WO2008042190A2 - Detection of neurodegenerative disease - Google Patents

Abstract

Provided are methods of assessing the absence or presence of a neurodegenerative disease in a subject comprising characterizing TDP-43 in a tissue sample from said subject. Also disclosed are methods for diagnosing a neurodegenerative disease in a subject, and methods for determining the efficacy of a drug against a neurodegenerative disease. Novel antibodies that bind to TDP-43 are also provided.

Description

DETECTION OF NEURODEGENERATIVE DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial Number 60/848,318, filed September 29, 2006, which is herein incorporated by reference in its entirety.

GOVERNMENT RIGHTS

[0002] Research leading to the disclosed invention was funded, in part, by the U.S. National Institutes of Health, Grant Nos. AG17586, to V.M.-Y. Lee, and AG10124, to J.Q. Trojanowski. Accordingly, the United States Government may have rights in the invention described herein.

FIELD OF THE INVENTION

[0003] 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.

BACKGROUND OF THE INVENTION

[0004] Neurodegenerative diseases are 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). Although significant advances have resulted in the identification of the misfolded disease proteins in many neurodegenerative disorders, the identity of the ubiquitinated disease protein(s) in UBIs (defined here as ubiquitinated cytoplasmic, nuclear and dystrophic neuritic inclusions) in FTLD-U, the most common form of frontotemporal dementias (FTDs) (J. R. Hodges et al, Ann. Neurol. 56, 399 (2004); A. M. Upton, C. L. White, 3rd, E. H. Bigio, Acta Neuropathol. (Berl) 108, 379 (2004); J. K. Johnson et al, Arch. Neurol. 62, 925 (2005); J. Shi et al., Acta Neuropathol. (Berl) 110, 501 (2005)), and amyotrophic lateral sclerosis (ALS) have remained enigmatic.

[0005] 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)). Clinically, FTDs present with progressive changes in social, behavioral, and/or language dysfunction (G. M. McKhann et al, Arch. Neurol. 58, 1803 (2001); D. Neary et al, Neurology 51, 1546 (1998); M. Grossman, J. Int. Neuropsychol. Soc. 8, 566 (2002)) and less commonly with parkinsonism or motor neuron disease (MND). J. R. Hodges et al, Ann. Neurol. 56, 399 (2004); C. Lomen-Hoerth, T. Anderson, B. Miller, Neurology 59, 1077 (2002). Conversely, ALS, a common form of MND, is often associated with FTD (C. Lomen-Hoerth, et al.) and UBIs as in FTLD-U (M. S. Forman et al, Ann. Neurol. 59, 952 (2006)). Thus, the clinical overlap and shared ubiquitin pathologies in FTLD-U and ALS syndromes suggest they represent different ends of a clinicopathological spectrum of the same neurodegenerative disorder similar to amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam tauopathy. J. Q. Trojanowski et al, Exp. Neurol. 176, 1 (2002).

[0006] Although diverse neuropathology underlies the clinical syndrome of FTDs, genetic, immunohistochemical, and biochemical data are incorporated into its current nosology (G. M. McKhann et al, Arch. Neurol. 58, 1803 (2001)), which broadly divides cases into those with tau-positive inclusions (e.g., Pick's disease [PiD], corticobasal degeneration [CBD], progressive supranuclear palsy [PSP], etc.), versus FTLD-U with UBIs. M. S. Forman et al, Ann. Neurol. 59, 952 (2006). More than 30% of FTD patients have a positive family history as exemplified by those with autosomal dominant inheritance linked to chromosome 17. However, FTD with parkinsonism linked to chromosome 17 (FTDP- 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.

[0007] Additionally, a number of these FTDP- 17 families do not develop tau pathology and lack MAPT gene mutations, but instead develop UBIs (designated as FTDP- 17U). C. L. Lendon et al, Neurology 50, 1546 (1998); R. Rademakers et al, Mol.Psychiatry 7, 1064 (2002); /. R. Mackenzie et al, Brain 129, 853 (2006). Recently, mutations that result in premature termination of the coding sequence for progranulin (PGRN) were identified and shown to be the disease-causing gene in FTDP- 17U. M. Baker et al, Nature. In press (2006). However, since PGRN is not incorporated into UBIs in FTDP-17U (id.), the identity of the disease protein in UBIs of sporadic and familial FTLD-U has remained enigmatic.

[0008] Ubiquitin-positive, tau- and α-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.

[0009] Pathological heterogeneity in the distribution and morphological characteristics of UBIs could signify that different disease proteins underlie FTLD-U variants or that a single protein is differentially modified in the variants. D. M. Sampathu et al, Am. J. Pathol. In press (2006). For example, at least three FTLD-U subtypes have been identified: Type 1 with a predominance of long neuritic profiles in superficial cortical layers; Type 2 with UBIs mainly in superficial and deep cortical layers; Type 3 with signature ring-shaped UBIs and short neuritic profiles predominantly in superficial cortex. Id. Immunohistochemical analyses with novel monoclonal antibodies (MAbs), generated by immunization of mice with high M₁- insoluble material prepared by biochemical fractionation of FTLD-U brains supports the distinction of these FTLD-U subtypes (id.), but these MAbs did not enable identification of the disease protein in the UBIs of FTLD-U (id.).

SUMMARY OF THE INVENTION

[0010] It has now been discovered that TAR DNA-binding protein (TDP-43) is the major disease protein in UBIs of FTLD-U and ALS that form the signature lesions of these disorders. The inventors have determined that pathologically-altered TDP-43 is present in all sporadic and familial FTLD-U as well as ALS cases.

[0011] Provided herein are methods of 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

[0012] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, there are shown in the figures exemplary embodiments of the invention; however, the invention is not limited to the specific methods, compositions, characteristics, and devices disclosed.

[0013] FIG. 1 depicts the use of immunohistochemical screening to identify TDP-43 as the major disease protein in UBIs of FTLD-U.

[0014] FIG. 2 shows the results of studies to identify protein spots for LC -MS/MS analyses.

[0015] FIG. 3 shows the spectrum of FTLD-U neuropathology detected by anti-TDP- 43 antibodes.

[0016] 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.

[0017] FIG. 5 provides the results of biochemical analyses of TDP-43 in sporadic and familial FTLD-U.

[0018] FIG. 6 depicts the results of studies demonstrating that pathological TDP-43 is hyperphosphorylated and ubiquitinated.

[0019] FIG. 7 illustrates the finding that UBIs and ALS are immunolabeled by anti- TDP-43 antibodies.

[0020] FIG. 8 demonstrates that hyperphosphorylated TDP-43 disease protein is recovered from multiple central nervous system regions of sporadic ALS cases.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0021] In the present disclosure the singular forms "a," "an," and "the" include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to "a tissue" is a reference to one or more tissues and equivalents thereof known to those skilled in the art, a reference to "a drug" is a reference to one or more drugs and equivalents thereof known to those skilled in the art, and so forth. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Where present, all ranges are inclusive and combinable.

[0022] TAR DNA-binding protein (TDP-43) has now been identified as the disease protein in UBIs of all subtypes of sporadic FTLD-U and familial FTDP- 17U as well as in ALS. This discovery provides a mechanistic link between dementia and motor neuron disease. Like beta amyloid and tau tangles that are the major pathological lesions in Alzheimer's disease (AD), TDP-43 is the equivalent in other neurodegenerative diseases, including FTD/ ALS.

[0023] Provided are methods of 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. Thus, 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.

[0024] In some embodiments, the neurodegenerative disease can be frontotemporal lobar degeneration. Alternatively, 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.

[0025] 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. For example, 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. As provided below, 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.

[0026] 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. Within the brain, the tissue can be of the frontal cortex, temporal cortex, hippocampus, or brain stem, or a combination thereof. As used herein the phrase "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.

[0027] 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 or a fragment thereof; and, determining the extent of binding of the antibody to the tissue. In preferred embodiments, the tissue has a lesion. The lesion may be a ubiquitin-positive, tau- and α-synuclein-negative inclusion ("UBI"). As provided herein (see Examples 2 and 4), 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. Preferably, the tissue with which the anti-TDP-43 antibody is contacted is tissue of the central nervous system (i.e., cerebrospinal tissue). Exemplary cerebrospinal tissue includes tissue of the hippocampus, neocortex, brain stem, and spinal cord.

[0028] 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). In preferred embodiments, the antibodies are purified antibodies that bind to TDP-43 or fragments thereof in ubiquitin-positive, tau- and α-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. Thus, also disclosed are novel anti-TDP-43 antibodies themselves, including purified antibodies that bind to TDP-43 or fragments thereof in ubiquitin-positive, tau- and α-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.

[0029] In accordance with the disclosed methods of diagnosing a neurodegenerative disease, 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.

[0030] 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. In preferred embodiments, 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. Lee, M. Goedert, J. Q. Trojanowski, Ann. Rev. Neurosci. 24, 1121 (2001); H. Fujiwara et al, Nat. Cell Biol. 4, 160 (2002). 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.

[0031] 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. In such cases, the cerebrospinal tissue may have a lesion. The lesion can be a ubiquitin-positive, tau- and α-synuclein-negative inclusion (UBI), and the UBI can be cytoplasmic, neuritic, or nuclear.

[0032] 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. For example, 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. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only, and should not be construed as limiting the appended claims From the present disclosure and these examples, one skilled in the art can ascertain certain characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. EXAMPLE 1: Identification ofTDP-43 as the Disease Protein in FTLD-U

[0034] 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. IE shows that 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 μm for FIG. IA-D.

[0035] Brain tissue collection and neuropathological assessment. 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 Munich, 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.

[0036] Antibodies. 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. Davies), 3) 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-α-synuclein: rat MAb 15G7 (4), and 6) anti-α-internexin (Zymed Laboratories Inc., San Francisco, CA).

[0037] Immunohistochemical staining. The harvesting, fixation, and further processing of the tissue specimens used herein were conducted as described previously (D. M. Sampathu et al, Am. J. Pathol, in press (2006)). Briefly, tissue blocks from representative brain regions (frontal and temporal cortices, hippocampus, basal ganglia, medulla and spinal cord) were fixed with either 70% ethanol in 150 mM NaCl or phosphate-buffered 3.65% formaldehyde, and paraffin-embedded. Immunohistochemistry was carried out as described (D. M. Sampathu et al.) with sections pretreated with formic acid (5 min) to enhance anti-TDP-43 immunoreactivity. Frozen sections (10 μm) 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% H₂O₂ 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).

[0038] Sequential biochemical fractionation, dephosphorylation and immunoblot analysis. Post-mortem brain tissue was dissected, weighed, and sequentially extracted with buffers of increasing strength as previously described (5). Briefly, gray matter was extracted at 5 mL/g (volume/weight) with low salt (LS) buffer (10 mM Tris, pH 7.5, 5 mM EDTA, 1 mM DTT, 10% sucrose, and a cocktail of protease inhibitors), high salt-Triton (TX) buffer (LS + 1% Triton X-100 + 0.5M NaCl), myelin floatation buffer (TX buffer containing 30% sucrose), and sarkosyl (SARK) buffer (LS + 1% N-Lauroyl-sarcosine + 0.5 M NaCl). 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)). Blots were developed with Renaissance Enhanced Luminol Reagents (NEN Life Science Product, Inc., Boston, MA), and digital images were acquired using a Fujifilm Intelligent Darkbox II (Fuji Systems USA, Stamford, CT). Where indicated, TDP-43 was dephosphorylated by dialysis (50 mM Tris, 0.2 mM EDTA, pH 8.0) and treated with Escherichia coli alkaline phosphatase (Sigma, St. Louis MO) for 2h at 56°C. [0039] Generation of novel MAbs. 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 μg 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 μg 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.

[0040] Two-Dimensional (2D)-PAGE. 2D-P AGE was performed with the ZOOM® IPGRunner™ 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.

[0041] Results. New MAbs generated to TX-100 and sarkosyl insoluble, but urea soluble, fractions of frontal gray matter of FTLD-U Type 1 (FIG. 1, case #11) and FTLD-U Type 2 (FIG. 1, case #18) brains were screened by immunohistochemistry to select those for further analysis that labeled UBIs in paraffin-embedded and frozen sections from these 2 cases. Of these, MAb 182 generated from case #11 was highly specific for UBIs in FTLD-U Type 1, while MAb 406 generated from case #18 labeled specifically UBIs in FTLD-U Type 2 cases (FIG. 1C, ID). All Type 1 (cases #1-12, Table 1, below) and Type 2 (case # 13-26, Table 1, below) cases were immunostained by MAb 182 and MAb 406, respectively. Both MAbs demonstrated exquisite specificity for their corresponding FTLD-U subtypes, but they did not detect any pathology in FTLD-U Type 3 (case # 27-47, Table 1, below), FTDP-17U (case # 48-53, Table 1, below) or in other neurodegenerative disorders including Alzheimer's disease (AD), dementia with Lewy bodies (DLB), PiD, CBD, PSP and multiple system atrophy (MSA) (not shown).

Table 1, provided below, lists the demographic characteristics of FTLD-U cases used in this study.

TABLE l

* Patient also had severe AD pathology (CERAD C, Braak & Braak stage V-VI). Age and disease duration are given in years. Abbreviations: M = male, F = female, nr = not recorded.

As used in Table 1, 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)).

[0042] To further characterize the disease protein(s) recognized by MAbs 182 and 406, immunoblot analyses were conducted on urea fractions, similar to those used for immunization, from FTLD-U Type 1 (case #11) and FTLD-U Type 2 (case #18). Significantly, MAb 182 recognized an additional band ~ 24 kD in the urea fraction of FTLD-U Type 1 (case #11) that is not present in FTLD-U Type 2 (case #18) or neuropathologically confirmed AD and normal brains (CO), whereas MAb 406 detected a specific band ~ 26 kD in FTLD-U Type 2, but not in FTLD-U Type 1, AD and CO (FIG. IE). As expected, anti-tau antibodies detected insoluble pathological tau in AD, but not in FTLD-U Type 1, Type 2 or CO (FIG. IE), and the 24 and 26 kD bands were not detected by these MAbs in FTLD-U Type 3 and FTDP- 17U cases (not shown). To determine the identity of the 24 and 26 kD protein bands recognized by MAbs 182 and 406, respectively, 2-D PAGE immunoblots were performed using urea fractions from case #11 and #18. MAbs 182 and 406 immunolabeled protein spots ~ 25 kD with a pi ~ 3.5 (FIG. 2 A, C). The corresponding protein spots were identified on duplicate Coomassie blue stained 20- PAGE gels (FIG. 2B, D), excised, trypsin digested, and analyzed by LC -MS/MS. 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.

[0043] 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. Y. Wang, I. F. Wang, J. Bose, C. K. Shen, Genomics 83, 130 (2004)), and it is expressed ubiquitously in a variety of tissues including heart, lung, liver, spleen, kidney, muscle, and brain (E. Buratti et al.). Since the same peptides were recovered from protein spots detected by MAbs 182 and 406, this suggests that both MAbs recognize specific conformations or post-translational modifications of a C-terminal breakdown and/or cleavage product of TDP-43 that are unique to FTLD-U Type 1 and 2, respectively.

EXAMPLE 2: Demonstrating that TDP-43 is present in Familial and Sporadic FTLD-U and Co-localizes with Ubiquitin

[0044] 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. 3C, 3F, 31, and 3L. All sections are from frontal cortex. (M-U) Conventional immunohistochemistry of UBIs in FTLD-U cases with anti- TDP-43 reveals robust staining of long-neuritic profiles in Type 1 (M), cytoplasmic neuronal inclusions in Type 2 (N), cytoplasmic, comma-shaped inclusions in Type 3 (O) and HDDD2 (P) in frontal cortex. Strong anti-TDP-43 staining of UBIs (arrowheads) in hippocampal dentate granule neurins is shown in FIG. 3Q and 2R. Note clearing of nuclear TDP-43 (arrows) of inclusion bearing neurons compared to normal nuclear TDP-43 immunoreactivity (*). 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 μm (A-P, and S), 25 μm (Q and R) and 20 μm (T and U).

[0045] Anti-TDP-43 strongly immunolabeled inclusions were detected by MAb 182 in FTLD-U Type 1 cases (FIG. 3A-3C). Similarly, the inclusions in FTLD-U Type 2 cases immunolabeled by MAb 406 also were positive for TDP-43 (FIG. 3D-3F). Surprisingly, anti- TDP-43 robustly labeled UBIs that were not detected by MAbs 182 and 406 in FTLD-U Type 3 cases (FIG. 3G-3I) as well as the UBIs in familial FTDP-17U cases (FIG. 3J-3L). Indeed, two- color immunofluorescence studies showed that anti-TDP-43 detected at least as many UBIs as anti-ubiquitin antibodies or MAbs 182 and 406 in all FTLD-U cases examined (not shown).

[0046] Single-label immunohistochemistry revealed robust anti-TDP-43 staining of neuronal UBIs and dystrophic neurites in affected cortical regions of FTLD-U Type 1 (FIG. 3M, FIG. 4A, 4E), Type 2 (FIG. 3N, FIG. 4B, 4F) and Type 3 (FIG. 30, FIG. 4C, 4G) cases with the distinct morphology and distribution pattern characteristic of each of these FTLD-U subtypes (18). TDP-43 positive UBIs resembling the morphology and distribution of those described for FTLDU Type 3 were detected in two separate FTDP- 17U pedigrees (UBC- 17 (16) and HDDD2 (14)) (FIG. 3P, FIG. 4D, 4H). Furthermore, strong immunostaining was also observed in the UBIs of hippocampal dentate granule cells in all FTLD-U subtypes (FIG. 3Q, 3R). Notably, while physiological TDP-43 was detectable in the nuclei of unaffected neurons (asterisks in FIG. 3 Q, 3R), TDP-43 was undetectable in nuclei of neurons with UBIs (arrows and arrowheads in FIG. 3 Q, 3R) suggesting that TDP-43 is redistributed from the nucleus to the cytoplasm in affected neurons. Since many FTLD-U patients also develop MND associated with UBIs in brainstem and spinal cord neurons, we tested whether these lesions also are TDP-43 positive. Significantly, 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). Furthermore, UBIs in all FTLD-U subtypes (n = 47) as well as FTDP- 17U (including a subset (UBC- 17 cases) with PGRN gene mutations) (n = 6) listed in Table 1, supra, were detected by both rabbit polyclonal antibodies (FIG. 3) and a mouse MAb (data not shown) specific for TDP- 43. In contrast, none of the pathological inclusions in other neurodegenerative disorders (i.e., AD, PD, DLB, MSA, PiD, PSP, CBD, FTDP- 17T and neuronal intermediate filament inclusions disease (NIFID)) were immunolabeled with these anti-TDP-43 antibodies (FIG. 4I-4T and data not shown). Collectively, these studies show that 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

[0047] To characterize pathological TDP-43 protein in FTLD-U cases biochemically, samples of cortical gray matter from FTLD-U and FTDP- 17U brains were sequentially extracted with buffers of increasing strength for immunoblotting.

[0048] Immunoprecipitation. Urea fractions were dialyzed into RIPA buffer (0.1 % SDS, 1% NP40, 0.5% sodium dexoycholate, 5 raM EDTA, 150 mM NaCl, 50 mM Tris, pH 8.0), pre-absorbed with Protein A Sepharose, and immunoprecipitated with polyclonal TDP-43 antibody conjugated to Protein A Sepharose CL-4B (GE Healthcare Bio-Sciences, Piscataway, NJ). 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.

[0049] 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. FIG. 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.

[0050] 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). To demonstrate that this disease-specific protein signature of TDP-43 is present in all FTLD-U subtypes and familial FTDP- 17U, analysis was conducted of urea fractions extracted from hippocampus or frontal cortex of multiple cases (FIG. 5B). Notably, this distinct TDP-43 banding pattern was observed in all FTLD-U types except in unaffected regions (e.g., cerebellum), and it was FTLD-U specific since it was not detected in CO or in other neurodegenerative disorders (e.g., AD, PD, FTDP- 17T, DLB, MSA) (FIG. 5B and data not shown). Thus, these data indicate that 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.

[0051] 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 α- 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).

[0052] Alkaline phosphatase treatment of urea fractions from FTLD-U brains followed by immunoblot analysis demonstrated that the FTLD-U specific 45 kD band collapsed into the 43 kD band after dephosphorylation (FIG. 6A). Furthermore, dephosphorylation of the two C- terminal fragments also increased the immunobanding complexity revealing at least 4 fragments (FIG. 6A). These data suggest that abnormal hyperphosphorylation of TDP-43 might play a role in FTLD-U pathogenesis. Since UBIs are defined by ubiquitin immunoreactivities, we asked if TDP-43 recovered from urea fractions of FTLD-U brains is a substrate for ubiquitination. Immunoprecipitation of TDP-43 using the rabbit polyclonal anti-TDP-43 antibody followed by immunoblot analyses with both anti-TDP-43 and anti-ubiquitin antibodies unequivocally demonstrated that TDP-43 is ubiquitinated (FIG. 6B). EXAMPLE 4: TDP-43 is the Disease Protein in UBIs of Sporadic ALS

[0053] It has been suggested that 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).

[0054] 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. FIG. 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 μm (FIG. 7A-P), and the scale bar in FIG. 7Q corresponds to 50 μm (FIG. 7Q-S).

[0055] Although none of the inclusions typical of ALS were detected by MAbs 182 and 406 (not shown), double-immunofluorescence demonstrated that all UBIs with different morphologies (e.g., skein-like, round and Lewy body like inclusions) in motor neurons of ALS were robustly double-labeled by both the anti-TDP-43 and anti-ubiquitin antibodies (FIG. 7A- 7L), and these findings were confirmed by single-label immunohistochemistry (FIG. 7M-7P). Since a significant number of ALS patients demonstrate UBIs in hippocampus as well as frontal and temporal cortices with or without clinical signs of FTD (/. R. Mackenzie, H. Feldman, Acta Neuropathol.(Berl) 105, 98 (2003)), it was questioned if these UBIs are TDP-43 positive, and the present studies revealed that anti-TDP-43 antibodies immunolabeled UBIs in hippocampal dentate granule cells as well as neurons in frontal and temporal cortices in a subset of the studied ALS cases (FIG. 7Q-7S, and data not shown).

[0056] To determine if the distinct pathological TDP-43 protein profile seen in FTLD- U also is present in ALS brains, immunoblots of urea fractions of spinal cord as well as frontal and temporal cortices of ALS cases were probed with anti-TDP-43 antibodies. 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 M₁- smear (***). FIG. 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.

[0057] 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 M₁- 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.

[0058] The inventors have identified 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. That TDP-43 is the major disease protein of UBIs in FTLD-U and ALS is supported by: (i) the presence of immunoreactive TDP-43 in UBIs of all FTLD-U subtypes, familial FTDP- 17U and classical ALS cases, but not in the ubiquitin-positive inclusions formed by disease proteins (e.g., tau, α- synuclein) characteristic of other neurodegenerative disorders; (ii) the detection of a disease- specific biochemical signature of pathologically altered TDP-43 proteins in urea fractions of FTLD-U, FTDP- 17U and ALS brains and spinal cords; and (iii) the demonstration that pathological TDP-43 protein is ubiquitinated and hyperphosphorylated in FTLD-U and ALS.

[0059] The disclosures of each patent, patent application and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.

Claims

What is Claimed:

1. A method of assessing the presence or absence of a neurodegenerative disease in a subject comprising characterizing TDP-43 in a tissue of said subject.

2. The method of claim 1 further comprising comparing the TDP-43 in said tissue with data for TDP-43 in a tissue of a subject in which said neurodegenerative disease is known to be absent.

3. The method of claim 1 further comprising comparing the TDP-43 in said tissue with data for TDP-43 in a tissue of a subject in which said neurodegenerative disease is known to be present.

4. The method of claim 1 wherein said characterizing comprises determining the presence or absence of one or more post-translational modifications of TDP-43.

5. The method of claim 1 wherein said characterizing comprises determining the phosphorylation state of TDP-43.

6. The method of claim 1 wherein said characterizing comprises determining the ubiquitination state of TDP-43.

7. The method of claim 1 wherein said characterizing comprises determining the presence or absence of C-terminal breakdown or cleavage fragments of TDP-43.

8. The method of claim 7 wherein said C-terminal fragments comprise fragments of from about 24 kD to about 26 kD.

9. The method of claim 1 wherein said neurodegenerative disease is frontotemporal lobar degeneration.

10. The method of claim 1 wherein said neurodegenerative disease is amyotrophic lateral sclerosis.

11. The method of claim 1 wherein said tissue is of the brain or spinal cord, or a combination thereof.

12. The method of claim 11 wherein said tissue is of the frontal cortex, temporal cortex, hippocampus, or brain stem, or a combination thereof.

13. A method for diagnosing a neurodegenerative disease in a subject comprising:

contacting a tissue of said subject with an antibody that binds to TDP-43; and,

determining the extent of binding of said antibody to said tissue.

14. The method according to claim 13 wherein said tissue has a lesion.

15. The method according to claim 14 wherein said lesion is a ubiquitin-positive, tau- and α- synuclein-negative inclusion.

16. The method according to claim 15 wherein said ubiquitin-positive, tau- and α-synuclein- negative inclusion is cytoplasmic, neuritic, or nuclear.

17. The method according to claim 13 wherein said antibody that binds to TDP-43 can bind to a fragment of TDP-43.

18. A purified antibody that binds to TDP-43 or fragments thereof in ubiquitin-positive, tau- and α-synuclein-negative inclusions in subjects having Type 1 ubiquitin-positive frontotemporal lobar degeneration.

19. A purified antibody that binds to TDP-43 or fragments thereof in ubiquitin-positive, tau- and α-synuclein-negative inclusions in subjects having Type 2 ubiquitin-positive frontotemporal lobar degeneration.

20. A method of determining efficacy of a drug against a neurodegenerative disease comprising determining whether said drug modulates the activity of a modified form of TDP-43.

21. The method according to claim 20 wherein said neurodegenerative disease is frontotemporal lobar degeneration.

22. The method according to claim 20 wherein said neurodegenerative disease is amyotrophic lateral sclerosis.

23. The method according to claim 20 wherein said modified form of TDP-43 is a hyperphosphorylated form of TDP-43.

24. The method according to claim 20 wherein said modified form of TDP-43 is a ubiquitinated form of TDP-43.

25. The method according to claim 20 wherein said modified form of TDP-43 is a C-terminal breakdown or cleavage fragment of TDP-43.

26. The method according to claim 20 comprising determining whether said drug modulates the activity of a modified form of TDP-43 in a cerebrospinal tissue of a subject

27. The method according to claim 26 wherein said cerebrospinal tissue has a lesion.

28. The method according to claim 27 wherein said lesion is a ubiquitin-positive, tau- and α- synuclein-negative inclusion.

29. The method according to claim 28 wherein said ubiquitin-positive, tau- and α-synuclein- negative inclusion is cytoplasmic, neuritic, or nuclear.