Validation of Novel Pathogenic Post-Translational Modifications of Huntingtin, and of Modifying Enzymes as Therapeutic Targets for Huntington's Disease

亨廷顿蛋白的新型致病性翻译后修饰的验证， 以及修饰酶作为亨廷顿病治疗靶点的研究进展 HD是一种进行性神经退行性疾病，由单一基因突变（CAG重复）引起 Huntingtin（Htt）的扩张。HD中最有效的治疗靶点仍然是Htt本身，而RNAi 降低HD患者中突变Htt的方法正在开发中。然而，这些方法 向中枢神经系统输送大分子的巨大障碍。小分子治疗仍然是一个重要的 替代.我们已经鉴定了近40个人内源性Htt的PTM（磷酸化和乙酰化）， 和小鼠大脑的蛋白质组学研究。我们发现PTM是成簇排列的，我们有 这些集群中某些站点之间串扰的初步证据。此外，我们发现， 几种PTM的残基消除了突变的Htt神经元毒性，表明PTM是Htt神经元毒性的调节剂。 突变体Htt毒性。这些数据表明，催化这些PTM的酶的鉴定将具有 为HD的疾病改善治疗产生明确分子靶点的潜力。我们现在提出下一个 这些研究中的一系列步骤，以确定其他PTM，并确定催化 修饰，作为HD合理治疗的潜在确定的分子靶点。我们将利用 现在我们新开发的HD iPSC衍生的永生化纹状体前体细胞系。在目标1中，我们将确定 使用我们的衍生自HD iPS细胞的新型纹状体前体细胞系（SPN）对Htt的新型PTM。这些可以 分化成具有中等多刺神经元表型的神经元。我们将产生PTM特异性抗体 高优先级PTM，以便使用免疫荧光评价细胞区室中的定位，以及 在SPN和人脑中进行细胞分级分离，然后进行蛋白质印迹。在目标2中，我们将确定激酶和其他 修饰酶在目标3中，我们将证实PTM和激酶在HD细胞发病机制中的作用及其在HD细胞中的作用。 作为治疗目标。我们将进行CRISPR/Cas9 PTM改变，以获得高排名的功能性基因。 PTMs，以及CRISPR/Cas9敲除纹状体前体神经元模型中最相关的激酶， 与Gene Edit BioLab和Xiao和Shihua Li合作，他们是基因编辑方面的专家， HD.综合起来，这些研究将进一步阐明突变体Htt的基础生物学。的识别 修饰酶具有为临床前治疗研究提供确定的分子靶标的潜力。

Validation of Novel Pathogenic Post-Translational Modifications of Huntingtin, and of Modifying Enzymes as Therapeutic Targets for Huntington's Disease HD is a progressive neurodegenerative disorder caused by a single gene mutation, the CAG repeat expansion in Huntingtin (Htt). The best-validated therapeutic target in HD remains Htt itself, and RNAi approaches are under development to lower mutant Htt in HD patients. However, these approaches have daunting obstacles of delivery large molecules to the CNS. Small molecule therapeutics remains an important alternative. We have identified near 40 PTMs (phosphorylation and acetylation) on endogenous Htt from human and mouse brain using quantitative proteomics. We find that the PTMs are arranged in clusters, and we have initial evidence for crosstalk among certain sites in these clusters.. Furthermore, we find that alteration of the residues for several of the PTMs abrogates mutant Htt neuronal toxicity, showing that PTMs are modulators of mutant Htt toxicity. These data indicate that identification of the enzymes catalyzing these PTMs will have potential for yielding defined molecular targets for disease-modifying therapy for HD. We now propose the next series of steps in these studies, in order to identify additional PTMs, and to identify enzymes that catalyze modifications, as potential defined molecular targets for rational therapeutics for HD. We will take advantage now of our newly developed HD iPSC-derived immortalized striatal precursor cell lines. In Aim 1, we will identify novel PTMs on Htt using our novel striatal precursor cell lines (SPNs) derived from HD iPS cells. These can be differentiated into neurons with a medium-spiny neuron phenotype. We will generate PTM-specific antibodies to high-priority PTMs, in order to evaluate localization in cellular compartments using immunofluorescence, and cell fractionation followed by Western blot, in SPNs and human brain. In Aim 2 we will identify kinases and other modifying enzymes. In Aim 3 we will confirm the role of PTMs and kinases in HD cellular pathogenesis and their validation as therapeutic targets. We will perform CRISPR/Cas9 PTM alterations for highly-ranked functional PTMs, as well as CRISPR/Cas9 knock-out of most relevant kinases in striatal precursor neuron model in collaboration with Gene Edit BioLab and Xiao and Shihua Li, who are expert at gene editing in relation to HD. Taken together, these studies will further elucidate the basic biology of mutant Htt. The identification of modifying enzymes has the potential to provide defined molecular targets for preclinical therapeutic studies.