| 研究課題/領域番号 |
26830052
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| 研究機関 | 独立行政法人理化学研究所 |
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研究代表者 |
HUI KAIWAN 独立行政法人理化学研究所, 脳科学総合研究センター, 国際特別研究員 (70721843)
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| 研究期間 (年度) |
2014-04-01 – 2016-03-31
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| キーワード | proteomics / autism / translation regulation |
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| 研究実績の概要 |
In order to identify activity-dependent newly synthesized proteins, various stimulation protocols including stimulus, dosage and stimulation time were examined for their effectiveness in inducing the synthesis of known upregulated proteins such as Arc and CaMKII-alpha/beta. Two different stimuli relevant to suspected deficiencies in autism spectrum disorder (ASD) have been chosen at this point for future experiments.
In addition, bioorthogonal labeling procedures have been examined for effectiveness in labeling newly synthesized proteins during neuronal activity stimulation. The isolation of these labeled proteins for identification and quantification by mass spectrometry has also been tested. Reproducibility between experiments is currently being examined.
Preliminary mass spectrometry experiments indicate that, consistent with experimental results using traditional methods like immunoblotting, protein synthesis is increased as a result of neuronal activity stimulation. As expected, some newly synthesized proteins during neuronal activity stimulation as identified by mass spectrometry are known FMRP targets. However many other newly synthesized proteins were also identified, many of which are presynaptic proteins.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
The principal experimental method being employed in this research project, QuaNCAT (quantitative noncanonical amino acid tagging), required some more optimization than originally anticipated. Specific issues which had to be addressed include labeling efficiency as well as non-specific binding, both of which could significantly affect experimental outcomes and interpretation. As such, though it was originally planned that experiments utilizing wild-type neurons could be completed within the first fiscal year of this grant proposal, part of this work is ongoing but is expected to be completed soon, with experiments using FMRP knockout neurons to commerce shortly.
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| 今後の研究の推進方策 |
Experiments designed to identify newly synthesized in wild-type and FMRP knockout (KO) neurons under basal condition and following neuronal activity stimulation will continue as outlined in the original research proposal. Findings from mass spectrometric experiments will be validated by traditional methods such as immunoblotting and/or immunofluorescence.
In order to examine the effect of Tsc2 heterozygosity on protein translation regulation and the rescuing effects on FMRP KO neurons, shRNA-mediated knockdown will be examined both in wild-type and FMRP KO neurons. Once sufficient knockdown effect is achieved, similar experiments will be performed to identify the effects of Tsc2 loss on newly synthesized proteins in both wild-type and FMRP KO neurons.
Going forward, it is expected that these experiments will yield valuable information on how two major known autism spectrum disorder (ASD) mutations affect protein translation regulation and proteomic changes which occur during neuronal activity stimulation.
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