Contrasting pathomechanisms of membrane versus cytosol alpha-synuclein excess

α-突触核蛋白（αS）的失调导致帕金森病（PD）。路易体痴呆（DLB）、PD痴呆（PDD）、多系统萎缩（MSA）以及阿尔茨海默病都以αS失衡为特征。虽然大多数病例是散发的，但一些家族性αS错义突变与突触核蛋白病有关，可以告诉我们病理机制。其中，E46K引起PD/DLB， G51D引起PD/MSA。αS与囊泡膜短暂结合，矛盾的是，E46K增加αS与囊泡膜的结合，而G51D则减少αS与囊泡膜的结合。我们试图测试一种有趣的可能性，即αS聚集上游的“早期干预”策略必须考虑潜在不平衡的确切性质：αS是在膜（e46k样）还是在细胞质（g51d样）中积累？我们将验证以下假设：(1)膜αS积累和细胞质αS积累是αS毒性的两种不同途径。(2)纠正这两种通路的药理策略不同。Dettmer实验室开创了通过家族PD/DLB突变体“扩增”策略触发细胞αS应激的策略。利用αS重复序列结构，构建E46K扩增3K序列（E35K+E46K+E61K），将fpd连接的E46K序列延伸至相邻的两个重复序列。我们已经证明3K确实加强了E46K的关键方面，包括增加膜结合，从而引起明显的细胞应激。我们新的关键创新是G51D扩增‘ 3D ’ （V40D+G51D+V65D），与E46K相反，它使αS大部分呈细胞质，从而也引起明显的细胞应激。我们将对3K和3D进行表征，以比较膜（3K）和细胞质相关(3D) αS胁迫。我们考虑在扩增系统中发现的策略，然后在患者iPSC神经元中进行确认，这对我们的两个目标是理想的：目标1:αS膜与细胞质积聚在细胞通路3K和3D上的对比效应将带来共享的和突变特异性的应激特征，这些特征在单突变体中太微妙了。a)关注人类iPSC神经元的3K、wt和3D转导，以执行TMT蛋白质组学和RNAseq b)通过Western blot确认E46K与G51D加等基因对照系的hiPSC神经元的显著变化。目的2：寻找特异性（或非特异性）减轻细胞质和膜αS相关应激的化合物。我们将建立3K和3D αS失衡的创新神经元报告细胞系。a)详细比较候选化合物b)进行中等通量小分子筛选比较c)在hiPSC E46K和G51D神经元中进行生化证实。这项工作遵循“阿尔茨海默病相关痴呆会议建议”中概述的研究重点。它特别侧重于解决阿尔茨海默病相关痴呆（ADRD）的优先事项，特别是路易体痴呆（DLB）和帕金森病（PD）痴呆（PDD）。

Dysregulation of α-synuclein (αS) results in Parkinson’s Disease (PD). Dementia with Lewy bodies (DLB), PD Dementia (PDD), Multi-System Atrophy (MSA), and also Alzheimer’s disease are likewise characterized by αS imbalance. While most cases are sporadic, several familial αS missense mutations are linked to synucleinopathy and can inform us about pathologic mechanisms. Among them, E46K causes PD/DLB while G51D causes PD/MSA. αS transiently binds to vesicle membranes and, paradoxically, E46K increases αS membrane association whereas G51D decreases it. We seek to test the intriguing possibility that ‘early intervention’ strategies upstream of αS aggregation must take into account the exact nature of the underlying disequilibrium: does αS accumulate at membranes (E46K-like) or in the cytosol (G51D-like)? We will test the hypotheses that: (1) Membrane αS accumulation and cytosol αS accumulation are two distinct pathways to αS toxicity. (2) Pharmacological strategies to correct the two pathways differ. The Dettmer lab has pioneered the strategy of triggering cellular αS stress by familial PD/DLB mutant ‘amplification’ strategies. We took advantage of the αS repeat structure to create the E46K amplification ‘3K’ (E35K+E46K+E61K), which extends fPD-linked E46K to the two neighboring repeats. We have demonstrated that 3K indeed accentuates key aspects of E46K, including increased membranes binding, thereby causing pronounced cellular stress. Our new key innovation is the G51D amplification ‘3D’ (V40D+G51D+V65D), which – opposite to E46K – renders αS largely cytosolic, thereby also causing pronounced cellular stress. We will characterize 3D alongside 3K to compare membrane- (3K) vs. cytosol-associated (3D) αS stress. We consider a strategy of discovery in amplified systems followed by confirmation in patient iPSC neurons ideal for our 2 Aims: Aim 1: Contrasting effects of αS membrane vs. cytosol accumulation on cellular pathways 3K and 3D will bring out shared and mutant-specific stress signatures that are too subtle in the single mutants. a) Focus on 3K, wt, and 3D transduction of human iPSC neurons to perform TMT proteomics and RNAseq b) Confirm compelling changes by Western blot in hiPSC neurons of E46K vs. G51D plus isogenic control lines Aim 2: Finding compounds specific (or not) to lessening cytosolic vs. membrane αS-related stress We will build upon innovative neuronal reporter cell lines of 3K and 3D αS imbalance. a) Compare in detail candidate compounds b) Perform comparative medium-throughput small-molecule screens c) Confirm findings biochemically in hiPSC E46K and G51D neurons. This work pursues research priorities outlined in “Recommendations of the Alzheimer's disease-related dementias conference”. It especially focuses on priorities that address Alzheimer's disease-related dementias (ADRD), specifically dementia with Lewy bodies (DLB) and Parkinson’s disease (PD) dementia (PDD).