The Role of miR-195 in Neuro-vascular Cross-talk and Regulation of Blood-Brain-Barrier Chien-Yuan Chen1, Suh-Hang H Juo2* 1Graduate Institute of Medicine, Kaohsiung Medical University, Taiwan; 2Graduate Institute of Biomedical Sciences, China Medical University, Taiwan
Cerebral vascular endothelial cells (EC) degeneration significantly contributes to blood-brain-barrier (BBB) dysfunction during the aging process. Functional interaction between neuro-vascular units can be essential to maintain BBB integrity. miR-195 is abundant in neuron and its levels are decreased with aging. We hypothesized that miR-195 not only possesses beneficial effect toward neuron but indirectly affects BBB integrity via neuron-secreted-exosomes. We first tested the effect of exosome secreted by miR-195-enriched-neuron (195-Neu-Exo.) on cerebral EC in vitro, and then measure BBB function in C57BL/6 mice. Neuron was pre-transfected by negative control, miR-195, and miR-195 inhibitor. Nanoparticle Tracking Analysis (NTA) software showed no significant differences in exosome number and size distribution between groups; which suggested that miR-195 may effect exosome component but not exosome size and number. 195-Neu-Exo significantly improved EC functional outcome: decreased BBB leakage and increased tight junction protein expression; as assessed by FITC-dextran assay, western blot, and confocal immunofluorescence. Furthermore, we found that tight junction proteins were catabolized by autophagy, but this regulation was suppressed after treated by 195-Neu-Exo. To investigate the effect of exosomal protein on EC autophagy, iTRAQ proteomic approach was used to identify exosome-loaded protein. Among the 112 identified protein, we focused on thrombospondin-1 (TSP-1), had found significantly decreased in 195-Neu-Exo. TSP-1 is a glycoprotein that influences multiple cellular functions including autophagy. We confirmed the pro-autophagy effect of TSP-1 in both cultured EC and C57BL/6 mice. BBB leakage was significantly increased in TSP-1 treated mice confirmed by Evans Blue permeability assay. Brain slices of the TSP-1 treated mice showed tight junction disruption and robust extravascular albumin accumulated around BBB. We conclude that miR-195 participates in BBB maintenance by altering TSP-1 protein in neuron exosome, and exosomal TSP-1 may play a key role in aging-induced-BBB-leakage by regulating EC autophagy efficiency.
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