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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

基本信息

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
项目类别：
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

项目摘要

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.

突变的亨廷顿蛋白外显子1 （mHTTex1）在亨廷顿病（HD）患者的大脑中积累