Dissecting the in vivo role of Huntingtin in Rab vesicle movement on microtubules

描述（由申请人提供）：亨廷顿舞蹈病（HD）是一种毁灭性的、显性遗传的神经退行性疾病，临床特征为舞蹈病和因纹状体神经元丧失而引起的认知障碍。目前还没有有效的治疗/治愈HD。目前使用的大多数治疗方法旨在溶解/解离聚集体和防止细胞死亡，这是疾病末期常见的神经病理学。虽然HD蛋白，亨廷顿蛋白（HTT）对生存能力至关重要，但HTT介导的关联的复杂性表明了多种功能。因此，挑战在于揭示HTT的主要功能，当它被破坏时就会引发疾病。先前的工作提出了一个诱人的建议，即在长而窄的轴突内破坏轴突运输是一个早期事件，导致蛋白质积聚，引发细胞死亡，最终导致在HD中观察到的神经元功能障碍。我们的长期目标是了解htt介导的轴突运输缺陷如何启动疾病途径。这里的目标是确定HTT如何影响特定囊泡亚类（Rab蛋白）的运输，这是我们追求这一目标的下一步。我们的中心假设是，HTT介导的轴突内兔囊泡运输的中断可能有助于HD观察到的早期神经病理。这个假设有两个明确的预测；1: HTT和Rab蛋白在相同的囊泡上，2:Rab囊泡使用动力蛋白-1和动力蛋白马达在微管（MT）上运动。在这种情况下，我们的具体目标是确定HTT如何影响轴突内mt依赖性运输的Rab蛋白。我们有5个特定的目标，1：确定HTT如何影响rabb蛋白，2：测试HTT和Rab11、Rab32和RabX4在同一个囊泡上的预测，3：测试Rab32和RabX4都在Rab11囊泡上的预测，4：测试Rab32和RabX4使用kineins -1和dynein马达在MT上运动的预测，5：测试突变HTT破坏rabb介导的功能的预测。一个全面的在体内的方法将被用来解剖HTT的生理作用在一个有机体的拉布囊泡运输。这项研究的基本原理是，一旦了解突变HTT对Rab转运的扰动如何引发HD疾病的机制，就可以开发针对HD的新的创新方法。因此，确定HTT在神经元中的正常功能将对开发有效的预防和治疗干预措施提供新的靶标途径产生重大影响，这是目前HD无法获得的。因此，在我们看来，我们的工作是创新的，因为它代表了一种新的和实质性的偏离现状，即使用活生物体中囊泡运动的体内动力学来详细描述HTT的作用的方法。拟议的研究意义重大，因为它有望垂直推进和扩展我们对疾病途径如何启动的理解，这将显著改变当前的知识。所获得的知识将极大地推动许多针对轴突缺陷或恢复Rab功能的药理学或遗传修饰剂的发展。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a devastating, dominantly inherited neurodegenerative disease clinically characterized by chorea and cognitive impairment due to loss of striatal neurons. Currently there are no effective treatments/cures for HD. Most therapeutic treatments currently used are aimed at dissolving/dissociating aggregates and preventing cell death, common neuropathology seen at the end stage of disease. Although the HD protein, huntingtin (HTT) is critical for viability, the complexity of HTT-mediated associations indicates multiple functions. Thus the challenge is to unravel the primary function of HTT, which when disrupted initiates disease. Previous work put forth a tantalizing proposal that disruption of axonal transport within long, narrow-caliber axons is an early event that causes protein accumulations that elicit cell death, ultimately resulting in neuronal dysfunction observed in HD. Our long-term goal is to understand how HTT-mediated axonal transport defects initiate disease pathways. The objective here, which is our next step in the pursuit of thi goal, is to determine how HTT influences the transport of a specific sub class of vesicles (Rab proteins). Our central hypothesis is that disruption of Rab vesicle transport within axons mediated by HTT can contribute to early neuropathology observed in HD. There are two clear predictions of this hypothesis; 1: HTT and Rab proteins are on the same vesicles and 2: Rab vesicles use kinesin-1 and dynein motors for movement on microtubules (MT). In this context our specific aim is to identify how HTT influences Rab proteins for MT-dependent transport within axons. We have 5 specific objectives, 1: determine how HTT influences Rab proteins, 2: test the prediction that HTT and Rab11, Rab32 and RabX4 are on the same vesicle, 3: test the prediction that Rab32 and RabX4 are both on the Rab11 vesicle, 4: test the prediction that Rab32 and RabX4 use kinesin-1 and dynein motors for movement on MT, and 5: test the prediction that mutant HTT disrupts Rab-mediated functions. A comprehensive in vivo approach will be used to dissect the physiological role of HTT in Rab vesicle transport in an organism. The rationale for the proposed research is that once the mechanisms of how HD disease is initiated by perturbations in Rab transport by mutant HTT are known, new and innovative approaches against HD can be developed. Therefore identifying how HTT normally functions in neurons will have a significant impact on providing novel target pathways for developing effective preventive and therapeutic interventions, which are currently unavailable for HD. Thus our work is innovative, in our opinion because it represents a new and substantive departure from the status quo, namely the approach of detailing the role of HTT using in vivo dynamics of vesicle movement in a living organism. The proposed research is significant, because it is expected to vertically advance and expand our understanding of how disease pathways initiate, which will significantly alter current knowledge. The knowledge acquired will dramatically propel the development of numerous pharmacological or genetic modifiers against axonal defects or to restore Rab function.