Increase Rab11 Activity as HD Therapy

描述（由申请人提供）：本申请响应赞助项目公告（NIH/CHDI），以验证亨廷顿舞蹈病（HD）的潜在治疗靶点。寻找治疗HD的主要目标是确定引起神经元功能障碍的早期事件。我们已经在HD神经元中发现了一种早期的脑功能障碍，即从回收核内体到质膜的货物运输。这种缺陷是由于突变体htt干扰了Rab11上的核苷酸交换，Rab11是一种小的GTPase，其功能是回收内体。我们发现症状前HD敲敲小鼠脑内Rab11 GEF活性受损。神经元细胞膜运输缺陷导致细胞死亡。Rab11活性是细胞稳态和存活所必需的。许多货物参与细胞内稳态循环使用Rab11依赖途径。这些货物可以是细胞特异性的。到目前为止，我们发现在表达内源性全长突变体htt的HD神经元中，三种重要的Rab11依赖性货物的再循环减慢。在HD神经元中受影响的一个神经元特异性货物是半胱氨酸/谷氨酸转运体。在HD神经元中，依赖EAAC1的半胱氨酸摄取受损，导致谷胱甘肽合成不足、ROS升高和细胞死亡。慢病毒在HD原代皮质神经元中传递显性活性Rab11，可修复这些缺陷。我们的体外数据为优势活性Rab11的营养效应提供了令人信服的证据，并指出Rab11是一个需要在体内验证的极好的靶点。我们将在两种表达全长突变体htt的HD小鼠模型中验证Rab11活性作为新的治疗靶点——HD140Q/140Q敲入和HD YAC 128转基因。我们培育了组成性表达显性活性Rab11的转基因小鼠（TgRab11小鼠）。9周的Tg小鼠在行为上与同窝对照组没有差异，将通过体内和体外研究进一步研究以排除副作用。在目标1中，我们将HD小鼠模型与TgRab11小鼠交叉。由于诱导表达模拟了HD患者的治疗条件，我们还将用两种转基因系杂交HD小鼠，这两种转基因系结合了Cre-loxP系统的元件，并将允许在症状前和症状性HD小鼠中严格调节dARab11的诱导表达。目的2将确定Rab11活性的增加是否能挽救HD表型。我们将评估体内运动行为、生化和解剖读数以及体外核内体循环和神经元存活。这些研究将证明Rab11活性水平的升高可增加HD患者的神经元存活，改善或延缓运动缺陷和神经病理。提高Rab11活性的体内验证将证明药物发现的努力是正确的，以筛选提高Rab11活性的小分子作为HD的治疗方法。

DESCRIPTION (provided by applicant): This application responds to a sponsored program announcement (NIH/CHDI) to validate potential therapeutic targets in Huntington's disease (HD). A major goal in finding treatments for (HD) is to identify early events causing neuronal dysfunction. We have identified an early brain dysfunction in the trafficking of cargoes from recycling endosomes back to the plasma membrane in HD neurons. This deficit is due to interference by mutant htt with nucleotide exchange on Rab11, a small GTPase which functions at recycling endosomes. We identify the impaired Rab11 GEF activity in brain of pre-symptomatic HD knock in mice. Defects in membrane trafficking in neurons cause cell death. Rab11 activity is required for cellular homeostasis and survival. Numerous cargoes involved in cellular homeostasis recycle using a Rab11 dependent pathway. These cargoes can be cell specific. So far we found that the recycling of three important Rab11 dependent cargoes is slowed in HD neurons expressing endogenous full-length mutant htt. One neuron specific cargo affected in HD neurons is the cysteine/glutamate transporter. In HD neurons, EAAC1 dependent uptake of cysteine is impaired leading to insufficient glutathione synthesis, elevated ROS and cell death. Lentivirus delivery of dominant active Rab11 in HD primary cortical neurons rescues these deficits. Our in vitro data provide compelling evidence for the trophic effect of dominant active Rab11 and point to Rab11 as a superb target needing in vivo validation. We will validate Rab11 activity as a novel therapeutic target in two HD mouse models that express full length mutant htt-- HD140Q/140Q knock-in and HD YAC 128 transgenic. We have generated transgenic mice that constitutively express dominant active Rab11 (TgRab11 mice). The Tg mice at 9 weeks show no difference in behavior to littermate controls and will be further studied using in vivo and in vitro studies to rule out side effects. In Aim 1, we will cross the HD mouse models with the TgRab11 mice. Since inducible expression simulates the condition for treatment of patients with HD, we will also cross HD mice with two transgenic lines that together incorporate the elements for a Cre-loxP system and will allow for tightly regulated inducible expression of dARab11 in presymptomatic and symptomatic HD mice. Aim 2 will be to determine if increased Rab11 activity rescues HD phenotypes. We will evaluate in vivo motor behavior and biochemical and anatomical readouts and in vitro endosome recycling and neuronal survival. These studies will demonstrate that raising levels of Rab11 activity increases neuronal survival and ameliorates or delays motor deficits and neuropathology in HD. In vivo validation of increasing Rab11 activity will justify a drug discovery effort to screen for small molecules that raise the activity of Rab11 as a therapy for HD.