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

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.