Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood–brain barrier injury through the Wnt/β-catenin signalling pathway

Abstract

Background Disruption of the blood–brain barrier (BBB) after a stroke can lead to brain injury and neurological impairment. Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2 (LMP2) in the pathophysiology of ischemia stroke. However, the relationship between the immunoproteasome LMP2 and the BBB remains unclear. Methods Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). Three days before MCAO, the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region. The rat brain microvascular endothelial cell (RBMVEC) line was exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions in vitro. The RNA interference-mediated knockdown of LMP2 or β-catenin was analysed in vivo and in vitro. Analysis of the quantity of extravasated Evans blue (EB) and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB. Immunofluorescence and Western blotting were employed to detect the expression of target proteins. Cell migration was evaluated using a scratch migration assay. The results of immunofluorescence, Western blotting and cell migration were quantified using the software ImageJ (Version 1.53m). Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference (LSD) test. Results Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins (occludin, claudin-1 and ZO-1) in the MCAO/R group compared with the sham group (P < 0.001). However, inhibition of the immunoproteasome LMP2 restored the expression of these proteins, resulting in higher levels of occludin, claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group (P < 0.001). In addition, inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability [e.g., the quantity of extravasated EB: LMP2-shRNA group (58.54 ± 7.37) µg/g vs. control-shRNA group (103.74 ± 4.32) µg/g, P < 0.001], and promoted the upregulation of Wnt-3a and β-catenin proteins in rats following MCAO/R. In vitro experiments, OGD/R induced marked upregulation of LMP2, proapoptotic protein Bax and cleaved caspase-3, and downregulation of occludin, claudin-1, ZO-1 and Bcl-2, as well as inhibition of the Wnt/β-catenin pathway Wnt-3a and β-catenin proteins in RBMVECs, compared with the control group under normal culture conditions (P < 0.001). However, silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R. Silencing of β-catenin by transfection of RBMVECs with β-catenin-siRNA aggravated the downregulation of tight junction proteins, and reduced the proliferation and migration of RBMVECs following OGD/R, compared with the control-siRNA group (P < 0.001). LMP2-siRNA and β-catenin-siRNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R. Conclusion This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced BBB injury, and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.

Brain endothelial cells exposure to malaria parasites links type I interferon signalling to antigen presentation, immunoproteasome activation, endothelium disruption, and cellular metabolism

“…Next, we explored the possibility that impaired transcription of Wnt/ ß -catenin target genes resulted from increased immunoproteasome activation, which is known to promote the destruction of ubiquitinated ß -catenin ( 34 ). We found that the immunoproteasome inhibitor (ONX) counteracts the downregulation of Wnt/ ß -catenin signaling pathway genes induced by Pb A-IE although this effect did not reach statistical significance in the case of the Axin2 gene ( Figures 3I–K ).…”

Brain endothelial cells exposure to malaria parasites links Type I interferon signalling to antigen presentation, immunoproteasome activation, endothelium disruption and cell metabolic adaptation

“…Next, we explored the possibility that impaired transcription of Wnt/ ß -catenin target genes resulted from increased immunoproteasome activation, which is known to promote the destruction of ubiquitinated ß -catenin [27]. We found that the immunoproteasome inhibitor (ONX) counteracts the downregulation of Wnt/ ß -catenin signaling pathway genes induced by Pb A-IE (Fig.…”

LMP2 deficiency causes abnormal metabolism, oxidative stress, neuroinflammation, myelin loss and neurobehavioral dysfunctions

“…Immunofluorescence was performed as described previously [ 22 ]. After blocking with 10% normal goat serum (Sigma-Aldrich, USA), slices were incubated with primary antibodies as following: rabbit anti-GFAP (1:300; Cell Signaling Technology, USA), rabbit anti-IBA1 (1:400; Abcam, Cambridge, MA, USA), rabbit anti-beta Amyloid (1:50; Abcam, Cambridge, MA, USA), and mouse anti-Olig1 (1:100; Santa Cruz, USA).…”

“…Approximately 30 μg of total protein was loaded on an SDS–polyacrylamide gel as described previously [ 22 ]. The primary antibodies were used as follows: rabbit anti-myelin basic protein (MBP) and rabbit anti-IRAK1 (1:1000; Abcam, Cambridge, MA, USA), mouse anti-IL-6 (1:500; Abcam, Cambridge, MA, USA), mouse anti-Olig1 and mouse anti-TNF-a (1:300; Santa Cruz, USA), mouse monoclonal anti-β-actin (1:3000; Cell Signaling Technology, USA).…”