Increase Rab11 Activity as HD Therapy

描述(由申请者提供)：本申请响应一项赞助项目公告(NIH/CHDI)，以验证亨廷顿病(HD)的潜在治疗靶点。寻找治疗(HD)的一个主要目标是确定导致神经元功能障碍的早期事件。我们发现，在HD神经元中，早期的大脑功能障碍是从回收内小体到质膜的货物运输过程中发生的。这种缺陷是由于突变的HTT干扰了Rab11上的核苷酸交换，Rab11是一种小的GTP酶，功能是回收内小体。我们发现在有症状的HD敲击小鼠的脑组织中，Rab11的全球环境基金活性受损。神经细胞膜运输的缺陷会导致细胞死亡。Rab11活性是细胞内稳态和生存所必需的。许多货物通过Rab11依赖的途径参与细胞内稳态循环。这些货物可以是特定于细胞的。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，三个重要的Rab11依赖的货物的回收速度放缓。HD神经元中受影响的一个神经元特异物是半胱氨酸/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，细胞死亡。慢病毒在HD初级皮质神经元中传递优势活性Rab11可以挽救这些缺陷。我们的体外数据为显性活性Rab11的营养作用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靶点。我们将在两个表达全长突变HTT的HD小鼠模型--HD140Q/140Q敲入和HD YAC 128转基因--中验证Rab11作为新的治疗靶点的作用。我们已经培育出了结构性表达显性活性Rab11的转基因小鼠(TgRab11小鼠)。出生9周的转基因小鼠在行为上与产仔对照组没有差异，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小鼠进行杂交。由于可诱导表达模拟了HD患者的治疗条件，我们还将使HD小鼠与两个转基因品系杂交，这两个转基因品系共同整合了Cre-loxP系统的元素，并将允许在症状前和有症状的HD小鼠中严格调控dARab11的诱导表达。目标2将确定增加的Rab11活性是否拯救HD表型。我们将评估体内的运动行为、生化和解剖学读数，以及体外内小体循环和神经元存活。这些研究将证明，提高Rab11活性水平可以增加神经元存活率，改善或延缓HD患者的运动障碍和神经病理。在体内验证增加的Rab11活性将证明药物发现工作是合理的，以筛选提高Rab11活性的小分子作为HD的治疗方法。 与公共卫生相关：亨廷顿蛋白的突变导致亨廷顿病(HD)。我们已经证明了突变的Huntingtin干扰了一个名为Rab11的小Rab GTP酶的功能，这是循环内小体的正常功能所必需的。这种功能障碍会导致神经元动态平衡失调，从而导致神经退化。我们建议确认Rab11作为HD的治疗靶点。我们将从基因上将显性活性Rab11引入两种HD小鼠模型中，并表明Rab11活性的增加是有益的，可以挽救这些小鼠的神经退行性变和运动功能障碍。

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.