Reduced Levels of A20 Protein Prompted RIPK1-dependent Apoptosis and Blood–brain Barrier Breakdown During Cerebral Ischemia Reperfusion Injury
Chaonan Yang, Yongjiao Wang, Xiaohui Wu, Min Gong , Ying Li
Abstract
Blood–brain barrier (BBB) leakage is an important cause of the exacerbation of pathological features of cerebral ischemia reperfusion injury (CIRI). However, the specific mechanism of BBB leakage is not clear. It was found that the CIRI resulted in RIPK1 activation and subsequent RIPK1-dependent apoptosis (RDA). Inhibition of RIPK1 significantly reduced BBB breakdown and brain damage. The aim of this study is to investigate the mechanism of RIPK1 in the BBB leakage during CIRI. It was discovered by proteomics that autophagy activation resulting from ischemia and reperfusion significantly downregulated the level of A20 protein. A20 is an important protein that regulates RIPK1 and RDA. It was hypothesized that activation of autophagy caused by ischemic reperfusion led to a decrease in A20 protein, which, in turn, caused the activation of RIPK1 and the occurrence of RDA, leading to leakage of the BBB. The findings in this study revealed the role of RIPK1 in the cell death and BBB leakage upon cerebral ischemia reperfusion injury, and these findings provide a novel perspective for the treatment of ischemic reperfusion.
Introduction
The BBB is an anatomical and biochemical barrier that protects the brain from potentially harmful substances [1]. The BBB is a highly selective membrane barrier in the brain microvasculature that facilitates transport between the systemic circulation and the central nervous system [2]. The BBB regulates homeostasis of the central nervous system (CNS) by forming a tightly regulated neurovascular unit (NVU) that includes endothelial cells (ECs), pericytes and astrocytic endfeet, which jointly maintain normal brain function [3–5]. The presence of the BBB is capable of preventing some substances, mostly harmful, from entering the brain tissue from blood. The BBB acts as a physical and metabolic barrier between the CNS and the peripheral circulation, exerting regulatory and protective effects on the microenvironment of the brain [6]. Under normal circumstances, the primary function of the BBB is to establish and maintain homeostasis in the CNS. Once the BBB is destroyed, the brain is particularly vulnerable to infection and damage [7–9].
Materials and methods
Antibodies and reagents
The following antibodies were employed: mouse anti-MLKL (Abcam), mouse anti-caspase 3 (Cell Signaling), mouse anti-cleaved caspase 3 (Cell Signaling), mouse anti-pRIPK1(S166) (Lifespan) and mouse anti-pMLKL(S345) (Novus). Secondary horseradish peroxidase HRP-conjugated antibodies were from Abcam.
Mice and treatments
The pathogen-free male wild-type C57BL/6J mice (n = 24), Ripk1D138N mice (n = 3) (on a C57BL/6J background) and Ripk3-/- mice (n = 3) (on a C57BL/6J background), 8 to 11 weeks of age, were provided by Model Organisms (Shanghai, China). Animal experiments were carried out in accordance with ARRIVE guidelines and of the China Animal Care and Use Committee of Tianjin Medical University (Application Number 201910017).
Results
After MCAO treatment, the Ripk1D138N mice showed remarkable ameliorating infarction area but Ripk3 knock out mice did not (Fig 1A). The finding ascertained that RDA played dominant role in the infarction instead of RIPK-1 associated necroptosis [39]. In this study, the activation of RIPK1 protein was investigated upon the phosphorylation-serine 166 [40] by western blotting (in Fig 1B and 1C). Result exhibited the wild-type and Ripk1D138N mice showed significant increase in the phosphorylated level of RIPK1(S166) but not the Ripk3-/- mice. The p-RIPK1 level was only increased 24 hours after MCAO treatment in Ripk3 knock out mice. This finding ascertained that the dead knockin allele D138N in RIPK1 is crucial for kinase activation which was contributed to the infarction volume.
Discussion
The BBB is central to the regulation of cerebral microcirculation due to its characteristic barrier properties and transport system. The BBB is principally composed of cerebral micro cells, which form tight junctions together and are interlaced with astrocytes, pericytes, and a basal lamina [43]. These cells possess specialized receptor-mediated transport mechanisms and barrier properties and contribute equally to the local control of cerebral microcirculation. CIRI can result in breakdown of the brain microvasculature and BBB, leading to increased BBB permeability [18]. CIRI results in brain edema, a primary event that affects both morbidity and mortality following CIRI. Edema increases intracerebral pressure (ICP) and leads to secondary ischemic injuries by impairing cerebral perfusion and oxygenation [44]. An additional consequence of BBB disruption is the infiltration of leukocytes into brain tissue, accompanied by microglial activation and inflammation [45]. BBB injury is recognized to play an important role in brain injury caused by ischemic reperfusion. Inhibition of BBB destruction and vascular endothelial cell death can greatly ameliorate brain injury [46]. At present, the mechanism of endothelial cell death induced by ischemia and reperfusion injury is not completely clear.
Conclusion
The findings in this study demonstrated that the death of cells induced by ischemic reperfusion injury was mediated mainly by the activation of RIPK1. The BBB leakage and infarct volume of RIPK1 mutant mice (Ripk1D138N) were significantly reduced compared to those of wild-type mice. RIPK1 is the main protein that mediates necroptosis; however, it was found that mice without the capacity for necroptosis (Ripk3-/-) did not show the same high survival rate as RIPK1 kinase mutant mice (Ripk1D138N). Additionally, the death of cells in Ripk3-/- mice was not significantly improved, indicating that RIPK1-mediated programmed necrosis was not the main cause of BBB injury. Further investigations in this study revealed that RDA might be responsible for the endothelial cell death that leads to BBB leakage after MCAO treatment. A20 was decreased during cerebral ischemia–reperfusion injury, and A20 acted as a key regulatory protein that inhibited RIPK1 and RDA. The decrease in A20 may be related to degradation during autophagy, and autophagy is known to be activated by CIRI.
Citation: Yang C, Wang Y, Wu X, Gong M, Li Y (2023) Reduced levels of A20 protein prompted RIPK1-dependent apoptosis and blood–brain barrier breakdown during cerebral ischemia reperfusion injury. PLoS ONE 18(8): e0290015. https://doi.org/10.1371/journal.pone.0290015
Editor: Divakar Sharma, Lady Hardinge Medical College, INDIA
Received: January 31, 2023; Accepted: August 1, 2023; Published: August 14, 2023
Copyright: © 2023 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: This study was supported by the National Natural Science Funding program (81771221, 81870967 and 82071384). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: BBB, blood brain barrier; CIRI, Cerebral Ischemia Reperfusion Injury; DND, Delayed Neuron Death; MCAO, Middle-Cerebral-Artery Occlusion.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0290015#abstract0
