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研究発表を行った学会；
・第89回日本薬理学会年会　2016年3月9日〜3月11日（横浜）

タイトル；Visualization of microautophagy and chaperone-mediated autophagy in primary cultured neurons.
発表者；佐藤　正寛　氏
　　　（熊本大学　大学院生命科学研究部　薬物活性学分野）
要旨；
Removal of misfolded proteins by protein degradation systems is important for various neuronal functions and survival. There are two major protein degradation systems: the ubiquitin-proteasome system and the autophagy-lysosome system. The latter is further classified into macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA). MA has been revealed to be involved in various functions and pathogenesis of various diseases. However, studies about mA and CMA are delayed by the absence of easy methods to assess their activities. Previously, we established a novel method to assess mA and CMA activity using GAPDH, a mA and CMA substrate, fused with HaloTag (HT) in AD293 cells. In this study, we attempted to assess CMA and mA activities in primary cultured neurons from rat cerebral cortex. We transfected GAPDH-HT to primary cultured rat cortical neurons using adeno-associated virus serotype 9 (AAV9) vectors. Expression of GAPDH-HT was mediated by synapsin I promoter, a highly neuron-specific promoter. Using this AAV9 vector, we could express GAPDH-HT in primary cultured cortex neurons without any toxicity. 24h after labeling with fluorescent dye-fused HT ligands, labeled GAPDH-HT translocated from cytosol to punctate structures. This punctate accumulation of GAPDH-HT reflects mA and CMA activities. siRNA-mediated knockdown of TSG101, a mA-related protein, and LAMP2A, a CMA-related protein partially, but significantly, decreased GAPDH-HT puncta. Mycophenolic acid, a CMA activator, significantly increased the punctate accumulation in TSG101-knocked down neurons, but not in LAMP2A-knocked down neurons. These findings suggest that mA and CMA activities can be separately detected using GAPDH-HT by the knockdown of CMA- and mA-related proteins, respectively, in primary neurons.