Mdivi-1 alleviates blood-brain barrier disruption and cell death in experimental traumatic brain injury by mitigating autophagy dysfunction and mitophagy activation

Abstract

Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission. Our previous studies proved that the inhibition of Drp1 may help attenuate traumatic brain injury (TBI)-induced functional outcome and cell death through maintaining normal mitochondrial morphology and inhibiting activation of apoptosis. However, the molecular mechanisms of Drp1 after TBI remain poorly understood. In this study, we investigated the role of mitochondrial division inhibitor 1 (Mdivi-1), a small molecule inhibitor of Drp1, in underlying mechanisms of general autophagy and mitochondria autophagy (mitophagy) after experimental TBI. In vivo, we found that autophagosomes accumulated in cortical neurons at 24h after TBI, owing to the enhanced autophagy indicated by the accumulation of LC3 and the decrease of p62; but Mdivi-1 reversed the enhancement. Mdivi-1 also alleviated the number of LC3 puncta and TUNEL-positive structures in cells, indicating that autophagy maybe involved in Mdivi-1's anti-apoptosis effects. Then, the expression level of mitochondrial dynamics related and mitophagy related proteins was assessed using the isolated mitochondria. The results showed that TBI-induced mitochondrial fission (represented by Drp1), mtDNA concentration down-regulation and PTEN induced putative kinase 1 (PINK1)-Parkin mediated mitophagy activation were all inhibited by Mdivi-1. In addition, TBI-induced blood-brain barrier (BBB) disruption and matrix metalloproteinases (MMP)-9 expression up-regulation were inhibited following Mdivi-1 treatment. In vitro, Mdivi-1 significantly alleviated the scratch injury-induced cell death, loss of mitochondrial membrane potential, reactive oxygen species (ROS) production and ATP reduction in primary cortical neurons (PCNs). Additionally, the lysosome inhibitor chloroquine (CQ) abrogated the Mdivi-1-induced decrease in autophagosomes accumulation and cell death at 24h both in the basal state and under the conditions of scratch cell injury. Together, these data demonstrate that Mdivi-1 mitigates TBI-induced BBB disruption and cell death at least in part by a mechanism involving inhibiting autophagy dysfunction and mitophagy activation.

Keywords: Autophagy; Blood-brain barrier; Dynamin-related protein 1; Mitophagy; Traumatic brain injury.