The diagnosis of PD might be in difficulty, especially in the early stages. Therefore, the identification of novel biomarkers is imperative for the diagnosis and monitoring disease progression in PD. DJ-1 and α-synuclein, are two proteins that are critically involved in the pathogenesis of PD, and they have been examined as disease biomarkers in studies. However, no study exists regarding DJ-1 in plasma neural-derived exosomes. In the present study, the levels of DJ-1 and α-synuclein in plasma neural-derived exosomes were studied together in order to investigate novel biomarkers for PD. DJ-1 and α-synuclein in plasma and plasma neural-derived exosomes of the patients with PD and controls were quantified by ELISAs. The data revealed that the levels of DJ-1 and α-synuclein in plasma neural-derived exosomes and the ratio of plasma neural-derived exosomal DJ-1 to total DJ-1 were significantly higher in patients with PD, compared with controls, while levels of the two proteins in plasma exhibited no difference between the patients with PD and controls. However, no relationship was identified between biomarkers and disease progression. In addition, significant positive correlations between DJ-1 and α-synuclein in plasma neural-derived exosomes were found in the patients with PD and in healthy individuals. We hypothesize that DJ-1 in plasma neural-derived exosomes may be used as a potential biomarker as α-synuclein in PD and they might participate in the mechanism of PD together.

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.

JOURNAL/nrgr/04.03/01300535-202407000-00033/figure1/v/2026-05-11T120643Z/r/image-tiff Accumulating evidence suggests that oxidative stress and the Wnt/β-catenin pathway participate in stroke-induced disruption of the blood-brain barrier. However, the potential links between them following ischemic stroke remain largely unknown. The present study found that cerebral ischemia leads to oxidative stress and repression of the Wnt/β-catenin pathway. Meanwhile, Wnt/β-catenin pathway activation by the pharmacological inhibitor, TWS119, relieved oxidative stress, increased the levels of cytochrome P450 1B1 (CYP1B1) and tight junction-associated proteins (zonula occludens-1 [ZO-1], occludin and claudin-5), as well as brain microvascular density in cerebral ischemia rats. Moreover, rat brain microvascular endothelial cells that underwent oxygen glucose deprivation/reoxygenation displayed intense oxidative stress, suppression of the Wnt/β-catenin pathway, aggravated cell apoptosis, downregulated CYP1B1 and tight junction protein levels, and inhibited cell proliferation and migration. Overexpression of β-catenin or knockdown of β-catenin and CYP1B1 genes in rat brain microvascular endothelial cells at least partly ameliorated or exacerbated these effects, respectively. In addition, small interfering RNA-mediated β-catenin silencing decreased CYP1B1 expression, whereas CYP1B1 knockdown did not change the levels of glycogen synthase kinase 3β, Wnt-3a, and β-catenin proteins in rat brain microvascular endothelial cells after oxygen glucose deprivation/reoxygenation. Thus, the data suggest that CYP1B1 can be regulated by Wnt/β-catenin signaling, and activation of the Wnt/β-catenin/CYP1B1 pathway contributes to alleviation of oxidative stress, increased tight junction levels, and protection of the blood-brain barrier against ischemia/hypoxia-induced injury.

Compelling evidence showed that both nucleotide‐binding oligomerization domain‐like receptor family, pyrin domain‐containing protein 3 (NLRP3) inflammasomes and the immunoproteasome participate in neuroinflammatory responses in cerebral ischaemia injury. Moreover, inhibition of either NLRP3 inflammasomes or the immunoproteasome attenuates both neuroinflammation and neurological deterioration during ischaemic stroke. However, the underlying mechanism between the immunoproteasome and NLRP3 inflammasomes under ischaemic stroke conditions remains to be established. In this study, using both in vitro and in vivo ischaemic models, we demonstrated that the immunoproteasome inhibition reduced the expressions of NLRP3 inflammasome‐associated proteins, including NLRP3, apoptosis‐associated speck‐like protein (ASC), caspase‐1 and mature cytokines (interleukin [IL]‐1β and IL‐18). It also downregulated the levels of nuclear factor (NF)‐κB and pyroptotic‐ and apoptotic‐related proteins, and improved cell viability. In addition, inhibition of NF‐κB by the small molecule inhibitor Bay‐11‐7082 led to lower levels of NLRP3 inflammasomes and cleaved caspase‐1 proteins in BV2 cells after oxygen‐glucose deprivation and reoxygenation. Together, these findings suggest that the immunoproteasome may be responsible for inducing the expression and activation of NLRP3 inflammasomes via the NF‐κB pathway. Therapeutic interventions that target activation of the immunoproteasome/NF‐κB/NLRP3 inflammasome pathway may provide novel prospects for the future treatment of ischaemic stroke.