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

描述（由申请人提供）：HD的最佳治疗靶点仍然是Htt本身。先前发现的扩展Htt的PTMs（例如S13/16和S421）是HD发病机制的重要调节剂。我们之前研究了Htt的蛋白水解裂解（Ratovitski等人，2007,2009,2011），最近研究了Htt的共价PTMs，特别是磷酸化。除了目前已知的PTM位点（在重要性一节中描述）之外，Htt很可能还有许多其他的PTM位点。我们计划系统和定量地表征Htt PTMs。此外，我们的实验包括使用人类HD iPS细胞进行持续的发现研究，以及从质谱法开始的分阶段项目，通过体外，然后在体内确认和功能验证。如果能识别和抑制相关激酶，增强毒性的磷酸化将特别有希望作为治疗靶点。在Aim 1中，我们将使用Htt- n586 - 82q小鼠、HD“敲入”小鼠和人类HD iPS细胞定义Htt PTMs，并确定Htt中polyQ的扩增是否会导致PTMs的变化。在Aim 2中，我们将对Htt PTMs对突变体Htt构象和细胞毒性的影响进行体外功能研究。在Aim 3中，我们将使用我们的N-586-82Q转基因小鼠模型或将编码Htt的带有改变的PTMs的病毒表达载体立体定向注射到野生型小鼠纹状体中，测试PTMs对体内突变Htt毒性的影响。这些研究将共同确定突变Htt中PTM的新位点，并在体外和体内功能上验证其在发病机制中的作用。这些位点将成为治疗发展的候选靶点。

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.