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.

亨廷顿病(HD)患者脑内亨廷顿蛋白突变基因外显子1(MHTTES 1)积聚 并与神经退行性变有关。本质上混乱的mHTTEX 1错误地折叠成一个 然而，不均匀的组装体混合物，致病的构象还没有得到很好的表征。这个 主要的限制因素是缺乏组装超纯mHTTEX 1结构、分子 识别它们的工具和研究它们神经毒性的模型。为此，我们制定了 组装mHTTEX 1的不同寡聚体、原纤维和纤维的协议，并生成了 针对特定结构的单抗(MAbs)。我们计划描述代表的约束力 利用生物物理和生化方法将单抗转化为不同组装体中的构象，探索 各单抗与其表位的相互作用会影响其在体外的错误折叠、播种和聚集，并检测 MHTTEX 1物种与选定单抗结合时的结构(目标1)。我们进一步开发了一种 研究结构上已知的mHTTES 1物种进入人类神经元及其相互作用的诊断平台 聚集成神经毒性集合体。有了这个模型，我们计划识别和描述 MHTTES 1的神经侵袭/神经毒性物种，绘制其致病构象图并确定其 使用生物物理方法的结构，包括低温EM。此外，我们计划发现神经元受体， 它们参与mHTTex1的进入，并识别相互作用的蛋白质，这些蛋白质被整合到 神经毒性集合体。此外，我们计划检查神经侵袭性mHTT物种在 HD患者的大脑与人类神经元和小鼠HD模型的生理学相关性验证 体外组装的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 以高分辨率鉴定神经毒性mHTtex 1的结构，这是一种生产它们的新途径，并可能 提供治疗发展的目标(目标2)。在目标3中，我们将研究细胞外mHTT在 HD小鼠中枢神经系统疾病。在一组实验中，我们计划将神经侵入性物种注入 无症状R6/2Q51 HD小鼠(51Qs表达人mHTtex 1)和 研究它们进入神经元、触发组装形成和加速疾病进展的能力。 此外，我们将确定AAV是否可以阻断mHTTES 1的任何致病构象- 在中枢神经系统中分泌的重组抗体的介导性传递抑制了进入、放大 以及注射物种的神经毒性。最后，鉴于mHTT存在于HD患者的脑脊液和血浆中 患者和小鼠模型，我们计划调查分泌型重组抗体的治疗效果 Q140 HD病原性mHTT集合体积聚及类HD病变进展的研究 表达全长mHTT的小鼠。这些研究将填补一些关于血管紧张素转换酶的作用的知识空白 细胞外mHTT在HD的发病机制中可能提供治疗靶点和试剂。