Investigating the impact of MLC-causing hepaCAM mutations on astrocyte morphology in the mouse visual cortex

Astrocytes are the most common glial cell type in the brain and have complex morphological features that allow them to interact with all brain cell types and structures. Disruptions to astrocyte morphology are hallmarks of numerous neurological disorders. Astrocytes express membrane proteins such as hepaCAM, which plays a critical role in astrocyte branching organization, astrocyte–astrocyte interactions, and astrocyte–synapse interactions. Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy with 25% of cases attributed to missense mutations in HEPACAM that disrupt homophilic cis and trans interactions between hepaCAM molecules. Despite the known link between MLC and hepaCAM, the impact of MLC-causing mutations in HEPACAM on in vivo astrocyte morphology remain unknown. Here, I used viral tools to express MLC-causing hepaCAM mutants in astrocytes of the developing mouse visual cortex and performed a comprehensive morphological analysis. I used established Imaris protocols to quantify neuropil infiltration volume (NIV) and astrocyte territory volume. To establish a new method for measuring in vivo astrocyte branching complexity, I developed an AI filament tracing protocol, DIVA Filament Analysis, to perform 3D Sholl analysis in Imaris. Notably, I observed significant morphological changes between astrocytes carrying G89S or Q56P mutations, which impair homophilic interactions in hepaCAM, and astrocytes under control conditions. These results suggest an important role for homophilic hepaCAM interaction in regulating astrocyte morphology in vivo and provide new insights into astrocyte dysfunction in MLC pathogenesis.