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

 描述（由申请人提供）：在亨廷顿病（HD）中，侧翼蛋白结构域调节突变亨廷顿（HTT）扩增polyQ链段的毒性。在哺乳动物中，富含脯氨酸的区域（PRR）位于polyQ延伸的C末端，并且它与polyQ延伸共同进化，随着正常HTT polyQ延伸在进化过程中延长而增加大小。在酵母模型系统中，在扩增的polyQ延伸的背景下HTT PRR的缺失干扰攻击基因组形成并增加突变HTT的N-末端片段的毒性。为了确定HTT PRR在HD小鼠模型中调节突变型HTT发病机制中的作用，我们产生了表达正常或突变型亨廷顿蛋白（htt）的小鼠HD基因（Hdh）的三种敲入等位基因，缺失PRR - HdhΔP、Hdh 140 Q ΔP和Hdh 3xFlag 140 Q ΔP。我们已经发现，与酵母研究的结果相反，突变体htt PRR的缺失改善了CAG 140敲入HD小鼠模型所表现出的几种表型，导致聚集体形成的显著延迟、聚集体构象的改变、纹状体Darpp-32表达的正常化和活性缺陷的挽救。使用这些新的小鼠模型，我们提出了三个互补的目的，以确定删除的htt PRR影响HD小鼠模型的发病机制。在目标1中，我们将测试的假设，删除PRR调制突变htt的毒性，通过改变关联的htt相互作用蛋白。我们提出 使用抗FLAG免疫亲和纯化富集纹状体和皮质中与3xFlag 140 Q Δ P-tt和3xFlag 140 Q-htt相关的蛋白，并使用质谱法表征它们的身份。此外，我们将表征从Hdh 140 Q/+和Hdh 140 Q ΔP/+脑中纯化的htt聚集体的蛋白组成，以确定140 Q-htt和140 Q ΔP-htt聚集体对细胞蛋白的差异螯合是否有助于发病机制。在目的2中，我们将通过对野生型、Hdh 140 Q/+和Hdh 140 Q ΔP/+纹状体基因表达进行RNA-seq分析来验证突变型htt PRR缺失会干扰突变型htt基因表达的假设，以鉴定在Hdh 140 Q/+脑中表达改变但在Hdh 140 Q ΔP/+脑中通过突变型htt PRR缺失恢复的那些基因。在目标3中，我们将通过表征Hdh 140 Q ΔP/ΔP、Hdh 140 Q ΔP/+、Hdh 140 Q ΔP/7 QhuPRR和Hdh 140 Q ΔP/20 QhuPRR小鼠随年龄增长的行为和神经病理学，从遗传学上测试htt PRR对攻击性基因组形成（顺式或反式）的作用，以及小鼠和人PRR在HD发病机制中的潜在差异。此外，我们还将对野生型、HdhΔP/ΔP、Hdh 140 Q/140 Q和Hdh 140 Q ΔP/140 Q ΔP小鼠的出生后早期（P5）原代皮层和纹状体神经元培养物中基于微管的转运和自噬（参与攻击体形成和蛋白质降解的两种途径）进行表征。

 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.