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: 10814676
• 负责人: Ali Khoshnan
• 金额: $ 62.98万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10814676
• 关键词: Acceleration; 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; 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; neural; neurotoxic; neurotoxicity; new therapeutic target; novel; physical state; polyglutamine; prevent; proteostasis; receptor; 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

项目成果

Amplification of neurotoxic HTTex1 assemblies in human neurons.

• DOI: 10.1016/j.nbd.2021.105517
• 发表时间: 2021-11
• 期刊: Neurobiology of disease
• 影响因子: 6.1
• 作者: Chongtham A;Isas JM;Pandey NK;Rawat A;Yoo JH;Mastro T;Kennedy MB;Langen R;Khoshnan A
• 通讯作者: Khoshnan A

Fluorescent protein tagging promotes phase separation and alters the aggregation pathway of huntingtin exon-1.

• DOI: 10.1016/j.jbc.2023.105585
• 发表时间: 2024-01
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Pandey, Nitin K;Varkey, Jobin;Ajayan, Anakha;George, Gincy;Chen, Jeannie;Langen, Ralf
• 通讯作者: Langen, Ralf