Pan Yongcheng, Tang Beisha, Li Xiao-Jiang, Li Shihua, Liu Qiong
Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, China.
Key Laboratory of Hunan Province in Neurodegenerative Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, China.
Front Aging Neurosci. 2023 Aug 10;15:1237018. doi: 10.3389/fnagi.2023.1237018. eCollection 2023.
Huntington's disease (HD) is caused by CAG trinucleotide repeats in the HTT gene. Selective neurodegeneration in the striatum is prominent in HD, despite widespread expression of mutant HTT (mHTT). Ras homolog enriched in the striatum (Rhes) is a GTP-binding protein enriched in the striatum, involved in dopamine-related behaviors and autophagy regulation. Growing evidence suggests Rhes plays a critical role in the selective striatal degeneration in HD, but its specific function in this context remains complex and controversial.
In this study, we utilized CRISPR/Cas9 to knockdown Rhes at different disease stages through adeno-associated virus (AAV) transduction in HD knock-in (KI) mice. Immunoblotting and immunofluorescence were employed to assess the impact of Rhes depletion on mHTT levels, neuronal loss, astrogliosis and autophagy activity.
Rhes depletion in 22-week-old HD KI mice (representing the presymptomatic stage) led to mHTT accumulation, reduced neuronal cell staining, and increased astrogliosis. However, no such effects were observed in 36-week-old HD KI mice (representing the symptomatic stage). Additionally, Rhes deletion in 22-week-old HD KI mice resulted in increased P62 levels, reduced LC3-II levels, and unchanged phosphorylation of mTOR and beclin-1, unchanged mTOR protein level, except for a decrease in beclin-1.
Our findings suggest that knockdown Rhes promotes striatal aggregation of mutant huntingtin by reducing autophagy activity in a mTOR-independent manner. Rhes plays a protective role during the presymptomatic stage of HD KI mice.
亨廷顿舞蹈症（HD）由HTT基因中的CAG三核苷酸重复序列引起。尽管突变型HTT（mHTT）广泛表达，但纹状体中的选择性神经变性在HD中很突出。富含纹状体的Ras同源物（Rhes）是一种富含纹状体的GTP结合蛋白，参与多巴胺相关行为和自噬调节。越来越多的证据表明，Rhes在HD的选择性纹状体变性中起关键作用，但其在这种情况下的具体功能仍然复杂且存在争议。
在本研究中，我们利用CRISPR/Cas9通过腺相关病毒（AAV）转导在HD基因敲入（KI）小鼠的不同疾病阶段敲低Rhes。采用免疫印迹和免疫荧光评估Rhes缺失对mHTT水平、神经元丢失、星形胶质细胞增生和自噬活性的影响。
22周龄HD KI小鼠（代表症状前阶段）中Rhes缺失导致mHTT积累、神经元细胞染色减少和星形胶质细胞增生增加。然而，在36周龄HD KI小鼠（代表症状阶段）中未观察到此类影响。此外，22周龄HD KI小鼠中Rhes缺失导致P62水平升高、LC3-II水平降低、mTOR和beclin-1的磷酸化不变、mTOR蛋白水平不变，但beclin-1减少。
我们的研究结果表明，敲低Rhes以mTOR非依赖的方式通过降低自噬活性促进突变型亨廷顿蛋白在纹状体中的聚集。Rhes在HD KI小鼠的症状前阶段起保护作用。
