| 研究実績の概要 |
In this proposal, we hypothesize that m6A modulates spatiotemporal regulation of neuronal gene expression at synapse, which in turn, regulate cellular response of neurons to learning-related stimuli to store information of past events. This cellular mechanism may play critically important roles in aging-, nutrition-, alcohol-dependent “memory dynamism”. To achieve this goal, we proposed three research aims and in the fiscal year of 2014, we are close to finishing the first aim: to identify m6A modification and its mRNA targets at synapses.
So far, we have established and performed experiments to identify synaptic m6A-RNA targets. To do so, we have isolated synaptoneurosomes from forebrains of adult mice. Successful enrichment of synaptic proteins was confirmed with western-blots. First, we were able to detect m6A immunoreactivity in the synaptic fraction of total RNA, suggesting that indeed m6A modification exist locally. To identify the specific RNA species bearing m6A modifications, we have performed immuno-capture of m6A-RNA from ribominus RNA fraction from total synaptic RNA pool followed by deep sequencing. Bioinformatics analysis has been set up for the genome-wide peak-callings of m6A-RNA population. The initial analysis validates the immunoprecipitation procedure and revealed a list of candidates of RNA involved in versatile synaptic transmission and plasticity pathways. We are currently repeating the experiments to further validate the initial results.
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| 今後の研究の推進方策 |
Our next aim is to elucidate environmental and activity-dependent dynamic regulation of m6A at synapse. Environmental cues will be introduced to cultured hippocampal neurons and their impact on m6A expression level will be analyzed. Whether m6A expression regulation occurs in a stimulus-, synapse-specific manner will be studied. At system level, animals under mild starving or high-sucrose diet will be analyzed for m6A expression brain-wise. Finally, to dissect the role of m6A regulation in synaptic plasticity and lasting memory, we will apply stimuli to trigger transcription-dependent and synapse-specific plasticity will be applied before m6A expression is examined. Neuronal plasticity and its interaction with environmental cues will be tested after manipulation of m6A level. Based on the phenotype, region-specific, conditional knock-out mouse model will be designed and generated for further investigation.
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