Dysfunctional astroglial exosome to (motor) neuron axon signaling in ALS

摘要 同时形成下行运动束和周围神经的运动神经元轴突功能障碍 肌萎缩侧索硬化症(ALS)中广泛可见肌束，通常先于MN胞体(和轴突)。 并在很大程度上促进了疾病的病理。尽管星形胶质细胞黏附信号是必不可少的 为了正确的轴突生长和引导，胶质细胞黏附分子是否以及如何在轴突中发挥作用 肌萎缩侧索硬化症的功能障碍/变性在很大程度上仍不清楚。虽然星形胶质细胞条件培养液(ACM) 来源于小鼠SOD1突变模型或来源于人类ALS患者的脑星形胶质细胞能够实质上 调节MN的健康和存活，ACM中的星形胶质细胞因子调节ALS条件下MN的存活 基本上仍未确定身份。胞外体(直径50-150 nm)，细胞外分泌物的主要类型 囊泡(EV)是从多泡小体(MVB，一种中间内涵体结构)中释放出来的 内体成熟。由中枢神经系统(CNS)细胞类型分泌的EV和Exosome已经出现 作为与神经退行性疾病发病机制有关的重要细胞间通路 包括肌萎缩侧索硬化症。星形胶质细胞来源的外切体如何影响ALS(运动神经元)存活，特别是轴突特性 在很大程度上仍未被探索。在这个项目中，我们打算研究新的刺激和保护作用。 星形胶质细胞外体，特别是外体黏附分子HepaCAM信号转导神经元，促进轴突的形成 生长、功能和(运动神经元)存活。我们还将确定功能失调的HepaCAM信号 从SOD1G93A星形胶质外体到(运动神经元)神经元参与MN轴突功能障碍和变性 在肌萎缩侧索硬化症。 根据我们的初步结果，我们提出了本项目的以下目标：目标1：确定 星形胶质外切体对(运动神经元)轴突生长和功能的影响；目的2：研究功能丧失 ALS模型中星形胶质细胞外切体向(运动神经元)传递信号的研究；目的3：阐明HepaCAM介导的 ALS模型中星形胶质外切体向(运动神经元)传递信号；我们已经产生了大量的 初步数据支持我们的理由，并证明拟议目标的可行性。我们将聘用 小鼠遗传学、原代神经元和星形胶质细胞培养、分子生物学、病毒注射、各种成像以及 生物化学方法来完成这些目标。这个项目的结果将揭示一个新的星云 外体HepaCAM介导的调控(运动神经元)轴突生长和存活的机制。它还将 提供有关ALS中星形胶质细胞外切体功能丧失的重要知识。这些研究 将极大地促进我们对ALS星形胶质细胞功能障碍的理解，并有助于发展新的星形胶质细胞- 基于神经保护策略。

Abstract Dysfunctions of motor neuron (MN) axons that form both descending motor tracts and peripheral nerve tracts are widely observed in amyotrophic lateral sclerosis (ALS), which often precede MN soma (and axon) degeneration and significantly contribute to disease pathology. Although astroglial adhesion signals are essential for proper axon growth and guidance, whether and how glial adhesion molecules play a role in axon dysfunction/degeneration in ALS remains largely unknown. Although astroglia conditioned medium (ACM) derived from the mouse SOD1 mutant model or from human ALS patient brain astroglia is able to substantially modulate health and survival of MNs, astroglial factors in ACM that modulate MN survival in ALS conditions remain essentially unidentified. Exosomes (50-150 nm in diameter), a major type of secreted extracellular vesicles (EVs), are released from multivesicular bodies (MVBs, an intermediate endosome structure) during endosome maturation. EVs and exosomes secreted from central nervous system (CNS) cell types have emerged as an important intercellular pathway that is implicated in the pathogenesis of neurodegenerative diseases including ALS. How astroglia-derived exosomes affect (motor) neuron survival especially axon properties in ALS remains largely unexplored. In this project, we intend to investigate novel stimulatory and protective roles of astroglial exosomes, especially exosomal adhesion molecule HepaCAM signaling to neurons, in promoting axon growth, functions, and (motor) neuron survival. We will also determine whether dysfunctional HepaCAM signaling from SOD1G93A astroglial exosomes to (motor) neurons contributes to MN axon dysfunction and degeneration in ALS. Based on our preliminary results, we propose the following aims in this project: Aim 1: Determine the effect of astroglial exosomes on (motor) neuron axon growth and functions; Aim 2: Investigate loss-of-function of astroglial exosome signaling to (motor) neurons in ALS models; Aim 3: Elucidate HepaCAM-mediated astroglial exosome signaling to (motor) neurons in ALS models; We have generated a large amount of preliminary data to support our rationales and to demonstrate feasibility for proposed aims. We will employ mouse genetics, primary neuron and astroglial cultures, molecular biology, virus injections, various imaging, and biochemical approaches to complete these aims. Outcomes from this project will reveal a new astroglial exosomal HepaCAM-mediated mechanism in modulating (motor) neuron axon growth and survival. It will also provide important knowledge about the loss-of-functional effect of astroglial exosomes in ALS. These studies will significantly advance our understanding of the astroglial dysfunction in ALS and help develop new astroglia- based neuroprotective strategies.