Fig. 4: In situ illustration and developmental dynamics of A-Exo. in the CNS.

a Schematic diagram of stereotaxic injections of AAV5-mCherry-Gfap-Cre into the motor cortex of hCD63-GFP^f/+Ai14-tdT^f/f mice at P1 or P21. Mice were collected for analysis at P6-8 or P28, respectively. b Representative confocal and Imaris images of tdT⁺ astroglia and hCD63-GFP⁺ puncta at P6 (green)–8 (blue) and P28 in AAV5-mCherry-Gfap-Cre-injected hCD63-GFP^f/+Ai14-tdT^f/+ mice. Yellow and white arrows indicate extracellularly or intracellularly localized hCD63-GFP⁺ puncta, respectively, based on their co-localization with tdT⁺ astroglia; Scale bar: 15 μm (i); 20 μm (iii); c Quantification of extracellularly localized hCD63-GFP⁺ puncta based on their co-localization with tdT⁺ astroglia; n = 18 images (3 images/mouse) from 6 mice/group; d Representative (from 3 replicates) HepaCAM immunoblot from in vivo isolated exosome fractions following the SEC purification. P3-5 (3 pups pooled as one sample) pups CNS tissues (brain and spinal cord) were used in exosome isolation. HepaCAM C-terminal antibody was used in detecting HepaCAM; A-Exo. from cultured WT or HepaCAM KO astrocytes were used as positive and negative controls; e Schematic view of AAV5-mCherry-Gfap-Cre virus injection into spinal cord of hCD63-GFP^f/+ mice (P90) and representative images of induced hCD63-GFP⁺ and mCherry signals in proximal and distal spinal cord sections from the injection site. Mice analyzed 2 weeks post-injection; Red dashed circles: hCD63-GFP⁺ area; White dashed circles: ventral horn gray matter area; mCherry signals are only visible within 500 μm from the injection site. Scale bar: 200 μm; f Quantification of the distance hCD63-GFP⁺ and mCherry signal traveled along spinal cord in AAV5-mCherry-Gfap-Cre injected hCD63-GFP^f/+ mice. n = 4 mice (injected at P90); g Schematic view of AAV5-mCherry-Gfap-Cre virus injection into spinal cords of hCD63-GFP^f/+Ai14-tdT^f/+ mice (P60) and the representative (from >3 injected mice) magnified image of induced hCD63-GFP⁺ and tdT⁺ astroglia; Yellow and white arrows indicate extracellularly or intracellularly localized hCD63-GFP⁺ puncta, respectively; Scale bar: 20 μm; h Representative (from 3 mice) immunoEM images of hCD63 labeling on spinal cord sections of AAV5-mCherry-Gfap-Cre-injected hCD63-GFP^f/+ mice. Intracellular immunogold signals (yellow arrows) are observed inside astroglia (astro., i) and in neuronal post-synaptic (indicated with black arrows, iv) dendritic compartment (D, iv). ii and iv are the magnified views of i and iii, respectively. A axonal terminal, Mt mitochondria; Scale bars, 100 nm. p value in (c) determined from two-tailed t-test. Data are presented as mean values ± SEM.