A novel transgenic reporter mouse line of astrocyte-derived small extracellular vesicles for investigating intercellular communication in vivo.

BACKGROUND

Small extracellular vesicles (sEVs) mediate intercellular communication in the central nervous system, regulating processes ranging from homeostatic maintenance to injury repair. Although astrocytes are a major source of sEVs in the brain, in vivo investigation of their endogenous and cell-specific signaling remains technically challenging.

NEW METHOD: To address this limitation, we developed a novel inducible transgenic reporter mouse line, GFAP-CD63-GFP, that enables specific labeling of astrocyte-derived sEVs. The mouse line was generated by crossing GFAP-CreERT2 mice with CD63-emGFPloxP/stop/loxP mice. This system enables Tamoxifen-inducible, astrocyte-specific expression of GFP-tagged CD63, a tetraspanin enriched in sEVs. The model allows visualization and quantification of astrocyte-derived CD63-positive sEVs in vivo.

RESULTS

Following Tamoxifen induction, GFP expression was robustly detected in the brain and spinal cord. Immunogold transmission electron microscopy further identified GFP-positive sEVs within neurons, providing ultrastructural evidence of astrocyte-to-neuron vesicle transfer. As a proof-of-concept, ischemic stroke significantly increased astrocyte-derived sEVs in the ipsilesional cerebral hemisphere and the stroke-impaired side of the spinal cord, accompanied by enhanced neuronal endocytosis.

COMPARISON WITH EXISTING

METHODS

Current approaches rely primarily on in vitro EV isolation or nonspecific membrane dyes. The GFAP-CD63-GFP model enables cell type-specific, temporally controlled, and in situ tracking of astrocyte-derived sEVs.

CONCLUSIONS

These findings provide the first in vivo demonstration of increased astrocyte-to-neuron sEV communication during post-stroke recovery. The GFAP-CD63-GFP reporter mouse thus provides a powerful platform for investigating astrocyte-derived sEV signaling under both physiological and pathophysiological conditions of the CNS.