A Novel Reporter Mouse for Astrocyte-Derived Extracellular Vesicles Reveals Enrichment of Cargo at Neuronal Mitochondria.

Extracellular vesicles (EVs) mediate intercellular transfer of lipids, proteins, and nucleic acids between cell types. We previously showed that astrocyte-derived EVs modulate neuronal mitochondria in vitro. Whether endogenous astrocytic EVs associate with neuronal mitochondria in vivo remained unknown. To address this, we generated an EV reporter mouse, Aldh1l1-Cre; CD9-tGFPfl/fl, which resulted in fluorescent labeling of astrocyte-derived CD9-positive EVs. Astrocyte-specific expression of CD9-tGFP was verified in brain tissue, where CD9-tGFP-positive particles comprised 13.2% ± 1.6% of total isolated EVs. In primary glial cultures, CD9-tGFP was restricted to astrocytes, localizing to vesicular compartments and cell protrusions (filopodia and cilia); 89.3% ± 2.2% of astrocyte-derived EVs were CD9-tGFP positive. In cortex, hippocampus, and cerebellum, CD9-tGFP was predominantly detected in astrocytic processes co-labeled with glutamate aspartate transporter 1 (GLAST1) and glial fibrillary acidic protein (GFAP), forming contacts with laminin-positive capillaries and parvalbumin-positive neurons. CD9-tGFP-labeled EVs were detected at capillaries and inside neurons, and STED microscopy revealed partial co-localization with neuronal mitochondria. Live-cell spinning disk confocal imaging and AI-assisted proximity analysis confirmed uptake of CD9-tGFP EVs by neuronal cells and cargo enrichment at mitochondria in vitro. Immunoblotting and imaging of isolated mitochondria established physical association with EV-derived CD9-tGFP in vivo, with 3-fold higher CD9-tGFP puncta density on synaptic mitochondria. Together, these findings validate the Aldh1l1-Cre; CD9-tGFPfl/fl reporter mouse as a tool for tracking astrocyte-derived EVs in vivo and provide evidence of preferential cargo enrichment at synaptic mitochondria.