Characterizing the Conformations and Neurotoxic Species of Huntingtin

亨廷顿蛋白的构象和神经毒性种类的表征

• 批准号: 10316055
• 负责人: Ali Khoshnan
• 金额: $ 52.2万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10316055
• 关键词: Affect; Antibodies; Antibody Therapy; Binding; Biochemical; Biological; Biological Assay; Biophysics; Brain; Classification; Collection; Cryoelectron Microscopy; Development; Diagnostic; Disease; Disease Progression; Epitopes; Exons; Human; Huntington Disease; Huntington gene; Huntington protein; In Vitro; Inherited; Injections; Intervention; Knowledge; Length; Libraries; Link; Maps; Measures; Mediating; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Monoclonal Antibodies; Mus; Nerve Block; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Outcome; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Physiological; Plasma; Production; Property; Proteins; Protocols documentation; Reagent; Recombinant Antibody; Recombinants; Research; Resolution; Role; Severity of illness; Specimen; Structure; Testing; Therapeutic; Time; Toxic effect; biophysical techniques; conformer; diagnostic assay; diagnostic platform; experimental study; extracellular; in vivo; innovation; insight; monomer; mouse model; mutant; neurotoxic; neurotoxicity; new therapeutic target; novel; physical state; polyglutamine; prevent; proteostasis; receptor; relating to nervous system; targeted treatment; therapeutic target; therapy development; tool

The exon-1 of mutant huntingtin protein (mHTTex1) accumulates in the brains of Huntington’s disease (HD) patients and is implicated in neurodegeneration. The intrinsically disordered mHTTex1 misfolds into a heterogeneous mixture of assemblies, however, the pathogenic conformers are not well characterized. The major limiting factors have been the lack of methods to assemble ultrapure mHTTex1 structures, molecular tools to identify them and models to investigate their neurotoxicity. Towards this end, we have developed protocols to assemble distinct oligomers, protofibrils and fibrils of mHTTex1 and have generated libraries of monoclonal antibodies (mAbs) to defined structures. We plan to characterize the binding of representative mAbs to conformations in various assemblies using biophysical and biochemical methods, explore whether the interaction of each mAb with its epitope affects the misfolding, seeding and aggregation in vitro, and examine the structures of mHTTex1 species upon binding to selected mAbs (aim 1). We have further developed a diagnostic platform to study the entry of structurally known mHTTex1 species into human neurons and their aggregation into neurotoxic assemblies. With this model, we plan to identify and characterize the neuroinvasive/neurotoxic species of mHTTex1, map their pathogenic conformations and determine their structures with biophysical methods including Cryo-EM. Moreover, we plan to discover the neuronal receptors, which participate in the entry of mHTTex1 and identify the interacting proteins, which are incorporated in the neurotoxic aggregates. In addition, we plan to examine for the presence of neuroinvasive mHTT species in the brains of HD patients and in human neuronal and mouse models of HD to validate the physiological relevance of the in vitro-assembled structures and any links to disease severity. These experiments may for the first time identify the structures of neurotoxic mHTTex1 at high resolution, a novel pathway for their production, and may provide targets for therapy development (aim 2). In aim 3, we will investigate the role extracellular mHTT in disease in the CNS of HD mice. In one set of experiments, we plan to inject neuroinvasive species of mHTTex1 into the brains of asymptomatic R6/2Q51 HD mice (express human mHTTex1 with 51Qs) and investigate their ability to enter neurons, trigger assembly formation and accelerate disease progression. Moreover, we will determine whether blocking any of the pathogenic conformations of mHTTex1 by AAV- mediated delivery of recombinant antibodies, which are secreted in the CNS, inhibits the entry, amplification and neurotoxicity of the injected species. Finally, given that mHTT is present in the CSF and plasma of HD patients and mouse models, we plan to investigate the therapeutic impacts of secreted recombinant antibodies on the accumulation of pathogenic mHTT assemblies and progression of HD-like pathology in the Q140 HD mice expressing full-length mHTT. These studies will fill some of the knowledge gaps on the role of extracellular mHTT in the pathogenesis of HD and may provide therapeutic targets and reagents.

突变亨廷顿蛋白（MHTTEX1）的外显子1在亨廷顿氏病（HD）的大脑中积聚 患者，隐含在神经退行性中。本质上干扰的MHTTEX1不折叠成一个 然而，组件的异质混合物，致病构象体的表征尚未得到很好的特征。这 主要限制因素是缺乏组装超纯MHTTEX1结构的方法，分子 识别它们和模型以研究其神经毒性的工具。为此，我们已经发展 组装MHTTEX1的不同低聚物，原纤维和原纤维的协议，并产生了库 对定义结构的单克隆抗体（mAb）。我们计划表征代表的结合 使用生物物理和生化方法的各种组件中构象的mAb，请探讨是否探讨 每个mAb及其表位的相互作用会在体外影响错误折叠，播种和聚集，并检查 MHTTEX1物种与选定的mAb结合后的结构（AIM 1）。我们进一步开发了 诊断平台研究结构已知的MHTTEX1物种进入人类神经元及其 聚集到神经毒性组件中。使用此模型，我们计划识别和表征 MHTTEX1的神经活动/神经毒性物种，绘制其致病构象并确定其 具有生物物理方法在内的结构，包括冷冻EM。此外，我们计划发现神经元接收器， 参与MHTTEX1的进入并识别相互作用的蛋白质，并纳入其中 神经毒性聚集体。此外，我们计划检查在 HD患者的大脑以及HD的人类神经元和小鼠模型，以验证物理相关性 体外组装结构以及与疾病严重程度的任何联系。这些实验可能是第一次 在高分辨率下确定神经毒性MHTTEX1的结构，这是其生产的新途径，可以 提供治疗开发的目标（AIM 2）。在AIM 3中，我们将研究细胞外MHTT的作用 HD小鼠中枢神经系统中的疾病。在一组实验中，我们计划注入神经脱落物种 MHTTEX1进入无症状R6/2Q51 HD小鼠的大脑（以51QS的表达人MHTTEX1）和 研究它们进入神经元，触发组装形成并加速疾病进展的能力。 此外，我们将确定是否通过AAV-来阻止MHTTEX1的任何致病构象 CNS中分泌的重组抗体的介导的递送，抑制进入，放大 注射物种的神经毒性。最后，鉴于MHTT在HD的CSF和血浆中呈现 患者和小鼠模型，我们计划研究分泌重组抗体的治疗影响 关于致病性MHTT组件的积累和Q140 HD中HD样病理的进展 表达全长MHTT的小鼠。这些研究将填补有关角色的一些知识差距 高清发病机理中的细胞外MHTT，可能提供治疗靶标和试剂。