Chaperone protection in Lewy body and Alzheimer’s dementias: determining the structural, molecular and cellular mechanisms of a novel, non-canonical Hsp70 action blocking a-synuclein oligomerization

路易体和阿尔茨海默氏痴呆中的伴侣保护：确定阻断 α-突触核蛋白寡聚化的新型非典型 Hsp70 作用的结构、分子和细胞机制

• 批准号: 10649331
• 负责人: DAVID A. AGARD
• 金额: $ 20.19万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10649331
• 关键词: Affect; Alzheimer' s Disease; Alzheimer' s disease model; Amyloid beta-Protein; Animal Disease Models; Animal Model; Binding; Binding Sites; Biochemical; Biological Assay; Biological Process; Cell model; Cell physiology; Cells; Client; Clinical; Cryoelectron Microscopy; Cytosol; Data; Dementia; Deposition; Development; Disease; Disease Progression; Ensure; Equilibrium; Future; Gene Mutation; Impairment; In Vitro; Lewy Bodies; Lewy Body Dementia; Lipid Binding; Lipids; Maps; Mediating; Membrane; Microscopic; Molecular; Molecular Chaperones; Molecular Structure; Mutate; Mutation; Mutation Analysis; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Neurons; Pathogenicity; Pathologic; Pathology; Pathway interactions; Persons; Play; Proteins; Publishing; Resolution; Role; Site; Societies; Stress; Testing; Therapeutic; Toxic effect; Translational Research; Validation; Vesicle; Visualization; abeta oligomer; alpha synuclein; cost; in vivo; monomer; mutant; neurotoxic; neurotoxicity; novel; novel therapeutic intervention; prion-like; protective pathway; protein folding; side effect; synuclein; targeted treatment; tau Proteins; tau aggregation; tau mutation; therapeutic target; tool

PROJECT SUMMARY/ABSTRACT In the US, Lewy body dementias (LBD) and Alzheimer’s disease (AD) affect over 7 million people. No treatment slows their inexorable decline. Broad evidence implicates malicious roles of α-synuclein (ASyn), tau and Abeta (A) proteins in these dementias. LBD and AD are characterized by the sequential misfolding of ASyn, tau and A into toxic oligomers and fibrils that propagate in prion-like fashion and accumulate in pathological deposits, with ASyn the predominating pathology in LBD and A and tau pathologies predominating in AD. ASyn or A precursor gene alterations cause LBD and AD respectively. Although central to disease, ASyn, A and tau mis- folding have been challenging to target therapeutically. ASyn, tau and A pathologies correlate with LBD and AD decline. Elevated levels of the stress-induced chaperone Hsp70 protects against ASyn misfolding and neu- rodegeneration in LBD cell and animal models. However, there is minimal mechanistic understanding of this important protective pathway. In particular, it is generally assumed that the canonical and promiscuous mode of Hsp70 action underlies Hsp70-ASyn protection. Consequently, translational investigations have been stymied by the anticipation of undesirable side effects of therapeutically targeting Hsp70 canonical actions. Our data contradict this assumption, providing a potentially more targeted and exploitable mechanism of Hsp70-mediated protection in disease. We find that Hsp70 blocks the earliest and most neurotoxic stage of ASyn misfolding, ASyn oligomerization, by interacting at a previously unknown site separate from its canonical site of action. It is therefore plausible that targeting this non-canonical mechanism would protect against disease. Importantly, this approach holds the potential to correct ASyn misfolding without disrupting critical cellular processes. Our central hypothesis is that this novel, non-canonical blockage of ASyn oligomerization by Hsp70 is protective in LBD and AD. The next steps to support and exploit this hypothesis are to determine the molecular mechanism of Hsp70’s engagement with ASyn, to validate this mechanism as protective, and test its action on additional AD/LBD pro- teins. In Aim 1, we will determine the critical molecular residues in Hsp70 for blockage of ASyn oligomerization and toxicity. We will use state-of-the-art cryo-EM microscopic approaches to capture how ASyn binds to Hsp70 with atomic resolution and validate the role of this binding through mutational analysis in LBD-relevant biochem- ical and cellular ASyn oligomerization and toxicity assays. This will directly test our hypothesis that non-canonical Hsp70 engagement of ASyn underlies its activity in blocking ASyn oligomers and in providing protection against ASyn toxicity in neurons. In Aim 2, we will determine the impact of Hsp70 non-canonical action on oligomeriza- tion of additional AD and LBD-relevant protein species, including lipid-bound ASyn and tau and A proteins. Confirming an effect of non-canonical Hsp70 action on multiple pathogenic proteins would greatly expand the therapeutic potential of this effect, and suggest that non-canonical Hsp70 plays a broader and so far unsuspected role in many additional biological processes.

项目摘要/摘要 在美国，路易体痴呆症（LBD）和阿尔茨海默病（AD）影响超过700万人。无治疗 减缓了它们不可阻挡的衰落广泛的证据表明α-突触核蛋白（ASyn），tau和Abeta的恶意作用 （A）痴呆症中的蛋白质。LBD和AD的特征在于ASyn、tau和LBD的顺序错误折叠。 一种能降解成有毒寡聚体和纤维的蛋白，以朊病毒样方式繁殖并在病理沉积物中积累， 其中ASyn是LBD中的主要病理学，而A β和tau病理学在AD中占主导地位。ASyn或A 前体基因改变分别引起LBD和AD。虽然疾病的核心，ASyn，A β和tau错误- 折叠对于治疗靶向是具有挑战性的。ASyn、tau和Asyn病理与LBD相关， AD下降。应激诱导的分子伴侣Hsp70水平升高可防止ASyn错误折叠和neu- 在LBD细胞和动物模型中的变性。然而，对这一点的机械理解很少 重要的保护途径。特别是，一般认为，规范和混杂的模式， Hsp70作用是Hsp70-ASyn保护的基础。因此，翻译研究受到了阻碍 通过预期治疗靶向Hsp70规范作用的不良副作用。我们的数据 与此假设相反，提供了一个潜在的更有针对性和可利用的机制，热休克蛋白70介导的 疾病的保护。我们发现Hsp70阻断了ASyn错误折叠的最早和最具神经毒性的阶段， ASyn寡聚化，通过在与其典型作用位点分开的先前未知位点相互作用。是 因此，靶向这种非经典机制可以预防疾病是合理的。重要的是这 这种方法具有纠正ASyn错误折叠而不破坏关键细胞过程的潜力。我们的中央 假设Hsp70对ASyn寡聚化的这种新的非典型阻断在LBD中是保护性的， ad.支持和利用这一假设的下一步是确定Hsp70的分子机制。 与ASyn合作，以验证这种机制的保护作用，并测试其对其他AD/LBD pro的作用。 teins。在目的1中，我们将确定Hsp70中阻断ASyn寡聚化的关键分子残基 和毒性。我们将使用最先进的冷冻电镜显微镜方法来捕捉ASyn如何与Hsp70结合。 并通过LBD相关生物化学中的突变分析验证这种结合的作用， 医学和细胞ASyn寡聚化和毒性测定。这将直接检验我们的假设，即非规范 Hsp70与ASyn的结合是其阻断ASyn寡聚体和提供抗Hsp70的保护作用的基础。 神经元中的ASyn毒性。在目标2中，我们将确定Hsp70非经典作用对寡聚化的影响- 另外的AD和LBD相关蛋白质种类，包括脂质结合的ASyn和tau和A β蛋白。 证实非典型Hsp70对多种致病蛋白的作用将极大地扩展Hsp70的作用机制。 这种作用的治疗潜力，并表明，非典型的Hsp70发挥更广泛的，迄今未被怀疑的作用。 在许多其他生物过程中的作用。