Validation of Novel Pathogenic Htt Post-Translational Modifications (PTMs)

描述（由申请人提供）：HD中最有效的治疗靶点仍然是Htt本身。先前鉴定的扩增Htt的PTM（例如S13/16和S421）是HD发病机制的重要调节剂。我们先前研究了Htt的蛋白水解裂解（Ratovitski等人，2007，2009，2011），并且最近一直在研究Htt的共价PTM，特别是磷酸化。除了目前已知的PTM位点（在意义部分中描述）之外，Htt很可能还有许多其他的PTM位点。我们计划系统地和定量地表征Htt PTM。此外，我们的实验包括使用人HD iPS细胞进行我们的持续发现研究，以及从质谱法开始进行发现并通过体外然后体内确认和功能验证进行的阶段性计划。如果相关的激酶能够被鉴定和抑制，那么增强毒性的磷酸化将特别有希望作为治疗靶点。在目标1中，我们将使用Htt-N586- 82 Q小鼠、HD“敲入”小鼠和人HD iPS细胞来定义Htt PTM，并将确定Htt中的polyQ扩增是否导致PTM的变化。在目标2中，我们将进行体外功能研究的影响，Htt PTMs的突变体Htt构象和细胞毒性。在目标3中，我们将使用我们的N-586- 82 Q转基因小鼠模型或立体定向注射编码具有改变的PTM的Htt的病毒表达载体到野生型小鼠的纹状体中，测试PTM对突变体Htt体内毒性的影响。这些研究将共同确定新的PTM突变体Htt的网站，并在功能上验证其在体外和体内的发病机制中的作用。这些位点将成为治疗开发的候选靶点。

DESCRIPTION (provided by applicant): The best validated therapeutic target in HD remains Htt itself. Previously identified PTMs of expanded Htt (e.g. S13/16 and S421) are important modulators of HD pathogenesis. We previously studied proteolytic cleavage of Htt (Ratovitski et al., 2007, 2009, 2011), and more recently have been studying covalent PTMs of Htt, especially phosphorylation. Htt is very likely to have many other sites of PTM besides the currently known ones (described in the Significance section). We plan to characterize Htt PTMs systematically and quantitatively. Furthermore, our experiments include the use of human HD iPS cells for our continuing discovery studies, and a staged program beginning with mass spectrometry for discovery and progressing through in vitro and then in vivo confirmation and functional validation. Phosphorylation which enhances toxicity will be especially promising as a therapeutic target, if relevant kinases can be identified and inhibited. In Aim 1, we will define Htt PTMs usin Htt-N586-82Q mice, HD "knock-in" mice and human HD iPS cells, and will determine whether the polyQ expansion in Htt leads to changes in PTMs. In Aim 2, we will conduct in vitro functional studies of the effects of Htt PTMs on mutant Htt conformation and cellular toxicity. In Aim 3, we will test the effects of PTMs on mutant Htt toxicity in vivo, using our N-586-82Q transgenic mouse model or stereotactic injection of viral expression vectors encoding Htt with altered PTMs into the striatum of wild-type mice. These studies taken together will identify novel sites of PTM in mutant Htt, and functionally validate their role in pathogenesis in vitro and in vivo. The sites will then be candidate targets for therapeutic development.