MLC-causing mutations alter hepaCAM protein interactome and astrocyte morphology

Astrocytes are morphologically complex glial cells that play critical roles in the brain. The morphology of astrocytes is essential for their function and is, in part, regulated by cell adhesion molecules (CAMs). One such molecule is hepaCAM, an astrocyte enriched, disease-linked CAM that helps astrocytes organize their interactions with other cells. Mutations in hepaCAM cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), a developmental and progressive brain disorder. Surprisingly, the cellular mechanisms of hepaCAM function, and how mutations disrupt this function, are unknown. The focus of our project is to determine how MLC-causing mutations alter hepaCAM interactions with other proteins and astrocyte morphology. For this project, we created two disease-mutant versions of hepaCAM using site-directed mutagenesis and cloned these into multiple expression vectors. By transfecting these plasmids into cultured rat astrocytes, we found that astrocytes with mutated hepaCAM had dysregulated hepaCAM localization and less complex morphology. Next, we tested if adeno-associated viruses (AAVs) containing these plasmids could express mutant hepaCAM in astrocytes in live mice. We observed robust expression in astrocytes for all AAVs. Using a proximal proteomics model known as BioID, we were able to identify key protein interaction differences between wild type and MLC mutated hepaCAM that can help illuminate the hepaCAM localization process and mechanism for MLC pathogenesis. These investigations are crucial to understanding MLC pathogenesis and basic astrocyte biology.