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

 描述(申请人提供)：在亨廷顿病(HD)中，侧翼蛋白结构域调节突变的亨廷顿(HTT‘S)扩展多聚Q延伸的毒性。在哺乳动物中，富含脯氨酸的区域(PRR)位于PolyQ伸展的C末端，它与PolyQ伸展一起进化，随着正常HTT PolyQ伸展的延长而增大。在酵母模型系统中，在扩展的多Q拉伸的背景下，HTT PRR的缺失干扰了侵袭体的形成，并增加了突变的HTT的N末端片段的毒性。为了确定HTTPRR在HD小鼠模型中调节突变HTT发病机制中的作用，我们产生了三个敲入的小鼠HD基因(Hdh)的等位基因，它们表达正常或突变的Huntingtin(Htt)，Prr-hdh-hdhΔP，Hdh140QΔP和Hdh3xFlag140QΔP的缺失。我们发现，与酵母研究的结果相反，突变的hTPRR的缺失改善了HD的CAG140敲入小鼠模型所表现的几种表型，导致聚集体形成显著延迟，聚集体构象改变，纹状体DARPP-32表达正常化，以及活性缺陷的挽救。利用这些新的小鼠模型，我们提出了三个互补的目标来确定HTT PRR缺失影响HD小鼠模型发病的机制。在目标1中，我们将检验这一假设，即PRR的缺失通过改变HTT相互作用蛋白的关联来调节突变的HTT的毒性。我们建议 采用抗FLAG免疫亲和纯化的方法，在纹状体和皮质中浓缩与3xFlag140QΔP-TT和3xFlag140Q-hTt相关的蛋白质，并用质谱仪鉴定它们的特性。此外，我们将对从Hdh140Q/+和Hdh140QΔP/+脑中纯化的HTT聚合体的蛋白质组成进行表征，以确定140Q-HTT和140QΔP-HTT聚合体对细胞蛋白的差异隔离是否在发病机制中起作用。在目标2中，我们将通过对野生型、Hdh140Q/+和Hdh140QΔP/+纹状体基因表达的RNA-SEQ分析来验证缺失突变的HTTPRR可以缓解突变的HTT对基因表达的干扰的假设，以确定哪些基因在Hdh140Q/+脑中的表达发生变化，但在Hdh140QΔP/+脑中的表达被突变的HTTPRR缺失恢复。在目标3中，我们将通过对Hdh140QΔP/ΔP、Hdh140QΔP/+、Hdh140QΔP/7QhuPRR和Hdh140QΔP/20QhuPRR小鼠的行为和神经病理特征的研究，从遗传学上检验HTTPRR在侵袭体形成(顺式或反式)中的作用，以及小鼠和人类PRR在HD发病机制中的潜在差异。此外，我们将在出生后早期(P5)的原代皮质和纹状体神经元培养中表征基于微管的转运和自噬(两条途径涉及侵袭体的形成和蛋白质的降解)，这些培养来自野生型、HDHΔP/ΔP、HDH 140Q/140Q和HDH 140QΔP/140QΔP小鼠。

 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.