{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T20:24:53Z","timestamp":1790713493438,"version":"4.1.0"},"posted":{"date-parts":[[2026,4,21]]},"group-title":"Neuroscience","reference-count":67,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T00:00:00Z","timestamp":1776729600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["1RF1AG078338-01"],"award-info":[{"award-number":["1RF1AG078338-01"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["1R21AG078601-01"],"award-info":[{"award-number":["1R21AG078601-01"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["1I01BX007282-01"],"award-info":[{"award-number":["1I01BX007282-01"]}],"id":[{"id":"https:\/\/ror.org\/05rsv9s98","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,4,21]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Extracellular vesicles (EVs) mediate intercellular transfer of lipids, proteins, and nucleic acids between nearly all cell types. We previously showed that astrocyte-derived EVs modulate neuronal mitochondria\n                  <jats:italic>in vitro<\/jats:italic>\n                  , but whether endogenous astrocytic EVs are trafficked to neuronal mitochondria\n                  <jats:italic>in vivo<\/jats:italic>\n                  remained unknown. To address this, we generated an EV reporter mouse, Aldh1l1-Cre; CD9-tGFP\n                  <jats:sup>fl\/fl<\/jats:sup>\n                  , in which astrocyte-secreted EVs are labeled with a CD9-turboGFP fusion protein (CD9-tGFP). Astrocyte-specific expression of CD9-tGFP was verified in brain tissue and isolated EVs, comprising 13.2 \u00b1 1.6% of total brain EVs. In primary glial cultures, CD9-tGFP was restricted to astrocytes, localizing to vesicular compartments and cell protrusions (filopodia and cilia), with 89.3 \u00b1 2.2% of astrocyte-derived EVs carrying the label. These EVs were enriched with the sphingolipid ceramide, consistent with its co-distribution with CD9-tGFP in astrocytic cell protrusions. In the cortex, hippocampus, and cerebellum, CD9-tGFP was predominantly detected in astrocytic processes co-labeled with GLAST1 and GFAP, forming contacts with laminin-positive capillaries and parvalbumin-positive neurons. CD9-tGFP-labeled EVs were detected inside capillaries and neurons, and super-resolution STED microscopy revealed partial overlap with neuronal mitochondria. Live-cell spinning disk confocal imaging and AI-assisted proximity analysis confirmed uptake of CD9-tGFP EVs by neuronal cells and trafficking of their cargo to mitochondria\n                  <jats:italic>in vitro<\/jats:italic>\n                  . Biochemical isolation of synaptic and non-synaptic mitochondria confirmed EV-derived cargo on mitochondria\n                  <jats:italic>in vivo<\/jats:italic>\n                  , with 3-fold higher association of CD9-tGFP with synaptic than non-synaptic mitochondria. Together, these findings validate the Aldh1l1-Cre; CD9-tGFP\n                  <jats:sup>fl\/fl<\/jats:sup>\n                  reporter mouse as a powerful tool for tracking astrocyte-derived EVs\n                  <jats:italic>in vivo<\/jats:italic>\n                  and provide direct evidence that their cargo is preferentially trafficked to synaptic mitochondria.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Graphical Abstract<\/jats:title>\n                  <jats:p>\n                    Astrocyte-derived extracellular vesicles target neuronal mitochondria\n                    <jats:italic>in vivo<\/jats:italic>\n                  <\/jats:p>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"718987v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n    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