Huntingtin proline-rich region modulation of Huntington's disease pathogenesis

亨廷顿蛋白脯氨酸富集区对亨廷顿病发病机制的调节

• 批准号: 8932828
• 负责人: Scott Zeitlin
• 金额: $ 34.56万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-22 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8932828
• 关键词: Affect; Affinity; Age; Age-Months; Alleles; Animal Model; Antibodies; Autophagocytosis; Behavior; Behavioral; Binding; Brain; C-terminal; Cell model; Cell physiology; Corpus striatum structure; Data; Deletion Mutation; Disease model; Evolution; Exhibits; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Targeting; Genes; Health; Human; Huntington Disease; Image; In Vitro; Knock-in Mouse; Learning; Life; Mammals; Mass Spectrum Analysis; Microtubules; Molecular Conformation; Mus; N-terminal; Neurodegenerative Disorders; Neurons; Organelles; Pathogenesis; Pathway interactions; Phenotype; Population; Proline-Rich Domain; Prosencephalon; Proteins; Proteomics; Role; Scaffolding Protein; Stretching; Tertiary Protein Structure; Testing; Time; Toxic effect; Trinucleotide Repeats; Yeast Model System; Yeasts; base; human Huntingtin protein; in vivo; mouse model; mutant; neuron loss; neuropathology; polyglutamine; postnatal; protein aggregate; protein degradation; research study; therapeutic target; transcriptome sequencing

 DESCRIPTION (provided by applicant): In Huntington's disease (HD), flanking protein domains modulate the toxicity of mutant Huntingtin's (HTT''s) expanded polyQ stretch. In mammals, a proline-rich region (PRR) is located at the C-terminal end of the polyQ stretch, and it has co-evolved with the polyQ stretch, increasing in size as the normal HTT polyQ stretch has lengthened during evolution. In yeast model systems, deletion of the HTT PRR in the context of an expanded polyQ stretch interferes with aggresome formation and increases toxicity of an N-terminal fragment of mutant HTT. To determine the role of the HTT PRR in modulating mutant HTT pathogenesis in mouse models for HD, we have generated three knock-in alleles of the mouse HD gene (Hdh) that express normal or mutant huntingtin (htt ) with deletions of the PRR - HdhΔP, Hdh140QΔP, and Hdh3xFlag140QΔP. We have found, in contrast to the results of the yeast studies, that deletion of the mutant htt PRR ameliorates several phenotypes exhibited by the CAG140 knock-in mouse model for HD, resulting in a significant delay in aggregate formation, alterations in aggregate conformation, normalization of striatal Darpp-32 expression, and the rescue of activity deficits. Using these new mouse models, we propose three complementary aims to determine the mechanisms by which deletion of the htt PRR affects HD mouse model pathogenesis. In Aim 1, we will test the hypothesis that deletion of the PRR modulates mutant htt's toxicity by altering the association of htt-interacting proteins. We propose using anti-FLAG immunoaffinity purification to enrich for proteins associating with 3xFlag140QΔP- tt and 3xFlag140Q-htt in the striatum and cortex, and characterize their identity using mass spectrometry. In addition, we will characterize the protein composition of htt aggregates purified from the Hdh140Q/+ and Hdh140QΔP/+ brain to determine if differential sequestration of cellular proteins by 140Q-htt and 140QΔP-htt aggregates contributes to pathogenesis. In Aim 2, we will test the hypothesis that deletion of the mutant htt PRR alleviates mutant htt's perturbation of gene expression by performing RNA-seq analysis of wild-type, Hdh140Q/+, and Hdh140QΔP/+ striatal gene expression to identify those genes whose expression is altered in the Hdh140Q/+ brain but restored by the mutant htt PRR deletion in the Hdh140QΔP/+ brain. In Aim 3, we will genetically test the role of the htt PRR on aggresome formation (in cis or in trans), and the potential difference between the murine and human PRR in HD pathogenesis by characterizing behavior and neuropathology in Hdh140QΔP/ΔP, Hdh140QΔP/+, Hdh140QΔP/7QhuPRR and Hdh140QΔP/20QhuPRR mice as they age. In addition, we will characterize microtubule-based transport and autophagy (two pathways involved in aggresome formation and protein degradation) in early postnatal (P5) primary cortical and striatal neuronal cultures generated from wild-type, HdhΔP/ΔP, Hdh140Q/140Q and Hdh140QΔP/140QΔP mice.

 描述（由适用提供）：在亨廷顿氏病（HD）中，侧翼蛋白质结构域调节了突变亨廷顿蛋白（HTT）扩展的Polyq strave的毒性。在哺乳动物中，富含脯氨酸的区域（PRR）位于Polyq伸展的C末端，并且与Polyq strave共同进化，随着正常的HTT Polyq伸展运动在进化过程中的延长，大小增加。在酵母模型系统中，在扩展的polyq伸展干扰中删除HTT PRR，并增加了多数的形成，并增加了突变体HTT的N末端片段的毒性。为了确定HTT PRR在HTT的HTT发病机理中的HTT PRR在HD模型中的作用，我们生成了三个敲入小鼠HD基因（HDH）的敲入等位基因，该等位基因表达了正常或突变的狩猎蛋白或突变的htttin（HTT），并用PRR -HDHΔP，HDHΔP，HDH2H140QΔP和HDH3XFLAG的作用。我们发现，与酵母研究的结果相反，缺失突变的HTT PRR可以改善HD的CAG140敲入小鼠模型暴露于HD的几种表型，从而显着延迟了聚集的形成，聚集会议的变化，骨料darpp-32表达的正常化的骨料会议的正常化和活动防御能力的促进。使用这些新的小鼠模型，我们提出了三个完整的目的，以确定HTT PRR缺失影响HD小鼠模型发病机理的机制。在AIM 1中，我们将检验以下假设：删除PRR通过改变HTT相互作用蛋白的关联来调节突变体HTT的毒性。我们建议 使用抗FLAG免疫亲和力纯化，以富集与纹状体和皮质中3xflag140QΔP-TT和3XFLAG140Q-HTT相关的蛋白质，并使用质谱法表征它们的身份。此外，我们将表征从HDH140Q/+和HDH140QΔP/+脑纯化的HTT聚集体的蛋白质组成，以确定140Q-HTT和140QΔP-HTT聚集物是否对细胞蛋白的差异隔离是否有助于病虫气。在目标2中，我们将检验以下假说：缺失HTT PRR的缺失通过对野生型，HDH140Q/+的RNA-SEQ分析，HDH140Q/+，HDH140QΔP/+纹状体基因表达来鉴定其表达的bri​​nts in+ brin in+ brint in+ brints in+ brints intery in ht tripty inter htt in htt rna-ndt the htt inter htt rne htt rna htt prr的htt prr htt htt htt扰动基因表达的扰动。 HDH140QΔP/+脑中的PRR缺失。在AIM 3中，我们将普遍测试HTT PRR在骨料形成（在顺式或trans中）中的作用，以及通过表征HDH140QΔP/ΔP中的行为和神经病理学来表征HDH140QΔP/HDH140QΔP/HDH140QΔP/7QHUPR和HDH140QΔP/δP中的行为和神经病理，通过表征鼠和人PRR之间的潜在差异。 HDH140QΔP/20QHUPRR小鼠随着年龄的增长。此外，我们还将表征基于微管的运输和自噬（在产后早期（P5）原发性皮质和纹状体神经元培养物中，由HDHΔP/δP/δP，HDH140Q/140Q/140Q和HDH140QΔPE产生的初级皮层和纹状体神经元培养物中的基于微管和自噬（涉及两种途径）。