Analysis of mRNA translation in cultured hippocampal neurons.
Synaptic plasticity, the ability of neuronal synapses to undergo morphological and biochemical changes in response to various stimuli, forms the underlying basis of long-term memory storage. Regulated mRNA translation at synapses is required for this plasticity. However, the mechanism by which translation at synapses is controlled and how the encoded proteins modulate persistent changes in synaptic morphology and functional integration in response to different input stimulations remain mostly unclear (Schuman et al., 2006; Sutton and Schuman, 2006). One approach to investigating the relationship between protein synthesis and plasticity is to identify factors, such as RNA binding proteins that control translation in the neurons and then determine the identities of the mRNAs to which they are bound. Molecular and cellular techniques have been employed in cultured neurons to study sequence-specific RNA-binding proteins, for example, the Cytoplasmic Polyadenylation Element Binding protein (CPEB) (Huang et al., 2002, 2003) and the Fragile-X Mental Retardation Protein (FMRP) (Vanderklish and Edelman, 2005; Zalfa et al., 2006) for their functions in localizing and regulating translation of mRNAs. Although several CPE-containing neuronal RNAs that undergo activity-dependent polyadenylation (Du and Richter, 2005; Wu et al., 1998) and FMRP-interacting mRNAs have been identified (Brown et al., 2001; Miyashiro et al., 2003), the validation of these targets whose translation is important for plasticity in vivo remains to be demonstrated (Darnell et al., 2005). In general, primary neurons in culture are difficult to manipulate. For example, they do not proliferate and their transfection efficiency is low ( approximately 1 to 10% of cells); this low efficiency is reduced even further as the cells age in culture, which hampers their practical use for biochemical analysis. When biochemical approaches are applied, they are often carried out in other more facile model systems, such as oocytes, in the case of CPEB, or in brains derived from knockout mice, for both CPEB and FMRP. However, the development of various viral delivery systems, shRNA knockdown techniques, reporter assays with high sensitivity, and neuron culture protocols have allowed investigators to analyze translational control in these cells, which may ultimately be used to investigate key mechanisms of synaptic plasticity. We have employed these procedures to investigate the function of CPEB3, a novel RNA-binding protein, in primary rat hippocampal neurons (Huang et al., 2006); here, we describe the experimental details of our methods, which could be used for any RNA binding protein.
Huang
Yi-Shuian
YS
Division of Neuroscience, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
Richter
Joel D
JD
D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D017930:Genes, Reporter; D006624:Hippocampus / Q000166:cytology* / Q000196:embryology / Q000378:metabolism*; D047468:Immunoprecipitation / Q000379:methods; D007553:Isotope Labeling / Q000379:methods; D008954:Models, Biological; D009473:Neuronal Plasticity / Q000502:physiology; D009474:Neurons / Q000378:metabolism*; D014176:Protein Biosynthesis; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000138:chemical synthesis; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013574:Synaptosomes / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014776:Virus Cultivation
C525080:CPEB3 protein, rat; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins