Contrasting pathomechanisms of membrane versus cytosol alpha-synuclein excess

膜与胞质 α-突触核蛋白过量的病理机制对比

• 批准号: 10195494
• 负责人: Ulf Dettmer
• 金额: $ 50.46万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10195494
• 关键词: 3-Dimensional; Address; Alzheimer' s Disease; Alzheimer' s disease related dementia; Binding; Biochemical; Biological Assay; Biology; Biopolymers; Brain Diseases; Caymans; Cell Culture Techniques; Cell Line; Cell membrane; Cells; Cellular Membrane; Cellular Stress; Cellular Structures; Cellular biology; Chemistry; Cytosol; Data; Defect; Dementia with Lewy Bodies; Disease; Early Intervention; Genetic; Growth; Human; Institutes; Intervention; Isradipine; Letters; Lewy Body Dementia; Libraries; Link; Membrane; Minor; Missense Mutation; Molecular Profiling; Multiple System Atrophy; Nature; Neurons; Nortriptyline; Parkinson Disease; Parkinson' s Dementia; Parkinsonian Disorders; Pathologic; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Pharmacology; Phenotype; Physiological; Proteins; Proteomics; Publishing; RNA; Rattus; Recommendation; Reporter; Research Priority; Series; Squalamine; Standardization; Stearoyl-CoA Desaturase; Stress; Structure; System; Testing; Toxic effect; Trifluoperazine; Variant; Vesicle; Western Blotting; Work; alpha synuclein; base; cell growth; comparative; crosslink; experimental study; induced pluripotent stem cell; inhibitor/antagonist; innovation; insight; medical schools; mutant; neuroblastoma cell; neuron loss; novel therapeutic intervention; perilipin; phthalocyanine; screening; small molecule; symposium; synucleinopathy; transcriptome sequencing

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).

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）以及阿尔茨海默病的特征同样是αS失衡。虽然大多数病例是散发性的，但几种家族性αS错义突变与突触核蛋白病有关，并可以为我们提供病理机制。其中，E46 K引起PD/DLB，而G51 D引起PD/MSA。αS瞬时结合囊泡膜，矛盾的是，E46 K增加αS膜结合，而G51 D减少它。我们试图测试有趣的可能性，即αS聚集上游的“早期干预”策略必须考虑到潜在的不平衡的确切性质：αS积累在膜（E46 K样）或在胞质溶胶（G51 D样）？我们将检验以下假设：（1）细胞膜αS积累和细胞质αS积累是αS毒性的两种不同途径。(2)纠正这两种途径的药理学策略不同。Dettmer实验室开创了通过家族性PD/DLB突变体“扩增”策略触发细胞αS应激的策略。我们利用αS重复序列的优势，创建了E46 K扩增“3 K”（E35 K + E46 K + E61 K），它将fPD连接的E46 K延伸到两个相邻的重复序列。我们已经证明，3 K确实加重了E46 K的关键方面，包括增加膜结合，从而引起明显的细胞应激。我们的新的关键创新是G51 D扩增“3D”（V40 D + G51 D + V65 D），与E46 K相反，它使αS主要出现在细胞溶质中，从而也引起明显的细胞应激。我们将对3D和3 K进行表征，以比较膜（3 K）与细胞溶质相关（3D）αS应力。我们考虑了在扩增系统中发现，然后在患者iPSC神经元中确认的策略，这对于我们的2个目标是理想的：目标1：对比αS膜与细胞溶质积累对细胞途径3 K和3D的影响，将产生共享的和mu特异性的应激特征，这些特征在单个突变体中过于微妙。a）关注人iPSC神经元的3 K、wt和3D转导以进行TMT蛋白质组学和RNAseq B）通过蛋白质印迹证实E46 K与G51 D加同基因对照系的hiPSC神经元中的引人注目的变化目的2：寻找对减轻胞质与膜α S相关应激具有特异性（或不特异性）的化合物我们将建立3 K和3D αS不平衡的创新神经元报告细胞系。a）详细比较候选化合物B）进行比较性中等通量小分子筛选c）在hiPSC E46 K和G51 D神经元中生物化学地确认发现。这项工作遵循“阿尔茨海默病相关痴呆会议建议”中概述的研究优先事项。它特别关注解决阿尔茨海默病相关痴呆症（ADRD）的优先事项，特别是路易体痴呆症（DLB）和帕金森病（PD）痴呆症（PDD）。

项目成果

A Brain-Penetrant Stearoyl-CoA Desaturase Inhibitor Reverses α-Synuclein Toxicity.

• DOI: 10.1007/s13311-022-01199-7
• 发表时间: 2022-04
• 期刊: NEUROTHERAPEUTICS
• 影响因子: 5.7
• 作者: Nuber, Silke;Chung, Chee Yeun;Tardiff, Daniel F.;Bechade, Pascal A.;McCaffery, Thomas D.;Shimanaka, Kazuma;Choi, Jeonghoon;Chang, Belle;Raja, Waseem;Neve, Esther;Burke, Christopher;Jiang, Xin;Xu, Ping;Khurana, Vikram;Dettmer, Ulf;Fanning, Saranna;Rhodes, Kenneth J.;Selkoe, Dennis J.;Scannevin, Robert H.
• 通讯作者: Scannevin, Robert H.

1,4-Diurea- and 1,4-Dithiourea-Substituted Aromatic Derivatives Selectively Inhibit α-Synuclein Oligomer Formation In Vitro.

• DOI: 10.1021/acsomega.3c07453
• 发表时间: 2024-01-09
• 期刊: ACS OMEGA
• 影响因子: 4.1
• 作者: Ganegamage, Susantha K.;Ramirez, Eduardo;Alnakhala, Heba;Tripathi, Arati;Nguyen, Cuong Calvin Duc;Zami, Ashique;Ostafe, Raluca;Tian, Shiliang;Dettmer, Ulf;Fortin, Jessica S.
• 通讯作者: Fortin, Jessica S.

The effects of KTKEGV repeat motif and intervening ATVA sequence on α-synuclein solubility and assembly.

KTKEGV 重复基序和干预 ATVA 序列对 α-突触核蛋白溶解度和组装的影响。

• DOI: 10.1111/jnc.15763
• 发表时间: 2023
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Brontesi,Lisa;Imberdis,Thibaut;Ramalingam,Nagendran;Dettmer,Ulf
• 通讯作者: Dettmer,Ulf

Lipotoxicity Downstream of α-Synuclein Imbalance: A Relevant Pathomechanism in Synucleinopathies?

• DOI: 10.3390/biom12010040
• 发表时间: 2021-12-28
• 期刊: Biomolecules
• 影响因子: 5.5
• 作者: Tripathi A;Fanning S;Dettmer U
• 通讯作者: Dettmer U

5-Nitro-1,2-benzothiazol-3-amine and N-Ethyl-1-[(ethylcarbamoyl)(5-nitro-1,2-benzothiazol-3-yl)amino]formamide Modulate α-Synuclein and Tau Aggregation.

• DOI: 10.1021/acsomega.3c02668
• 发表时间: 2023-06-06
• 期刊: ACS omega
• 影响因子: 4.1
• 作者: Ramirez E;Ganegamage SK;Elbatrawy AA;Alnakhala H;Shimanaka K;Tripathi A;Min S;Rochet JC;Dettmer U;Fortin JS
• 通讯作者: Fortin JS