Characterization of Dopamine Neuron Axonal Mitochondria Specialization and its Relevance to Parkinson’s Disease

项目摘要 帕金森病（Parkinson's disease，PD）是最常见的运动神经退行性疾病， 神经系统疾病的患病率、残疾调整生命年和死亡率增长最快，而且越来越多 重大公共卫生问题。它的核心运动症状是由多巴胺的功能障碍和死亡引起的 (DA)黑质神经元（SNpc）。这些神经元特别脆弱， 了解这种增强的脆弱性的分子基础可能对疾病的发展至关重要- 修改治疗。由于神经退行性变的不可逆性，神经保护性治疗可能 在针对早期更可逆的神经元功能障碍时最有效。在PD，一个这样的早期 病理特征是SNpc DA神经元轴突变性，轴突比胞体早丢失 并且其变性与多巴胺替代疗法功效的下降更密切相关 (the PD的主要对症治疗）。因为轴突损失可能在细胞死亡之前被靶向， 其机制可能是开发神经保护疗法所必需的。 先前的工作表明，轴突线粒体是动态调节和专门支持轴突， 功能和生存。在这个建议中，我试图确定轴突线粒体的机制， 专门促进SNpc DA神经元轴突的功能和存活，并确定如何 这些过程的改变可能导致PD病理生理学。在目标1中，我将开发一部小说 分子标记和体积成像平台，用于表征线粒体的基线特征， SNpc DA神经元的轴突，如它们在不同纹状体亚区的密度，并量化它们的空间分布。 与突触前结构的关系在目标2中，我将实施免疫纯化策略（Mitotag）， 分离SNpc DA神经元轴突和体树突线粒体，进行蛋白质组学研究 和功能测定以鉴定轴突线粒体的分子和功能特化。在目标3中，我 然后将在病毒PD小鼠模型中进行类似的实验，以确定轴突的改变是如何发生的。 线粒体可能导致轴突变性。以这些发现为指导，我将进行 过表达或敲低实验，以评估是否挽救轴突线粒体缺陷， 减轻PD的轴突变性。 通过深入研究线粒体如何专门维持轴突生存的基础生物学 并探讨它们的功能如何在疾病中受到干扰，这一建议将有助于阐明基本原理 神经元的维持和功能，同时可能确定疾病的早期机制， 在不可逆的细胞死亡之前被靶向。

Project Summary Parkinson’s disease (PD) is the most common motor neurodegenerative disease and, as one of the fastest growing neurological disorders in prevalence, disability-adjusted life years, and deaths, an increasingly significant public health issue. Its core motor symptoms are caused by the dysfunction and death of dopamine (DA) neurons in the Substantia Nigra pars compacta (SNpc). These neurons are particularly vulnerable, and understanding the molecular underpinnings of this enhanced vulnerability may be critical for developing disease- modifying treatments. Due to the irreversible nature of neurodegeneration, neuroprotective therapies are likely to be most effective when targeting early, more reversible forms of neuronal dysfunction. In PD, one such early pathological feature is the degeneration of SNpc DA neuron axons, which are lost earlier than the cell bodies and whose degeneration more closely correlates with the decline of efficacy of dopamine replacement therapy (the primary symptomatic treatment for PD). Because axon loss may be targeted prior to cell death, identifying its mechanisms may be essential for developing neuroprotective therapies. Prior work indicates that axonal mitochondria are dynamically regulated and specialized to support axonal function and survival. In this proposal, I seek to identify mechanisms by which axonal mitochondria are specialized to promote the function and survival of SNpc DA neuron axons, and to identify how alterations in these processes may contribute to PD pathophysiology. In Aim 1, I will develop a novel molecular labeling and volumetric imaging platform for characterizing baseline features of mitochondria in the axons of SNpc DA neurons, such as their density across different striatal subregions, and quantify their spatial relationship to presynaptic structures. In Aim 2, I will implement an immunopurification strategy (Mitotag) for separately isolating axonal and somatodendritic mitochondria from SNpc DA neurons, and carry out proteomics and functional assays to identify the molecular and functional specialization of axonal mitochondria. In Aim 3, I will then carry out similar experiments in a viral PD mouse model to identify how alterations to axonal mitochondria may contribute to axonal degeneration. Using these findings as a guide, I will then carry out overexpression or knockdown experiments to evaluate whether rescuing axonal mitochondrial defects could alleviate axonal degeneration in PD. By delving into the fundamental biology of how mitochondria are specialized to sustain axonal survival and probing how their functions are perturbed in disease, this proposal will help shed light into basic principles of neuronal maintenance and function while potentially identifying early mechanisms of disease that can be targeted before irreversible cell death.