Endoplasmic reticulum (ER) stress is a common feature of Parkinson’s disease (PD), and several PD-related genes are responsible for ER dysfunction. Recent studies suggested LRRK2-G2019S, a pathogenic mutation in the PD-associated gene LRRK2, cause ER dysfunction, and could thereby contribute to the development of PD. It remains unclear, however, how mutant LRRK2 influence ER stress to control cellular outcome. In this study, we identified the mechanism by which LRRK2-G2019S accelerates ER stress and cell death in astrocytes. To investigate changes in ER stress response genes, we treated LRRK2-wild type and LRRK2-G2019S astrocytes with tunicamycin, an ER stress-inducing agent, and performed gene expression profiling with microarrays. The XBP1 SUMOylation and PIAS1 ubiquitination were performed using immunoprecipitation assay. The effect of astrocyte to neuronal survival were assessed by astrocytes-neuron coculture and slice culture systems. To provide in vivo proof-of-concept of our approach, we measured ER stress response in mouse brain. Microarray gene expression profiling revealed that LRRK2-G2019S decreased signaling through XBP1, a key transcription factor of the ER stress response, while increasing the apoptotic ER stress response typified by PERK signaling. In LRRK2-G2019S astrocytes, the transcriptional activity of XBP1 was decreased by PIAS1-mediated SUMOylation. Intriguingly, LRRK2-GS stabilized PIAS1 by increasing the level of small heterodimer partner (SHP), a negative regulator of PIAS1 degradation, thereby promoting XBP1 SUMOylation. When SHP was depleted, XBP1 SUMOylation and cell death were reduced. In addition, we identified agents that can disrupt SHP-mediated XBP1 SUMOylation and may therefore have therapeutic activity in PD caused by the LRRK2-G2019S mutation. Our findings reveal a novel regulatory mechanism involving XBP1 in LRRK2-G2019S mutant astrocytes, and highlight the importance of the SHP/PIAS1/XBP1 axis in PD models. These findings provide important insight into the basis of the correlation between mutant LRRK2 and pathophysiological ER stress in PD, and suggest a plausible model that explains this connection.

中文翻译：

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小异源二聚体（SHP）通过调节 XBP1 SUMOylation 加重帕金森病相关 LRRK2 突变星形胶质细胞的内质网应激

内质网 (ER) 应激是帕金森病 (PD) 的一个共同特征，几个 PD 相关基因导致 ER 功能障碍。最近的研究表明，LRRK2-G2019S 是 PD 相关基因 LRRK2 的致病突变，导致 ER 功能障碍，从而可能导致 PD 的发展。然而，目前尚不清楚突变的 LRRK2 如何影响内质网应激以控制细胞结果。在这项研究中，我们确定了 LRRK2-G2019S 加速星形胶质细胞内质网应激和细胞死亡的机制。为了研究内质网应激反应基因的变化，我们用衣霉素（一种内质网应激诱导剂）处理 LRRK2 野生型和 LRRK2-G2019S 星形胶质细胞，并用微阵列进行基因表达谱分析。XBP1 SUMOylation 和 PIAS1 泛素化使用免疫沉淀法进行。通过星形胶质细胞-神经元共培养和切片培养系统评估星形胶质细胞对神经元存活的影响。为了提供我们方法的体内概念验证，我们测量了小鼠大脑中的 ER 应激反应。微阵列基因表达谱显示，LRRK2-G2019S 通过 XBP1（内质网应激反应的关键转录因子）减少信号传导，同时增加以 PERK 信号传导为代表的细胞凋亡内质网应激反应。在 LRRK2-G2019S 星形胶质细胞中，PIAS1 介导的 SUMOylation 降低了 XBP1 的转录活性。有趣的是，LRRK2-GS 通过增加小异二聚体伙伴 (SHP)（PIAS1 降解的负调节因子）的水平来稳定 PIAS1，从而促进 XBP1 SUMOylation。当 SHP 耗尽时，XBP1 SUMOylation 和细胞死亡减少。此外，我们确定了可以破坏 SHP 介导的 XBP1 SUMOylation 的药物，因此可能对由 LRRK2-G2019S 突变引起的 PD 具有治疗活性。我们的研究结果揭示了 LRRK2-G2019S 突变星形胶质细胞中涉及 XBP1 的新调控机制，并强调了 SHP/PIAS1/XBP1 轴在 PD 模型中的重要性。这些发现提供了对突变 LRRK2 与 PD 病理生理 ER 应激之间相关性基础的重要见解，并提出了一个合理的模型来解释这种联系。