Role of autophagy in blood-brain barrier disruption under ischemic and diabetic conditions

Abstract

Autophagy is a conserved pathway that involves degradation and recycling of damaged or unnecessary organelles, but excessive autophagy can induce cell damage and death under pathological conditions. The integrity of blood-brain barrier (BBB), which shows selective permeability for substances to brain, is significantly damaged under pathological conditions such as ischemic stroke or diabetes, contributing to the damage of neurovascular unit. There have been attempts to identify the mechanism of ischemic and diabetic BBB disruption, but the precise mechanism remains unknown. Here, I investigated if autophagy involves BBB dysfunction under ischemic and diabetic conditions. In vitro BBB of brain endothelial cell (bEnd.3) was exposed to oxygen-glucose deprivation (OGD), an in vitro ischemic condition. OGD significantly decreased cell viability and impaired cellular permeability in bEnd.3 cells. Formation of microtubule-associated protein light chain 3 (LC3)-II, a molecular marker for autophagy induction, was significantly increased in OGD-exposed bEnd.3 cells, and inhibition of autophagy by 3-methyladenine (3-MA) restored the OGD-induced cellular dysfunctions. The level of occludin, a tight junction (TJ) protein in BBB, was significantly decreased following OGD exposure, and reversed by 3-MA. Occludin was found to be co-localized with LC3 puncta in bEnd.3 cells after OGD, as determined by immunofluorescence staining. The involvement of autophagy in ischemic damage in BBB cells was also confirmed in in vivo rat ischemic model with middle cerebral artery occlusion (MCAO). MCAO induced the conversion of LC-I to LC3-II and occludin degradation in ipsilateral brain capillaries, and these were restored by in vivo 3-MA administration. To investigate the diabetic brain endothelial cell dysfunction, cellular alterations were examined following exposure to methylglyoxal (MG), an endogenous metabolite that is significantly increased under diabetic conditions. The cell viability of bEnd.3 cells was decreased by MG in a dose-dependent manner, and the permeability was significantly increased at a concentration of 1 mM MG. MG treatment also decreased the level of TJ proteins in bEnd.3 cells. Significant increases of c-Jun N-terminal kinases (JNK) phosphorylation and LC3-II conversion were observed in MG-exposed BBB cells. Interestingly, the decrease of cell viability of bEnd.3 cells was enhanced by co-exposure to OGD and MG, suggesting that increased susceptibility of cellular damage. In conclusion, I suggest that an excessive autophagy activation could be a new mechanism of occludin degradation in BBB under ischemic condition, and that autophagy might be involved in MG-induced brain endothelial dysfunctions.