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.

中文翻译：

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αB-晶状体蛋白抑制星形细胞自噬有助于α-突触核蛋白包涵体的形成

帕金森病 (PD) 的特征是多巴胺能神经元的慢性丧失和黑质中一些剩余神经元内存在蛋白质包涵体（路易体）。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包涵体。然而，星形胶质细胞蛋白聚集形成的潜在分子机制仍然很大程度上未知。在这里，我们研究了 αB-晶状体蛋白 (CRYAB)，一种小的热休克蛋白，在星形胶质细胞中 α-突触核蛋白包涵体形成中的作用。进行小干扰 RNA (siRNA) 介导的 CRYAB (siCRYAB) 敲低或 CRYAB 过表达以研究 CRYAB 对人胶质母细胞瘤细胞系 U251 细胞自噬的影响。共免疫沉淀 (co-IP) 和免疫印迹用于剖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。使用 CRYAB 转基因小鼠和过表达人 α-突触核蛋白 A30P 突变形式的转基因小鼠来检查 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 会导致自噬活性显着增强。相反，外源 CRYAB 通过与 BAG3 结合破坏了 BAG3-HSPB8-HSC70 复合物的组装，从而抑制了自噬活性。此外，CRYAB 调节的自噬与 PD 发病机制有关。敲除 CRYAB 显着促进了 α-突触核蛋白预制原纤维 (PFF) 的细胞质清除。反过来，星形胶质细胞中 CRYAB 的选择性过表达显着抑制了自噬，导致表达人 α-突触核蛋白 A30P 突变体的转基因小鼠脑中 α-突触核蛋白聚集体的积累。该研究揭示了 CRYAB 作为星形细胞自噬的天然抑制剂的新功能，并表明 CYRAB 的敲低可能提供针对蛋白病（如突触核蛋白病）的治疗靶点。