Extended Data Fig. 4: AAV-BI30-mediated transfer facilitated nSMase2 knockdown and reduced EV release in brain endothelial cells.
a Immunoblots showing the downregulation of the nSMase2 (top) and Rab27a (bottom) in Neuro 2a (N2a) cells. β-actin was used as the loading control. b Immunoblots of EV proteins as indicated. Small EVs were collected from equal volumes of supernatants from N2a cells treated with shRNA. β-actin from N2a cells was used as the loading control. The samples shown were obtained from the same experiment and the blots were processed in parallel. c Immunoblots showing the downregulation of the nSMase2 (top) in SVEC4-10 cells and the expression of EV proteins (bottom) as indicated. β-actin in cells was used as the loading control. d Quantification of the nSMase2 in N2a cells. (n = 4 biological replicates, one-way ANOVA with Dunnett’s correction). e Quantification of the protein Rab27a in N2a cells. (n = 4 biological replicates, unpaired two-sided t-test). f Quantification of EV proteins from N2a cells treated with shRNA. (n = 3 biological replicates, one-way ANOVA with Dunnett’s correction). g Quantification of the nSMase2 in SVEC4-10 cells. (n = 3 biological replicates, unpaired two-sided t-test). h Quantification of EV proteins in SVEC4-10 cells. (n = 3 biological replicates, unpaired two-sided t-test). i Representative images showing AAV-BI30-mediated FLAG expression in the brain of mice following intravenous injection. Brain sections from the indicated 8-week-old mice were analyzed 5 weeks after injection. Scale bar, 1 mm. j Representative images showing the inter-organ distribution of AAV-BI30-mediated FLAG expression in mice following intravenous injection. Scale bar, 200 μm. k Specificity of AAV-BI30-mediated transduction of brain endothelial cells in vivo. Images of FLAG expression in the cortex of mice injected with AAV-BI30, co-stained with Lectin, PDGFRβ, GFAP, Iba1, and NeuN. Scale bar, 25 μm. l Immunoblots (left) and quantification (right) of nSMase2 expression in brain microvascular endothelial cells (BMVECs) from mice following intravenous injection. BMVECs were isolated 5 weeks after injection. β-actin from BMVECs was used as the loading control. (n = 4 per group, unpaired two-sided t-test). m-n EV characterization in the CSF 5 weeks following intravenous injection by nano flow cytometry. Representative nano-flow cytometry plots (m) and (n) percentage of endothelial CD31+ small EVs in the total CD63+ small EVs for the indicated groups. (n = 4 per group, unpaired two-sided t-test). Data are presented as mean ± SEM. ns, not significant. Abbreviations: nSMase2, neutral sphingomyelinase 2; shRNA, short hairpin RNA; Scr, a short hairpin RNA targeting scramble sequences; sh nSMase2, short hairpin RNA targeting nSMase2; sh1, a short hairpin RNA targeting nSMase2 with sequence 1; sh2, a short hairpin RNA targeting nSMase2 with sequence 2; sh3, a short hairpin RNA targeting nSMase2 with sequence 3; shRab27a, a short hairpin RNA targeting RAB27A; AAV-sh nSMase2, an engineered adeno-associated virus 9 targeting endothelial cells throughout the central nervous system with short hairpin RNA targeting neutral sphingomyelinase 2 (nSMase2); AAV-sh Scr, an engineered adeno-associated virus 9 targeting endothelial cells throughout the central nervous system with short hairpin RNA targeting scramble sequences.
