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 突触的形成，以及调节突触的激发。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反应，星形胶质细胞产生局部钙尖峰， 促进三磷酸腺苷等神经活性递质的释放。星形胶质细胞释放ATP对 维持神经元放电、突触成熟和可塑性。先前对单一培养的星形胶质细胞的研究表明 溶酶体经历强烈的胞吐作用，并在谷氨酸能刺激下释放三磷酸腺苷。这是怎么回事 溶酶体胞吐过程发生在与神经元突触相连的星形胶质细胞内 仍然不为人知。关于影响人口贩运的细胞骨架组织和 星形胶质细胞分支中溶酶体的运输。然而，细胞骨架组织中的扰动 星形胶质细胞损伤钙反应，减少ATP释放，导致神经发育障碍 和早发性神经变性。这些数据表明，对溶酶体贩运的监管是 对维持星形胶质细胞与神经元的相互作用至关重要。我的初步数据显示神经元放电 限制星形胶质细胞中溶酶体的运动，并可能促进非星形胶质细胞中的溶酶体胞吐 细胞自主方式。具体地说，我发现在发育中的星形胶质细胞中，溶酶体表现为短程 受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，溶酶体 在很大程度上是不活动的，它们的运动性对突触活动不敏感。药理学上的微扰 细胞骨架显示，这种溶酶体在星形胶质细胞中的锚定很可能是由于微管的切换。 到肌动蛋白细丝。基于这些数据，我推测随着星形胶质细胞的成熟，溶酶体在 由于从微管到肌动蛋白细胞骨架轨迹的转换，突触周围区。此本地化可能 定位溶酶体进行活性依赖的分泌，释放支持成熟的内容 突触隔间。为了验证这一假设，我将(目标1)定义溶酶体定位的机制 星形胶质细胞分支和(目标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同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。综合起来，这些目标将 将星形胶质细胞中的溶酶体定义为在突触成熟过程中发挥关键作用的信号细胞器。知识 从这项研究中获得的将阐明星形胶质细胞是如何成为 三方突触及其对星形胶质细胞功能障碍在突触形成中的作用 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.