FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2137048103> ?p ?o ?g. }

• W2137048103 endingPage "194" @default.
• W2137048103 startingPage "182" @default.
• W2137048103 abstract "TAR DNA-binding protein (TDP-43) has been recently described as a major pathological protein in both frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. However, little is known about the relative abundance and distribution of different pathological TDP-43 species, which include hyperphosphorylated, ubiquitinated, and N-terminally cleaved TDP-43. Here, we developed novel N-terminal (N-t) and C-terminal (C-t)-specific TDP-43 antibodies and performed biochemical and immunohistochemical studies to analyze cortical, hippocampal, and spinal cord tissue from frontotemporal dementia with ubiquitin-positive inclusions and amyotrophic lateral sclerosis cases. C-t-specific TDP-43 antibodies revealed similar abundance, morphology, and distribution of dystrophic neurites and neuronal cytoplasmic inclusions in cortex and hippocampus compared with previously described pan-TDP-43 antibodies. By contrast, N-t-specific TDP-43 antibodies only detected a small subset of these lesions. Biochemical studies confirmed the presence of C-t TDP-43 fragments but not extreme N-t fragments. Surprisingly, immunohistochemical analysis of inclusions in spinal cord motor neurons in both diseases showed that they are N-t and C-t positive. TDP-43 inclusions in Alzheimer's disease brains also were examined, and similar enrichment in C-t TDP-43 fragments was observed in cortex and hippocampus. These results show that the composition of the inclusions in brain versus spinal cord tissues differ, with an increased representation of C-t TDP-43 fragments in cortical and hippocampal regions. Therefore, regionally different pathogenic processes may underlie the development of abnormal TDP-43 proteinopathies. TAR DNA-binding protein (TDP-43) has been recently described as a major pathological protein in both frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. However, little is known about the relative abundance and distribution of different pathological TDP-43 species, which include hyperphosphorylated, ubiquitinated, and N-terminally cleaved TDP-43. Here, we developed novel N-terminal (N-t) and C-terminal (C-t)-specific TDP-43 antibodies and performed biochemical and immunohistochemical studies to analyze cortical, hippocampal, and spinal cord tissue from frontotemporal dementia with ubiquitin-positive inclusions and amyotrophic lateral sclerosis cases. C-t-specific TDP-43 antibodies revealed similar abundance, morphology, and distribution of dystrophic neurites and neuronal cytoplasmic inclusions in cortex and hippocampus compared with previously described pan-TDP-43 antibodies. By contrast, N-t-specific TDP-43 antibodies only detected a small subset of these lesions. Biochemical studies confirmed the presence of C-t TDP-43 fragments but not extreme N-t fragments. Surprisingly, immunohistochemical analysis of inclusions in spinal cord motor neurons in both diseases showed that they are N-t and C-t positive. TDP-43 inclusions in Alzheimer's disease brains also were examined, and similar enrichment in C-t TDP-43 fragments was observed in cortex and hippocampus. These results show that the composition of the inclusions in brain versus spinal cord tissues differ, with an increased representation of C-t TDP-43 fragments in cortical and hippocampal regions. Therefore, regionally different pathogenic processes may underlie the development of abnormal TDP-43 proteinopathies. Frontotemporal lobar degeneration (FTLD) is the diagnostic term for a group of clinically, genetically, and neuropathologically heterogeneous neurodegenerative disorders characterized by prominent atrophy of the frontal and anterior temporal lobes. FTLD is the second most common neurodegenerative cause of dementia after Alzheimer's Disease (AD) under age 65.1Snowden JS Neary D Mann DM Frontotemporal dementia.Br J Psychiatry. 2002; 180: 140-143Crossref PubMed Scopus (311) Google Scholar, 2Cairns NJ Bigio EH Mackenzie IR Neumann M Lee VMY Hatanpaa KJ White III, CL Schneider JA Grinberg LT Halliday G Duyckaerts C Lowe JS Holm IE Tolnay M Okamoto K Yokoo H Murayama S Woulfe J Munoz DG Dickson DW Ince PG Trojanowski JQ Mann DM Neuropathologic diagnostic and nosologic criteria for frontotemporal lobar degeneration: consensus of the Consortium for Frontotemporal Lobar Degeneration.Acta Neuropathol. 2007; 114: 5-22Crossref PubMed Scopus (902) Google Scholar The most prevalent clinical form of FTLD is frontotemporal dementia, which primarily manifests as changes in social and personal behavior, including disinhibition, and a progressive disorder of language.3McKhann GM Albert MS Grossman M Miller B Dickson D Trojanowski JQ Clinical and pathological diagnosis of frontotemporal dementia: report of the Work Group on Frontotemporal Dementia and Pick's Disease.Arch Neurol. 2001; 58: 1803-1809Crossref PubMed Scopus (1320) Google Scholar Affected individuals can develop a movement disorder including Parkinsonism and/or motor neuron disease (MND). The neuropathology of FTLD can be broadly divided into those with tau-positive inclusions known as tauopathies, and those with ubiquitin-positive, tau-negative, and synuclein-negative inclusions (UBIs) referred to as FTLD-U, which is the most common underlying neuropathology in FTLD.3McKhann GM Albert MS Grossman M Miller B Dickson D Trojanowski JQ Clinical and pathological diagnosis of frontotemporal dementia: report of the Work Group on Frontotemporal Dementia and Pick's Disease.Arch Neurol. 2001; 58: 1803-1809Crossref PubMed Scopus (1320) Google Scholar Recent studies also classified FTLD-U cases into four major subtypes based on the distribution of UBIs detected by immunohistochemistry (IHC).4Sampathu DM Neumann M Kwong LK Chou TT Micsenyi M Truax A Bruce J Grossman M Trojanowski JQ Lee VMY Pathological heterogeneity of frontotemporal lobar degeneration with ubiquitin-positive inclusions delineated by ubiquitin immunohistochemistry and novel monoclonal antibodies.Am J Pathol. 2006; 169: 1343-1352Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 5Neumann M Mackenzie IR Cairns NJ Boyer PJ Markesbery WR Smith CD Taylor JP Kretzschmar HA Kimonis VE Forman MS TDP-43 in the ubiquitin pathology of frontotemporal dementia with VCP gene mutations.J Neuropathol Exp Neurol. 2007; 66: 152-157Crossref PubMed Scopus (275) Google Scholar, 6Mackenzie IR Baborie A Pickering-Brown S Du Plessis D Jaros E Perry RH Neary D Snowden JS Mann DM Heterogeneity of ubiquitin pathology in frontotemporal lobar degeneration: classification and relation to clinical phenotype.Acta Neuropathol. 2006; 112: 539-549Crossref PubMed Scopus (289) Google Scholar Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease that involves selective death of the upper and lower motor neurons, leading to muscle atrophy, paralysis, and death.7Pasinelli P Brown RH Molecular biology of amyotrophic lateral sclerosis: insights from genetics.Nat Rev Neurosci. 2006; 7: 710-723Crossref PubMed Scopus (911) Google Scholar Approximately 90% of ALS cases are sporadic and have no known cause, but the remaining 10% have a genetic basis.8Gros-Louis F Gaspar C Rouleau GA Genetics of familial and sporadic amyotrophic lateral sclerosis.Biochim Biophys Acta. 2006; 1762: 956-972Crossref PubMed Scopus (210) Google Scholar Significantly, some ALS patients also develop a dementia consistent with FTLD.9Talbot K Ansorge O Recent advances in the genetics of amyotrophic lateral sclerosis and frontotemporal dementia: common pathways in neurodegenerative disease.Hum Mol Genet. 2006; 15: R182-R187Crossref PubMed Scopus (78) Google Scholar The neuropathology of ALS is characterized by UBIs formed by abnormal accumulations of ubiquitin-positive filamentous skeins or compact, round Lewy body-like inclusions in the cytoplasm of degenerating motor neurons.10Leigh PN Whitwell H Garofalo O Buller J Swash M Martin JE Gallo JM Weller RO Anderton BH Ubiquitin-immunoreactive intraneuronal inclusions in amyotrophic lateral sclerosis: morphology, distribution, and specificity.Brain. 1991; 114: 775-788Crossref PubMed Scopus (280) Google Scholar Moreover, there is evidence showing that ALS is a multisystem neurodegenerative disease, with pathological inclusions present in additional regions, including hippocampal and neocortical structures.11Wightman G Anderson VE Martin J Swash M Anderton BH Neary D Mann D Luthert P Leigh PN Hippocampal and neocortical ubiquitin-immunoreactive inclusions in amyotrophic lateral sclerosis with dementia.Neurosci Lett. 1992; 139: 269-274Crossref PubMed Scopus (231) Google Scholar, 12Geser F Brandmeir NJ Kwong LK Martinez-Lage M Elman LB McCluskey L Xie SX Lee VMY Trojanowski JQ Evidence of multisystem disorder in whole-brain map of pathological TDP-43 in amyotrophic lateral sclerosis.Arch Neurol. 2008; 65: 636-641Crossref PubMed Scopus (231) Google Scholar Recently, TAR-DNA binding protein (TDP-43) was identified as the major disease protein in UBIs of sporadic and familial forms of FTLD-U and of sporadic and familial forms of ALS with the notable exception of autosomal dominantly inherited ALS caused by SOD1 mutations.13Mackenzie IR Bigio EH Ince PG Geser F Neumann M Cairns NJ Kwong LK Forman MS Ravits J Stewart H Eisen A McClusky L Kretzschmar HA Monoranu CM Highley JR Kirby J Siddique T Shaw PJ Lee VMY Trojanowski JQ Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations.Ann Neurol. 2007; 61: 427-434Crossref PubMed Scopus (727) Google Scholar, 14Tan CF Eguchi H Tagawa A Onodera O Iwasaki T Tsujino A Nishizawa M Kakita A Takahashi H TDP-43 immunoreactivity in neuronal inclusions in familial amyotrophic lateral sclerosis with or without SOD1 gene mutation.Acta Neuropathol. 2007; 113: 535-542Crossref PubMed Scopus (249) Google Scholar, 15Neumann M Sampathu DM Kwong LK Truax AC Micsenyi MC Chou TT Bruce J Schuck T Grossman M Clark CM McCluskey LF Miller BL Masliah E Mackenzie IR Feldman H Feiden W Kretzschmar HA Trojanowski JQ Lee VMY Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4517) Google Scholar These important findings suggest that FTLD-U and ALS represent a clinicopathological spectrum of the same neurodegenerative disorder. Furthermore, the presence of pathological TDP-43 in these diseases was rapidly confirmed by others.16Davidson Y Kelley T Mackenzie IR Pickering-Brown S Du Plessis D Neary D Snowden JS Mann DM Ubiquitinated pathological lesions in frontotemporal lobar degeneration contain the TAR DNA-binding protein TDP-43.Acta Neuropathol. 2007; 113: 521-533Crossref PubMed Scopus (268) Google Scholar, 17Arai T Hasegawa M Akiyama H Ikeda K Nonaka T Mori H Mann D Tsuchiya K Yoshida M Hashizume Y Oda T TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Biochem Biophys Res Commun. 2006; 351: 602-611Crossref PubMed Scopus (1898) Google Scholar TDP-43 is a ubiquitously expressed nuclear protein that has been reported to facilitate RNA splicing and transcriptional repression,18Buratti E Dork T Zuccato E Pagani F Romano M Baralle FE Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping.EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (499) Google Scholar, 19Buratti E Baralle FE Multiple roles of TDP-43 in gene expression, splicing regulation, and human disease.Front Biosci. 2008; 13: 867-878Crossref PubMed Scopus (384) Google Scholar although much of the biology and functions of TDP-43 remain enigmatic. Pathological TDP-43 is distinct from its normal counterpart because it is hyperphosphorylated, ubiquitinated, and cleaved to generate C-terminal (C-t) fragments in affected brain and spinal cord of FTLD-U and ALS.15Neumann M Sampathu DM Kwong LK Truax AC Micsenyi MC Chou TT Bruce J Schuck T Grossman M Clark CM McCluskey LF Miller BL Masliah E Mackenzie IR Feldman H Feiden W Kretzschmar HA Trojanowski JQ Lee VMY Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4517) Google Scholar Furthermore, these N-terminal (N-t) truncated TDP-43 species are only recovered in sarkosyl-insoluble fractions, suggesting that they could serve as a nidus for the aggregation and formation of UBIs. To test this hypothesis, we generated antibodies specific to the N-t and C-t of TDP-43 and used them to assess the relative abundance and distribution of full-length (FL) and N-t truncated C-t fragments of TDP-43. Our findings demonstrate that although FL-TDP-43 pathology is similarly represented in spinal cord motor neurons of FTLD-U and ALS cases, brain UBIs contain predominantly C-t fragments. Our data suggest that the pathogenic mechanisms of brain and spinal cord TDP-43 inclusion formation may be different. Frozen brain tissues and fixed, paraffin-embedded tissue blocks were obtained from the Center for Neurodegenerative Disease Research Brain Bank at the University of Pennsylvania. Consent for autopsy was obtained from legal representatives for all subjects in accordance with local institutional review board requirements. All cases were diagnosed by trained neuropathologists in accordance with published guidelines for ALS20Brooks BR Miller RG Swash M Munsat TL El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis.Amyotroph Lateral Scler Other Motor Neuron Disord. 2000; 1: 293-299Crossref PubMed Scopus (4035) Google Scholar and different pathological variants of FTLD.2Cairns NJ Bigio EH Mackenzie IR Neumann M Lee VMY Hatanpaa KJ White III, CL Schneider JA Grinberg LT Halliday G Duyckaerts C Lowe JS Holm IE Tolnay M Okamoto K Yokoo H Murayama S Woulfe J Munoz DG Dickson DW Ince PG Trojanowski JQ Mann DM Neuropathologic diagnostic and nosologic criteria for frontotemporal lobar degeneration: consensus of the Consortium for Frontotemporal Lobar Degeneration.Acta Neuropathol. 2007; 114: 5-22Crossref PubMed Scopus (902) Google Scholar, 3McKhann GM Albert MS Grossman M Miller B Dickson D Trojanowski JQ Clinical and pathological diagnosis of frontotemporal dementia: report of the Work Group on Frontotemporal Dementia and Pick's Disease.Arch Neurol. 2001; 58: 1803-1809Crossref PubMed Scopus (1320) Google Scholar Three disease groups were defined, representing FTLD-U with and without MND and ALS cases. Within the FTLD-U cases, they were further subdivided into subtypes I, II, and III4Sampathu DM Neumann M Kwong LK Chou TT Micsenyi M Truax A Bruce J Grossman M Trojanowski JQ Lee VMY Pathological heterogeneity of frontotemporal lobar degeneration with ubiquitin-positive inclusions delineated by ubiquitin immunohistochemistry and novel monoclonal antibodies.Am J Pathol. 2006; 169: 1343-1352Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar (Supplemental Table 1, see http://ajp.amjpathol.org). Additionally, two cases of subtype IV5Neumann M Mackenzie IR Cairns NJ Boyer PJ Markesbery WR Smith CD Taylor JP Kretzschmar HA Kimonis VE Forman MS TDP-43 in the ubiquitin pathology of frontotemporal dementia with VCP gene mutations.J Neuropathol Exp Neurol. 2007; 66: 152-157Crossref PubMed Scopus (275) Google Scholar with VCP mutations were also used, but because of limited availability of tissue, the data are only shown in Supplemental Figure 1 (see http://ajp.amjpathol.org). Age-matched normal cases were included as nondisease controls. Demographics of the patients, including diagnoses, age at onset, disease duration, and gender are summarized in Supplemental Table 1. In addition, cases diagnosed with a progressive dementia and showing typical Alzheimer's disease (AD) pathology (CERAD stage C and Braak & Braak stages V–VI) by neuropathological examination were studied. These cases fulfilled the criteria for high likelihood of AD according to the National Institute on Aging criteria.21Hyman BT Trojanowski JQ Consensus recommendations for the postmortem diagnosis of Alzheimer disease from the National Institute on Aging and the Reagan Institute Working Group on diagnostic criteria for the neuropathological assessment of Alzheimer disease.J Neuropathol Exp Neurol. 1997; 56: 1095-1097Crossref PubMed Scopus (683) Google Scholar Two polyclonal antibodies (pAb) were produced by immunizing rabbits (Covance Research Products, Inc., Denver, PA). The first (designated as N-t TDP-43) was raised against a synthetic peptide near the N-t of human TDP-43 corresponding to amino acid residues 6 to 24. The other antibody (designated as C-t TDP-43) was raised against an extreme C-terminal synthetic peptide, corresponding to amino acid residues 394 to 414 of human TDP-43. The resulting immune sera were affinity purified, and their specificities were characterized using recombinant full-length and C-t truncated T-7 tagged human TDP-43 (ProteinTech Group, Chicago, IL), QBI293 cell lysates, and sarkosyl-insoluble fractions from all three types of FTLD-U brains. A murine C-t monoclonal antibody (mAb) was generated by immunizing mice with the same synthetic peptide used for the C-t TDP-43 pAb, as previously described.22Tu PH Galvin JE Baba M Giasson B Tomita T Leight S Nakajo S Iwatsubo T Trojanowski JQ Lee VMY Glial cytoplasmic inclusions in white matter oligodendrocytes of multiple system atrophy brains contain insoluble alpha-synuclein.Ann Neurol. 1998; 44: 415-422Crossref PubMed Scopus (586) Google Scholar Fusion was conducted by using spleen lymphocytes from immunized BALB/c mice and SP2 cells to produce hybridomas. Resulting hybridoma supernatants were screened by enzyme-linked immunosorbent assay using plates coated with the immunizing peptide, and their specificity was determined by immunoblot and IHC on paraffin-embedded sections of FTLD-U cortex known to contain TDP-43-positive inclusions. The harvesting, fixation, and further processing of the tissue specimens used in this study were conducted as previously described.4Sampathu DM Neumann M Kwong LK Chou TT Micsenyi M Truax A Bruce J Grossman M Trojanowski JQ Lee VMY Pathological heterogeneity of frontotemporal lobar degeneration with ubiquitin-positive inclusions delineated by ubiquitin immunohistochemistry and novel monoclonal antibodies.Am J Pathol. 2006; 169: 1343-1352Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 15Neumann M Sampathu DM Kwong LK Truax AC Micsenyi MC Chou TT Bruce J Schuck T Grossman M Clark CM McCluskey LF Miller BL Masliah E Mackenzie IR Feldman H Feiden W Kretzschmar HA Trojanowski JQ Lee VMY Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4517) Google Scholar Briefly, fresh tissues from brain and spinal cord of FTLD-U or ALS cases were fixed with either 70% ethanol in 150 mmol/L NaCl or phosphate-buffered 3.65% formaldehyde, infiltrated with paraffin, and cut into 6-μm serial sections. Antigen retrieval was done by boiling the sections in 10 mmol/L citrate buffer (pH 6.0) in a microwave oven. IHC was performed using the avidin-biotin complex (ABC) detection system (Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine as described previously.4Sampathu DM Neumann M Kwong LK Chou TT Micsenyi M Truax A Bruce J Grossman M Trojanowski JQ Lee VMY Pathological heterogeneity of frontotemporal lobar degeneration with ubiquitin-positive inclusions delineated by ubiquitin immunohistochemistry and novel monoclonal antibodies.Am J Pathol. 2006; 169: 1343-1352Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar Briefly, sections were deparaffinized and rehydrated, endogenous peroxidases were quenched with 5% H2O2 in methanol for 30 minutes, and sections were blocked in 0.1 mol/L Tris with 2% fetal bovine serum for 5 minutes. Primary antibodies were incubated for either 1 or 2 hours at room temperature or overnight at 4°C. The following primary antibodies were used: mouse anti-ubiquitin (1510; 1:40,000; Chemicon, Temecula, CA), mAb and affinity-purified rabbit polyclonal anti-TDP-43 (TDP-43 mAb and pan TDP-43 pAb; 1:50,000; Abnova Corp., Taiwan; and 1:4500; ProteinTech Group, respectively), C-t TDP-43 pAb (1:60,000), N-t TDP-43 pAb (1:60,000), and C-t TDP-43 mAb supernatant (1:30). After washing, sections were sequentially incubated with biotinylated secondary antibodies for 1 hour and avidin-biotin complex for 1 hour. Bound antibody complexes were visualized by incubating sections in a solution containing 100 mmol/L Tris, pH 7.6, 0.1% Triton X-100, 1.4 mmol/L diaminobenzidine, 10 mmol/L imidazole, and 8.8 mmol/L H2O2. Sections were then lightly counterstained with hematoxylin, dehydrated, and coverslipped. Double-labeling immunofluorescence (IF) analyses were performed as previously described15Neumann M Sampathu DM Kwong LK Truax AC Micsenyi MC Chou TT Bruce J Schuck T Grossman M Clark CM McCluskey LF Miller BL Masliah E Mackenzie IR Feldman H Feiden W Kretzschmar HA Trojanowski JQ Lee VMY Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4517) Google Scholar using Alexa Fluor 488- and 594-conjugated secondary antibodies (Molecular Probes, Eugene, OR), treated for autofluorescence with Sudan Black solution,23Schnell SA Staines WA Wessendorf MW Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue.J Histochem Cytochem. 1999; 47: 719-730Crossref PubMed Scopus (544) Google Scholar and coverslipped with Vectashield-DAPI mounting medium (Vector Laboratories). Digital images were obtained using an Olympus BX 51 microscope (Tokyo, Japan) equipped with bright-field and fluorescence light sources using a ProgRes C14 digital camera (Jenoptik AG, Jena, Germany) and Adobe Photoshop, version 9.0 (Adobe Systems, San Jose, CA) or digital camera DP71 (Olympus) and DP manager (Olympus). Semiquantitative analysis of pan TDP-43, C-t TDP-43, or N-t TDP-43 pAb-immunoreactive pathology was performed on sections with the most robust pathology from neocortex, hippocampus, and spinal cord following a modified protocol described previously.24Cairns NJ Neumann M Bigio EH Holm IE Troost D Hatanpaa KJ Foong C White III, CL Schneider JA Kretzschmar HA Carter D Taylor-Reinwald L Paulsmeyer K Strider J Gitcho M Goate AM Morris JC Mishra M Kwong LK Stieber A Xu Y Forman MS Trojanowski JQ Lee VMY Mackenzie IR TDP-43 in familial and sporadic frontotemporal lobar degeneration with ubiquitin inclusions.Am J Pathol. 2007; 171: 227-240Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar For all TDP-43 antibodies, the severity of neocortical and hippocampal pathology was rated semiquantitatively: 0, no inclusions; 1, rare; 2, mild; 3, moderate; and 4, severe. The scoring system for lower motor neuron cytoplasmic inclusions was based on total numbers in a single section of spinal cord. A total score including filamentous, round, or irregular shaped inclusions or small dense granules was calculated using the following scoring system: 0, none; 1, 1 inclusion per section; 2, 2–5 inclusions per section; 3, 6–10 inclusions per section; and 4, >10 inclusions per section. Sections were primarily scored by one investigator (L.M.I.), with validation performed by different researchers (K.U., M.N., J.Q.T.). Statistical nonparametric analysis of neuropathological scores was performed using Kruskal-Wallis test followed by Dunn's multiple comparison post test. Postmortem brain tissue from FTLD-U and control cases was dissected, weighed, and sequentially extracted with buffers of increasing strength, as described previously.15Neumann M Sampathu DM Kwong LK Truax AC Micsenyi MC Chou TT Bruce J Schuck T Grossman M Clark CM McCluskey LF Miller BL Masliah E Mackenzie IR Feldman H Feiden W Kretzschmar HA Trojanowski JQ Lee VMY Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4517) Google Scholar For Western blot analysis, protein extracts were resolved by Tris-glycine 5 to 20% gradient SDS-polyacrylamide gel electrophoresis, 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 Products, 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 (20 mmol/L Tris and 0.2 mmol/L EDTA, pH 8.0) and treated with Escherichia coli alkaline phosphatase (Sigma-Aldrich, St. Louis, MO) for 2 hours at 56°C. The identification of one or more extreme C-t fragments recovered from the sarkosyl-insoluble fractions of FTLD-U brains15Neumann M Sampathu DM Kwong LK Truax AC Micsenyi MC Chou TT Bruce J Schuck T Grossman M Clark CM McCluskey LF Miller BL Masliah E Mackenzie IR Feldman H Feiden W Kretzschmar HA Trojanowski JQ Lee VMY Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4517) Google Scholar led us to investigate the relative abundance of FL, C-t, or N-t TDP-43 fragments in UBIs of FTLD-U and ALS. To accomplish this, we generated pAbs that recognize epitopes at the N-t and extreme C-t of the TDP-43 protein (Figure 1A). To demonstrate the specificities of these antibodies to detect the N-t and C-t of the TDP-43 protein, we tested these TDP-43 pAbs using recombinant full-length or C-t truncated TDP-43 protein in immunoblots. A commercially available TDP-43 rabbit pAb (pan TDP-43) that was raised to recombinant TDP-43 and used in previous published reports15Neumann M Sampathu DM Kwong LK Truax AC Micsenyi MC Chou TT Bruce J Schuck T Grossman M Clark CM McCluskey LF Miller BL Masliah E Mackenzie IR Feldman H Feiden W Kretzschmar HA Trojanowski JQ Lee VMY Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4517) Google Scholar, 17Arai T Hasegawa M Akiyama H Ikeda K Nonaka T Mori H Mann D Tsuchiya K Yoshida M Hashizume Y Oda T TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Biochem Biophys Res Commun. 2006; 351: 602-611Crossref PubMed Scopus (1898) Google Scholar was included in all studies as a positive control. Although both N-t and C-t TDP-43 pAbs recognize FL-TDP-43, N-t and not C-t TDP-43 pAb recognizes the C-t truncated TDP-43 fragment (Figure 1B). Furthermore, both N-t and C-t TDP-43 pAbs detected endogenous TDP-43 from QBI293 cell extracts (Figure 1C), and indirect IF studies confirm that the N-t- and C-t-specific antibodies detect the endogenous nuclear TDP-43 protein in human QBI293 cells (data not shown). Previous studies have identified a disease-specific biochemical signature of pathologically altered TDP-43 in sarkosyl-insoluble but urea-soluble extracts of gray matter from sporadic and familial FTLD-U cases of different subtypes.15Neumann M Sampathu DM Kwong LK Truax AC Micsenyi MC Chou TT Bruce J Schuck T Grossman M Clark CM McCluskey LF Miller BL Masliah E Mackenzie IR Feldman H Feiden W Kretzschmar HA Trojanowski JQ Lee VMY Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4517) Google Scholar To evaluate the specificity and affinity of our antibodies in detecting this pathological signature, we conducted immunoblot analyses on urea fraction extracted from frontal cortices of FTLD-U type I-III cases (Figure 1, D–F). Although both C-t- and N-t-specific antibodies detected FL-TDP-43, C-t but not N-t TDP-43 pAb detected C-t fragments from all three subtypes. Interestingly, the C-t pAb showed less affinity for FL-TDP-43 compared with the N-t and pan TDP-43 pAbs, but it detected the C-t fragments better than the pan TDP-43 pAb. Both C-t and N-t pAbs detected phosphorylated FL TDP-43 at 45 kDa, which upon dephosphorylation collapsed into the 43-kDa protein band (Figure 1, D–F). Unexpectedly, although the C-t TDP-43 pAb was raised to a nonphosphorylated synthetic peptide, dephosphorylation of the C-t fragments reduced the affinity of the antibody for these fragments. Finally, the C-t detected much more high Mr ubiquitinated smear than the N-t TDP-43 pAb from all three FTLD-U subtypes (Figure 1, D–F). Thus, these termini-specific pAbs exhibit the desirable specificities for IHC studies described below. To determine whether TDP-43 inclusions in different brain regions of FTLD-U cases comprise mostly FL or C-t fragment of TDP-43, we conducted IHC studies using serial sections from FTLD-U cases (Supplemental Table 1) and probed with C-t, N-t, or pan TDP-43 pAbs. Analysis of hippocampal tissue sections from FTLD-U cases showed robust staining of UBIs in granule cells of the dentate gyrus with the C-t and the pan TDP-43 pAb but not the N-t TDP-43 pAb (Figure 2, A–F). Consistent with previous observations,15Neumann M Sampathu DM Kwong LK Truax AC Micsenyi MC Chou TT Bruce J Schuck T Grossman M Clark CM McCluskey LF Miller BL Masliah E Mackenzie IR Feldman H Feiden W Kretzschmar HA Trojanowski JQ Lee VMY Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4517) Google Scholar nuclear clearance of endogenous TDP-43 was observed when cells contained TDP-43 inclusions (arrowheads), but most albeit not all nuclei of normal cells were immunostained (compare wit" @default.
• W2137048103 created "2016-06-24" @default.
• W2137048103 creator A5003243009 @default.
• W2137048103 creator A5020029308 @default.
• W2137048103 creator A5021394368 @default.
• W2137048103 creator A5025921750 @default.
• W2137048103 creator A5033623570 @default.
• W2137048103 creator A5037327989 @default.
• W2137048103 creator A5046120768 @default.
• W2137048103 creator A5047108669 @default.
• W2137048103 creator A5056969742 @default.
• W2137048103 creator A5062512325 @default.
• W2137048103 creator A5065852445 @default.
• W2137048103 creator A5074080979 @default.
• W2137048103 creator A5084210639 @default.
• W2137048103 date "2008-07-01" @default.
• W2137048103 modified "2023-10-10" @default.
• W2137048103 title "Enrichment of C-Terminal Fragments in TAR DNA-Binding Protein-43 Cytoplasmic Inclusions in Brain but not in Spinal Cord of Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis" @default.
• W2137048103 cites W1951130496 @default.
• W2137048103 cites W1964347491 @default.
• W2137048103 cites W1966943597 @default.
• W2137048103 cites W1967932847 @default.
• W2137048103 cites W2002093269 @default.
• W2137048103 cites W2003179114 @default.
• W2137048103 cites W2003618511 @default.
• W2137048103 cites W2004395649 @default.
• W2137048103 cites W2010528085 @default.
• W2137048103 cites W2014670147 @default.
• W2137048103 cites W2027460468 @default.
• W2137048103 cites W2031964319 @default.
• W2137048103 cites W2035678859 @default.
• W2137048103 cites W2042930414 @default.
• W2137048103 cites W2051490456 @default.
• W2137048103 cites W2052888134 @default.
• W2137048103 cites W2053362200 @default.
• W2137048103 cites W2077083529 @default.
• W2137048103 cites W2077792283 @default.
• W2137048103 cites W2080861984 @default.
• W2137048103 cites W2091173932 @default.
• W2137048103 cites W2092646825 @default.
• W2137048103 cites W2093276554 @default.
• W2137048103 cites W2094741094 @default.
• W2137048103 cites W2104658204 @default.
• W2137048103 cites W2107754763 @default.
• W2137048103 cites W2127282494 @default.
• W2137048103 cites W2128815118 @default.
• W2137048103 cites W2133068803 @default.
• W2137048103 cites W2134051267 @default.
• W2137048103 cites W2136057227 @default.
• W2137048103 cites W2136305293 @default.
• W2137048103 cites W2146913804 @default.
• W2137048103 cites W2152585035 @default.
• W2137048103 cites W2158787641 @default.
• W2137048103 cites W2161938104 @default.
• W2137048103 cites W2169092675 @default.
• W2137048103 cites W2171303338 @default.
• W2137048103 cites W4247755733 @default.
• W2137048103 doi "https://doi.org/10.2353/ajpath.2008.080003" @default.
• W2137048103 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2438296" @default.
• W2137048103 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18535185" @default.
• W2137048103 hasPublicationYear "2008" @default.
• W2137048103 type Work @default.
• W2137048103 sameAs 2137048103 @default.
• W2137048103 citedByCount "281" @default.
• W2137048103 countsByYear W21370481032012 @default.
• W2137048103 countsByYear W21370481032013 @default.
• W2137048103 countsByYear W21370481032014 @default.
• W2137048103 countsByYear W21370481032015 @default.
• W2137048103 countsByYear W21370481032016 @default.
• W2137048103 countsByYear W21370481032017 @default.
• W2137048103 countsByYear W21370481032018 @default.
• W2137048103 countsByYear W21370481032019 @default.
• W2137048103 countsByYear W21370481032020 @default.
• W2137048103 countsByYear W21370481032021 @default.
• W2137048103 countsByYear W21370481032022 @default.
• W2137048103 countsByYear W21370481032023 @default.
• W2137048103 crossrefType "journal-article" @default.
• W2137048103 hasAuthorship W2137048103A5003243009 @default.
• W2137048103 hasAuthorship W2137048103A5020029308 @default.
• W2137048103 hasAuthorship W2137048103A5021394368 @default.
• W2137048103 hasAuthorship W2137048103A5025921750 @default.
• W2137048103 hasAuthorship W2137048103A5033623570 @default.
• W2137048103 hasAuthorship W2137048103A5037327989 @default.
• W2137048103 hasAuthorship W2137048103A5046120768 @default.
• W2137048103 hasAuthorship W2137048103A5047108669 @default.
• W2137048103 hasAuthorship W2137048103A5056969742 @default.
• W2137048103 hasAuthorship W2137048103A5062512325 @default.
• W2137048103 hasAuthorship W2137048103A5065852445 @default.
• W2137048103 hasAuthorship W2137048103A5074080979 @default.
• W2137048103 hasAuthorship W2137048103A5084210639 @default.
• W2137048103 hasBestOaLocation W21370481031 @default.
• W2137048103 hasConcept C105702510 @default.
• W2137048103 hasConcept C142724271 @default.
• W2137048103 hasConcept C153911025 @default.
• W2137048103 hasConcept C169760540 @default.
• W2137048103 hasConcept C190062978 @default.
• W2137048103 hasConcept C2776939681 @default.
• W2137048103 hasConcept C2778641062 @default.

• next