Increase Rab11 Activity as HD Therapy

描述（由申请人提供）：本申请是对一项申办项目公告（NIH/CHDI）的回应，旨在验证亨廷顿病（HD）的潜在治疗靶点。寻找HD治疗方法的一个主要目标是识别导致神经元功能障碍的早期事件。我们已经确定了一个早期的脑功能障碍的运输货物从回收内体回到质膜在HD神经元。这种缺陷是由于突变体htt干扰Rab 11上的核苷酸交换，Rab 11是一种小的GTT，其功能是回收内体。我们确定了在症状前HD敲除小鼠脑中Rab 11 GEF活性受损。神经元中膜运输的缺陷导致细胞死亡。Rab 11活性是细胞稳态和存活所必需的。许多参与细胞内稳态的货物使用Rab 11依赖性途径再循环。这些货物可以是细胞特异性的。到目前为止，我们发现，三个重要的Rab 11依赖货物的回收在表达内源性全长突变htt的HD神经元中减慢。HD神经元中受影响的一种神经元特异性货物是半胱氨酸/谷氨酸转运蛋白。在HD神经元中，半胱氨酸的EAAC 1依赖性摄取受损，导致谷胱甘肽合成不足、ROS升高和细胞死亡。在HD初级皮质神经元中显性活性Rab 11的慢病毒递送挽救了这些缺陷。我们的体外数据为显性活性Rab 11的营养作用提供了令人信服的证据，并指出Rab 11是一个需要体内验证的极好靶点。我们将在两种表达全长突变htt的HD小鼠模型--HD 140 Q/140 Q敲入和HD YAC 128转基因小鼠模型中验证Rab 11活性作为新的治疗靶点。我们已经产生了组成型表达显性活性Rab 11的转基因小鼠（TgRab 11小鼠）。9周时的Tg小鼠与同窝对照小鼠的行为无差异，将使用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TgRab 11小鼠杂交。由于诱导型表达模拟了HD患者的治疗条件，我们还将HD小鼠与两个转基因系杂交，这两个转基因系共同整合了Cre-loxP系统的元件，并将允许dARab 11在症状前和症状性HD小鼠中严格调控诱导型表达。目的2将是确定Rab 11活性增加是否能挽救HD表型。我们将评估体内运动行为、生化和解剖学读数以及体外内体再循环和神经元存活。这些研究将证明，Rab 11活性水平的提高可增加神经元存活，改善或延迟HD中的运动缺陷和神经病理学。增加Rab 11活性的体内验证将证明药物发现工作的合理性，以筛选提高Rab 11活性的小分子作为HD的治疗方法。 公共卫生相关性：亨廷顿蛋白的突变导致亨廷顿病（HD）。我们已经证明，突变亨廷顿干扰了一个小的Rab GTdR的功能，称为Rab 11，这是所需的回收内体的正常功能。这种功能障碍导致神经元内稳态的紊乱，从而引起神经变性。我们建议验证Rab 11作为HD的治疗靶点。我们将在遗传学上将显性活性Rab 11引入两种HD小鼠模型，并表明Rab 11活性的增加是有益的，可以挽救这些小鼠的神经变性和运动功能障碍。

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. PUBLIC HEALTH RELEVANCE: A mutation in huntingtin causes Huntington's disease (HD). We have shown that mutant huntingtin interferes with the function of a small Rab GTPase called Rab11, which is required for the normal function of recycling endosomes. This dysfunction leads to disturbances in neuronal homeostasis that cause neurodegeneration. We propose to validate Rab11 as a therapeutic target for HD. We will genetically introduce a dominant active Rab11 into two mouse models of HD and show that an increase in the activity of Rab11 is beneficial and can rescue neurodegeneration and motor dysfunction in these mice.