| 研究実績の概要 |
During FY2016, I began to use QuaNCAT (quantitative non-canonical amino acid tagging) to identify newly synthesized proteins in response to BDNF treatment in wild-type neurons. As expected, more proteins were reproducibly upregulated by BDNF compared to those which were downregulated. Consistent with neuronal stimulation leading to structural changes at synaptic spines, GO term enrichment analysis showed that the group of newly synthesized proteins upregulated by BDNF treatment was associated with regulation of actin polymerization and synaptic vesicle cycling, while downregulated proteins were associated with protein folding and proteasome. Moreover, several proteins known to be upregulated by BDNF were identified as expected, demonstrating the validity of the methodology.
Following the examination of BDNF stimulation on wild-type neurons, I proceeded to examine for differences between FMRP knockout and wild-type neurons following BDNF stimulation. As expected from its role as a translation suppressor, FMRP knockout neurons showed an increased number of newly synthesized proteins upregulated by BDNF treatment. Interestingly, many of the differentially upregulated proteins were not known FMRP targets.
I have also tested shRNA sequences to significantly reduce Tsc2 expression in primary cultured neurons. SuNSET experiments revealed that global protein translation rate is reduced in Tsc2 knocked down neurons compared to scrambled control, despite Tsc2 being a negative mTOR regulator and should therefore increase global translation when reduced in expression.
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