For knockdown of Smpd1 in brain endothelial cells in vivo, Smpd1 miR RNAi or control miR RNAi (50 µg/50 µL) was diluted in 50 µL of 10% glucose solution. Solution was mixed and briefly centrifuged. In a separate tube, 6.4 µL in vivo-jetPEI was diluted in 50 µL of a 10% glucose solution. The volume was adjusted to 100 µl, using RNase free water, and solutions were briefly mixed. The 100 µL in vivo-jetPEI solution was then added to miR RNAi solution. The solutions were then incubated for 15 min at RT, to allow complexes to form. Smpd1 miR RNAi or control miR RNAi solutions were rapidly injected into tail vein twice a week for 3 weeks in 12-month-old Slco1c1-creERT2;Smpd1ox/ox mice or 17-month-old WT mice (200 µL final volume per mouse).

Although many reports have revealed dysfunction of endothelial cells in aging, resulting in blood-brain barrier (BBB) breakdown, the underlying mechanism or mechanisms remain to be explored. Here, we find that acid sphingomyelinase (ASM) is a critical factor for regulating brain endothelial barrier integrity. ASM is increased in brain endothelium and/or plasma of aged humans and aged mice, leading to BBB disruption by increasing caveolae-mediated transcytosis. Genetic inhibition and endothelial-specific knockdown of ASM in mice ameliorated BBB breakdown and neurocognitive impairment during aging. Using primary mouse brain endothelial cells, we found that ASM regulated the caveolae-cytoskeleton interaction through protein phosphatase 1-mediated ezrin/radixin/moesin (ERM) dephosphorylation and apoptosis. Moreover, mice with conditional ASM overexpression in brain endothelium accelerated significant BBB impairment and neurodegenerative change. Overall, these results reveal a novel role for ASM in the control of neurovascular function in aging, suggesting that ASM may represent a new therapeutic target for anti-aging.