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

项目摘要/摘要 在美国，路易体痴呆(LBD)和阿尔茨海默病(AD)影响着700多万人。不接受治疗 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)蛋白质在这些痴呆中。LBD和AD的特征是ASyn、tau和ASyn的顺序错误折叠 一种有毒的低聚物和纤维的，它们以类病毒的方式繁殖，并在病理性沉积中积累， 腰椎退行性变以Asyn为主，AD以A、和tau病变为主。ASyn或A 前体基因改变分别导致LBD和AD。虽然Asyn、A和tau是疾病的中心，但它们是错误的- 折叠一直是治疗靶点的挑战。Asyn、tau和A病理与腰椎病和 广告下滑。应激诱导的伴侣蛋白Hsp70水平升高可保护ASyn错误折叠和Neu-2 LBD细胞和动物模型中的退行性变。然而，人们对此的机械性理解很少。 重要的保护途径。具体地说，通常假设规范和混杂模式 HSP70的作用是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 与这一假设相矛盾，提供了一种潜在的更有针对性和更可利用的Hsp70介导的机制 对疾病的保护。我们发现，Hsp70阻止了ASyn错误折叠的最早和最具神经毒性的阶段， ASyn寡聚，通过在与其正则作用位点分离的先前未知的位置上相互作用。它是 因此，以这种非规范机制为目标可以预防疾病，这似乎是合理的。重要的是，这 这种方法有可能纠正ASyn的错误折叠，而不会扰乱关键的细胞过程。我们的中央 假设Hsp70对ASyn寡聚的这种新的、非典型的阻断在LBD和LBD中具有保护作用 广告。支持和利用这一假说的下一步是确定热休克蛋白70‘S的分子机制 与ASyn合作，以验证此机制是否具有保护性，并测试其对其他AD/LBD PRO- 泰恩斯。在目标1中，我们将确定Hsp70中阻断ASyn齐聚的关键分子残基 和毒性。我们将使用最先进的冷冻-EM显微方法来捕捉ASyn如何与Hsp70结合 并通过突变分析验证这种结合在LBD相关生物化学中的作用- 细胞和细胞ASyn寡聚及毒性分析。这将直接测试我们的假设，即非正则 ASyn的HSP70参与是其阻断ASyn寡聚体和提供保护作用的基础 ASyn对神经元的毒性。在目标2中，我们将确定Hsp70的非正则作用对寡聚化的影响。 其他与AD和LBD相关的蛋白质种类，包括与脂质结合的Asyn和tau和A蛋白质。 确认Hsp70对多种致病蛋白的非典型作用将大大扩展 这种效应的治疗潜力，并表明非典型性Hsp70发挥了更广泛的作用，到目前为止还没有被怀疑 在许多额外的生物过程中发挥作用。

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.