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

描述(由申请人提供)：HD最有效的治疗靶点仍然是Htt本身。已发现的扩张型Htt的PTM(如S13/16和S421)是HD发病的重要调节因子。我们以前研究了Htt的蛋白水解性切割(Ratovitski等人，2007、2009、2011)，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(在重要性部分中描述)，HTT很可能还有许多其他的PTM部位。我们计划对Htt PTMS进行系统和定量的表征。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，以及从质谱学开始的分阶段计划，通过体外发现和进展，然后是体内确认和功能验证。如果相关的激酶能够被识别和抑制，增强毒性的磷酸化将特别有希望成为治疗的靶点。在目标1中，我们将在Htt-N586-82Q小鼠、HD“敲入”小鼠和人类HD iPS细胞中定义Htt PTM，并将确定Htt中的多聚Q扩增是否导致PTM的变化。在目标2中，我们将对Htt PTMS对突变的Htt构象和细胞毒性的影响进行体外功能研究。在目标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的病毒表达载体立体定向注射到野生型小鼠的纹状体内，以测试PTMS对突变型Htt毒性的影响。这些研究将在突变型Htt中确定PTM的新位点，并从功能上验证它们在体外和体内的发病机制中的作用。然后，这些部位将成为治疗开发的候选目标。

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.