Fig 1: HepaCAM is essential for early postnatal CST axon growth and expands axon growth cone size.a Diagram of CM-DiI dye injections at the motor cortex to label layer V pyramidal neurons and descending CST axons; PD pyramidal decussation; green dashed box indicates postnatal CST growth (as shown in c); b Representative (from >10 injected mice) image to show the CM-DiI labeling in the motor cortex 2 days following the injection; Scale bar: 1 mm; Representative images (c) and quantification (d) of CM-DiI-labeled CST axons in the spinal cord of WT (i) and HepaCAM KO (ii) mice. Orange arrows indicate the pyramidal decussation; yellow lines indicate the beginning and ending points for the CST axon length measurement; The image was generated by superimposing images of serial longitudinal sections, which are shown in Supplementary Fig. 5a. Scale bar: 1 mm; n = 8 mice for WT and 9 mice for HepaCAM KO; Representative images (e) and quantification (f) of axon growth cone size of control (i) cortical neurons or cortical neurons treated with WT (ii) or HepaCAM KO (iii) A-Exo. C center domain (white circle), P peripheral domain (growth cone area outside of the center domain); Scale bars: 20 µm. Number of neurons quantified in each group shown in the graph (4–14 neurons/replicate, 3 biological replicates)/group; Representative images (g) and quantification of axon growth cone size (h) of cortical neurons grown on either PDL alone (i) or PDL/HepaCAM-ECD (ii) coating. n = 17 neurons (5–8 neurons/replicate, 3 biological replicates)/group; Scale bar: 20 µm; i Quantification of axon growth rate of cortical neurons treated with HepaCAM ECD and A-Exo. Number of neurons quantified in each group shown in the graph (7–9 neurons/replicate, 3 biological replicates)/group; live-cell imaging was performed at DIV 3 for 2 h; p value in (d, h) determined from two-tailed t-test; p values in (f, i) determined using one-way ANOVA followed by a Tukey post hoc test. Data are presented as mean values ± SEM.
Fig 2: In situ illustration and developmental dynamics of A-Exo. in the CNSa, Schematic diagram of stereotaxic injections of AAV5-mCherry-Gfap-Cre into the motor cortex of CD63-GFPf/+Ai14-tdTf/f mice at P1 or P21. Mice were collected for analysis at P8 or P28, respectively. b, Representative confocal and Imaris images of tdT+ astroglia and CD63-GFP+ puncta at P8 and P28 in AAV5-mCherry-Gfap-Cre-injected CD63-GFPf/+Ai14-tdTf/+ mice. Yellow and white arrows indicate extracellularly or intracellularly localized CD63-GFP+ puncta, respectively, based on their co-localization with tdT+ astroglia; c, Quantification of extracellularly localized CD63-GFP+ puncta based on their co-localization with tdT+ astroglia; n = 9 images from 3 mice/group; d, Representative image of HepaCAM immunostaining signals co-localized with CD63-GFP+ puncta signals from spinal cord sections of 4-OHT-injected Slc1a3-CreER+CD63-GFPf/+ mice (P7). e, Schematic view of AAV5-mCherry-Gfap-Cre virus injection into spinal cord of CD63-GFPf/+ mice and representative images of induced CD63-GFP+ and mCherry signals in proximal and distal spinal cord sections from the injection site. Mice analyzed 2 weeks post-injection; Red dashed circles: CD63-GFP+ area; White dashed circles: ventral horn grey matter area; mCherry signals are only visible within 500 µm from the injection site. Scale bar: 200 µm; f, Quantification of the distance CD63-GFP+ signal traveled along spinal cord in AAV5-mCherry-Gfap-Cre injected CD63-GFPf/+ mice. n = 4 mice (injected at P90); g, Representative immunoEM images of hCD63 labeling on spinal cord sections of AAV5-mCherry-Gfap-Cre-injected CD63-GFPf/+ mice. Intracellular immunogold signals (yellow arrows) are observed inside astroglia (astro., subpanel i) and in neuronal post-synaptic (indicated with black arrows, subpanel iv) dendritic compartment (D, subpanel iv). Subpanels ii and iv are the magnified views of subpanels i and iii, respectively. A: axonal terminal; Mt: mitochondria; Scale bars, 100 nm. p value in c determined from two-tailed t test.
Fig 3: 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-GFPf/+Ai14-tdTf/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-GFPf/+Ai14-tdTf/+ 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-GFPf/+ 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-GFPf/+ mice. n = 4 mice (injected at P90); g Schematic view of AAV5-mCherry-Gfap-Cre virus injection into spinal cords of hCD63-GFPf/+Ai14-tdTf/+ 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-GFPf/+ 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.
Fig 4: Surface expression of HepaCAM essentially and sufficiently mediates stimulatory effects of A-Exo. on axon growthRepresentative images (a) and quantification (b) of ßIII-tubulin+ neuronal axon (white arrows) length following equal amount (1µg) of WT and HepaCAM-depleted A-Exo. treatment. Subpanels: i, control; ii, WT A-Exo.; iii, HepaCAM KO A-Exo.; HepaCAM-depleted A-Exo. were prepared from HepaCAM KO astrocyte cultures as described in materials and methods. Scale bar: 100 µm; n = 23–34 neurons (> 3 biological replicates)/group; Representative images (c) and quantification (d) of ßIII-tubulin+ neuronal axon (white arrows) length following co-treatment with HepaCAM antibody (ProteinTech) and A-Exo. Subpanels: i, control (1 x PBS); ii, IgG alone; iii, A-Exo. + IgG; iv, A-Exo. + HepaCAM ab; 8 µg ab/coverslip (12 mm diameter) was used in the treatment. Scale bar: 100 µm; n=10–14 neurons (= 2 biological replicates)/group; Representative images (e) and quantification (f) of ßIII-tubulin+ neuronal axon (white arrows) length following HepaCAM ECD coating. Subpanels: i, PDL alone; ii, PDL + BSA (4 µg); iii, PDL + HepaCAM ECD (4 µg); Scale bar: 100 µm; n=12 neurons (= 2 biological replicates)/group; 1µg A-Exo. was used in each experiment; p values in b, d, and f determined using one-way ANOVA followed by a Tukey post-hoc test.
Fig 5: Surface expression of HepaCAM essentially and sufficiently mediates stimulatory effects of A-Exo. on axon growth.Representative images (a) and quantification (b) of ßIII-tubulin+ neuronal axon (white arrows) length following equal amount (1 µg) of WT and HepaCAM-depleted A-Exo. treatment. i control; ii WT A-Exo.; iii HepaCAM KO A-Exo.; HepaCAM-depleted A-Exo. were prepared from HepaCAM KO astrocyte cultures as described in materials and methods. Scale bar: 100 µm; Number of neurons quantified in each group shown in the graph (5–8 neurons/replicate, >3 biological replicates)/group; Representative images (c) and quantification (d) of ßIII-tubulin+ neuronal axon (white arrows) length following co-treatment with HepaCAM antibody (ProteinTech) and A-Exo. i control (1 x PBS); ii IgG alone; iii A-Exo. + IgG; iv A-Exo. + HepaCAM ab; 8 µg ab/coverslip (12 mm diameter) was used in the treatment. Scale bar: 100 µm; Number of neurons quantified in each group shown in the graph (7–9 neurons/replicate, =2 biological replicates)/group; Representative images (e) and quantification (f) of ßIII-tubulin+ neuronal axon (white arrows) length following HepaCAM ECD coating. i PDL alone; ii PDL + BSA (4 µg); iii PDL + HepaCAM ECD (4 µg); Scale bar: 100 µm; Number of neurons quantified in each group shown in the graph (8–12 neurons/replicate, =3 biological replicates)/group; 1 µg A-Exo. was used in each experiment; p values in (b, d, f) determined using one-way ANOVA followed by a Tukey post hoc test. Data are presented as mean values ± SEM.
Supplier Page from Sino Biological, Inc. for Human HEPACAM Protein (ECD, His Tag)