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Matrin 3 Is a Component of Neuronal Cytoplasmic Inclusions of Motor Neurons in Sporadic Amyotrophic Lateral Sclerosis

2017; Elsevier BV; Volume: 188; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2017.10.007

ISSN

1525-2191

Autores

Mikiko Tada, Hiroshi Doi, Shigeru Koyano, Shun Kubota, Ryoko Fukai, Shunta Hashiguchi, Noriko Hayashi, Yuko Kawamoto, Misako Kunii, Kenichi Tanaka, Keita Takahashi, Yuki Ogawa, Ryo Iwata, Shoji Yamanaka, Hideyuki Takeuchi, Fumiaki Tanaka,

Tópico(s)

Alzheimer's disease research and treatments

Resumo

Mutations in the MATR3 gene have been identified as a cause of familial amyotrophic lateral sclerosis, but involvement of the matrin 3 (MATR3) protein in sporadic amyotrophic lateral sclerosis (SALS) pathology has not been fully assessed. We immunohistochemically analyzed MATR3 pathology in the spinal cords of SALS and control autopsy specimens. MATR3 immunostaining of the motor neuron nuclei revealed two distinct patterns: mild and strong staining. There were no differences in the ratio of mild versus strong nuclear staining between the SALS and control cases. MATR3-containing neuronal cytoplasmic inclusions (NCIs) were observed in 60% of SALS cases. Most motor neurons with MATR3-positive NCIs exhibited a mild nuclear staining pattern. Although 16.8% of NCIs positive for transactivating response region DNA–binding protein 43 (TDP-43) were estimated as double-labeled by MATR3, no MATR3-positive or TDP-43–negative NCIs were observed. Although a previous study found that MATR3-positive NCIs are present only in cases with C9orf72 hexanucleotide repeat expansion, ubiquitin-positive granular NCIs were not observed in the cerebellum, which have been reported as specific to C9orf72-related ALS. Six ALS cases were confirmed to be negative for the GGGGCC hexanucleotide. Our results reveal that MATR3 is a component of TDP-43–positive NCIs in motor neurons, even in SALS, and indicate the broader involvement of MATR3 in ALS pathology and the heterogeneity of TDP-43–positive NCIs. Mutations in the MATR3 gene have been identified as a cause of familial amyotrophic lateral sclerosis, but involvement of the matrin 3 (MATR3) protein in sporadic amyotrophic lateral sclerosis (SALS) pathology has not been fully assessed. We immunohistochemically analyzed MATR3 pathology in the spinal cords of SALS and control autopsy specimens. MATR3 immunostaining of the motor neuron nuclei revealed two distinct patterns: mild and strong staining. There were no differences in the ratio of mild versus strong nuclear staining between the SALS and control cases. MATR3-containing neuronal cytoplasmic inclusions (NCIs) were observed in 60% of SALS cases. Most motor neurons with MATR3-positive NCIs exhibited a mild nuclear staining pattern. Although 16.8% of NCIs positive for transactivating response region DNA–binding protein 43 (TDP-43) were estimated as double-labeled by MATR3, no MATR3-positive or TDP-43–negative NCIs were observed. Although a previous study found that MATR3-positive NCIs are present only in cases with C9orf72 hexanucleotide repeat expansion, ubiquitin-positive granular NCIs were not observed in the cerebellum, which have been reported as specific to C9orf72-related ALS. Six ALS cases were confirmed to be negative for the GGGGCC hexanucleotide. Our results reveal that MATR3 is a component of TDP-43–positive NCIs in motor neurons, even in SALS, and indicate the broader involvement of MATR3 in ALS pathology and the heterogeneity of TDP-43–positive NCIs. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized clinically by progressive weakness of skeletal muscles and neuropathologically by degeneration of upper and lower motor neurons. Ubiquitinated inclusions in the cytoplasm of motor neurons are the pathologic hallmark of ALS and the related disorder frontotemporal lobar degeneration (FTLD). Transactivating response region DNA–binding protein 43 (TDP-43) is a major component of these inclusions in most ALS cases.1Arai 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 (1891) Google Scholar, 2Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4499) Google Scholar Although mutations in TARDBP, the gene encoding TDP-43, have been identified as a cause of familial ALS (FALS),3Kabashi E. Valdmanis P.N. Dion P. Spiegelman D. McConkey B.J. Vande Velde C. Bouchard J.P. Lacomblez L. Pochigaeva K. Salachas F. Pradat P.F. Camu W. Meininger V. Dupre N. Rouleau G.A. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis.Nat Genet. 2008; 40: 572-574Crossref PubMed Scopus (1228) Google Scholar, 4Sreedharan J. Blair I.P. Tripathi V.B. Hu X. Vance C. Rogelj B. Ackerley S. Durnall J.C. Williams K.L. Buratti E. Baralle F. de Belleroche J. Mitchell J.D. Leigh P.N. Al-Chalabi A. Miller C.C. Nicholson G. Shaw C.E. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis.Science. 2008; 21: 1668-1672Crossref Scopus (1967) Google Scholar most sporadic ALS (SALS), FALS, and FTLD cases with TDP-43–positive inclusions are negative for TARDBP mutations.1Arai 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 (1891) Google Scholar, 2Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4499) Google Scholar Instead, the C9orf72 mutation is the most common genetic cause of FALS with TDP-43–positive inclusions in white patients.5DeJesus-Hernandez M. Mackenzie I.R. Boeve B.F. Boxer A.L. Baker M. Rutherford N.J. Nicholson A.M. Finch N.A. Flynn H. Adamson J. Kouri N. Wojtas A. Sengdy P. Hsiung G.Y. Karydas A. Seeley W.W. Josephs K.A. Coppola G. Geschwind D.H. Wszolek Z.K. Feldman H. Knopman D.S. Petersen R.C. Miller B.L. Dickson D.W. Boylan K.B. Graff-Radford N.R. Rademakers R. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (3410) Google Scholar In addition, fused in sarcoma/translated in liposarcoma (FUS/TLS) and ubiquilin 2 (UBQLN2) are present in neuronal cytoplasmic inclusions (NCIs) in SALS and FTLD cases without mutations in the encoding genes FUS6Deng H.X. Zhai H. Bigio E.H. Yan J. Fecto F. Ajroud K. Mishra M. Ajroud-Driss S. Heller S. Sufit R. Siddique N. Mugnaini E. Siddique T. FUS-immunoreactive inclusions are a common feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis.Ann Neurol. 2010; 67: 739-748PubMed Google Scholar, 7Vance C. Rogelj B. Hortobágyi T. De Vos K.J. Nishimura A.L. Sreedharan J. Hu X. Smith B. Ruddy D. Wright P. Ganesalingam J. Williams K.L. Tripathi V. Al-Saraj S. Al-Chalabi A. Leigh P.N. Blair I.P. Nicholson G. de Belleroche J. Gallo J.M. Miller C.C. Shaw C.E. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6.Science. 2009; 323: 1208-1211Crossref PubMed Scopus (1941) Google Scholar and UBQLN2,8Deng H.X. Chen W. Hong S.T. Boycott K.M. Gorrie G.H. Siddique N. Yang Y. Fecto F. Shi Y. Zhai H. Jiang H. Hirano M. Rampersaud E. Jansen G.H. Donkervoort S. Bigio E.H. Brooks B.R. Ajroud K. Sufit R.L. Haines J.L. Mugnaini E. Pericak-Vance M.A. Siddique T. Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia.Nature. 2011; 477: 211-215Crossref PubMed Scopus (876) Google Scholar whereas superoxide dismutase 1 (SOD1) accumulation is restricted to cases with mutations in SOD1.9Da Cruz S. Bui A. Saberi S. Lee S.K. Stauffer J. McAlonis-Downes M. Schulte D. Pizzo D.P. Parone P.A. Cleveland D.W. Ravits J. Misfolded SOD1 is not a primary component of sporadic ALS.Acta Neuropathol. 2017; 134: 97-111Crossref PubMed Scopus (56) Google Scholar The fact that the products of FALS-related genes are involved in SALS pathology indicates that these proteins play essential roles in the disease progression of FALS and SALS. Matrin 3 (MATR3) is a 125-kDa inner nuclear matrix protein that binds to DNA and RNA.10Belgrader P. Dey R. Berezney R. Molecular cloning of matrin 3. A 125- kilodalton protein of the nuclear matrix contains an extensive acidic domain.J Biol Chem. 1991; 266: 9893-9899PubMed Google Scholar, 11Nakayasu H. Berezney R. Nuclear matrins: identification of the major nuclear matrix proteins.Proc Natl Acad Sci U S A. 1991; 88: 10312-10316Crossref PubMed Scopus (163) Google Scholar MATR3 contains a nuclear localization signal,12Hisada-Ishii S. Ebihara M. Kobayashi N. Kitagawa Y. Bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells.Biochem Biophys Res Commun. 2007; 354: 72-76Crossref PubMed Scopus (26) Google Scholar two zinc finger domains predicted to bind DNA, and two RNA recognition motifs. A missense mutation in a domain-less region of MATR3 causes vocal cord and pharyngeal weakness with distal myopathy.13Senderek J. Garvey S.M. Krieger M. Guergueltcheva V. Urtizberea A. Roos A. Elbracht M. Stendel C. Tournev I. Mihailova V. Feit H. Tramonte J. Hedera P. Crooks K. Bergmann C. Rudnik-Schoneborn S. Zerres K. Lochmuller H. Seboun E. Weis J. Beckmann J.S. Hauser M.A. Jackson C.E. Autosomal-dominant distal myopathy associated with a recurrent missense mutation in the gene encoding the nuclear matrix protein, matrin 3.Am J Hum Genet. 2009; 84: 511-518Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 14Muller T.J. Kraya T. Stoltenburg-Didinger G. Hanisch F. Kornhuber M. Stoevesandt D. Senderek J. Weis J. Baum P. Deschauer M. Zierz S. Phenotype of matrin-3-related distal myopathy in 16 German patients.Ann Neurol. 2014; 76: 669-680Crossref PubMed Scopus (61) Google Scholar Recently, mutations in MATR3 were identified as a cause of FALS15Johnson J.O. Pioro E.P. Boehringer A. Chia R. Feit H. Renton A.E. Pliner H.A. Abramzon Y. Marangi G. Winborn B.J. Gibbs J.R. Nalls M.A. Morgan S. Shoai M. Hardy J. Pittman A. Orrell R.W. Malaspina A. Sidle K.C. Fratta P. Harms M.B. Baloh R.H. Pestronk A. Weihl C.C. Rogaeva E. Zinman L. Drory V.E. Borghero G. Mora G. Calvo A. Rothstein J.D. Drepper C. Sendtner M. Singleton A.B. Taylor J.P. Cookson M.R. Restagno G. Sabatelli M. Bowser R. Chio A. Traynor B.J. ITALSGENMutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis.Nat Neurosci. 2014; 17: 664-666Crossref PubMed Scopus (318) Google Scholar and were also detected in SALS.16Lin K.P. Tsai P.C. Liao Y.C. Chen W.T. Tsai C.P. Soong B.W. Lee Y.C. Mutational analysis of MATR3 in Taiwanese patients with amyotrophic lateral sclerosis.Neurobiol Aging. 2015; 36: 2005.e1-2005.e4Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 17Marangi G. Lattante S. Doronzio P.N. Conte A. Tasca G. Monforte M. Patanella A.K. Bisogni G. Meleo E. La Spada S. Zollino M. Sabatelli M. Matrin 3 variants are frequent in Italian ALS patients.Neurobiol Aging. 2017; 49: 218.e1-218.e7Abstract Full Text Full Text PDF Scopus (28) Google Scholar In that report, densely stained nuclei of motor neurons and glial cells by MATR3 immunostaining were described as a pathologic finding associated with SALS cases; MATR3-positive NCIs were not observed in FALS or SALS cases, except in one patient harboring C9orf72 hexanucleotide repeat expansion. Moreover, MATR3 has not been detected in TDP-43–positive NCIs.15Johnson J.O. Pioro E.P. Boehringer A. Chia R. Feit H. Renton A.E. Pliner H.A. Abramzon Y. Marangi G. Winborn B.J. Gibbs J.R. Nalls M.A. Morgan S. Shoai M. Hardy J. Pittman A. Orrell R.W. Malaspina A. Sidle K.C. Fratta P. Harms M.B. Baloh R.H. Pestronk A. Weihl C.C. Rogaeva E. Zinman L. Drory V.E. Borghero G. Mora G. Calvo A. Rothstein J.D. Drepper C. Sendtner M. Singleton A.B. Taylor J.P. Cookson M.R. Restagno G. Sabatelli M. Bowser R. Chio A. Traynor B.J. ITALSGENMutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis.Nat Neurosci. 2014; 17: 664-666Crossref PubMed Scopus (318) Google Scholar Except for the single report mentioned above, MATR3 pathology in motor neurons has not been investigated in SALS. Our aim was to elucidate the involvement of MATR3 pathology in SALS. The immunoreactivity of MATR3 was investigated in motor neurons of lumbar spinal cords from 15 autopsied SALS cases, and it was also investigated whether MATR3 co-localizes in TDP-43–positive NCIs. On the basis of our results, we discuss the pathologic role of MATR3 in SALS. The brains and spinal cords of 15 SALS and seven control cases for which autopsies were performed from 2004 to 2015 at the Yokohama City University Hospital were analyzed. This study was approved by the institutional review board of Yokohama City University School of Medicine. The following information from the medical records and clinical summaries was reviewed: age at onset, sex, initial symptoms, and disease duration. It was clinically and neuropathologically confirmed that control cases did not have motor neuron disease. Clinical features of SALS cases (cases 1 to 15) and control cases (cases 16 to 22) are summarized in Table 1.Table 1Clinical Findings of the Cases Used in This StudyDiseaseCase no.Age at death, yearsSexInitial symptom (muscle weakness)Disease duration, monthsALS164MUpper limb30254MUpper limb24372MUpper limb3454MUpper limb48587MUpper limb29663MUpper limb12772FUpper limb31864MUpper limb23961MLower limb81065MLower limb481179MLower limb751286MBulbar211379MBulbar221469FBulbar71578MBulbar9Sarcoidosis1653MNANADLBCL1737MNANACCA1889FNANAPD1971MNANAAD2084FNANACPA2177FNANASarcoidosis2280FNANAF, female; M, male; AD, Alzheimer disease; ALS, amyotrophic lateral sclerosis; CCA, cortical cerebellar atrophy; CPA, cardiac pulmonary arrest; DLBCL, diffuse large B-cell lymphoma; NA, not applicable; PD, Parkinson disease. Open table in a new tab F, female; M, male; AD, Alzheimer disease; ALS, amyotrophic lateral sclerosis; CCA, cortical cerebellar atrophy; CPA, cardiac pulmonary arrest; DLBCL, diffuse large B-cell lymphoma; NA, not applicable; PD, Parkinson disease. For six cases in which DNA materials were available, GGGGCC hexanucleotide expansion in C9orf72 was screened for using repeat-primed PCR, as reported previously.5DeJesus-Hernandez M. Mackenzie I.R. Boeve B.F. Boxer A.L. Baker M. Rutherford N.J. Nicholson A.M. Finch N.A. Flynn H. Adamson J. Kouri N. Wojtas A. Sengdy P. Hsiung G.Y. Karydas A. Seeley W.W. Josephs K.A. Coppola G. Geschwind D.H. Wszolek Z.K. Feldman H. Knopman D.S. Petersen R.C. Miller B.L. Dickson D.W. Boylan K.B. Graff-Radford N.R. Rademakers R. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (3410) Google Scholar Fragment analysis was performed using an ABI PRISM 3500xl (Life Technologies, Carlsbad, CA) and the GeneMapper Software version 3.5 (Applied Biosystems, Foster City, CA). We used samples from patients with the C9orf72 hexanucleotide repeat expansion as controls which were provided from Japanese Consortium for Amyotrophic Lateral Sclerosis Research (Nagoya, Japan). Full-length MATR3 (NM_018834.5) cDNA (pFN21ASDA0723) was obtained from Kazusa DNA Research Institute (Chiba, Japan). A part of SH-SY5Y or HEK293T cells on 24-well plates were transfected with 0.5 μg of pFN21ASDA0723 vector using Lipofectamine LTX transfection reagent (Thermo Fisher Scientific, Waltham, MA). The cells with or without transfection were then washed twice with ice-cold phosphate-buffered saline (PBS), fixed for 20 minutes in 4% paraformaldehyde and PBS, washed twice with PBS, and permeabilized for 5 minutes with 0.1% Triton X-100 and PBS. Immunostaining was performed using the four anti-MATR3 antibodies listed in Table 2. Goat polyclonal Alexa Fluor 488–conjugated anti-mouse or anti-rabbit IgG (A-11017 or A-11034; Thermo Fisher Scientific) was used as the secondary antibody. Halo-tagged MATR3 was also visualized using the Halo-tag TMR ligand (Promega Corporation, Madison, WI).Table 2Antibodies Used for Immunohistochemical AnalysisAntibodyTypeDilutionCatalog no.Immunogen (target region)SourceMatrin 3Rabbit polyclonal1:200A300-590AHuman MATR3 (aa475–aa500)Bethyl LaboratoriesMatrin 3Rabbit monoclonal1:100EPR10635 (B)Not availableAbcam (Cambridge, UK)Matrin 3Rabbit polyclonal1:100AV40922Human MATR3 (aa2–aa51)Sigma-Aldrich (St. Louis, MO)Matrin 3Mouse monoclonal1:100SC-81318Human MATR3 (internal region)Santa Cruz Biotechnology (Santa Cruz, CA)pTDP-43 (pSer409/410)Mouse monoclonal1:1000TIP-PTD-M01Human TDP-43 [aa405-aa414 (with phosphorylated aa409 and aa410)]CosmoBio (Tokyo, Japan)TDP-43 (full-length)Mouse monoclonal1:200060019-2-lgHuman TDP-43-GST fusion protein: catalog no. Ag1231Proteintech (Chicago, IL)UbiquitinRabbit polyclonal1:1000Z0458Cow ubiquitinDako (Carpinteria, CA)aa, amino acid; MATR3, matrin 3; pTDP-43, phosphorylated transactivating response region DNA–binding protein 43; TDP-43, transactivating response region DNA–binding protein 43. Open table in a new tab aa, amino acid; MATR3, matrin 3; pTDP-43, phosphorylated transactivating response region DNA–binding protein 43; TDP-43, transactivating response region DNA–binding protein 43. For Western blotting, SH-SY5Y or HEK293T cells grown in 10-cm culture dishes were washed twice with ice-cold PBS, lyzed with radioimmunoprecipitation assay buffer [50 mmol/L Tris hydrochloride (pH 7.5), 150 mmol/L sodium calcium, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mmol/L EDTA, and complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)], and then briefly sonicated. Cell lysates were subjected to SDS-PAGE followed by Western blotting with four anti-MATR3 antibodies listed in Table 2. Horseradish peroxidase –conjugated sheep polyclonal anti-mouse IgG or donkey polyclonal anti-rabbit IgG (NA931V or NA934V; GE Healthcare UK Ltd., Buckinghamshire, UK) was used as the secondary antibody. As a control, human MATR3 was prepared as follows. HEK293T cells in 10-cm dishes were transfected with 14 μg of vector pFN21ASDA0723. Twenty-four hours after transfection, MATR3 was purified using the Halo-Tag Mammalian Pull-Down system (G6504; Promega Corporation). After elution of MATR3, the Halo-tag was removed by TEV protease (Promega Corporation) cleavage. Each brain and spinal cord was fixed in 20% neutral buffered formalin (Muto Pure Chemicals, Tokyo, Japan) for 7 to 13 days, embedded in paraffin, and sectioned at a thickness of 6 μm. Sections from the frontal pole, middle frontal gyrus, motor cortex, temporal pole, superior and the middle temporal gyrus, visual cortex, anterior cingulate gyrus, hypothalamus, amygdala, hippocampal formation, striatum, globus pallidus, thalamus, midbrain (including the substantial nigra and the red nucleus), upper pons at the level of the locus coeruleus, lower pons at the level of the motor nucleus of the facial nerve, medulla oblongata at the level of the hypoglossal nucleus, cerebellum (including dentate nucleus), cervical spinal cord, thoracic spinal cord, lumbar spinal cord, and sacral spinal cord were stained with hematoxylin and eosin, as well as by the Klüver-Barrera method for conventional neuropathologic studies. For immunohistochemical staining of MATR3 and TDP-43, lumbar spinal cord (at the lumbar enlargement) sections of each case were autoclaved at 121°C for 10 minutes, incubated in 100% formic acid for 5 minutes, and then incubated in 1% hydrogen peroxide for 20 minutes. Sections were immunostained with the primary antibodies described in Table 2, using the avidin-biotin-peroxidase complex method (ABC Elite; Vector Laboratories, Burlingame, CA). For double staining of MATR3 and TDP-43, Alexa Fluor 568–conjugated anti-mouse IgG antibody (A-11004) and Alexa Fluor 488–conjugated anti-rabbit IgG (A-11008) (Molecular Probes, Eugene, OR) were used as secondary antibodies. The sections of lumbar spinal cord were stained with hematoxylin and eosin to assess the extent and severity of neuronal loss and gliosis, which were graded independently as follows: 0, none; 1, mild; 2, moderate; or 3, severe. Immunostained preparations were used to evaluate the subcellular distribution of NCIs that contained TDP-43 or MATR3. To evaluate the frequency of TDP-43–positive and MATR3-positive NCIs of lower motor neurons, the number of anterior horn neurons were counted in two sections from the lumbar spinal cord (L4 or L5). Numerical comparisons were performed and assessed with the U-test. Differences were considered significant at P < 0.05. All 15 SALS cases were diagnosed with ALS by the conventional neuropathologic evaluation. TDP-43–positive NCIs (skein-like inclusions and round inclusions) were present in the spinal anterior horn cells of all ALS cases (Table 318Brettsschneider J. Tredici K.D. Toledo J.B. Robinson J.L. Irwin D.J. Grossman M. Suh E. Van Deerlin V.M. Wood E.M. Baek Y. Kwong L. Lee E. Elman L. McCluskey L. Fang L. Feldengut S. Ludolph A.C. Lee V.M. Braak H. Trojanowski J.Q. Stages of pDP-43 pathology in amyotrophic lateral sclerosis.Ann Neurol. 2013; 74: 20-38Crossref PubMed Scopus (620) Google Scholar and Supplemental Figure S1). None of the control cases exhibited TDP-43–positive deposits in brains or spinal cords.Table 3Pathologic Findings in ALS and Control CasesCase no.Brain weight, gStage of TDP-43∗Stages of pTDP-43 pathology: stage 1, agranular motor cortex, bulbar and spinal somatomotor neurons; stage 2, reticular formation and precerebellar nuclei; stage 3, prefrontal neocortex and basal ganglia; and stage 4, anteromedial areas of the temporal lobe and hippocampal formation (according to the methods described in the article by Brettschneider et al18).Neuronal loss and gliosis†Neuronal loss and gliosis of the anterior horn: 0, none; 1, mild; 2, moderate; and 3, severe.AHC with NCI/total AHC, n (%)Nuclear staining pattern of cells having MATR3-positive NCIsMild staining nuclei of AHC, n (%)Strong staining nuclei of AHC, n (%)pTDP-43MATR3ALS cases 11425429/37 (24)1/45 (2)Mild 128 (62)17 (38) 21230139/40 (22)0/43 (0)20 (47)23 (53) 31270433/23 (13)2/18 (9)Mild 1, strong 16 (33)12 (67) 41530433/22 (14)0/33 (0)1 (3)32 (97) 51300115/45 (11)1/55 (2)Mild 135 (64)20 (36) 615002130/64 (47)5/64 (8)Mild 4, strong 129 (45)35 (55) 714502318/23 (78)0/36 (0)13 (36)23 (64) 81330425/37 (14)1/34 (3)Mild 121 (43)28 (57) 913201216/42 (38)1/31 (3)Mild 18 (26)23 (74) 1013403319/48 (40)2/43 (4)Strong 216 (37)27 (63) 111370422/23 (8)0/24 (0)5 (21)19 (79) 121020426/39 (15)0/30 (0)2 (7)28 (93) 131350422/40 (5)0/30 (0)11 (16)19 (84) 1413104211/50 (22)2/43 (4)Mild 1, strong 119 (44)24 (56) 151160229/75 (12)3/59 (5)Mild 325 (42)34 (58)Control cases 16150000028 (62)17 (38) 17105500020 (47)23 (53) 1810800006 (33)12 (67) 1913900001 (3)32 (97) 20121000035 (64)20 (36) 21115000029 (45)35 (55) 22105000013 (36)23 (64)AHC, anterior horn cell; NCI, neuronal cytoplasmic inclusion; pTDP-43, phosphorylated transactivating response region DNA–binding protein 43; TDP-43, transactivating response region DNA–binding protein 43.∗ Stages of pTDP-43 pathology: stage 1, agranular motor cortex, bulbar and spinal somatomotor neurons; stage 2, reticular formation and precerebellar nuclei; stage 3, prefrontal neocortex and basal ganglia; and stage 4, anteromedial areas of the temporal lobe and hippocampal formation (according to the methods described in the article by Brettschneider et al18Brettsschneider J. Tredici K.D. Toledo J.B. Robinson J.L. Irwin D.J. Grossman M. Suh E. Van Deerlin V.M. Wood E.M. Baek Y. Kwong L. Lee E. Elman L. McCluskey L. Fang L. Feldengut S. Ludolph A.C. Lee V.M. Braak H. Trojanowski J.Q. Stages of pDP-43 pathology in amyotrophic lateral sclerosis.Ann Neurol. 2013; 74: 20-38Crossref PubMed Scopus (620) Google Scholar).† Neuronal loss and gliosis of the anterior horn: 0, none; 1, mild; 2, moderate; and 3, severe. Open table in a new tab AHC, anterior horn cell; NCI, neuronal cytoplasmic inclusion; pTDP-43, phosphorylated transactivating response region DNA–binding protein 43; TDP-43, transactivating response region DNA–binding protein 43. Subcellular localization of MATR3 was evaluated in the spinal cords of SALS and control cases by anti-MATR3 immunostaining. The immunoreactivity of anti-MATR3 antibodies was examined in SH-SY5Y and HEK293T cells. It was confirmed that all four anti-MATR3 antibodies reacted with MATR3 in both cell lines and were suitable for use in Western blotting and immunohistochemistry (Supplemental Figures S2 and S3). However, specificity of AV40922 was lower than that of the other antibodies. In immunohistochemistry, only a single antibody (A300-590A, Bethyl Laboratories, Montgomery, TX) detected MATR3-positive inclusions in the SALS cases tested (Figure 1). Therefore, this antibody was used for further evaluations. MATR3 in the nuclei of motor neurons exhibits two distinct immunostaining patterns: mild and strong (Figure 2). There were no significant differences in the frequency of mild nuclear staining between SALS (35.0% ± 17.8%) and control cases (42.7% ± 22.8%) (Figure 2 and Table 3). This was also true for the frequency of strong nuclear staining (64.9% ± 17.8% for SALS versus 57.2% ± 22.8% for control). Glial cells also exhibited mild and strong staining patterns in SALS and control cases. These results indicate that the strong nuclear staining of motor neuron and glial cells described in the original report15Johnson J.O. Pioro E.P. Boehringer A. Chia R. Feit H. Renton A.E. Pliner H.A. Abramzon Y. Marangi G. Winborn B.J. Gibbs J.R. Nalls M.A. Morgan S. Shoai M. Hardy J. Pittman A. Orrell R.W. Malaspina A. Sidle K.C. Fratta P. Harms M.B. Baloh R.H. Pestronk A. Weihl C.C. Rogaeva E. Zinman L. Drory V.E. Borghero G. Mora G. Calvo A. Rothstein J.D. Drepper C. Sendtner M. Singleton A.B. Taylor J.P. Cookson M.R. Restagno G. Sabatelli M. Bowser R. Chio A. Traynor B.J. ITALSGENMutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis.Nat Neurosci. 2014; 17: 664-666Crossref PubMed Scopus (318) Google Scholar was not a specific pathologic finding of SALS. Importantly, MATR3-positive NCIs were observed in 2% to 9% of motor neurons in nine of 15 SALS cases (Table 3). Although 16 of 18 MATR3-positive NCIs (88.9%) were round in shape, 2 of 18 (11.1%) were skein-like in shape (Figure 1), similar to those observed in TDP-43 positive NCIs.2Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4499) Google Scholar Furthermore, most of the motor neurons with MATR3-positive NCIs had a mild nuclear staining pattern (Table 3 and Figure 1).Figure 2Matrin 3 (MATR3) staining pattern of lumbar spinal cords in sporadic amyotrophic lateral sclerosis (SALS) and control cases. A: Immunohistochemical staining with anti-MATR3 antibody in the lumbar cord of SALS case 14. Nuclei of motor neurons exhibit both strong (arrowheads) and mild (arrows) staining patterns. A subset of motor neurons have MATR3-positive neuronal cytoplasmic inclusions (NCIs). B: Immunohistochemical staining with anti-MATR3 antibody in the lumbar cord of control case 17. As in SALS case 14, the nuclei of motor neurons exhibit both strong (arrowheads) and mild (arrows) staining patterns. C: Nucleus of motor neuron showing strong staining pattern (case 15). D: Nucleus of motor neuron showing mild staining pattern with NCI (case 15). Scale bars: 50 μm (A and B); 10 μm (C and D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm whether these SALS cases included C9orf72-related ALS, ubiquitin-positive and TDP-43–negative cytoplasmic inclusions were studied in the cerebellum and hippocampus5DeJesus-Hernandez M. Mackenzie I.R. Boeve B.F. Boxer A.L. Baker M. Rutherford N.J. Nicholson A.M. Finch N.A. Flynn H. Adamson J. Kouri N. Wojtas A. Sengdy P. Hsiung G.Y. Karydas A. Seeley W.W. Josephs K.A. Coppola G. Geschwind D.H. Wszolek Z.K. Feldman H. Knopman D.S. Petersen R.C. Miller B.L. Dickson D.W. Boylan K.B. Graff-Radford N.R. Rademakers R. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (3410) Google Scholar, 19Al-Sarraj S. King A. Troakes C. Smith B. Maekawa S. Bodi I. Rogelj B. Al- Chalabi A. Hortobagyi T. Shaw C.E. P62 positive, TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the pathology of C9orf72-linked FTLD and MND/ALS.Acta Neuropathol. 2011; 122: 691-702Crossref PubMed Scopus (363) Google Scholar, 20Mackenzie I.R. Arzberger T. Kremmer E. Troost D. Lorenzl S. Mori K. Weng S.M. Haass C. Kretzschmar H.A. Edbauer D. Neumann M. Dipeptide repeat protein pathology in C9ORF72 mutation cases: clinico-pathological correlations.Acta Neuropathol. 2013; 126: 859-879Crossref PubMed Scopus (242)

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