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:
    10814676
  • 负责人:
  • 金额:
    $ 62.98万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

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.
突变型亨廷顿蛋白外显子1在亨廷顿病患者脑内的蓄积 患者并与神经退行性疾病有关。本质上无序的mHTTex 1错误折叠成 然而,由于组装体的异质混合物,致病性构象异构体没有很好地表征。的 主要的限制因素是缺乏组装超纯mHTTex 1结构、分子结构和分子结构的方法。 工具来识别它们和模型来研究它们的神经毒性。为此,我们制定了 组装mHTTex 1的不同寡聚体、原纤维和原纤维的方案,并产生了mHTTex 1的文库。 单克隆抗体(mAb)的定义结构。我们计划将代表的约束力 使用生物物理学和生物化学方法对各种组装体中的mAb进行构象分析,探索 每个mAb与其表位的相互作用影响体外错误折叠、接种和聚集,并检查 mHTTex 1物质与选定mAb结合后的结构(目的1)。我们进一步发展了一个 诊断平台,以研究结构已知的mHTTex 1物种进入人类神经元及其 聚集成神经毒性集合体。有了这个模型,我们计划识别和表征 mHTTex 1的神经侵袭性/神经毒性物种,绘制其致病构象并确定其 生物物理学方法,包括Cryo-EM。此外,我们计划发现神经元受体, 其参与mHTTex 1的进入并鉴定相互作用的蛋白质,所述相互作用的蛋白质被掺入到 神经毒性聚集体。此外,我们计划检查是否存在神经侵袭性mHTT物种, HD患者的大脑以及HD的人类神经元和小鼠模型中,以验证生理相关性 以及与疾病严重程度的任何联系。这些实验可能首次 以高分辨率鉴定神经毒性mHTTex 1的结构,这是一种新的产生途径, 为制定治疗方案提供目标(目标2)。在目标3中,我们将研究细胞外mHTT在 HD小鼠CNS中的疾病。在一组实验中,我们计划注射神经侵入性物种, mHTTex 1进入无症状R6/2 Q51 HD小鼠(表达人mHTTex 1和51 Qs)的脑中, 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,我们将确定是否通过AAV阻断mHTTex 1的任何致病性构象。 在CNS中分泌的重组抗体的介导递送抑制了细胞的进入、扩增、 和神经毒性。最后,鉴于mHTT存在于HD的CSF和血浆中, 患者和小鼠模型,我们计划研究分泌的重组抗体的治疗作用 对Q140 HD中致病性mHTT组件的积累和HD样病理学的进展的影响 表达全长mHTT的小鼠。这些研究将填补一些知识空白的作用 细胞外mHTT在HD发病机制中的作用,并可提供治疗靶点和试剂。

项目成果

期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Amplification of neurotoxic HTTex1 assemblies in human neurons.
  • DOI:
    10.1016/j.nbd.2021.105517
  • 发表时间:
    2021-11
  • 期刊:
  • 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:
  • 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
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Ali Khoshnan其他文献

Ali Khoshnan的其他文献

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{{ truncateString('Ali Khoshnan', 18)}}的其他基金

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:
    10447659
  • 财政年份:
    2021
  • 资助金额:
    $ 62.98万
  • 项目类别:
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
  • 财政年份:
    2021
  • 资助金额:
    $ 62.98万
  • 项目类别:
Validation of IKKbeta as a therapeutic target for HD
验证 IKKbeta 作为 HD 治疗靶点
  • 批准号:
    8697149
  • 财政年份:
    2011
  • 资助金额:
    $ 62.98万
  • 项目类别:

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