RNA Interference Reveals a Requirement for Myocyte Enhancer Factor 2A in Activity-dependent Neuronal Survival

RNA interference (RNAi) provides a powerful method of gene silencing in eukaryotic cells, including proliferating mammalian cells. However, the utility of RNAi as a method of gene knock-down in primary postmitotic mammalian neurons remained unknown. Here, we asked if RNAi might be utilized to allow the assessment of the biological function of a specific gene in the nervous system. We employed a U6 promoter-driven DNA template approach to induce hairpin RNA-triggered RNAi to characterize the role of the transcription factor myocyte enhancer factor 2A (MEF2A) in the neuronal activity-dependent survival of granule neurons of the developing rat cerebellum. We found that the expression of MEF2A hairpin RNAs leads to the efficient and specific inhibition of endogenous MEF2A protein expression in primary cerebellar granule neurons. We also found that RNAi of MEF2A reduces significantly MEF2 response element-mediated transcription in granule neurons and inhibits activity-dependent granule neuron survival. Taken together, our RNAi experiments have revealed that MEF2A plays a critical role in activity-dependent neuronal survival. In addition, our findings indicate that RNAi does operate in postmitotic mammalian neurons and thus offers a rapid genetic method of studying gene function in the development and function of the mammalian nervous system. RNA interference (RNAi) provides a powerful method of gene silencing in eukaryotic cells, including proliferating mammalian cells. However, the utility of RNAi as a method of gene knock-down in primary postmitotic mammalian neurons remained unknown. Here, we asked if RNAi might be utilized to allow the assessment of the biological function of a specific gene in the nervous system. We employed a U6 promoter-driven DNA template approach to induce hairpin RNA-triggered RNAi to characterize the role of the transcription factor myocyte enhancer factor 2A (MEF2A) in the neuronal activity-dependent survival of granule neurons of the developing rat cerebellum. We found that the expression of MEF2A hairpin RNAs leads to the efficient and specific inhibition of endogenous MEF2A protein expression in primary cerebellar granule neurons. We also found that RNAi of MEF2A reduces significantly MEF2 response element-mediated transcription in granule neurons and inhibits activity-dependent granule neuron survival. Taken together, our RNAi experiments have revealed that MEF2A plays a critical role in activity-dependent neuronal survival. In addition, our findings indicate that RNAi does operate in postmitotic mammalian neurons and thus offers a rapid genetic method of studying gene function in the development and function of the mammalian nervous system. RNA interference (RNAi) 1The abbreviations used for: RNAi, RNA interference; siRNA, small interfering RNA, MEF2, myocyte enhancer factor 2; MRE, MEF2 response element; hpRNA, hairpin RNA; DIV, daysin vitro ; cdk2, cyclin-dependent kinase 2. 1The abbreviations used for: RNAi, RNA interference; siRNA, small interfering RNA, MEF2, myocyte enhancer factor 2; MRE, MEF2 response element; hpRNA, hairpin RNA; DIV, daysin vitro ; cdk2, cyclin-dependent kinase 2.is the process of gene silencing whereby double-stranded RNA induces the homology-dependent degradation of its cognate mRNA. Since its discovery in Caenorhabditis elegans (1Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11567) Google Scholar), RNAi has become a powerful and widely used tool for the analysis of gene function in many systems, including fungi, plants, andDrosophila (2Cogoni C. Irelan J.T. Schumacher M. Schmidhauser T.J. Selker E.U. Macino G. EMBO J. 1996; 15: 3153-3163Crossref PubMed Scopus (291) Google Scholar, 3Misquitta L. Paterson B.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1451-1456Crossref PubMed Scopus (290) Google Scholar). Biochemical studies have revealed that cells contain an evolutionarily conserved RNAi machinery that triggers the cleavage of double-stranded RNAs into 21- or 22-nucleotide small interfering RNAs (siRNAs). The siRNAs then hybridize to their cognate mRNA, thereby inducing the specific degradation of the target mRNA.An ultimate goal of RNAi studies is to knock down mammalian gene expression in the intact organism in a tissue-specific manner. Recently, the introduction of synthetic siRNAs in mammalian cells has been demonstrated to induce specific gene silencing (4Harborth J. Elbashir S.M. Bechert K. Tuschl T. Weber K. J. Cell Sci. 2001; 114: 4557-4565Crossref PubMed Google Scholar). However, although RNAi has been used successfully in a number of cell lines (4Harborth J. Elbashir S.M. Bechert K. Tuschl T. Weber K. J. Cell Sci. 2001; 114: 4557-4565Crossref PubMed Google Scholar, 5Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3942) Google Scholar, 6Lee N.S. Dohjima T. Bauer G. Li H. Li M.J. Ehsani A. Salvaterra P. Rossi J. Nat. Biotechnol. 2002; 20: 500-505Crossref PubMed Scopus (137) Google Scholar, 7Miyagishi M. Taira K. Nat. Biotechnol. 2002; 20: 497-500Crossref PubMed Scopus (677) Google Scholar, 8Paddison P.J. Caudy A.A. Bernstein E. Hannon G.J. Conklin D.S. Genes Dev. 2002; 16: 948-958Crossref PubMed Scopus (1306) Google Scholar, 9Paul C.P. Good P.D. Winer I. Engelke D.R. Nat. Biotechnol. 2002; 20: 505-508Crossref PubMed Scopus (749) Google Scholar, 10Yu J.Y. DeRuiter S.L. Turner D.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6047-6052Crossref PubMed Scopus (952) Google Scholar, 11Sui G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google Scholar, 12Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8078) Google Scholar, 13Caplen N.J. Parrish S. Imani F. Fire A. Morgan R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9742-9747Crossref PubMed Scopus (923) Google Scholar), it remained unknown whether RNAi-mediated gene silencing operates in primary postmitotic cells, a question that is of particular significance to studies of the nervous system. To begin to address this issue, it is essential to assess whether the RNAi machinery functions efficiently in postmitotic mammalian neurons.In this study, we addressed the question of the utility of RNAi in mammalian neurons by specifically disrupting the endogenous expression of a transcription factor in primary granule neurons cultured from the developing rat cerebellum. In previous studies employing dominant negative strategies, members of the myocyte enhancer factor 2 (MEF2) family of proteins have been suggested to contribute to neuronal survival (14Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar, 15Li M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. M.E. J. 2001; PubMed Google Scholar, S. Li Z. C. E. J. E. S.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, S. K. S.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). However, a dominant interfering of a transcription factor has the to the of proteins the family of the transcription factor of this approach does allow the assessment of the function of a specific family The MEF2 transcription and in the nervous X. S. 1996; PubMed Scopus Google J. J. 15: PubMed Google Scholar). the MEF2 MEF2A is in cerebellar granule neurons and is in neurons neuronal (14Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar, 15Li M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. M.E. J. 2001; PubMed Google Scholar). However, the specific role of MEF2A in activity-dependent granule neuron survival remained to be to the role of MEF2A in activity-dependent granule neuron survival by the RNAi We employed a DNA G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google in siRNAs from a RNA hairpin the of the U6 We found that the expression of MEF2A reduces the expression of endogenous MEF2A protein in primary cerebellar granule neurons and thereby inhibits transcription and granule neuron survival. findings RNAi be employed to gene expression and function in primary postmitotic neurons of the developing mammalian nervous system. the MEF2 transcription MEF2A is for activity-dependent cerebellar granule neuron whether RNAi might be in primary mammalian we used a DNA RNAi method to target the transcription factor MEF2A in cerebellar granule neurons. To assess whether RNAi specific gene silencing in primary neurons by of RNAi of we also the transcription factor MEF2A and in cerebellar granule neurons and X. S. 1996; PubMed Scopus Google Scholar, J. J. 15: PubMed Google Scholar, T. T. Genes Dev. 1999; PubMed Scopus Google Scholar, D.S. H. Dev. PubMed Scopus Google and as in G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google and that target the MEF2A and mRNA, The target have including the members of the MEF2 and We whether specifically the expression of the cognate gene in a neuronal cell cells the or the an expression MEF2A or 2 The expression of MEF2A and in cells by analysis a that MEF2A or a to The expression of MEF2A in cells to a in the of MEF2A protein 2 the expression of 2 the expression of in cells in the of expression to 2 MEF2A expression 2 indicate that the MEF2A and the expression of MEF2A and and then the utility of RNAi to knock-down endogenous gene expression in primary of cerebellar granule neurons. granule neurons from rat and 2 a method Y. M. Bonni A. 2002; PubMed Scopus Google the or RNAi an expression to a to MEF2A or a to and a to used to neurons. In granule neurons and neurons the endogenous MEF2A or in the and in neurons the endogenous MEF2A of granule neurons the the expression of endogenous assess the of RNAi silencing in the granule we the of neurons that a of expression of endogenous MEF2A or neurons in experiments of granule neurons endogenous MEF2A of the MEF2A granule neurons endogenous MEF2A In the of the RNAi, the in endogenous and granule neurons the expression of endogenous MEF2A expression in granule neurons the MEF2A the expression of endogenous in granule neurons findings indicate that RNAi efficiently and endogenous gene expression in cerebellar granule that RNAi of MEF2A triggers the knock-down of MEF2A expression in granule neurons to the biological role of endogenous MEF2A in cerebellar granule neurons. of the MEF2 family of transcription transcription in neurons the that is found the of (14Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar, 15Li M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. M.E. J. 2001; PubMed Google Scholar). The inhibition of transcription in granule neurons by dominant interfering of MEF2 triggers that members of the MEF2 family activity-dependent survival of granule neurons (14Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. M.E. J. 2001; PubMed Google Scholar). the specific role of the family MEF2A in transcription and neuronal survival remained to be characterize the biological role of MEF2A in cerebellar granule we the of MEF2A transcription in granule neurons neuron 2 a of the gene a gene to as a for granule neuron then to of that induce and the of Cell Dev. 16: PubMed Scopus Google Scholar). the expression of the gene The of the gene significantly in neurons the MEF2A RNAi as neurons the U6 the expression of the to the of the that MEF2A transcription in cerebellar granule neurons.In a of we the of MEF2A RNAi the survival of cerebellar granule neurons 2 the RNAi the that if in survival and neurons However, we found that the expression of MEF2A significantly the survival of cerebellar granule neurons and To the of a of MEF2A cell survival to the of hpRNA, we the of the gene cyclin-dependent kinase 2 The to specifically the expression G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google Scholar). MEF2A hpRNA, the expression of in granule neurons activity-dependent granule neuron survival. Taken together, that MEF2A is for activity-dependent cerebellar granule neuron we have found that the RNAi machinery functions efficiently in primary postmitotic mammalian neurons. a the RNAi method to the specific function of the transcription factor MEF2A in cerebellar granule neurons. We found that knock-down of MEF2A transcription and granule neuron survival. that MEF2A plays a critical role in the development of cerebellar granule neurons. In knock-down of MEF2A in neurons the of that activity-dependent neuronal findings have of its the RNAi method a rapid of analysis of gene function in primary of mammalian neurons. of the of RNAi in the expression of a target assess the biological role of a particular of a protein a that is to by dominant negative RNAi operates in primary postmitotic mammalian RNAi is to be of utility in the of the nervous in the intact mammalian organism and thus or a gene RNA interference (RNAi) 1The abbreviations used for: RNAi, RNA interference; siRNA, small interfering RNA, MEF2, myocyte enhancer factor 2; MRE, MEF2 response element; hpRNA, hairpin RNA; DIV, daysin vitro ; cdk2, cyclin-dependent kinase 2. 1The abbreviations used for: RNAi, RNA interference; siRNA, small interfering RNA, MEF2, myocyte enhancer factor 2; MRE, MEF2 response element; hpRNA, hairpin RNA; DIV, daysin vitro ; cdk2, cyclin-dependent kinase 2.is the process of gene silencing whereby double-stranded RNA induces the homology-dependent degradation of its cognate mRNA. Since its discovery in Caenorhabditis elegans (1Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11567) Google Scholar), RNAi has become a powerful and widely used tool for the analysis of gene function in many systems, including fungi, plants, andDrosophila (2Cogoni C. Irelan J.T. Schumacher M. Schmidhauser T.J. Selker E.U. Macino G. EMBO J. 1996; 15: 3153-3163Crossref PubMed Scopus (291) Google Scholar, 3Misquitta L. Paterson B.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1451-1456Crossref PubMed Scopus (290) Google Scholar). Biochemical studies have revealed that cells contain an evolutionarily conserved RNAi machinery that triggers the cleavage of double-stranded RNAs into 21- or 22-nucleotide small interfering RNAs (siRNAs). The siRNAs then hybridize to their cognate mRNA, thereby inducing the specific degradation of the target mRNA. ultimate goal of RNAi studies is to knock down mammalian gene expression in the intact organism in a tissue-specific manner. Recently, the introduction of synthetic siRNAs in mammalian cells has been demonstrated to induce specific gene silencing (4Harborth J. Elbashir S.M. Bechert K. Tuschl T. Weber K. J. Cell Sci. 2001; 114: 4557-4565Crossref PubMed Google Scholar). However, although RNAi has been used successfully in a number of cell lines (4Harborth J. Elbashir S.M. Bechert K. Tuschl T. Weber K. J. Cell Sci. 2001; 114: 4557-4565Crossref PubMed Google Scholar, 5Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3942) Google Scholar, 6Lee N.S. Dohjima T. Bauer G. Li H. Li M.J. Ehsani A. Salvaterra P. Rossi J. Nat. Biotechnol. 2002; 20: 500-505Crossref PubMed Scopus (137) Google Scholar, 7Miyagishi M. Taira K. Nat. Biotechnol. 2002; 20: 497-500Crossref PubMed Scopus (677) Google Scholar, 8Paddison P.J. Caudy A.A. Bernstein E. Hannon G.J. Conklin D.S. Genes Dev. 2002; 16: 948-958Crossref PubMed Scopus (1306) Google Scholar, 9Paul C.P. Good P.D. Winer I. Engelke D.R. Nat. Biotechnol. 2002; 20: 505-508Crossref PubMed Scopus (749) Google Scholar, 10Yu J.Y. DeRuiter S.L. Turner D.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6047-6052Crossref PubMed Scopus (952) Google Scholar, 11Sui G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google Scholar, 12Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8078) Google Scholar, 13Caplen N.J. Parrish S. Imani F. Fire A. Morgan R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9742-9747Crossref PubMed Scopus (923) Google Scholar), it remained unknown whether RNAi-mediated gene silencing operates in primary postmitotic cells, a question that is of particular significance to studies of the nervous system. To begin to address this issue, it is essential to assess whether the RNAi machinery functions efficiently in postmitotic mammalian neurons. In this study, we addressed the question of the utility of RNAi in mammalian neurons by specifically disrupting the endogenous expression of a transcription factor in primary granule neurons cultured from the developing rat cerebellum. In previous studies employing dominant negative strategies, members of the myocyte enhancer factor 2 (MEF2) family of proteins have been suggested to contribute to neuronal survival (14Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar, 15Li M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. M.E. J. 2001; PubMed Google Scholar, S. Li Z. C. E. J. E. S.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, S. K. S.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). However, a dominant interfering of a transcription factor has the to the of proteins the family of the transcription factor of this approach does allow the assessment of the function of a specific family The MEF2 transcription and in the nervous X. S. 1996; PubMed Scopus Google J. J. 15: PubMed Google Scholar). the MEF2 MEF2A is in cerebellar granule neurons and is in neurons neuronal (14Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar, 15Li M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. M.E. J. 2001; PubMed Google Scholar). However, the specific role of MEF2A in activity-dependent granule neuron survival remained to be We to the role of MEF2A in activity-dependent granule neuron survival by the RNAi We employed a DNA G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google in siRNAs from a RNA hairpin the of the U6 We found that the expression of MEF2A reduces the expression of endogenous MEF2A protein in primary cerebellar granule neurons and thereby inhibits transcription and granule neuron survival. findings RNAi be employed to gene expression and function in primary postmitotic neurons of the developing mammalian nervous system. the MEF2 transcription MEF2A is for activity-dependent cerebellar granule neuron survival. whether RNAi might be in primary mammalian we used a DNA RNAi method to target the transcription factor MEF2A in cerebellar granule neurons. To assess whether RNAi specific gene silencing in primary neurons by of RNAi of we also the transcription factor MEF2A and in cerebellar granule neurons and X. S. 1996; PubMed Scopus Google Scholar, J. J. 15: PubMed Google Scholar, T. T. Genes Dev. 1999; PubMed Scopus Google Scholar, D.S. H. Dev. PubMed Scopus Google and as in G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google and that target the MEF2A and mRNA, The target have including the members of the MEF2 and We whether specifically the expression of the cognate gene in a neuronal cell cells the or the an expression MEF2A or 2 The expression of MEF2A and in cells by analysis a that MEF2A or a to The expression of MEF2A in cells to a in the of MEF2A protein 2 the expression of 2 the expression of in cells in the of expression to 2 MEF2A expression 2 indicate that the MEF2A and the expression of MEF2A and and then the utility of RNAi to knock-down endogenous gene expression in primary of cerebellar granule neurons. granule neurons from rat and 2 a method Y. M. Bonni A. 2002; PubMed Scopus Google the or RNAi an expression to a to MEF2A or a to and a to used to neurons. In granule neurons and neurons the endogenous MEF2A or in the and in neurons the endogenous MEF2A of granule neurons the the expression of endogenous assess the of RNAi silencing in the granule we the of neurons that a of expression of endogenous MEF2A or neurons in experiments of granule neurons endogenous MEF2A of the MEF2A granule neurons endogenous MEF2A In the of the RNAi, the in endogenous and granule neurons the expression of endogenous MEF2A expression in granule neurons the MEF2A the expression of endogenous in granule neurons findings indicate that RNAi efficiently and endogenous gene expression in cerebellar granule that RNAi of MEF2A triggers the knock-down of MEF2A expression in granule neurons to the biological role of endogenous MEF2A in cerebellar granule neurons. of the MEF2 family of transcription transcription in neurons the that is found the of (14Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar, 15Li M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. M.E. J. 2001; PubMed Google Scholar). The inhibition of transcription in granule neurons by dominant interfering of MEF2 triggers that members of the MEF2 family activity-dependent survival of granule neurons (14Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. M.E. J. 2001; PubMed Google Scholar). the specific role of the family MEF2A in transcription and neuronal survival remained to be characterize the biological role of MEF2A in cerebellar granule we the of MEF2A transcription in granule neurons neuron 2 a of the gene a gene to as a for granule neuron then to of that induce and the of Cell Dev. 16: PubMed Scopus Google Scholar). the expression of the gene The of the gene significantly in neurons the MEF2A RNAi as neurons the U6 the expression of the to the of the that MEF2A transcription in cerebellar granule neurons.In a of we the of MEF2A RNAi the survival of cerebellar granule neurons 2 the RNAi the that if in survival and neurons However, we found that the expression of MEF2A significantly the survival of cerebellar granule neurons and To the of a of MEF2A cell survival to the of hpRNA, we the of the gene cyclin-dependent kinase 2 The to specifically the expression G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google Scholar). MEF2A hpRNA, the expression of in granule neurons activity-dependent granule neuron survival. Taken together, that MEF2A is for activity-dependent cerebellar granule neuron we have found that the RNAi machinery functions efficiently in primary postmitotic mammalian neurons. a the RNAi method to the specific function of the transcription factor MEF2A in cerebellar granule neurons. We found that knock-down of MEF2A transcription and granule neuron survival. that MEF2A plays a critical role in the development of cerebellar granule neurons. In knock-down of MEF2A in neurons the of that activity-dependent neuronal findings have of its the RNAi method a rapid of analysis of gene function in primary of mammalian neurons. of the of RNAi in the expression of a target assess the biological role of a particular of a protein a that is to by dominant negative RNAi operates in primary postmitotic mammalian RNAi is to be of utility in the of the nervous in the intact mammalian organism and thus or a gene To whether RNAi might be in primary mammalian we used a DNA RNAi method to target the transcription factor MEF2A in cerebellar granule neurons. To assess whether RNAi specific gene silencing in primary neurons by of RNAi of we also the transcription factor MEF2A and in cerebellar granule neurons and X. S. 1996; PubMed Scopus Google Scholar, J. J. 15: PubMed Google Scholar, T. T. Genes Dev. 1999; PubMed Scopus Google Scholar, D.S. H. Dev. PubMed Scopus Google Scholar). The and as in G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google and that target the MEF2A and mRNA, The target have including the members of the MEF2 and We whether specifically the expression of the cognate gene in a neuronal cell cells the or the an expression MEF2A or 2 The expression of MEF2A and in cells by analysis a that MEF2A or a to The expression of MEF2A in cells to a in the of MEF2A protein 2 the expression of 2 the expression of in cells in the of expression to 2 MEF2A expression 2 indicate that the MEF2A and the expression of MEF2A and and We then the utility of RNAi to knock-down endogenous gene expression in primary of cerebellar granule neurons. granule neurons from rat and 2 a method Y. M. Bonni A. 2002; PubMed Scopus Google the or RNAi an expression to a to MEF2A or a to and a to used to neurons. In granule neurons and neurons the endogenous MEF2A or in the and in neurons the endogenous MEF2A of granule neurons the the expression of endogenous To assess the of RNAi silencing in the granule we the of neurons that a of expression of endogenous MEF2A or neurons in experiments of granule neurons endogenous MEF2A of the MEF2A granule neurons endogenous MEF2A In the of the RNAi, the in endogenous and granule neurons the expression of endogenous MEF2A expression in granule neurons the MEF2A the expression of endogenous in granule neurons findings indicate that RNAi efficiently and endogenous gene expression in cerebellar granule neurons. The that RNAi of MEF2A triggers the knock-down of MEF2A expression in granule neurons to the biological role of endogenous MEF2A in cerebellar granule neurons. of the MEF2 family of transcription transcription in neurons the that is found the of (14Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar, 15Li M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. M.E. J. 2001; PubMed Google Scholar). The inhibition of transcription in granule neurons by dominant interfering of MEF2 triggers that members of the MEF2 family activity-dependent survival of granule neurons (14Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. M.E. J. 2001; PubMed Google Scholar). the specific role of the family MEF2A in transcription and neuronal survival remained to be To characterize the biological role of MEF2A in cerebellar granule we the of MEF2A transcription in granule neurons neuron 2 a of the gene a gene to as a for granule neuron then to of that induce and the of Cell Dev. 16: PubMed Scopus Google Scholar). the expression of the gene The of the gene significantly in neurons the MEF2A RNAi as neurons the U6 the expression of the to the of the that MEF2A transcription in cerebellar granule neurons. In a of we the of MEF2A RNAi the survival of cerebellar granule neurons 2 the RNAi the that if in survival and neurons However, we found that the expression of MEF2A significantly the survival of cerebellar granule neurons and To the of a of MEF2A cell survival to the of hpRNA, we the of the gene cyclin-dependent kinase 2 The to specifically the expression G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google Scholar). MEF2A hpRNA, the expression of in granule neurons activity-dependent granule neuron survival. Taken together, that MEF2A is for activity-dependent cerebellar granule neuron survival. To we have found that the RNAi machinery functions efficiently in primary postmitotic mammalian neurons. a the RNAi method to the specific function of the transcription factor MEF2A in cerebellar granule neurons. We found that knock-down of MEF2A transcription and granule neuron survival. that MEF2A plays a critical role in the development of cerebellar granule neurons. In knock-down of MEF2A in neurons the of that activity-dependent neuronal survival. findings have of its the RNAi method a rapid of analysis of gene function in primary of mammalian neurons. of the of RNAi in the expression of a target assess the biological role of a particular of a protein a that is to by dominant negative RNAi operates in primary postmitotic mammalian RNAi is to be of utility in the of the nervous in the intact mammalian organism and thus or a gene We Y. for E. for the and R. for the