Enhanced mTORC1 signaling and protein synthesis in pathologic alpha-synuclein cellular and animal models of Parkinson’s disease

Pathologic alpha-synuclein plays an important role in the pathogenesis of alpha-synucleinopathies such as Parkinson’s disease (PD). Disruption of proteostasis is thought to be central to pathologic alpha-synuclein (alpha-syn) toxicity; however, the molecular mechanism of this deregulation is poorly understood. Here we report that pathologic alpha-syn activates the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) leading to enhanced mRNA translation via binding tuberous sclerosis protein (TSC) 2 and destabilizing the TSC1-TSC2 complex. Genetic and pharmacologic inhibition of mTOR and protein synthesis rescue the dopamine neuron loss, behavioral deficits, and aberrant biochemical signaling in the alpha-syn preformed fibril (PFF) and Drosophila alpha-syn transgenic models of pathologic alpha-syn induced degeneration. Our findings establish a potential molecular mechanism by which pathologic alpha-syn activates mTORC1 leading to enhanced protein synthesis and concomitant neurodegen..., In vitro translation (IVT) assay
Translation extract was prepared from human neuroblastoma (SHSY-5Y) or HEK293 cells grown in 175 cm2 flask to achieve ~80% confluence. The growing cells were harvested using trypsinization and neutralization with complete DMEM media. The cell pellets were washed with 10 mL PBS to remove residual media from the cell pellet. After PBS wash, the cell pellets were kept in ice and lysed immediately in freshly made translation extract lysis buffer (30 mM HEPES-KOH, 100 mM potassium acetate, 2 mM magnesium acetate, 5 mM DTT, 1 mg/mL Pefabloc SC). The cell pellet from two 175 cm2 flasks was lysed in 500 mL lysis buffer. The lysis was performed using mechanical homogenizer and Corning PYREX Tissue Grinder in ice. A total of twenty strokes were applied to homogenize the cell pellet. The lysates were centrifuged at 12500 rpm for 20 mins at 4°C. After centrifugation, the clear cytoplasmic fractions were collected from the top of the centrifuge tube. The cytoplasmic ..., , # Enhanced mTORC1 signaling and Protein Synthesis in Pathologic alpha-Synuclein Cellular and Animal models of Parkinson’s Disease

[https://doi.org/10.5061/dryad.51c59zwf1](https://doi.org/10.5061/dryad.51c59zwf1)

In this study, we examined the pathological effect of a-synuclein in enhanced mRNA translation and mTORC1 activity in Parkinson’s disease models. We used complementary proteomic approaches and mouse primary neuron cellular and animal models of PD to identify and characterize the pathologic a-synuclein interactome. We demonstrated that the highest biological processes that interact with pathologic a-synuclein include RNA processing and translation initiation. Pathologic α-synuclein was found to bind with the tuberous sclerosis protein (TSC) 2, resulting in the destabilization of the TSC1-TSC2 complex. This destabilization ultimately triggered the activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). The activated mTORC1 complex plays a pivotal role in au...

致病型α-突触核蛋白（pathologic alpha-synuclein）在帕金森病（Parkinson’s disease, PD）等α-突触核蛋白病（alpha-synucleinopathies）的发病机制中发挥重要作用。蛋白质稳态失衡被认为是致病型α-突触核蛋白（后文简称α-突触核蛋白）毒性的核心机制，但这种调控异常的分子机制尚不清楚。本研究发现，致病型α-突触核蛋白可通过结合结节性硬化蛋白2（TSC2）并破坏TSC1-TSC2复合物的稳定性，激活雷帕霉素机制靶蛋白复合物1（mTOR complex 1, mTORC1），进而增强mRNA翻译过程。在α-突触核蛋白预形成纤丝（alpha-syn preformed fibril, PFF）诱导的退行性变模型以及果蝇α-突触核蛋白转基因模型中，对mTOR及蛋白质合成进行遗传与药理学抑制，可挽救多巴胺能神经元丢失、行为缺陷以及异常的生化信号通路。本研究的发现确立了致病型α-突触核蛋白激活mTORC1、进而增强蛋白质合成并伴随神经退行性变的潜在分子机制……
## 体外翻译（in vitro translation, IVT）实验
翻译提取物的制备：将人神经母细胞瘤细胞（SHSY-5Y）或HEK293细胞接种于175 cm²培养瓶中，待细胞汇合度达到约80%时收集。使用胰酶消化收集处于增殖期的细胞，并用含完全培养基的DMEM终止消化。用10 mL PBS洗涤细胞沉淀以去除残留培养基。PBS洗涤完成后，将细胞沉淀置于冰上，立即用新鲜配制的翻译提取物裂解缓冲液（30 mM HEPES-KOH、100 mM 乙酸钾、2 mM 乙酸镁、5 mM 二硫苏糖醇（DTT）、1 mg/mL Pefabloc SC）进行裂解。将两个175 cm²培养瓶的细胞沉淀置于500 mL裂解缓冲液中完成裂解，裂解过程在冰上通过机械匀浆器与Corning PYREX组织研磨器进行，共施加20次研磨以充分匀浆细胞沉淀。将裂解液在4℃下以12500 rpm离心20分钟，离心完成后从离心管上层收集清亮的细胞质组分。细胞质……
# 帕金森病致病型α-突触核蛋白细胞与动物模型中增强的mTORC1信号通路与蛋白质合成
[https://doi.org/10.5061/dryad.51c59zwf1](https://doi.org/10.5061/dryad.51c59zwf1)
本研究针对帕金森病模型中α-突触核蛋白对mRNA翻译增强及mTORC1活性的病理效应展开了探究。我们采用互补蛋白质组学方法，结合小鼠原代神经元细胞模型与PD动物模型，对致病型α-突触核蛋白的相互作用组进行了鉴定与表征。研究证实，与致病型α-突触核蛋白相互作用的富集度最高的生物学过程包括RNA加工与翻译起始。研究发现致病型α-突触核蛋白可结合结节性硬化蛋白2（TSC2），进而破坏TSC1-TSC2复合物的稳定性。这种复合物稳定性的破坏最终会激活雷帕霉素机制靶蛋白复合物1（mTORC1）。激活的mTORC1复合物在……中发挥关键作用