invention concerne également des méthodes de diagnostic d'une maladie neurodégénérative chez un sujet, ainsi que des méthodes visant à déterminer l'efficacité d'un médicament pour lutter contre une maladie neurodégénérative. L'invention concerne également de nouveaux anticorps qui se lient à TDP-43.
PCT/US2007/020795 2006-09-29 2007-09-26 Détection de maladies neurodégénératives WO2008042190A2 (fr)

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WO2011005628A1 (fr) * 2009-03-26 2011-01-13 The Trustees Of The University Of Pennsylvania Détection d'une maladie neurodégénérative
WO2013064934A1 (fr) 2011-11-04 2013-05-10 International Centre For Genetic Engineering And Biotechnology - Icgeb Modèle cellulaire pour le criblage de molécules affectant l'agrégation de tdp-43
US10060933B2 (en) 2011-06-22 2018-08-28 Universite Laval Methods for diagnosis and treatment of amyotrophic lateral sclerosis based on an increased level of interaction between TDP-43 polypeptide and NF-KB P65 polypeptide
US10202443B2 (en) 2014-12-05 2019-02-12 UNIVERSITé LAVAL TDP-43-binding polypeptides useful for the treatment of neurodegenerative diseases
WO2023194565A1 (fr) 2022-04-08 2023-10-12 Ac Immune Sa Molécules de liaison anti-tdp-43
US11938150B2 (en) 2015-11-02 2024-03-26 Neuvivo, Inc. Treatment of neurodegenerative disease with sodium chlorite

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8354236B2 (en) 2006-09-29 2013-01-15 The Trustees Of The University Of Pennsylvania Detection of neurodegenerative disease
WO2011005628A1 (fr) * 2009-03-26 2011-01-13 The Trustees Of The University Of Pennsylvania Détection d'une maladie neurodégénérative
US10060933B2 (en) 2011-06-22 2018-08-28 Universite Laval Methods for diagnosis and treatment of amyotrophic lateral sclerosis based on an increased level of interaction between TDP-43 polypeptide and NF-KB P65 polypeptide
WO2013064934A1 (fr) 2011-11-04 2013-05-10 International Centre For Genetic Engineering And Biotechnology - Icgeb Modèle cellulaire pour le criblage de molécules affectant l'agrégation de tdp-43
US10202443B2 (en) 2014-12-05 2019-02-12 UNIVERSITé LAVAL TDP-43-binding polypeptides useful for the treatment of neurodegenerative diseases
US11938150B2 (en) 2015-11-02 2024-03-26 Neuvivo, Inc. Treatment of neurodegenerative disease with sodium chlorite
US11938147B2 (en) 2015-11-02 2024-03-26 Neuvivo, Inc. Treatment of neurodegenerative disease with sodium chlorite
US11938148B2 (en) 2015-11-02 2024-03-26 Neuvivo, Inc. Treatment of neurodegenerative disease with sodium chlorite
US11938149B2 (en) 2015-11-02 2024-03-26 Neuvivo, Inc. Treatment of neurodegenerative disease with sodium chlorite
WO2023194565A1 (fr) 2022-04-08 2023-10-12 Ac Immune Sa Molécules de liaison anti-tdp-43

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