Characterization of Alternative Isoforms and Inclusion Body of the TAR DNA-binding Protein-43
2009; Elsevier BV; Volume: 285; Issue: 1 Linguagem: Inglês
10.1074/jbc.m109.022012
ISSN1083-351X
AutoresYoshinori Nishimoto, Daisuke Ito, Takuya Yagi, Yoshihiro Nihei, Yoshiko Tsunoda, Norihiro Suzuki,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoTAR DNA-binding protein-43 (TDP-43) has been recently identified as a major component of the ubiquitinated inclusions found in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis, diseases that are collectively termed TDP-43 proteinopathies. Several amyotrophic lateral sclerosis-linked mutations of the TDP-43 gene have also been identified; however, the precise molecular mechanisms underlying the neurodegeneration remain unclear. To investigate the biochemical characteristics of TDP-43, we examined truncation, isoforms, and cytoplasmic inclusion (foci) formation using TDP-43-expressing cells. Under apoptosis, caspase-3 generates two 35-kDa (p35f) and 25-kDa (p25f) fragments. However, in caspase-3(−/−) cells, novel caspase-3-independent isoforms of these two variants (p35iso and p25iso) were also detected under normal conditions. With a deletion mutant series, the critical domains for generating both isoforms were determined and applied to in vitro transcription/translation, revealing alternate in-frame translation start sites downstream of the natural initiation codon. Subcellular localization analysis indicated that p35 (p35f and p35iso) expression leads to the formation of stress granules, cellular structures that package mRNA and RNA-binding proteins during cell stress. After applying proteasome inhibitors, aggresomes, which are aggregates of misfolded proteins, were formed in the cytoplasm of cells expressing p35. Collectively, this study demonstrates that the 35-kDa isoforms of TDP-43 assemble in stress granules, suggesting that TDP-43 plays an important role in translation, stability, and metabolism of mRNA. Our findings provide new biological and pathological insight into the development of TDP-43 proteinopathies. TAR DNA-binding protein-43 (TDP-43) has been recently identified as a major component of the ubiquitinated inclusions found in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis, diseases that are collectively termed TDP-43 proteinopathies. Several amyotrophic lateral sclerosis-linked mutations of the TDP-43 gene have also been identified; however, the precise molecular mechanisms underlying the neurodegeneration remain unclear. To investigate the biochemical characteristics of TDP-43, we examined truncation, isoforms, and cytoplasmic inclusion (foci) formation using TDP-43-expressing cells. Under apoptosis, caspase-3 generates two 35-kDa (p35f) and 25-kDa (p25f) fragments. However, in caspase-3(−/−) cells, novel caspase-3-independent isoforms of these two variants (p35iso and p25iso) were also detected under normal conditions. With a deletion mutant series, the critical domains for generating both isoforms were determined and applied to in vitro transcription/translation, revealing alternate in-frame translation start sites downstream of the natural initiation codon. Subcellular localization analysis indicated that p35 (p35f and p35iso) expression leads to the formation of stress granules, cellular structures that package mRNA and RNA-binding proteins during cell stress. After applying proteasome inhibitors, aggresomes, which are aggregates of misfolded proteins, were formed in the cytoplasm of cells expressing p35. Collectively, this study demonstrates that the 35-kDa isoforms of TDP-43 assemble in stress granules, suggesting that TDP-43 plays an important role in translation, stability, and metabolism of mRNA. Our findings provide new biological and pathological insight into the development of TDP-43 proteinopathies. IntroductionAmyotrophic lateral sclerosis (ALS) 2The abbreviations used are: ALSamyotrophic lateral sclerosisTDP-43TAR DNA-binding protein-43FTLD-Ufrontotemporal lobar degeneration with ubiquitin-positive inclusionsCTFC-terminal fragmentSGstress granuleSOD-1Cu,Zn-superoxide dismutase-1CFTRcystic fibrosis transmembrane conductance regulatorMEFmouse embryonic fibroblastSMNsurvival motor neuronNLSnuclear localization signalIBNCinclusion body under normal conditionIBPIinclusion body under proteasome inhibitionMTOCmicrotubule organizing centerGFPgreen fluorescent proteinsALSsporadic ALSfALSfamilial ALSCMVcytomegalovirusZbenzyloxycarbonylFMKfluoromethyl ketoneaaamino acid(s)IBinclusion bodyPBprocessing bodyRFPred fluorescent proteinPABPpoly(A)-binding proteinHuRHu antigen RG3BPGAP SH3 domain-binding protein. was first reported in 1869 by the French neurologist Jean-Martin Charcot and is one of the most serious neurological diseases. ALS is characterized by progressive degeneration of upper and lower motor neurons, and although the vast majority of ALS cases are sporadic (sALS), almost 10% appear to be familial (fALS). Although mutations in the gene encoding the antioxidant enzyme Cu,Zn-superoxide dismutase-1 (SOD-1) have been detected in 20% of fALS patients (1Shaw C.E. Enayat Z.E. Chioza B.A. Al-Chalabi A. Radunovic A. Powell J.F. Leigh P.N. Ann. Neurol. 1998; 43: 390-394Crossref PubMed Scopus (142) Google Scholar), the cause of sALS and fALS not associated with SOD-1 remains unclear. Recently, two research groups have identified TDP-43 as a major component of ubiquitinated neuronal cytoplasmic and intranuclear inclusions identified in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), as well as in sALS (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. 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Neurol. 2007; 64: 1388-1394Crossref PubMed Scopus (147) Google Scholar).TDP-43 is a ubiquitously expressed nuclear protein that was originally identified as a binding protein of the human immunodeficiency virus, type-1 TAR DNA element (11Ou S.H. Wu F. Harrich D. García-Martínez L.F. Gaynor R.B. J. Virol. 1995; 69: 3584-3596Crossref PubMed Google Scholar). Evidence suggests that TDP-43 is involved in the regulation of RNA splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) and survival motor neuron (SMN) (12Buratti E. Dörk T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (494) Google Scholar, 13Bose J.K. Wang I.F. Hung L. Tarn W.Y. Shen C.K. J. Biol. Chem. 2008; 283: 28852-28859Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Loss of functional TDP-43 is known to affect nuclear membrane stability and induce apoptosis via phosphorylation of the retinoblastoma protein (14Ayala Y.M. Misteli T. Baralle F.E. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3785-3789Crossref PubMed Scopus (191) Google Scholar).Limited biochemical evidence has demonstrated the mechanisms underlying the molecular pathogenesis of TDP-43 proteinopathy. Using phosphorylation-specific antibodies, Hasegawa et al. demonstrated that TDP-43 is abnormally phosphorylated in the brain tissue of FTLD-U and ALS patients (15Hasegawa M. Arai T. Nonaka T. Kametani F. Yoshida M. Hashizume Y. Beach T.G. Buratti E. Baralle F. Morita M. Nakano I. Oda T. Tsuchiya K. Akiyama H. Ann. Neurol. 2008; 64: 60-70Crossref PubMed Scopus (494) Google Scholar). Zhang et al. focused on proteolytic cleavage and demonstrated that caspase-3 can mediate cleavage of TDP-43 to generate 25- and 35-kDa fragments when progranulin (a candidate gene for familial FTLD-U) is down-regulated (16Zhang Y.J. Xu Y.F. Dickey C.A. Buratti E. Baralle F. Bailey R. Pickering-Brown S. Dickson D. Petrucelli L. J. Neurosci. 2007; 27: 10530-10534Crossref PubMed Scopus (312) Google Scholar). They also reported that the 25-kDa C-terminal fragment (CTF) of caspase-cleaved TDP-43 leads to the formation of toxic cytoplasmic inclusions within cells (17Zhang Y.J. Xu Y.F. Cook C. Gendron T.F. Roettges P. Link C.D. Lin W.L. Tong J. Castanedes-Casey M. Ash P. Gass J. Rangachari V. Buratti E. Baralle F. Golde T.E. Dickson D.W. Petrucelli L. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7607-7612Crossref PubMed Scopus (439) Google Scholar). More recently, Igaz et al. identified the cleavage site for the lowest molecular mass TDP-43 CTF (∼22 kDa) in cortical urea extracts of FTLD-U brain and demonstrated that this fragment can recapitulate some of the pathological features of TDP-43 proteinopathy (18Igaz L.M. Kwong L.K. Chen-Plotkin A. Winton M.J. Unger T.L. Xu Y. Neumann M. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2009; 284: 8516-8524Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). However, little is still known about the biochemical process of the generation of truncated forms of TDP-43 and the characteristics of cytoplasmic inclusion formation of these fragments.In this study, we first verified the caspase-3-dependent generation of CTFs of TDP-43 protein (p35f and p25f), which were abolished in caspase-3(−/−) mouse embryonic fibroblasts (MEFs). Furthermore, we identified two novel caspase-3-independent isoforms (p35iso and p25iso). Site-specific mutagenesis and in vitro transcription/translation revealed that these isoforms were generated from an alternative translation start site. Our results also show that the p35 forms of TDP-43 (p35f and p35iso) accumulate in two types of cytoplasmic foci, stress granules (SGs) under normal conditions and aggresomes under proteasome inhibition. These findings reveal novel biochemical properties of the TDP-43 protein and suggest the possibility that RNA quality control via the function of SGs is involved in the development of TDP-43 proteinopathy.DISCUSSIONA number of studies have shown that truncation of accumulated neurotoxic proteins associated with neurodegeneration, including the amyloid precursor protein, polyglutamine, and Tau, is critical for neurodegeneration processes (41Sisodia S.S. Price D.L. FASEB J. 1995; 9: 366-370Crossref PubMed Scopus (224) Google Scholar, 42DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2284) Google Scholar, 43Wellington C.L. Singaraja R. Ellerby L. Savill J. Roy S. Leavitt B. Cattaneo E. Hackam A. Sharp A. Thornberry N. Nicholson D.W. Bredesen D.E. Hayden M.R. J. Biol. Chem. 2000; 275: 19831-19838Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 44Mende-Mueller L.M. Toneff T. Hwang S.R. 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Yoshida M. Hashizume Y. Oda T. Biochem. Biophys. Res. Commun. 2006; 351: 602-611Crossref PubMed Scopus (1835) Google Scholar, 18Igaz L.M. Kwong L.K. Chen-Plotkin A. Winton M.J. Unger T.L. Xu Y. Neumann M. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2009; 284: 8516-8524Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Zhang et al. have suggested that proteolytic cleavage generates the 25- and 35-kDa CTFs present in cultured cells, leading to redistribution of TDP-43 from its nuclear location to the cytoplasm and is associated with cell death through a toxic gain of function (16Zhang Y.J. Xu Y.F. Dickey C.A. Buratti E. Baralle F. Bailey R. Pickering-Brown S. Dickson D. Petrucelli L. J. Neurosci. 2007; 27: 10530-10534Crossref PubMed Scopus (312) Google Scholar, 17Zhang Y.J. Xu Y.F. Cook C. Gendron T.F. Roettges P. Link C.D. Lin W.L. Tong J. Castanedes-Casey M. Ash P. Gass J. Rangachari V. Buratti E. Baralle F. Golde T.E. Dickson D.W. Petrucelli L. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7607-7612Crossref PubMed Scopus (439) Google Scholar). Furthermore, Sreedharan et al. have demonstrated that a mutation linked to fALS enhanced fragmentation of TDP-43 in Chinese hamster ovary cells (5Sreedharan 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. Science. 2008; 319: 1668-1672Crossref PubMed Scopus (1927) Google Scholar), suggesting that truncated TDP-43 is critical in neurodegeneration related to TDP-43 proteinopathies.Our present study presents several important insights into the proteolytic cleavage and isoforms of TDP-43. Our study identified TDP-43 variant forms: two caspase-3-dependent fragments and two novel isoforms. Under normal conditions, the novel isoforms, p35iso (aa 85–414) and p25iso (aa ∼170–414), are translated from methionine 85 and aa 160–169, respectively, and apoptosis generates two fragments, p35f and p25f, via caspase-3 activation.The N-terminal amino acids of p25f and p25iso remain unclear. Based on the prediction of caspase-3 cleavage consensus sites (DXXD), p25f should comprise aa 220–414, with a molecular weight of appropriate 19,719, which is obviously smaller than 25 kDa. However, electromobility of overexpressed aa 170–414 (molecular weight: 25,635) of cDNA was compatible to that of the endogenous 25-kDa fragments seen on SDS-PAGE (Fig. 3H). Fig. 2 (B and C) shows no discrepancy in the size between p25f and p25iso on SDS-PAGE. Moreover, well characterized monoclonal anti-TDP-43 antibody, 2E2-D3, could not recognize aa 208–414 and the smaller C-terminal residues of human TDP-43 (26Zhang H.X. Tanji K. Mori F. Wakabayashi K. Neurosci. Lett. 2008; 434: 170-174Crossref PubMed Scopus (35) Google Scholar) but could detect both endogenous p25f and p25iso (Fig. 2C), indicating that the N terminus of p25f and p25iso is further upstream than previously reported (16Zhang Y.J. Xu Y.F. Dickey C.A. Buratti E. Baralle F. Bailey R. Pickering-Brown S. Dickson D. Petrucelli L. J. Neurosci. 2007; 27: 10530-10534Crossref PubMed Scopus (312) Google Scholar, 18Igaz L.M. Kwong L.K. Chen-Plotkin A. Winton M.J. Unger T.L. Xu Y. Neumann M. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2009; 284: 8516-8524Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 28Nonaka T. Kametani F. Arai T. Akiyama H. Hasegawa M. Hum. Mol. Genet. 2009; 18: 3353-3364Crossref PubMed Scopus (226) Google Scholar). Therefore, we suggest that DVMD (aa 216–219) may not be the true cleavage site of TDP-43 for generation of p25f and that additional unknown protease(s) activated by caspase-3 may generate p25f under apoptosis. We cannot however exclude the other possibility that p25iso and p25f are identical and caspase-3-dependent signaling suppresses degradation and/or enhances translation of p25iso, resulting in the accumulation of p25iso under staurosporine treatment.Recently, Igaz et al. have reported two major fragments of ∼22 and 24 kDa that were recognized in immunoprecipitation studies using an anti-TDP-43 antibody and cortical extracts of FTLD-U brain tissue (18Igaz L.M. Kwong L.K. Chen-Plotkin A. Winton M.J. Unger T.L. Xu Y. Neumann M. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2009; 284: 8516-8524Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Both this group and Nonaka et al. identified several cleavage sites (aa 208, 219, and 247) for generation of the smaller 22-kDa CTF of TDP-43 by protein sequencing (18Igaz L.M. Kwong L.K. Chen-Plotkin A. Winton M.J. Unger T.L. Xu Y. Neumann M. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2009; 284: 8516-8524Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 28Nonaka T. Kametani F. Arai T. Akiyama H. Hasegawa M. Hum. Mol. Genet. 2009; 18: 3353-3364Crossref PubMed Scopus (226) Google Scholar). As several smaller fragments (<25 kDa) appear following longer exposure in this study (Fig. 1A), several cleavage sites as well as alternate initial codons appear to exist within aa 160–250 for generating smaller fragments and isoforms.Another important finding was the characterization of TDP-43-containing IBs that appear in cells expressing p35. Two distinct IBs (IBPI and IBNC) were identified in either the presence or absence of proteasome inhibitors. IBPI demonstrated properties similar to aggresomes, which are formed by the assembly of misfolded proteins at the MTOC, and contained the redistributed intermediate filament protein vimentin in addition to other pathogenic proteins including mutant forms of huntingtin, α-synuclein, parkin, and prion (29Kopito R.R. Trends Cell Biol. 2000; 10: 524-530Abstract Full Text Full Text PDF PubMed Scopus (1583) Google Scholar, 30García-Mata R. Bebök Z. Sorscher E.J. Sztul E.S. J. Cell Biol. 1999; 146: 1239-1254Crossref PubMed Scopus (502) Google Scholar, 31Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1764) Google Scholar, 32Junn E. Lee S.S. Suhr U.T. Mouradian M.M. J. Biol. Chem. 2002; 277: 47870-47877Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 33Tanaka M. Kim Y.M. Lee G. Junn E. Iwatsubo T. Mouradian M.M. J. Biol. 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SGs, which are induced by stress events, including hypoxia, heat shock, and arsenite, are multimolecular aggregates present in the cytosol and are responsible for the recruitment of ribonucleoprotein and mRNA. SGs are thought to function in the protection of mRNA against harmful stress events and to arrest translation, preventing the accumulation of misfolded proteins (36Kedersha N. Stoecklin G. Ayodele M. Yacono P. Lykke-Andersen J. Fritzler M.J. Scheuner D. Kaufman R.J. Golan D.E. Anderson P. J. Cell Biol. 2005; 169: 871-884Crossref PubMed Scopus (1025) Google Scholar, 38Kedersha N. Anderson P. Methods Enzymol. 2007; 431: 61-81Crossref PubMed Scopus (478) Google Scholar). We found that p35 forms a part of these SGs and may facilitate the assembly of SGs when overexpressed, similar to other SG-associated proteins: G3BP, TIA-1/TIAR, and SMN (38Kedersha N. Anderson P. Methods Enzymol. 2007; 431: 61-81Crossref PubMed Scopus (478) Google Scholar, 47Tourrière H. Chebli K. Zekri L. 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As both are localized to the cytoplasm but lack the NLS, they subsequently form aggregates with a structure comparable to intracellular inclusions pathologically observed in the brain tissue of FTLD-U and ALS patients. p35 is also involved in SG formation. Recent evidence suggests that pathological aggregations of toxic proteins, including polyglutamine, and that β-amyloid are not directly linked to pathogenesis and rather might be neuroprotective (50Arrasate M. Mitra S. Schweitzer E.S. Segal M.R. Finkbeiner S. Nature. 2004; 431: 805-810Crossref PubMed Scopus (1584) Google Scholar, 51Taylor J.P. Tanaka F. Robitschek J. Sandoval C.M. Taye A. Markovic-Plese S. Fischbeck K.H. Hum. Mol. Genet. 2003; 12: 749-757Crossref PubMed Scopus (359) Google Scholar, 52Ito D. Suzuki N. Brain. 2009; 132: 8-15Crossref PubMed Scopus (111) Google Scholar, 53Orr H.T. Nature. 2004; 431: 747-748Crossref PubMed Scopus (26) Google Scholar, 54Selkoe D.J. Science. 2002; 298: 789-791Crossref PubMed Scopus (3328) Google Scholar), inappropriate SG assembly may be induced by p35, thereby chronically disturbing RNA stability and translation arrest, which is a key process of pathogenesis, compared with pathological aggregations such as neuronal cytoplasmic and skein-like inclusions in TDP- 43 proteinopathy. Interestingly, a recent study has shown that SMN, a protein associated with autosomal recessive motor neuron disease and spinal muscular atrophy, facilitates SG formation, and that loss-of-function of SMN in motor neurons may influence neurodegeneration via the disruption of RNA stability through SG function (49Hua Y. Zhou J. FEBS Lett. 2004; 572: 69-74Crossref PubMed Scopus (122) Google Scholar). Moreover, it has been reported that editing of the RNA of GluR2, a subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, is markedly decreased in the motor neurons of sALS patients (55Kawahara Y. Ito K. Sun H. Aizawa H. Kanazawa I. Kwak S. Nature. 2004; 427: 801Crossref PubMed Scopus (448) Google Scholar, 56Nishimoto Y. Yamashita T. Hideyama T. Tsuji S. Suzuki N. Kwak S. Neurosci. Res. 2008; 61: 201-206Crossref PubMed Scopus (44) Google Scholar). Therefore, dysfunction of RNA quality control systems may be a common feature of the pathological processes occurring in the development of motor neuron diseases.Further investigations are required to determine whether fALS-linked mutant TDP-43 affects the formation and RNA metabolism of SGs. Moreover, although future studies are needed to elucidate the precise molecular pathogenesis of TDP-43 proteinopathies, our study contributes significantly toward a greater understanding for the treatment of the serious neurological diseases ALS and FTLD-U. IntroductionAmyotrophic lateral sclerosis (ALS) 2The abbreviations used are: ALSamyotrophic lateral sclerosisTDP-43TAR DNA-binding protein-43FTLD-Ufrontotemporal lobar degeneration with ubiquitin-positive inclusionsCTFC-terminal fragmentSGstress granuleSOD-1Cu,Zn-superoxide dismutase-1CFTRcystic fibrosis transmembrane conductance regulatorMEFmouse embryonic fibroblastSMNsurvival motor neuronNLSnuclear localization signalIBNCinclusion body under normal conditionIBPIinclusion body under proteasome inhibitionMTOCmicrotubule organizing centerGFPgreen fluorescent proteinsALSsporadic ALSfALSfamilial ALSCMVcytomegalovirusZbenzyloxycarbonylFMKfluoromethyl ketoneaaamino acid(s)IBinclusion bodyPBprocessing bodyRFPred fluorescent proteinPABPpoly(A)-binding proteinHuRHu antigen RG3BPGAP SH3 domain-binding protein. was first reported in 1869 by the French neurologist Jean-Martin Charcot and is one of the most serious neurological diseases. ALS is characterized by progressive degeneration of upper and lower motor neurons, and although the vast majority of ALS cases are sporadic (sALS), almost 10% appear to be familial (fALS). Although mutations in the gene encoding the antioxidant enzyme Cu,Zn-superoxide dismutase-1 (SOD-1) have been detected in 20% of fALS patients (1Shaw C.E. Enayat Z.E. Chioza B.A. Al-Chalabi A. Radunovic A. Powell J.F. Leigh P.N. Ann. Neurol. 1998; 43: 390-394Crossref PubMed Scopus (142) Google Scholar), the cause of sALS and fALS not associated with SOD-1 remains unclear. Recently, two research groups have identified TDP-43 as a major component of ubiquitinated neuronal cytoplasmic and intranuclear inclusions identified in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), as well as in sALS (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. Science. 2006; 314: 130-133Crossref PubMed Scopus (4383) Google Scholar, 3Arai T. Hasegawa M. Akiyama H. Ikeda K. Nonaka T. Mori H. Mann D. Tsuchiya K. Yoshida M. Hashizume Y. Oda T. Biochem. Biophys. Res. Commun. 2006; 351: 602-611Crossref PubMed Scopus (1835) Google Scholar). Missense mutations in TDP-43 have been found in autosomal dominant ALS families, suggesting that mutant TDP-43 may be a primary cause of motor neuron degeneration (4Van Deerlin V.M. Leverenz J.B. Bekris L.M. Bird T.D. Yuan W. Elman L.B. Clay D. Wood E.M. Chen-Plotkin A.S. Martinez-Lage M. Steinbart E. McCluskey L. Grossman M. Neumann M. Wu I.L. Yang W.S. Kalb R. Galasko D.R. Montine T.J. Trojanowski J.Q. Lee V.M. Schellenberg G.D. Yu C.E. Lancet Neurol. 2008; 7: 409-416Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar, 5Sreedharan 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. Science. 2008; 319: 1668-1672Crossref PubMed Scopus (1927) Google Scholar, 6Rutherford N.J. Zhang Y.J. Baker M. Gass J.M. Finch N.A. Xu Y.F. Stewart H. Kelley B.J. Kuntz K. Crook R.J. Sreedharan J. Vance C. Sorenson E. Lippa C. Bigio E.H. Geschwind D.H. Knopman D.S. Mitsumoto H. Petersen R.C. Cashman N.R. Hutton M. Shaw C.E. Boylan K.B. Boeve B. Graff-Radford N.R. Wszolek Z.K. Caselli R.J. Dickson D.W. Mackenzie I.R. Petrucelli L. Rademakers R. PLoS Genet. 2008; 4: e1000193Crossref PubMed Scopus (367) Google Scholar, 7Kabashi 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. Nat. Genet. 2008; 40: 572-574Crossref PubMed Scopus (1206) Google Scholar, 8Gitcho M.A. Baloh R.H. Chakraverty S. Mayo K. Norton J.B. Levitch D. Hatanpaa K.J. White 3rd, C.L. Bigio E.H. Caselli R. Baker M. Al-Lozi M.T. Morris J.C. Pestronk A. Rademakers R. Goate A.M. Cairns N.J. Ann. Neurol. 2008; 63: 535-538Crossref PubMed Scopus (519) Google Scholar, 9Yokoseki A. Shiga A. Tan C.F. Tagawa A. Kaneko H. Koyama A. Eguchi H. Tsujino A. Ikeuchi T. Kakita A. Okamoto K. Nishizawa M. Takahashi H. Onodera O. Ann. Neurol. 2008; 63: 538-542Crossref PubMed Scopus (328) Google Scholar). Importantly, pathological analysis revealed that abnormal accumulation of TDP-43 does not occur in fALS cases with SOD-1 mutations, suggesting that the pathological process in sALS is distinct from those associated with SOD-1 mutations. Currently, FTLD-U, sALS, and fALS-linked TDP-43 mutations are classified together as TDP-43 proteinopathies (10Neumann M. Kwong L.K. Sampathu D.M. Trojanowski J.Q. Lee V.M. Arch. Neurol. 2007; 64: 1388-1394Crossref PubMed Scopus (147) Google Scholar).TDP-43 is a ubiquitously expressed nuclear protein that was originally identified as a binding protein of the human immunodeficiency virus, type-1 TAR DNA element (11Ou S.H. Wu F. Harrich D. García-Martínez L.F. Gaynor R.B. J. Virol. 1995; 69: 3584-3596Crossref PubMed Google Scholar). Evidence suggests that TDP-43 is involved in the regulation of RNA splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) and survival motor neuron (SMN) (12Buratti E. Dörk T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (494) Google Scholar, 13Bose J.K. Wang I.F. Hung L. Tarn W.Y. Shen C.K. J. Biol. Chem. 2008; 283: 28852-28859Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Loss of functional TDP-43 is known to affect nuclear membrane stability and induce apoptosis via phosphorylation of the retinoblastoma protein (14Ayala Y.M. Misteli T. Baralle F.E. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3785-3789Crossref PubMed Scopus (191) Google Scholar).Limited biochemical evidence has demonstrated the mechanisms underlying the molecular pathogenesis of TDP-43 proteinopathy. Using phosphorylation-specific antibodies, Hasegawa et al. demonstrated that TDP-43 is abnormally phosphorylated in the brain tissue of FTLD-U and ALS patients (15Hasegawa M. Arai T. Nonaka T. Kametani F. Yoshida M. Hashizume Y. Beach T.G. Buratti E. Baralle F. Morita M. Nakano I. Oda T. Tsuchiya K. Akiyama H. Ann. Neurol. 2008; 64: 60-70Crossref PubMed Scopus (494) Google Scholar). Zhang et al. focused on proteolytic cleavage and demonstrated that caspase-3 can mediate cleavage of TDP-43 to generate 25- and 35-kDa fragments when progranulin (a candidate gene for familial FTLD-U) is down-regulated (16Zhang Y.J. Xu Y.F. Dickey C.A. Buratti E. Baralle F. Bailey R. Pickering-Brown S. Dickson D. Petrucelli L. J. Neurosci. 2007; 27: 10530-10534Crossref PubMed Scopus (312) Google Scholar). They also reported that the 25-kDa C-terminal fragment (CTF) of caspase-cleaved TDP-43 leads to the formation of toxic cytoplasmic inclusions within cells (17Zhang Y.J. Xu Y.F. Cook C. Gendron T.F. Roettges P. Link C.D. Lin W.L. Tong J. Castanedes-Casey M. Ash P. Gass J. Rangachari V. Buratti E. Baralle F. Golde T.E. Dickson D.W. Petrucelli L. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7607-7612Crossref PubMed Scopus (439) Google Scholar). More recently, Igaz et al. identified the cleavage site for the lowest molecular mass TDP-43 CTF (∼22 kDa) in cortical urea extracts of FTLD-U brain and demonstrated that this fragment can recapitulate some of the pathological features of TDP-43 proteinopathy (18Igaz L.M. Kwong L.K. Chen-Plotkin A. Winton M.J. Unger T.L. Xu Y. Neumann M. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 2009; 284: 8516-8524Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). However, little is still known about the biochemical process of the generation of truncated forms of TDP-43 and the characteristics of cytoplasmic inclusion formation of these fragments.In this study, we first verified the caspase-3-dependent generation of CTFs of TDP-43 protein (p35f and p25f), which were abolished in caspase-3(−/−) mouse embryonic fibroblasts (MEFs). Furthermore, we identified two novel caspase-3-independent isoforms (p35iso and p25iso). Site-specific mutagenesis and in vitro transcription/translation revealed that these isoforms were generated from an alternative translation start site. Our results also show that the p35 forms of TDP-43 (p35f and p35iso) accumulate in two types of cytoplasmic foci, stress granules (SGs) under normal conditions and aggresomes under proteasome inhibition. These findings reveal novel biochemical properties of the TDP-43 protein and suggest the possibility that RNA quality control via the function of SGs is involved in the development of TDP-43 proteinopathy.
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