Mitochondrial Regulation of Cortical Astrocyte Morphogenesis and Organization

Abstract

Dysfunctional mitochondrial dynamics are a hallmark of devastating neurodevelopmental disorders such as childhood refractory epilepsy. However, the role of glial mitochondria in proper brain development is not well understood. Astrocytes, the most abundant glial cell in the brain, control crucial neurodevelopmental processes such as ion homeostasis, synapse formation, and blood-brain barrier establishment. Using genetic, microscopy, and molecular tools, I identified mitochondrial fission gatekeeping protein, Dynamin-related protein 1 (Drp1), as a critical regulator of astrocyte morphological and functional development in the postnatal cortex. Astrocytes are highly morphologically complex cells that develop thousands of ramified processes postnatally in the cortex, and the formation of these distal compartments allows astrocytes to perform key functions in brain development. Through a microscopy time-course characterization of astrocyte mitochondrial development in vivo, we show that astrocyte mitochondria undergo extensive division while populating astrocyte distal branches during postnatal cortical development. A candidate-based reverse genetic screen revealed that various regulators of mitochondrial dynamics, including fission, fusion, and trafficking, control distinct aspects of astrocyte morphogenesis in vitro and in vivo. Importantly, loss of mitochondrial fission regulator, Dynamin-related protein 1 (Drp1), decreases mitochondrial localization to distal astrocyte processes, and this mitochondrial mislocalization reduces peripheral astrocyte morphological complexity. In addition to their morphological complexity, astrocytes regulate brain homeostasis by tiling into functionally non-overlapping and evenly dispersed domains that are coupled by gap-junctions in the cortex. Astrocyte-specific reduction or deletion of Drp1 disrupts this astrocyte network organization, resulting in astrocytes clustering throughout the cortex. Drp1-deficient astrocytes with disrupted organization also lose astrocytic gap junction protein Connexin 43. Lastly, Drp1 cKO astrocytes upregulate markers of reactivity, indicating mitochondrial fission is critical for the structural and molecular homeostasis of the cortical astrocyte network. These findings uncover a crucial role for mitochondrial fission in coordinating astrocytic morphogenesis and organization, revealing astrocytic mitochondria dynamics as a critical step in neurodevelopment. We show mitochondrial fission regulates the localization of distal mitochondria in astrocytes, and the mislocalization of this distal mitochondrial subpopulation disrupts astrocyte morphological and functional development. Therefore, we developed a method to isolate and study distal astrocyte mitochondria for downstream proteomic analysis. We combined existing methods using a cell-type specific mitochondrial reporter mouse with astrocyte perisynaptic compartment isolation protocols to isolate whole and distal astrocyte mitochondria from Drp1 cWT and cKO mice. We validated astrocyte mitochondrial isolations through functional oxygen consumption and qualitative proteomic analyses. This method allows for investigating how mitochondrial dynamics generate mitochondrial heterogeneity within astrocytes. Our findings open new lines of investigation into how astrocyte mitochondria orchestrate astrocyte-astrocyte network organization and function in development, maturation, and disease.