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

ABSTRACT 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‐tGFP fl/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‐tGFP fl/fl reporter mouse as a tool for tracking astrocyte‐derived EVs in vivo and provide evidence of preferential cargo enrichment at synaptic mitochondria.