Tan, C. X. | bioRxiv Science

bioRxiv2023

Astrocytic LRRK2 Controls Synaptic Connectivity through ERM Phosphorylation

Astrocytes are highly complex cells that mediate critical roles in synapse formation and maintenance by establishing thousands of direct contacts with synapses through their perisynaptic processes. Here, we found that the most common Parkinsonism gene mutation, LRRK2 G2019S, enhances the phosphorylation of the ERM proteins (Ezrin, Radixin, and Moesin), components of the perisynaptic astrocyte processes in a subset of cortical astrocytes. The ERM hyperphosphorylation was accompanied by decreased astrocyte morphological complexity and reduced excitatory synapse density and function. Dampening ERM phosphorylation levels in LRRK2 G2019S mouse astrocytes restored both their morphology and the excitatory synapse density in the anterior cingulate cortex. To determine how LRRK2 mutation impacts Ezrin interactome, we used an in vivo BioID proteomic approach, and we found that astrocytic Ezrin interacts with Atg7, a master regulator of autophagy. The Ezrin/Atg7 interaction is inhibited by Ezrin phosphorylation, thus diminished in LRRK2 G2019S astrocytes. Importantly, the Atg7 function is required to maintain proper astrocyte morphology. Our data provide a molecular pathway through which the LRRK2 G2019S mutation alters astrocyte morphology and synaptic density in a brain-region-specific manner.

neuroscience↗

bioRxiv2023

GEARBOCS: An Adeno Associated Virus Tool for In Vivo Gene Editing in Astrocytes

CRISPR/Cas9-based genome engineering enables rapid and precise gene manipulations in the CNS. Here, we developed a non-invasive astrocyte-specific method utilizing a single AAV vector, which we named GEARBOCS (Gene Editing in AstRocytes Based On CRISPR/Cas9 System). We verified GEARBOCS specificity to mouse cortical astrocytes and demonstrated its utility for three types of gene manipulations: knockout (KO); tagging (TagIn); and reporter knock-in (GeneTrap) strategies. Next, we deployed GEARBOCS in two test cases. First, we determined that astrocytes are a necessary source of the synaptogenic factor Sparcl1 for thalamocortical synapse maintenance in the mouse primary visual cortex. Second, we determined that cortical astrocytes express the synaptic vesicle associated Vamp2 protein and found that it is required for maintaining excitatory and inhibitory synapse numbers in the visual cortex. These results show that the GEARBOCS strategy provides a fast and efficient means to study astrocyte biology in vivo. MotivationAstrocytes are indispensable for brain development, function, and health. However, molecular tools to study astrocyte biology and function in vivo have been largely limited to genetically modified mice. Here, we developed a CRISPR/Cas9-based gene editing strategy within a single AAV vector that enables efficient genome manipulations in astrocytes. We designed and optimized this easy-to-use viral tool to understand gene expression, protein localization and function in astrocytes in vivo.

neuroscience↗