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.
Ilangovan
Raju
R
Department of Medicine and Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Marshall
William L
WL
Hua
Yimin
Y
Zhou
Jianhua
J
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D000906:Antibodies; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004330:Drosophila; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D034622:RNA Interference; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; D015398:Signal Transduction / Q000502:physiology
D000590:Amino Acid Chloromethyl Ketones; D000906:Antibodies; D061945:Caspase Inhibitors; D017362:Cyclic AMP Response Element-Binding Protein; D015853:Cysteine Proteinase Inhibitors; D009419:Nerve Tissue Proteins; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D020169:Caspases