14-3-3 phosphorylation in Parkinson's disease

项目摘要 与帕金森病（PD）相关的两种关键蛋白质是α-突触核蛋白（αsyn）和LRRK 2。突变 这两种基因中的任何一种都会导致PD的常染色体显性形式，GWAS研究指出了这两种基因的变体 作为发展特发性PD的风险因素。虽然这两种蛋白质导致相似的病理结果， 这两种蛋白质引起神经元损伤，并且它们在疾病过程中如何相互作用还不清楚。 我们的关键发现是，14-3-3θ是αsyn和LRRK 2的主要调节因子，可能是缺失的一环 在αsyn和LRRK 2之间。14-3- 3 s是多功能、高表达的脑蛋白，充当伴侣， 影响蛋白质运输并调节其结合配偶体的酶活性。我们的研究强调了 这些关键蛋白在PD中的作用及其与αsyn和LRRK 2的相互作用。我们观察到 14-3-3θ作为一个伴侣，以减少α-syn聚集和细胞间的传输，而同样的14-3-3 同种型降低突变体LRRK 2的激酶活性和毒性。 虽然我们的数据清楚地指出了14-3-3功能障碍在帕金森病中的潜在关键作用，但一个关键因素是， 目前的问题是14-3-3θ的内源性功能如何在PD中受损。我们建议 14-3-3θ的异常磷酸化是关键的病理生理事件， 磷酸化促进LRRK 2和αsyn在疾病中的作用。为了支持这一假设，我们最近 已发表的数据表明，在洗涤剂不溶性的蛋白质中，S232的14-3-3θ磷酸化显著升高， 来自人PD和DLB脑的级分。S232磷酸化的增加与认知功能相关。 下降和病理严重程度的措施，与S232磷酸化在发病机制中的作用一致 神经退行性疾病此外，我们还观察到线粒体应激促进14-3-3θ 在培养物中S232的磷酸化。S232 D拟磷酸突变体在神经毒素中失去保护作用 和α-syn文化模型。 基于这些数据，我们假设氧化应激是过量14-3-3θ的关键上游诱导物， 磷酸化，导致αsyn和LRRK 2毒性。我们最近创建了一个条件性敲入（KI）14-3-3θ S232 D小鼠系将作为理解14-3-3磷酸化在PD中的影响的关键工具。 在目标1中，我们将测试14-3-3θ磷酸化如何改变与αsyn的相互作用并调节随后的αsyn。 病理在目的2中，我们将测试14-3-3磷酸化对其与LRRK 2和LRRK 2相互作用的影响。 功能在目标3中，我们将研究14-3-3θ磷酸化是否在散发性PD的早期发生， 氧化应激通过过度的14-3-3磷酸化介导毒性。

PROJECT SUMMARY Two critical proteins linked to Parkinson’s disease (PD) are alpha-synuclein (αsyn) and LRRK2. Mutations in either gene cause autosomal dominant forms of PD, and GWAS studies have pointed to variants in both genes as risk factors for developing idiopathic PD. Although both proteins lead to a similar pathological outcome, how these two proteins cause neuronal injury and how they interact in the disease process are not well understood. Our key discovery is that 14-3-3θ is a major regulator of both αsyn and LRRK2 and could be the missing link between αsyn and LRRK2. 14-3-3s are multifunctional, highly expressed brain proteins that act as chaperones, affect protein trafficking, and modulate enzymatic activity of their binding partners. Our research has highlighted the role of these critical proteins in PD and their interplay with both αsyn and LRRK2. We have observed that 14-3-3θ acts as a chaperone to reduce αsyn aggregation and cell-to-cell transmission, and this same 14-3-3 isoform reduces the kinase activity and toxicity of mutant LRRK2. While our data clearly points to a potentially critical role for 14-3-3 dysfunction in Parkinson’s disease, a key question that remains is how 14-3-3θ’s endogenous functions could become impaired in PD. We propose that aberrant phosphorylation of 14-3-3θ is the critical pathophysiologic event, and that increases in 14-3-3θ phosphorylation promotes LRRK2 and αsyn effects in disease. In support of this hypothesis, we recently published data showing that 14-3-3θ phosphorylation at S232 is dramatically elevated in the detergent-insoluble fractions from human PD and DLB brains. This increase in S232 phosphorylation correlates with cognitive decline and pathological severity measures, consistent with a role of S232 phosphorylation in the pathogenesis of neurodegeneration. In addition, we have observed that mitochondrial stress promotes 14-3-3θ phosphorylation at S232 in culture. The S232D phosphomimetic mutant loses its protective effects in neurotoxin and αsyn culture models. Based on these data, we hypothesize that oxidative stress is a key upstream inducer of excessive 14-3-3θ phosphorylation, leading to αsyn and LRRK2 toxicity. We recently created a conditional knock-in (KI) 14-3-3θ S232D mouse line that will serve as the critical tool to understand the impact of 14-3-3 phosphorylation in PD. In Aim 1, we will test how 14-3-3θ phosphorylation alters interactions with αsyn and modulates subsequent αsyn pathology. In Aim 2, we will test the impact of 14-3-3 phosphorylation on its interaction with LRRK2 and LRRK2 function. In Aim 3, we will examine whether 14-3-3θ phosphorylation occurs early in sporadic PD and whether oxidative stress mediates toxicity via excessive 14-3-3 phosphorylation.