TDP-43 Depletion Induces Neuronal Cell Damage through Dysregulation of Rho Family GTPases

The 43-kDa TAR DNA-binding protein (TDP-43) is known to be a major component of the ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Although TDP-43 is a nuclear protein, it disappears from the nucleus of affected neurons and glial cells, implicating TDP-43 loss of function in the pathogenesis of neurodegeneration. Here we show that the knockdown of TDP-43 in differentiated Neuro-2a cells inhibited neurite outgrowth and induced cell death. In knockdown cells, the Rho family members RhoA, Rac1, and Cdc42 GTPases were inactivated, and membrane localization of these molecules was reduced. In addition, TDP-43 depletion significantly suppressed protein geranylgeranylation, a key regulating factor of Rho family activity and intracellular localization. In contrast, overexpression of TDP-43 mitigated the cellular damage caused by pharmacological inhibition of geranylgeranylation. Furthermore administration of geranylgeranyl pyrophosphate partially restored cell viability and neurite outgrowth in TDP-43 knockdown cells. In summary, our data suggest that TDP-43 plays a key role in the maintenance of neuronal cell morphology and survival possibly through protein geranylgeranylation of Rho family GTPases. The 43-kDa TAR DNA-binding protein (TDP-43) is known to be a major component of the ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Although TDP-43 is a nuclear protein, it disappears from the nucleus of affected neurons and glial cells, implicating TDP-43 loss of function in the pathogenesis of neurodegeneration. Here we show that the knockdown of TDP-43 in differentiated Neuro-2a cells inhibited neurite outgrowth and induced cell death. In knockdown cells, the Rho family members RhoA, Rac1, and Cdc42 GTPases were inactivated, and membrane localization of these molecules was reduced. In addition, TDP-43 depletion significantly suppressed protein geranylgeranylation, a key regulating factor of Rho family activity and intracellular localization. In contrast, overexpression of TDP-43 mitigated the cellular damage caused by pharmacological inhibition of geranylgeranylation. Furthermore administration of geranylgeranyl pyrophosphate partially restored cell viability and neurite outgrowth in TDP-43 knockdown cells. In summary, our data suggest that TDP-43 plays a key role in the maintenance of neuronal cell morphology and survival possibly through protein geranylgeranylation of Rho family GTPases. The 43-kDa TAR DNA-binding protein (TDP-43) 2The abbreviations used are: TDP-4343-kDa TAR DNA-binding proteinALSamyotrophic lateral sclerosissiRNAsmall interfering RNAMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-caboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumGGPPgeranylgeranyl pyrophosphateMVAmevalonic acidPIpropidium iodideTUNELterminal deoxynucleotidyltransferase dUTP nick end labelingGAPDHglyceraldehyde-3-phosphate dehydrogenase. 2The abbreviations used are: TDP-4343-kDa TAR DNA-binding proteinALSamyotrophic lateral sclerosissiRNAsmall interfering RNAMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-caboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumGGPPgeranylgeranyl pyrophosphateMVAmevalonic acidPIpropidium iodideTUNELterminal deoxynucleotidyltransferase dUTP nick end labelingGAPDHglyceraldehyde-3-phosphate dehydrogenase. has recently been identified as a major component of the ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (1Neumann 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 (4411) Google Scholar, 2Arai 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 (1848) Google Scholar). Subsequently several point mutations located in the glycine-rich domain of TDP-43 have been identified as the disease-causing mutations of familial and sporadic ALS (3Gitcho 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 (521) Google Scholar, 4Yokoseki A. Shiga A. Tan C.F. Tagawa A. Kaneko H. 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Yu C.E. Lancet Neurol. 2008; 7: 409-416Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar). TDP-43 has been shown to be a fundamental component of ubiquitin-positive neuronal cytoplasmic and intranuclear inclusions as well as that of neuronal dystrophic neurites in the affected neurons or glial cells in these neurodegenerative diseases. TDP-43 is known to regulate gene transcription, exon splicing, and exon inclusion through interactions with RNA, heterogeneous nuclear ribonucleoproteins, and nuclear bodies (8Ayala Y.M. Pantano S. D舗Ambrogio A. Buratti E. Brindisi A. Marchetti C. Romano M. Baralle F.E. J. Mol. Biol. 2005; 348: 575-588Crossref PubMed Scopus (269) Google Scholar, 9Buratti E. Brindisi A. Giombi M. Tisminetzky S. Ayala Y.M. Baralle F.E. J. Biol. Chem. 2005; 280: 37572-37584Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 10Wang I.F. Reddy N.M. Shen C.K. Proc. Natl. Acad. Sci. 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However, the physiological function of TDP-43 in the central nervous system has not been fully elucidated, and it remains unclear how this protein is implicated in the pathogenesis of neurodegeneration. 43-kDa TAR DNA-binding protein amyotrophic lateral sclerosis small interfering RNA 3-(4,5-dimethylthiazol-2-yl)-5-(3-caboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium geranylgeranyl pyrophosphate mevalonic acid propidium iodide terminal deoxynucleotidyltransferase dUTP nick end labeling glyceraldehyde-3-phosphate dehydrogenase. 43-kDa TAR DNA-binding protein amyotrophic lateral sclerosis small interfering RNA 3-(4,5-dimethylthiazol-2-yl)-5-(3-caboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium geranylgeranyl pyrophosphate mevalonic acid propidium iodide terminal deoxynucleotidyltransferase dUTP nick end labeling glyceraldehyde-3-phosphate dehydrogenase. The Rho family of GTPases are members of the Ras superfamily and are known for regulating actin cytoskeletal dynamics (15Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (886) Google Scholar, 16Burridge K. Wennerberg K. Cell. 2004; 116: 167-179Abstract Full Text Full Text PDF PubMed Scopus (1503) Google Scholar, 17Jaffe A.B. Hall A. Annu. Rev. Cell Dev. Biol. 2005; 21: 247-269Crossref PubMed Scopus (2332) Google Scholar, 18Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5200) Google Scholar). RhoA, Rac1, and Cdc42, the most studied proteins of this family, also modulate functions such as cell movement, motility, transcription, cell growth, and cell survival (18Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5200) Google Scholar). In neurons, RhoA, Rac1, and Cdc42 have been shown to regulate neurite outgrowth (19Just I. Selzer J. Wilm M. von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (877) Google Scholar, 20Bradke F. Dotti C.G. Science. 1999; 283: 1931-1934Crossref PubMed Scopus (425) Google Scholar, 21Ahmed I. Calle Y. Iwashita S. Nur-E-Kamal A. Mol. Cell. Biochem. 2006; 281: 17-25Crossref PubMed Scopus (15) Google Scholar). Although TDP-43 is localized in the nucleus of unaffected neurons, nuclear staining of this protein is significantly reduced in neurons bearing ubiquitin inclusions (1Neumann 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 (4411) Google Scholar, 2Arai 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 (1848) Google Scholar, 22Cairns N.J. Neumann M. Bigio E.H. Holm I.E. Troost D. Hatanpaa K.J. Foong C. White 3rd, C.L. Schneider J.A. Kretzschmar H.A. Carter D. Taylor-Reinwald L. Paulsmeyer K. Strider J. Gitcho M. Goate A.M. Morris J.C. Mishra M. Kwong L.K. Stieber A. Xu Y. Forman M.S. Trojanowski J.Q. Lee V.M. Mackenzie I.R. Am. J. Pathol. 2007; 171: 227-240Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar), suggesting that loss of TDP-43 function may play a role in neurodegeneration. In this study, we used small interfering RNA (siRNA) to investigate the effect of TDP-43 loss of function on cell death and neurite outgrowth and elucidated a novel relation between TDP-43 and the activities of RhoA, Rac1, and Cdc42. The oligonucleotide siRNA duplex was synthesized by Takara Bio (Shiga, Japan). The siRNA sequences were as follows: scrambled (control) siRNA-set1, 5′-GAAUCAGAUGCACAUGAGUTT-3′; -set2, 5′-ACGGCCUAAUCUAACAGACTT-3′; TDP-43 siRNA-set1, 5′-GAACGAUGAACCCAUUGAATT-3′; -set2, 5′-CCAAUGCUGAACCUAAGCATT-3′. Unless otherwise mentioned, set 1 siRNA was used for TDP-43 knockdown throughout the experiments. The pEGFP-Rac1 construct was produced as described elsewhere (23Nakagawa M. Fukata M. Yamaga M. Itoh N. Kaibuchi K. J. Cell Sci. 2001; 114: 1829-1838Crossref PubMed Google Scholar, 24Watanabe T. Wang S. Noritake J. Sato K. Fukata M. Takefuji M. Nakagawa M. Izumi N. Akiyama T. Kaibuchi K. Dev. Cell. 2004; 7: 871-883Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). Mouse TDP-43 (GenBankTM accession number NM_145556) cDNA was amplified by PCR from mouse brain cDNA using the following primers: 5′-GTGCTTCCTCCTTGTGCTTC-3′ and 5′-CCACACTGAACAAACCAATCTG-3′. The PCR product was cloned into the pCR-BluntII-TOPO vector (Invitrogen), and the entire coding region of mouse TDP-43 was inserted in-frame into either the KpnI and XbaI sites of the pcDNA3.1/V5His vector (Invitrogen) or the KpnI and BamHI sites of the pDsRed-Monomer-Hyg-N1 vector (Clontech). An siRNA-resistant form of the TDP-43 gene was generated by changing the targeted sequence of the siRNA to 5′-GAATGACGAGCCAATTGAA-3′ (mutated nucleotides are underlined) using the KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan). Neuro-2a cells (American Type Culture Collection, Manassas, VA), a line derived from mouse neuroblastoma, were maintained as described previously (25Niwa J. Yamada S. Ishigaki S. Sone J. Takahashi M. Katsuno M. Tanaka F. Doyu M. Sobue G. J. Biol. Chem. 2007; 282: 28087-28095Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The transfection of siRNA into Neuro-2a cells was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer舗s instructions. the transfection of the and cells were using Lipofectamine (Invitrogen) according to the manufacturer舗s instructions. the Neuro-2a cells, the was to and and cells were for the the cells were for with or for with pyrophosphate was to the the siRNA and cells were for Neuro-2a cells in were using Japan). The of the neurite was with Japan). of the of cells were the of siRNA (Invitrogen) was with TDP-43 siRNA or The of transfection was using (Clontech). neurite outgrowth in TDP-43 knockdown cells, we performed a the siRNA transfection the was The 3-(4,5-dimethylthiazol-2-yl)-5-(3-caboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell was on the differentiated Neuro-2a cells using the Cell according to the manufacturer舗s instructions. was in a The were in 6 for cell differentiated Neuro-2a cells were with and with propidium iodide using the kit (Invitrogen) according to the manufacturer舗s instructions. were performed using the kit The of staining in cells was by and and the Neuro-2a cells were on The activity of differentiated Neuro-2a cells was using the of transfection or according to the manufacturer舗s instructions. was in the and the was in 6 for mRNA were by PCR as described M. H. M. M. K. H. K. Y. Tanaka F. Doyu M. Sobue G. J. Neurosci. 2006; PubMed Scopus Google Scholar). RNA from Neuro-2a cells was into PCR was and the product was by the system the expression of glyceraldehyde-3-phosphate was The following were and for TDP-43 and and for were performed as described M. H. Doyu M. M. C. Y. A. Sobue G. Nat. PubMed Scopus Google Scholar). Neuro-2a cells were in a we used the kit according to the manufacturer舗s instructions. Cell were by and by with used were as follows: mouse mouse 1 1 mouse as a as a membrane protein mouse mouse and pyrophosphate and we used the Rho domain of the Rho protein with and region of the protein with kinase 1 were performed in the of and according to the manufacturer舗s instructions. Neuro-2a cells, were with and siRNA siRNA or were by with for of were with and with a Neuro-2a cells were with siRNA on the was to the the of the cells were by and the cells were and was by with of mouse or mouse for by of protein proteins were by on a The were and in for The were and proteins were on a to a for this using neurite were by of and for or and the for were using the was as the effect of TDP-43 of siRNA were into Neuro-2a cells. The of TDP-43 siRNA transfection was by and and cell we cell in differentiated Neuro-2a cells of siRNA The viability of knockdown cells was significantly by of siRNA with the of a siRNA-resistant form of TDP-43 was with set 1 a the siRNA-resistant form of TDP-43 the of cell viability The was with staining and The knockdown of TDP-43 significantly the number of cells However, the number of cells for or was not and In the was between knockdown and cells the of TDP-43 depletion on cellular we performed a the of of cells the of neurite outgrowth was significantly inhibited in cells and of neurite outgrowth in Neuro-2a cells. and the number of the cells for and cells using of TDP-43 significantly the number of cells with not that with with or activity of Neuro-2a cells with TDP-43 siRNA or of neurite outgrowth in Neuro-2a cells with TDP-43 siRNA or of the neurite of cells were the transfection of of cells the of in cells, neurite outgrowth was or of Neuro-2a cells the transfection of RNA or TDP-43 not the of neuronal cell death and the caused by TDP-43 we on the Rho family are of neurite outgrowth and cell RhoA, Rac1, and Cdc42 activities were by In cells, the activities of RhoA, Rac1, and Cdc42 were with the and of was also by a in the of the phosphorylation of is by Rho kinase J. M. K. N. M. T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) that of Rho family GTPases to neuronal we the of C. a of Rho family on differentiated Neuro-2a cells. a this reduced cell viability and inhibited neurite outgrowth and Rho and Ras family be to the cell membrane to functions P. Hall A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the intracellular of Rho family proteins to the by TDP-43 regulates that the of RhoA, Rac1, and Cdc42, not or in the membrane were in the TDP-43 knockdown cells The of Rho of Rho was not by TDP-43 knockdown The siRNA-resistant form of TDP-43 the in the of Rho family GTPases The also that was significantly reduced in TDP-43 knockdown cells in with cells of Neuro-2a cells. Neuro-2a cells with and siRNA siRNA or were by with was significantly reduced in the TDP-43 cells with the cells that knockdown of TDP-43 and with the cells. membrane localization to members of the Rho family of GTPases have to be by to the Ras family such as that modulate geranylgeranylation of Rho family a of geranylgeranylation, has been shown to cell death through the of Rho family GTPases T. I. D. I. H. S. Y. T. M. Y. A. J. Neurosci. PubMed Google Scholar, V. F. D. J. J. Neurosci. PubMed Scopus Google Scholar). In differentiated Neuro-2a cells, inhibited neurite outgrowth and reduced cell viability in a of also the of Rho GTPases In contrast, the of geranylgeranylation, the in the of RhoA, Rac1, and Cdc42 caused by TDP-43 knockdown In addition, restored viability and neurite outgrowth in cells and that geranylgeranylation to be the molecular of TDP-43 cellular TDP-43 regulates geranylgeranylation of Rho GTPases. that TDP-43 the of Rho neurite and cell viability in of TDP-43 in Neuro-2a cells on cellular of the membrane of Neuro-2a cells with TDP-43 or neurite of Neuro-2a cells with or the viability of Neuro-2a cells with the of or these suggest that TDP-43 regulates the activities of Rho family members through protein geranylgeranylation. the effect of TDP-43 depletion on protein geranylgeranylation using acid of into or was significantly in the Neuro-2a cells and suggesting that the knockdown of TDP-43 geranylgeranylation of Rho family reduced of into Rac1, this geranylgeranylation of the Rho family is by and geranylgeranyl pyrophosphate is for of the expression of these However, the knockdown of TDP-43 not the protein expression of or geranylgeranyl pyrophosphate not In the study, we that knockdown of TDP-43 neurite outgrowth and cell death in differentiated Neuro-2a cells, suggesting that loss of TDP-43 function plays a role in neurodegeneration. the molecular by TDP-43 depletion neuronal cell we the between TDP-43 and Rho family GTPases. morphology is in through the of the of the key of the actin is the Rho family of RhoA, Rac1, and Cdc42 (15Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (886) Google Scholar, 16Burridge K. Wennerberg K. Cell. 2004; 116: 167-179Abstract Full Text Full Text PDF PubMed Scopus (1503) Google Scholar, 17Jaffe A.B. Hall A. Annu. Rev. Cell Dev. Biol. 2005; 21: 247-269Crossref PubMed Scopus (2332) Google Scholar). Although Rho family has a effect on cell morphology and plays role in the of neuronal we that of Rho family proteins by C. suppressed neurite outgrowth and reduced cell viability as reported previously (19Just I. Selzer J. Wilm M. von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (877) Google Scholar, 20Bradke F. Dotti C.G. Science. 1999; 283: 1931-1934Crossref PubMed Scopus (425) Google Scholar, 21Ahmed I. Calle Y. Iwashita S. Nur-E-Kamal A. Mol. Cell. Biochem. 2006; 281: 17-25Crossref PubMed Scopus (15) Google Scholar, T. M. H. Aktories K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H. Aktories K. J. 2006; PubMed Scopus Google Scholar). that the activities of RhoA, Rac1, and Cdc42 were in cells, suggesting that knockdown of TDP-43 inhibition of neurite outgrowth and cell death through the of Rho family GTPases. was of by TDP-43 knockdown in the we used of the of Neuro-2a cells in the of is known to be N. T. M. J. Res. 2001; Full Text Full Text PDF PubMed Google Scholar, M. M. C. Res. 2005; PubMed Scopus Google Scholar). In of this we that effect on in Neuro-2a cells the this has been shown to in cells Tan J. 2001; PubMed Scopus Google Scholar, X. L. J.C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). TDP-43 regulate the activities of the Rho molecules have been shown to regulate the activity of and Cdc42, are of regulating GTPases the molecular by TDP-43 regulates and Cdc42, we our to the that membrane localization is the key factor to these members of the Rho and Ras as molecular between and (18Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5200) Google Scholar, N. Hall A. Genet. Full Text PDF PubMed Scopus Google Scholar, K. Wang Y. N. J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). with a small proteins to with the membrane with and P. Hall A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). that the such as the Ras family, and the geranylgeranyl such as the Rho or the family P. Hall A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Cell Biol. PubMed Scopus Google Scholar). In the study, we that knockdown of TDP-43 RhoA, Rac1, and Cdc42 not the intracellular of or TDP-43 knockdown also inhibited the of a of the into and in differentiated Neuro-2a cells. In contrast, the expression of Rho functions by Rho family GTPases from and in the Y. Kaibuchi K. Y. H. S. T. A. Y. Google Scholar, A. Biochem. J. 2005; PubMed Scopus Google Scholar), was not significantly by TDP-43 In addition, inhibition of geranylgeranylation by the of TDP-43 the of the geranylgeranylation restored cell viability and neurite outgrowth in Neuro-2a cells. Furthermore in cells, overexpression of TDP-43 restored the membrane localization of Rho GTPases as well as cell viability and neurite suggest that TDP-43 depletion Rho family GTPases through inhibition of protein geranylgeranylation. geranylgeranylation of Rho family members is by using produced by geranylgeranyl pyrophosphate as the J. Carter D.R. C. J. Res. 1998; Full Text Full Text PDF PubMed Google Scholar). of and and the is also a component of protein J. Biol. Chem. Full Text PDF PubMed Google Scholar). the mRNA and protein expression of and geranylgeranyl pyrophosphate in Neuro-2a cells. However, the expression of these were not by TDP-43 that TDP-43 depletion the activities of these cytoplasmic inclusions are a of ALS and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Although the of these has not been fully elucidated, have identified TDP-43 as the major component of the neuronal inclusions in ALS and frontotemporal lobar degeneration with ubiquitin-positive inclusions (1Neumann 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 (4411) Google Scholar, 2Arai 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 (1848) Google Scholar). has been a of loss or of function of TDP-43 neuronal and cell death. Although TDP-43 is a nuclear protein, the in ALS and frontotemporal lobar degeneration with ubiquitin-positive TDP-43 disappears from the of the affected neurons (1Neumann 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 (4411) Google Scholar, 2Arai 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 (1848) Google Scholar). that loss of nuclear TDP-43 may neuronal it is also that neuronal inclusions has been reported that TDP-43 depletion to of cyclin-dependent kinase 6 protein and by of the cell and in human cells (14Ayala Y.M. Misteli T. Baralle F.E. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3785-3789Crossref PubMed Scopus (193) Google Scholar). In the study, knockdown of TDP-43 Rho family GTPases and induced cell death in differentiated Neuro-2a cells. Although this is not the the of TDP-43 from the nucleus to the our suggest that loss of function of TDP-43 may neuronal degeneration through of Rho family GTPases. In summary, we have that TDP-43 depletion neurite outgrowth and neuronal cell death. possibly from a reduced membrane localization of Rho family GTPases to the inhibition of protein geranylgeranylation.