Journal of neuroscience methods · 2026
A novel transgenic reporter mouse line of astrocyte-derived small extracellular vesicles for investigating intercellular communication in vivo.
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Liu Zhongwu, Zhang Yi, Kemper Amy, Li Yanfeng, Dudley Andrew C, Chopp Michael, Zhang Zheng Gang
Аннотация
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.
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.