2013 Fiscal Year Research-status Report
| Project/Area Number |
25830007
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Research Institution | University of Toyama |
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Principal Investigator |
SHEHATA Mohammad 富山大学, 医学薬学研究部(医学), 助教 (60444197)
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| Project Period (FY) |
2013-04-01 – 2015-03-31
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| Keywords | GFP-LC3 / LC3 interaction domain |
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| Research Abstract |
We planned to monitor autophagic activity in the hippocampus after fear memory consolidation and reconsolidation. Using GFP-LC3 Tg mice, we could not find changes in the number of autophagosomes in hippocampus after fear memory consolidation or reconsolidation. The reason might be that autophagic activity changes by memory processes is not as drastic as starvation or electroconvulsive shock as described in “progress status reasons” section. Therefore, we designed a modified version of a method to detect autophagic activity based on the degradation kinetics of the autophagy target protein; p62 (Larsen et al., autophagy, 2010). In the modified method, instead of using full length p62, only a truncated p62 that contains the LC3 interaction domain (LIR) is fused to GFP to detect autophagosomes. LIR-GFP is expected to increase the specificity and sensitivity of the method by excluding the p62 domains that interact with cellular processes other than autophagy. Also, mCherry protein will be co-expressed as an internal control. A lentivirus-vector expressing LIR-GFP and mCherry has been constructed and tested for functionality in cell culture system. For the FY2014, we planned to analyze the effects of autophagy modulator drugs on fear memory processes. To fulfill this purpose, we have established reconsolidation protocol using intra-hippocampal (CA1) injection of anisomycin. This is a primary step to test whether autophagy activators or inhibitors could affect memory destabilization and/or reconsolidation.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
For the FY2013, we planned to monitor autophagic activity in the hippocampus after fear memory consolidation and reconsolidation. We planned to use GFP-LC3 Tg mice which have been used to monitor autophagic induction in mice brains after starvation (Alirezaei et al., 2010, Autophagy). In our hands, GFP-LC3 Tg mice were useful in detecting increase of autophagic activities after electroconvulsive shock (ECS) (unpublished). However, in the hippocampus we could not find changes in the number of autophagosomes after fear memory consolidation or reconsolidation. The reason might be that autophagic activity changes by memory processes is not as drastic as starvation or ECS, although several staining protocols have been used to enhance the sensitivity of autophagosomes detection in GFP-LC3 Tg brain slices. We can only found some changes in the basolateral amygdala where there is a tendency for c-fos expressing neurons to have lower autophagosomes and lower total GFP-LC3. Also, there is a tendency for central amygdala, which receives inhibitory buttons, to have higher total GFP-LC3 signal compared to other amygdala regions. Therefore, this method did not give satisfactory conclusions to answer whether autophagic activity is changed after contextual fear memory consolidation or retrieval. Therefore, we designed another method to monitor autophagy in mice brains based on the degradation kinetics of an autophagy target protein (p62) as described in the “summary of research achievements” section.
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| Strategy for Future Research Activity |
The effect of autophagy modulators on contextual fear memory, especially memory destabilization and reconsolidation, will be tested. The widely used autophagy modulation drugs including rapamycin (enhancer) and wortmannin (inhibitor) are also inhibitors of mTOR activity, which is known to plays important role in synaptic plasticity and memory. Therefore, specific mTOR-independent autophagic enhancers will be used, namely, 10-NCP (chlorophenoxazine derivative) (Tsvetkov et al., PNAS, 2010) or the tat-beclin1 peptide (Shoji-Kawata, et al., Nature, 2013). As mTOR-independent autophagic inhibitors, we plan to use spautin-1 (Liu et al., Cell, 2011) or molecular knockdown by lentiviral delivery of short-hairpin RNA against Atg7 (shATG7) (Shehata et al., J Neurosci, 2012). Finally, the degradation of several synaptic proteins will be monitored with and without autophagy modulators and how this correlates with contextual fear memory. In addition, we will employ the developed autophagy monitoring method described in the “summary of research achievements” to monitor autophagic activities after neuronal stimulation by ECS. Therefore, a lentivirus expressing LIR-GFP and mCherry will be prepared and injected into mice hippocampus and degradation kinetics of the LIR-GFP will be monitored as measure of autophagic activity. According to the success of the method, it should be modified to monitor autophagic activity after fear memory consolidation and reconsolidation.
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| Expenditure Plans for the Next FY Research Funding |
平成25年度、309円の助成金が未支出となったが、これは、購入予定であった、脳定位注入に使用するポリエチレンチューブの在庫がなかったため購入できなかったことによる。
平成25年度に購入できなかったポリエチレンチューブを、平成26年度に購入する。
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Research Products
(1 results)
