Inhibition of Apoptosis by Z-VAD-fmk in SMN-depleted S2 Cells

Spinal muscular atrophy is an autosomal recessive motor neuron degenerative disorder, caused by the loss of telomeric copy of the survival motor neuron gene (SMN1). To better understand how motor neurons are targeted in Spinal muscular atrophy patients, it is important to study the role of SMN protein in cell death. In this report, we employed RNA interference (RNAi) to study the loss-of-function of SMN in Drosophila S2 cells. A 601-base pair double-stranded RNA (dsRNA) of Drosophila SMN (dSMN) was used for silencing the dSMN. Our data indicate that dSMN RNAi resulted in more than 90% reduction of both RNA and protein. Further analysis of S2 cells by cell death ELISA and flow cytometry assays revealed that reduction of dSMN expression significantly increased apoptosis. The cell death mediated by SMN depletion is caspase-dependent and specifically due to the activation of the endogenous caspases, DRONC and DRICE. Significantly, the effect of dSMN RNAi was reversed by a peptide caspase inhibitor, Z-VAD-fmk. These results suggest that dSMN is involved in signal pathways of apoptotic cell death in Drosophila. Hence, the model system of reduced SMN expression by RNAi in Drosophila could be exploited for identification of therapeutic targets. Spinal muscular atrophy is an autosomal recessive motor neuron degenerative disorder, caused by the loss of telomeric copy of the survival motor neuron gene (SMN1). To better understand how motor neurons are targeted in Spinal muscular atrophy patients, it is important to study the role of SMN protein in cell death. In this report, we employed RNA interference (RNAi) to study the loss-of-function of SMN in Drosophila S2 cells. A 601-base pair double-stranded RNA (dsRNA) of Drosophila SMN (dSMN) was used for silencing the dSMN. Our data indicate that dSMN RNAi resulted in more than 90% reduction of both RNA and protein. Further analysis of S2 cells by cell death ELISA and flow cytometry assays revealed that reduction of dSMN expression significantly increased apoptosis. The cell death mediated by SMN depletion is caspase-dependent and specifically due to the activation of the endogenous caspases, DRONC and DRICE. Significantly, the effect of dSMN RNAi was reversed by a peptide caspase inhibitor, Z-VAD-fmk. These results suggest that dSMN is involved in signal pathways of apoptotic cell death in Drosophila. Hence, the model system of reduced SMN expression by RNAi in Drosophila could be exploited for identification of therapeutic targets. The autosomal recessive disease, spinal muscular atrophy (SMA) 1The abbreviations used are: SMA, spinal muscular atrophy; dSMN, drosophila survival of motor neuron protein; RNAi, RNA interference; dsRNA, double-stranded RNA; GST, glutathione S-transferase; HA, hemagglutinin; Z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; ELISA, enzyme-linked immunosorbent assay; CAT, chloramphenicol acetyltransferase; DIAP1, Drosophila inhibitor of apoptosis protein 1; SMN, survival of motor neuron; RT, reverse transcription; GFP, green fluorescent protein; dPsn, Drosophila Presenilin; h, human; FACS, fluorescence-activated cell sorter. occurs in 1:6,000 live births and is one of the most common genetic causes of infant death, resulting from death of motor neurons in anterior horn cells. SMA is clinically categorized as types I, II, and III based on age of onset and clinical progression. Type I (severe) infants have onset of weakness within the first six months of life and typically die within the first two years. Type II (intermediate) children experience symptoms before age two and never walk unassisted. Type III (mild) patients exhibit less severe clinical symptoms with a highly variable age of onset. It was found that the survival of motor neuron (SMN) gene is the SMA-determining gene and is deleted, converted, or mutated in >95% of SMA patients. There are two human SMN genes, SMN1 and SMN2 (1Lefebvre S. Burglen L. Reboullet S. Clermont O. Burlet P. Viollet L. Benichou B. Cruaud C. Millasseau P. Zeviani M. Le Paslier D. Frezal F. Cohen D. Weissenbach J. Munnich A. Melki J. Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (3145) Google Scholar). However, only loss of the SMN1 gene causes the disease. SMN2 is maintained in most of the SMA patients, in many cases with multiple copies. However, 80% of SMN2 products are the alternatively spliced SMNΔ7 that lacks exon 7 and is unstable, whereas the primary product of SMN1 is full-length SMN mRNA. It has been demonstrated that clinical severity of SMA disease is inversely correlated with the number of SMN2 copies in SMA patients (2Vitali T. Sossi V. Tiziano F. Zappata S. Giuli A. Paravatou-Petsotas M. Neri G. Brahe C. Hum. Mol. Genet. 1999; 8: 2525-2532Crossref PubMed Scopus (61) Google Scholar), suggesting that disease progression is inversely dosage-dependent on full-length SMN proteins produced. Mice have only a single copy of the SMN gene that does not undergo alternative splicing of exon 7 and therefore is a functional SMN1 homologue. Homozygous knockout of the mouse SMN gene is embryonic lethal at a very early developmental stage (3Schrank B. Gotz R. Gunnersen J.M. Ure J.M. Toyka K.V. Smith A.G. Sendtner M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9920-9925Crossref PubMed Scopus (562) Google Scholar). By crossing mice transgenic for human SMN2 into a SMN+/– heterozygous mouse, animals with an hSMN2/– genotype were born. These mice did not die in utero, implying SMN2 can rescue the lethality of homozygous knockout. A single copy of human SMN2 was sufficient to restore mouse viability from SMN–/– lethality; however, severe weakness developed and the mice died within few days after birth. Mice with eight copies of hSMN2, on the other hand, appear normal, providing additional evidence that SMN2 functions as a disease modifying gene by producing low levels of full-length SMN, explaining the correlation between disease severity and SMN2 copy numbers (4Monani U.R. Coovert D.D. Burghes A.H. Hum. Mol. Genet. 2000; 9: 2451-2457Crossref PubMed Scopus (117) Google Scholar). SMN homologues have been also isolated from Caenorhabditis elegans (5Miguel-Aliaga I. Culetto E. Walker D.S. Baylis H.A. Sattelle D.B. Davies K.E. Hum. Mol. Genet. 1999; 8: 2133-2143Crossref PubMed Scopus (106) Google Scholar), Danio rerio (zebrafish) (6Bertrandy S. Burlet P. Clermont O. Huber C. Fondrat C. Thierry-Mieg D. Munnich A. Lefebvre S. Hum. Mol. Genet. 1999; 8: 775-782Crossref PubMed Scopus (76) Google Scholar), Schizosaccharomyces pombe (7Paushkin S. Charroux B. Abel L. Perkinson R.A. Pellizzoni L. Dreyfuss G. J. Biol. Chem. 2000; 275: 23841-23846Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 8Owen N. Doe C.L. Mellor J. Davies K.E. Hum. Mol. Genet. 2000; 9: 675-684Crossref PubMed Scopus (56) Google Scholar, 9Hannus S. Buhler D. Romano M. Seraphin B. Fischer U. Hum. Mol. Genet. 2000; 9: 663-674Crossref PubMed Scopus (64) Google Scholar), and recently from Drosophila (10Miguel-Aliaga I. Chan Y.B. Davies K.E. van den Heuvel M. FEBS Lett. 2000; 486: 99-102Crossref PubMed Scopus (50) Google Scholar). Biochemically, the SMN proteins from these species retain a number of properties identified in the human SMN, including RNA binding activity and self-association. Defects of SMN proteins in C. elegans, D. rerio, and S. pombe affect cell viability and growth. Drosophila models have been generated by ectopic over-expression of human SMN protein domains that may function as dominant negatives to disrupt the endogenous dSMN protein, resulting in abnormal positioned wings and legs or in many cases, pupal lethality (10Miguel-Aliaga I. Chan Y.B. Davies K.E. van den Heuvel M. FEBS Lett. 2000; 486: 99-102Crossref PubMed Scopus (50) Google Scholar). However, inactivation of function of a protein using dominant negative mutants can yield misleading results if the expressed protein has additional functions or if it interacts with its endogenous counterpart (11Hudson D.F. Morrison C. Ruchaud S. Earnshaw W.C. Trends Cell Biol. 2002; 12: 281-287Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). During the last few years, RNA interference has emerged as a promising and powerful technology to investigate the loss-of-function of a protein by specifically suppressing expression of a gene in a population of cells. Thus, RNAi was used to investigate the mechanisms of SMN-deficiency-induced cell death in a cell culture model, the insect S2 cells. Our results showed that cells with SMN expression suppressed by RNAi underwent apoptosis. Apoptosis occurred via a caspase pathway-dependent process by activation of DRONC, an apical caspase in Drosophila, which further activated its effector caspase, DRICE. Furthermore, Z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), which is a cell-permeable caspase inhibitor, efficiently blocked cell death induced by SMN deficiency. Taken together, these results strongly suggest that investigation of factors that mediate SMN and caspase-dependent apoptosis may provide clues on functions of the SMN protein and targets for identification of small molecules/drugs for SMA therapy. Antibodies—To generate antibodies specific for Drosophila SMN, a plasmid construct containing full-length of dSMN was fused to glutathione-S-transferase in pGEX-4T1 (Amersham Biosciences). Fusion protein was purified according to the manufacturer's instructions. Anti-dSMN antibodies (dSMNAb) were raised against the fusion protein in rabbits and affinity purified by the GST or GST-fusion proteins that were covalently linked to glutathione Sepharose-4B resin (12Bar-Peled M. Raikhel N.V. Anal. Biochem. 1996; 241: 140-142Crossref PubMed Scopus (66) Google Scholar). Antibodies specific for the human SMN are from Santa Cruz Biotechnology (Santa Cruz, CA) or Transduction Laboratory (Lexington, KY). Anti-HA antibody 12CA5 is from Roche Molecular Diagnostics. The monoclonal anti-tubulin antibody is from Oncogene (San Diego, CA). The polyclonal rabbit anti-DRONC, rabbit anti-DRICE, and monoclonal mouse anti-DIAP1 (Drosophila inhibitor of apoptosis protein 1) antibodies were a generous gift from Dr. Bruce A. Hay (CalTech, Pasadena, CA). Cell Culture and Apoptosis Induction—S2 cells were grown in Schneider's Drosophila media (Invitrogen) with and of at apoptotic as a cells were with for 7 the were of dSMN with was into and of a Drosophila expression containing the PubMed Scopus Google or the of the protein in cells or in the S2 cells that were induced by for was by analysis with antibody or a construct for human SMN was generated in of in S2 cells was by with antibodies as RNAi in S2 and Z-VAD-fmk products containing for the dSMN and reverse product and and Drosophila gene and reverse product were and (dsRNA) were generated by in with and with The products were and by at for and to at The products were on a to the of as a single The plasmid were into cells by using (Invitrogen) according to the manufacturer's instructions. was by using of Z-VAD-fmk Diego, CA) in the culture RNA was isolated using the (Invitrogen) from cells with dSMN or of RNA was To the endogenous dSMN and a was using from dSMN and the for for and for for and The to two and of which was used as an for expression and were on an by CA). were grown in cell culture and with of dSMN and cells were and with of and of and and the of protein was by of protein from and of dSMN cells were for in PubMed Scopus Google and on and the were on The were with antibodies against dSMN, anti-DRONC, full-length anti-DRICE, and anti-DIAP1 I. L. H.A. Hay Cell Biol. 2002; PubMed Scopus Google by In cases, were with and and with anti-tubulin for The signal was by were on with and with for with with in for and blocked with in for one The cells were with primary or antibodies at and antibodies were for one The of proteins was by for were with and expression plasmid as and for with for flow and cells with and cells were in cells were by for in binding to which was according to the manufacturer's The cells were on a flow using and and the The resulting data was using CA). Cell ELISA for apoptotic cell death using a we ELISA assays as in the manufacturer's using a Cell is based on the using mouse monoclonal antibodies against and ELISA a in of and in the of cell and at Cell to cells were used for ELISA of Drosophila SMN investigate expression of Drosophila SMN (dSMN) protein, two antibodies against protein were raised in rabbits and The dSMN gene was into and into cells. analysis that both dSMN antibodies specifically a single in cells with the full-length Drosophila SMN not in cells with The was also as by analysis with antibody The of is than that of the human SMN, with the of dSMN was also into a Drosophila and into Drosophila S2 cells. The dSMN protein expression was in cell with antibody and the was in cells with By using expression of dSMN was in cells than in cells with 1) as by The antibodies the endogenous dSMN protein in S2 cells as a single with a of the of both The were and with anti-tubulin antibody of proteins A and were RNAi of SMN in S2 the effect of depletion of dSMN protein in S2 a of double-stranded RNA (dsRNA) of the dSMN gene was into the cells. The analysis demonstrated that of of dSMN in cells its endogenous after The of endogenous dPsn, was not cells with of suppressed 90% of its whereas the of dSMN expression was results were in two other RNAi of dSMN expression was by the dSMN protein. The of the endogenous dSMN protein was reduced to 90% in dSMN RNAi cell and the was in cells with There was between the of in and cells Furthermore, we also the effect of RNAi after and h, and the of dSMN expression was only whereas the of expression was reduced to 90% after not suggesting the of dSMN mRNA. of SMN in S2 is on expression of Drosophila SMN protein in we to the of dSMN protein in S2 cells. of both dSMN and in the were generated and into cells. h, protein expression was induced by of for revealed of dSMN and proteins to the for human SMN was also as in of in the of of S2 cells purified we identified the of the endogenous Drosophila SMN in and it was However, was in the dSMN was expressed the endogenous of the dSMN protein expression was in cells The could not be in cells with of dSMN of to and in cells with both and may be due to of over-expression or cells to the of dSMN in S2 investigate how motor neurons die in SMA patients, it is important to understand the mechanisms of cell death induced by SMN deficiency. we used RNAi to SMN expression in S2 cells. were in dSMN cells after The cells were with and for apoptosis by flow a for of cells were with a low of that has been to apoptosis J. Cell Biol. 2002; PubMed Scopus Google Scholar), whereas S2 cells were used as negative The of cells an in on the cell of cells in to an apoptotic Furthermore, was an in apoptosis in the dSMN cells with the apoptosis of the cells with the and is of In these the apoptosis in SMN was whereas the apoptosis in the cells was The between SMN and apoptosis within of the was found to have a of by the was induced in a of cells using D. of of cells was by not The cell population was identified by analysis of the flow and as M. J. 2000; PubMed Scopus Google Scholar). cells were of the The cell death ELISA increased apoptosis in these cells of dSMN dSMN Apoptosis and by a the apoptosis resulting from the depletion of SMN in S2 cells is caspase-dependent or we by to the activation of the endogenous using and It was demonstrated P. J. J. 2000; PubMed Scopus Google Scholar, Hay J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Google Scholar, L. P. M. J. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google that these two with and the DRONC was an that the effector caspase DRICE. Our results indicate that whereas was of the DRONC in the cell its and were in dSMN RNAi cell The first product of DRONC may be due to which has been demonstrated Hay J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Google Scholar), and the product is the resulting product of which is with results in S2 cells I. Hay J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of full-length DRONC correlated very with the activation of caspase due to dSMN DRONC interacts with we also of using to has been found for DRONC, results indicate that was of in of its products and were in cell of RNAi and these suggest that are activated in dSMN RNAi cell Furthermore, Z-VAD-fmk reversed the effect of dSMN RNAi as by ELISA and flow assays The of apoptosis in the cells was whereas in the dSMN apoptosis was is with the increased apoptosis with the dSMN In the apoptosis in was whereas the apoptosis in the was Thus, was effect of Z-VAD-fmk on binding to apoptotic S2 cells. In the the of cells increased from to a to that of cells an effect of the caspase inhibitor, Z-VAD-fmk the survival of S2 cells with of dSMN. cell were from dSMN RNAi, and dSMN RNAi with cells and were for DRONC and protein. The results that was of DRONC and products in from Z-VAD-fmk cells However, the endogenous expression of DIAP1, a caspase inhibitor, which to the of 1999; PubMed Scopus Google was by dSMN RNAi and this that cell death may be of the effect of dSMN cell of for DRONC protein, was of DRONC The DRONC product was in from dSMN RNAi cells There was of DRONC in from dSMN RNAi and cells. cell were for protein. The full-length protein was in from cells of its products were in RNAi from cells with Z-VAD-fmk was of the is as a protein and of and between dSMN and of the for SMN proteins is the of in the However, it is if are the SMN protein in the In this report, we generated specific antibodies against the Drosophila SMN protein. demonstrated by that in insect S2 and dSMN were in the The was as in of the S2 cells However, only dSMN in cells not in cells of was in these suggesting that dSMN may not be a protein to due to between exon 7 of dSMN and which has been to a role for E. F. FEBS Lett. PubMed Scopus Google Scholar). The of dSMN in the from cells may a between dSMN and which has been demonstrated in a by and in binding and J. in recently identified an between SMN and a of the and we showed that the between these two proteins is between human and Drosophila and J. in These data and J. in suggest that dSMN and proteins are and in in with of domains and of and domains between human and Drosophila SMN SMN in spinal muscular atrophy is caused by death of motor However, the mechanisms of induced cell death are not of SMN expression from S. pombe to mice as as in a cell lethal (3Schrank B. Gotz R. Gunnersen J.M. Ure J.M. Toyka K.V. Smith A.G. Sendtner M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9920-9925Crossref PubMed Scopus (562) Google Scholar, I. Culetto E. Walker D.S. Baylis H.A. Sattelle D.B. Davies K.E. Hum. Mol. Genet. 1999; 8: 2133-2143Crossref PubMed Scopus (106) Google Scholar, S. Charroux B. Abel L. Perkinson R.A. Pellizzoni L. Dreyfuss G. J. Biol. 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J. Cell Biol. 2002; PubMed Scopus Google Scholar), and of in apoptosis has been demonstrated in Drosophila J. P. R. J.M. J. Cell Biol. 1999; PubMed Scopus Google Scholar). it has also been demonstrated in Drosophila cells that the role of also as or in apoptosis to be of J. Cell Biol. 2002; PubMed Scopus Google Scholar). In of has been in Drosophila cells apoptosis N. M. Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). are to of in apoptotic cell death in S2 cells. In the cells with Z-VAD-fmk and of dSMN in increased survival a in and in caspase However, the of apoptosis as by is by Z-VAD-fmk data is with that of 2002; PubMed Scopus Google have that caspase by Z-VAD-fmk does not on the cell In to these other 2002; PubMed Scopus Google Scholar, M. P. Mol. PubMed Google Scholar), the death of cells was not Thus, the caspase-dependent apoptosis induced by SMN can be blocked by Z-VAD-fmk, suggesting targets for to depletion of SMN protein in SMA patients. SMA Drosophila and of SMA is a disease caused by apoptotic cell death of motor However, expression of SMN protein in as to only motor neurons are targeted in SMA patients. It has been that SMN properties and interacts with factors including N. T. 1997; PubMed Scopus Google and J. C.L. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), both of which in the apoptotic process are In this study we demonstrated that apoptosis in S2 cells is caspase-dependent and could be blocked by a caspase inhibitor Z-VAD-fmk. that the specific between SMN and apoptotic pathways may and are or in motor resulting in more of these cells to low levels of SMN protein than other cell are in this to involved in the activation of caspase DRONC to the apoptotic in Drosophila cells. These proteins may be targeted for for SMA disease. Dr. Bruce A. Hay and R. for anti-DRONC, and J. and for and Dr. T. for Dr. for