Dynactin function in axons, synapses, and neurodegenerative disease

描述(申请人提供)：轴突运输中断被认为发生在许多神经疾病的早期，包括周围神经疾病和神经退行性疾病。运输缺陷导致疾病的机制包括细胞器向远端轴突的传递障碍，逆行神经营养信号的缺陷，以及突触终末内突触小泡周期的中断。然而，轴突运输的改变导致疾病的方式尚不清楚。简单的模式生物，如果蝇，黑腹果蝇，允许遗传操作与轴突运输和突触生理学的分析相结合，它们正在为周围神经和神经退行性疾病的病理生理学提供见解。这项提议的长期目标是了解神经疾病中细胞内转运事件是如何改变的。 以确定治疗发展的新靶点。逆行轴突运输是由动力蛋白/动力蛋白复合体介导的。P150Glued dynactin亚单位在两种不同的、不重叠的常染色体显性神经退行性疾病中发生突变：一种是导致一种名为遗传性运动神经病7B型(HMN7B)的运动神经元疾病，另一种是佩里综合征，特征是帕金森症、抑郁症、换气不足和体重减轻。HMN7B和Perry突变在p150Glued CAP-Gly微管(MT)结合区内相距近12个残基。我们的初步结果表明，这些突变不同地影响了P150与MTS的相互作用和突触上的P150功能。此外，我们发现HMN7B突变破坏了突触最远端(称为末端突触)MT介导的逆行运输的启动，并阻止了神经肌肉接头(NMJ)的神经递质释放。在目标1中，我们将确定HMN7B和Perry综合征突变是否扰乱了与P150结合伙伴的不同蛋白质相互作用，以及它们是否导致轴突运输或逆行信号转导的缺陷。在目标2中，我们将研究NMJ TBS逆行转运的启动，以确定动态MT+末端通过P150与末端结合蛋白-1(EB1)之间的相互作用捕获囊泡时是否发生起始作用。EB1是“MT+末端的主要调节因子”。我们还将检验P150上的HMN7B而不是Perry突变破坏TBS逆行启动的假设，并确定这种缺陷是否源于TBS微管动力学的改变。在Aim3中，我们将调查 P150的突变如何通过结合钙和FM1-43成像、超微结构分析和电生理学来改变突触传递。总之，这些研究将有助于阐明P150 CAP-Gly结构域在神经元中的功能，并有望揭示神经退行性疾病的细胞类型特异性机制。

DESCRIPTION (provided by applicant): Disruption of axonal transport is thought to occur early in the course of many neurological diseases including peripheral neuropathies and neurodegenerative diseases. Proposed mechanisms whereby transport defects lead to disease include impairment of organelle delivery to distal axons, defective retrograde neurotrophic signaling, and disruption of the synaptic vesicle cycle within the synaptic terminal. However, the way alterations in axonal transport cause disease is unclear. Simple model organisms such as the fruitfly, Drosophila melanogaster, allow genetic manipulations to be combined with analysis of axonal transport and synaptic physiology, and they are providing insights into the pathophysiology of peripheral nerve and neurodegenerative diseases. The long-term goal of this proposal is to understand how intracellular trafficking events are altered in neurological diseases in order to identify novel targets for therapeutic development. Retrograde axonal transport is mediated by the dynein/dynactin protein complex. The p150Glued dynactin subunit is mutated in two distinct, non-overlapping autosomal dominant neurodegenerative diseases: one that causes a motor neuron disease called Hereditary Motor Neuropathy type 7B (HMN7B), and the other that is called Perry Syndrome, characterized by parkinsonism, depression, hypoventilation, and weight loss. The HMN7B and Perry mutations are as close as 12 residues apart within the p150Glued CAP-Gly microtubule (MT)-binding domain. Our Preliminary Results suggest that these mutations differentially affect p150 interactions with MTs and p150 function at synapses. Furthermore, we find that HMN7B mutations disrupt the initiation of retrograde MT-mediated transport at the distal-most end of synapses (called terminal boutons) and block neurotransmitter release at the neuromuscular junction (NMJ). In Aim 1, we will determine if HMN7B and Perry Syndrome mutations disrupt distinct protein interactions with p150 binding partners and also whether they cause defects in axonal transport or retrograde signaling. In Aim 2, we will study the initiation of retrograde transport at NMJ TBs to determine if initiation occur when dynamic MT plus-ends capture vesicles through interactions between p150 and end binding protein-1 (EB1), a "master regulator of MT plus-ends". We will also test the hypothesis that HMN7B but not Perry mutations in p150 disrupt retrograde initiation at TBs, and determine if this defect is due to an alteration in microtubule dynamics at TBs. In Aim3, we will investigate how mutations in p150 alter synaptic transmission by combining calcium and FM1-43 imaging, ultrastructural analysis and electrophysiology. Together these studies will help elucidate the function of the p150 CAP-Gly domain in neurons, and hold promise for shedding light on the mechanisms of cell-type specificity of neurodegenerative disease.