Lu Shen-Zhao, Guo Yong-Shun, Liang Pei-Zhou, Zhang Shu-Zhen, Yin Shu, Yin Yan-Qing, Wang Xiao-Min, Ding Fei, Gu Xiao-Song, Zhou Jia-Wei
1Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031 China.
2School of Future Techology, University of Chinese Academy of Sciences, Beijing, 100049 China.
Transl Neurodegener. 2019 Jan 18;8:3. doi: 10.1186/s40035-018-0143-7. eCollection 2019.
Parkinson's disease (PD) is characterized by a chronic loss of dopaminergic neurons and the presence of proteinaceous inclusions (Lewy bodies) within some remaining neurons in the substantia nigra. Recently, astroglial inclusion body has also been found in some neurodegenerative diseases including PD. However, the underlying molecular mechanisms of how astroglial protein aggregation forms remain largely unknown. Here, we investigated the contribution of αB-crystallin (CRYAB), a small heat shock protein, in α-synuclein inclusion formation in astrocytes.
Small interfering RNA (siRNA)-mediated CRYAB (siCRYAB) knockdown or CRYAB overexpression was performed to investigate the impact of CRYAB on the autophagy in human glioblastoma cell line U251 cells. Co-immunoprecipitation (co-IP) and immunoblotting were used to dissect the interaction among multiple proteins. The clearance of α-synuclein in vitro was evaluated by immunocytochemistry. CRYAB transgenic mice and transgenic mice overexpressing A30P mutant form of human α-synuclein were used to examine the influence of CRYAB to α-synuclein accumulation in vivo.
We found that knockdown of CRYAB in U251 cells or primary cultured astrocytes resulted in a marked augmentation of autophagy activity. In contrast, exogenous CRYAB disrupted the assembly of the BAG3-HSPB8-HSC70 complex via binding with BAG3, thereby suppressing the autophagy activity. Furthermore, CRYAB-regulated autophagy has relevance to PD pathogenesis. Knockdown of CRYAB remarkably promoted cytoplasmic clearance of α-synuclein preformed fibrils (PFFs). Conversely, selective overexpression of CRYAB in astrocytes markedly suppressed autophagy leading to the accumulation of α-synuclein aggregates in the brain of transgenic mice expressing human α-synuclein A30P mutant.
This study reveals a novel function for CRYAB as a natural inhibitor of astrocytic autophagy and shows that knockdown of CYRAB may provide a therapeutic target against proteinopathies such as synucleinopathies.
帕金森病（PD）的特征是多巴胺能神经元慢性丧失，以及黑质中一些剩余神经元内存在蛋白质内含物（路易小体）。最近，在包括PD在内的一些神经退行性疾病中也发现了星形胶质细胞内含物。然而，星形胶质细胞蛋白质聚集形成的潜在分子机制在很大程度上仍不清楚。在此，我们研究了小热休克蛋白αB-晶状体蛋白（CRYAB）在星形胶质细胞中α-突触核蛋白内含物形成中的作用。
采用小干扰RNA（siRNA）介导的CRYAB（siCRYAB）敲低或CRYAB过表达来研究CRYAB对人胶质母细胞瘤细胞系U251细胞自噬的影响。采用免疫共沉淀（co-IP）和免疫印迹法分析多种蛋白质之间的相互作用。通过免疫细胞化学评估体外α-突触核蛋白的清除情况。使用CRYAB转基因小鼠和过表达人α-突触核蛋白A30P突变体形式的转基因小鼠来研究CRYAB对体内α-突触核蛋白积累的影响。
我们发现，在U251细胞或原代培养的星形胶质细胞中敲低CRYAB会导致自噬活性显著增强。相反，外源性CRYAB通过与BAG3结合破坏了BAG3-HSPB8-HSC70复合物的组装，从而抑制自噬活性。此外，CRYAB调节的自噬与PD发病机制相关。敲低CRYAB显著促进了α-突触核蛋白原纤维（PFFs）的细胞质清除。相反，在星形胶质细胞中选择性过表达CRYAB显著抑制自噬，导致在表达人α-突触核蛋白A30P突变体的转基因小鼠大脑中α-突触核蛋白聚集体积累。
本研究揭示了CRYAB作为星形胶质细胞自噬天然抑制剂的新功能，并表明敲低CYRAB可能为针对诸如突触核蛋白病等蛋白质病提供治疗靶点。
