Project Summary / Abstract The overall goal of this proposal is to reveal central regulators of microglial attributes that can impact synapses. Synaptic dysfunction is tightly linked to declining cognition and neuronal health during aging. A long- standing mystery in neuroscience is why some CNS neurons are more vulnerable to age-associated synapse loss and neurodegenerative disease. Microglia are well-positioned to influence synapses throughout the lifespan, being equipped to induce synapse formation, synapse elimination and alter synapse composition through multiple mechanisms. Moreover, synapse-relevant attributes of microglia change during aging, including their cell process motility, phagocytic behaviors, and production of inflammatory factors. We and others recently discovered that microglia exhibit region-specific phenotypes, raising the possibility that microglial regulation of synaptic health varies. In addition, our preliminary data indicate that ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) microglia begin to proliferate and produce inflammatory factors during midlife in mice, and months before microglia in other basal ganglia nuclei. These “pockets” of early inflammation are likely detrimental to synaptic function of nearby dopamine neurons, which are highly vulnerable to functional decline and degenerative disease during aging. Here, we will investigate the possibility that microglial lysosomes can simultaneously regulate multiple synapse-relevant attributes of these cells, and that regional differences in lysosome function give rise to regional variation in microglial aging and synapse vulnerability. Lysosomes are typically viewed as purely degradative organelles, but new data show that they are intimately involved in membrane recycling and intracellular signaling that can shape cell properties. Indeed, lysosomes play a central role in regulating macrophage phenotype, including their inflammatory profiles, phagocytosis, and responses to aging. Our findings suggest that lysosomes play similar regulatory roles within microglia; the region-specific phenotypes and responses to aging that we observe within VTA/SNc microglia were accompanied by prominent differences in lysosome abundance, expression of lysosome component genes, and rates of lysosome overload with protein-lipid aggregates, compared to microglia in other basal ganglia nuclei. Nonetheless, surprising little is known about microglial lysosomes in vivo. Focusing on the basal ganglia and working in mice, we will use multidisciplinary approaches to define the composition and functional status of microglial lysosomes across brain regions and lifespan (Aim 1). We developed novel methods to manipulate microglial lysosome overload and will determine how this shapes synapse-relevant microglial attributes during aging (Aim 2). Finally, we will directly measure how microglial lysosome overload affects lifespan synaptic integrity and function in distinct brain re...