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

亨廷顿病(HD)是一种临床上以遗传为主的破坏性神经退行性疾病。 以舞蹈症和纹状体神经元丧失所致的认知障碍为特征。目前没有 HD的有效治疗/治疗。目前使用的大多数治疗方法都是针对 溶解/解离聚集体和防止细胞死亡，终末期常见的神经病理 疾病的威胁。尽管HD蛋白Huntingtin(HTT)对生存至关重要，但HTT介导的复杂性 关联表示多种功能。因此，挑战是解开HTT的主要功能，它 当受到干扰时，就会引发疾病。先前的工作提出了一个诱人的建议，即轴突的破坏 长而窄口径轴突内的运输是导致蛋白质积累的早期事件，从而引发细胞 死亡，最终导致HD患者的神经元功能障碍。我们的长期目标是了解 HTT介导的轴突运输缺陷启动了疾病通路。这里的目标是，这是我们的下一步 在追求这一目标的过程中，确定HTT如何影响特定亚类囊泡的运输 (RAB蛋白)。我们的中心假设是HTT介导的RAB囊泡在轴突内运输的中断 有助于观察到HD的早期神经病理改变。对这一假设有两个明确的预测：1： HTT和Rab蛋白位于相同的囊泡上，2：Rab囊泡使用kinesin-1和dynein马达 微管运动(MT)。在这种情况下，我们的具体目标是确定HTT如何影响Rab蛋白 用于轴突内的MT依赖的运输。我们有5个具体目标，1：确定HTT如何影响RAB 蛋白质，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：检验Rab32和RabX4的预测 在MT上使用kinesin-1和dynein马达进行运动，以及5：测试突变HTT破坏的预测 RAB介导的功能。一种全面的活体方法将被用来剖析 HTT在Rab中的囊泡在生物体中的运输。提出这项研究的理由是，一旦 突变HTT在RAB运输中的扰动如何引发HD疾病的机制是已知的，新的 还可以开发针对HD的创新方法。因此，确定HTT如何在 神经元将在为开发有效的预防措施提供新的靶向途径方面发挥重要作用 和治疗性干预，目前尚不能用于HD。因此，我们的工作是创新的，在我们的 意见，因为它代表了对现状的新的实质性偏离，即 使用活体生物体内囊泡运动的动力学来详细说明HTT的作用。建议数 研究意义重大，因为它有望垂直推进并扩大我们对 疾病途径启动，这将极大地改变目前的知识。所获得的知识将 极大地推动了许多针对轴突缺陷的药物或遗传修饰剂的开发 或恢复RAB功能(IMPACT)。

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 initiates disease pathways. The objective here, which is our next step in the pursuit of this 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 (impact).