Defining the Role of Astrocytic Lysosome trafficking and Exocytosis in Regulating Synapse Maturation

摘要星形胶质细胞是突触的重要组成部分，提供必要的代谢支持，调节神经元的功能， 突触形成和调节突触放电。星形胶质细胞支持突触的机制 功能在很大程度上是未知的。作为对神经元活动的响应，星形胶质细胞产生局部钙峰， 促进神经活性递质的释放，如ATP。从星形胶质细胞释放ATP是至关重要的， 维持神经元放电、突触成熟和可塑性。先前对单一培养星形胶质细胞的研究表明， 溶酶体经历强烈的胞吐作用并响应于谷氨酸能刺激而释放ATP。这如何 溶酶体胞吐过程发生在与神经元突触连接的星形胶质细胞内 仍然未知。关于影响贩运的细胞骨架组织和 溶酶体在星形胶质细胞分支中的转运。然而，在细胞骨架组织的扰动， 星形胶质细胞损害钙反应并减少ATP释放，导致神经发育受损 和早发性神经退化这些数据表明，溶酶体运输的调节是 维持星形胶质细胞-神经元相互作用的关键。我的初步数据表明神经元放电 限制了星形胶质细胞中溶酶体的移动性，并可能促进非- 细胞自主的方式具体来说，我发现在发育中的星形胶质细胞中， 由突触活动抑制的双向运动。然而，在成熟的星形胶质细胞分支中， 大部分是不动的，它们的运动性对突触活动不敏感。药物干扰 细胞骨架显示，星形胶质细胞中溶酶体的锚定可能是由于微管的转换， 到肌动蛋白丝。基于这些数据，我假设随着星形胶质细胞的成熟，溶酶体在星形胶质细胞中积累。 由于从微管到肌动蛋白细胞骨架轨道的转换，突触周区室。这种定位可以 定位溶酶体进行活性依赖性分泌，释放支持成熟的内容物， 突触间室为了验证这一假设，我将（目的1）定义溶酶体定位的机制， 星形胶质细胞分支和（目的2）确定突触活动对星形胶质细胞中溶酶体胞吐的影响。 我将使用一个强大的系统来共培养神经元和星形胶质细胞，以研究这一过程的动力学， 在活细胞成像中使用尖端方法的高时空分辨率。结合起来，这些目标 将星形胶质细胞中的溶酶体定义为在突触成熟中起关键作用的信号细胞器。知识 从这项研究中获得的信息将阐明星形胶质细胞是如何成为神经细胞的关键组成部分的新的分子途径。 三方突触，启发我们理解星形胶质细胞功能障碍如何可能有助于突触 神经发育和神经退行性疾病的缺陷。

ABSTRACT Astrocytes are critical components of synapses, providing essential metabolic support, regulating synapse formation, and modulating synaptic firing. The mechanisms by which astrocytes support synaptic function are largely unknown. In response to neuronal activity, astrocytes produce local calcium spikes that promote the release of neuroactive transmitters, such as ATP. ATP release from astrocytes is essential for sustaining neuronal firing, synapse maturation, and plasticity. Prior work in monoculture astrocytes revealed that lysosomes undergo robust exocytosis and release ATP in response to glutamatergic stimulation. How this process of lysosome exocytosis occurs within astrocytes that are synaptically connected with neurons remains unknown. Even less is known regarding the cytoskeletal organization that impacts the trafficking and transport of lysosomes in astrocyte branches. However, perturbances in the cytoskeletal organization of astrocytes impairs calcium responses and reduces ATP release, leading to impairments in neurodevelopment and early onset neurodegeneration. These data suggest that the regulation of lysosome trafficking is essential in maintaining astrocyte-neuron interactions. My preliminary data suggests that neuronal firing restricts the mobility of lysosomes in astrocytes and may promote lysosome exocytosis in astrocytes in a non- cell autonomous manner. Specifically, I find that in developing astrocytes, lysosomes display short-range bidirectional motility that is dampened by synaptic activity. However, in mature astrocyte branches, lysosomes are largely immobile, and their motility is insensitive to synaptic activity. Pharmacological perturbations to the cytoskeleton revealed that this anchoring of lysosomes in astrocytes is likely due to a switch from microtubules to actin filaments. Based on these data, I hypothesize that as astrocytes mature, lysosomes accumulate in perisynaptic compartments due to a switch from microtubule to actin cytoskeletal tracks. This localization may position lysosomes to undergo activity-dependent secretion, releasing contents that support the maturation of synaptic compartments. To test this hypothesis, I will (Aim 1) define mechanisms of lysosome positioning in astrocytic branches and (Aim 2) determine the impact of synaptic activity on lysosome exocytosis in astrocytes. I will use a robust system to coculture neurons and astrocytes to investigate the dynamics of this process with high spatiotemporal resolution using cutting edge methodology in live cell imaging. Combined, these aims will define lysosomes in astrocytes as signaling organelles that play crucial roles in synaptic maturation. Knowledge gained from this study will elucidate new molecular pathways for how astrocytes are key components of the tripartite synapse and enlighten our understanding of how astrocyte dysfunction may contribute to synaptic deficiencies in neurodevelopmental and neurodegenerative disorders.