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Objective: Drug-resistant epilepsy causes great clinical danger and still lacks effective treatments.Methods: Here, we used multifaceted approaches combining electrophysiology, optogenetics, and chemogenetics in a classic phenytoin-resistant epilepsy model to reveal the key target of subicular pyramidal neurons in phenytoin resistance.Results: In vivo neural recording showed that the firing rate of pyramidal neurons in the subiculum, but not other hippocampal subregions, could not be inhibited by phenytoin in phenytoin-resistant rats. Selective inhibition of subicular pyramidal neurons by optogenetics or chemogenetics reversed phenytoin resistance, whereas selective activation of subicular pyramidal neurons induced phenytoin resistance. Moreover, long-term low-frequency stimulation at the subiculum, which is clinically feasible, significantly inhibited the subicular pyramidal neurons and reversed phenytoin resistance. Furthermore, in vitro electrophysiology revealed that off-target use of phenytoin on sodium channels of subicular pyramidal neurons was involved in the phenytoin resistance, and clinical neuroimaging data suggested the volume of the subiculum in drug-resistant patients was related to the usage of sodium channel inhibitors.Interpretation: These results highlight that the subicular pyramidal neurons may be a key switch control of drug-resistant epilepsy and represent a new potential target for precise treatments. ANN NEUROL 2019;86:626-640. [ABSTRACT FROM AUTHOR]

Objective: Lupus nephritis (LN) is a major determinant of morbidity and mortality in systemic lupus erythematosus (SLE). Pim‐1 regulates lymphocyte proliferation and activation. The role of Pim‐1 in autoimmune disease remains unclear. This study was undertaken to test the hypothesis that inhibition of Pim‐1 would have therapeutic potential in patients with LN. Methods: Pim‐1 expression was analyzed in lupus‐prone (NZB × NZW)F1 mice (n = 6), human peripheral blood mononuclear cells (PBMCs) from SLE patients (n = 10), and glomeruli from patients with LN (n = 8). The therapeutic effect of the Pim‐1 inhibitor AZD1208 was assessed in the same murine lupus model (n = 10 mice per group). In vitro analysis was conducted to explore the mechanisms of action of Pim‐1 in mouse and human podocytes after Pim‐1 expression had been induced by anti–double‐stranded DNA (anti‐dsDNA) antibody–positive serum. Finally, MRL/lpr mice were used to confirm the therapeutic effects of Pim‐1 inhibition in vivo (n = 10 mice per group). Results: Up‐regulation of Pim‐1 was seen in renal lysates from diseased (NZB × NZW)F1 mice and in PBMCs from patients with SLE and renal biopsy tissue from patients with LN, relative to their control counterparts (each P < 0.05). The Pim‐1 inhibitor AZD1208 reduced the severity of proteinuria, glomerulonephritis, renal immune complex deposits, and serum anti‐dsDNA antibody levels, concomitant with the suppression of NFATc1 expression and NLRP3 inflammasome activation, in diseased (NZB × NZW)F1 mice (each P < 0.05 versus controls). Moreover, in mouse and human podocytes, Pim‐1 knockdown with targeted small interfering RNA (siRNA) suppressed NFATc1 and NLRP3 inflammasome signaling in the presence of anti‐dsDNA–positive serum (each P < 0.05 versus control siRNA). Mechanistically, Pim‐1 modulated NLRP3 inflammasome activation through intracellular Ca2+ (P < 0.05 versus normal controls). The therapeutic effect of Pim‐1 blockade was replicated in MRL/lpr mice. Conclusion: These data identify Pim‐1 as a critical regulator of LN pathogenesis in patients with SLE. Targeting of the Pim‐1/NFATc1/NLRP3 pathway might therefore have therapeutic potential in human LN. [ABSTRACT FROM AUTHOR]

Objective Development of proteinuria in lupus nephritis (LN) is associated with podocyte dysfunction. The NLRP3 inflammasome has been implicated in the pathogenesis of LN. The purpose of this study was to investigate whether NLRP3 inflammasome activation is involved in the development of podocyte injury in LN. Methods A fluorescence-labeled caspase 1 inhibitor probe was used to detect the activation of NLRP3 inflammasomes in podocytes derived from lupus-prone NZM2328 mice and from renal biopsy tissues obtained from patients with LN. MCC950, a selective inhibitor of NLRP3, was used to treat NZM2328 mice. Proteinuria, podocyte ultrastructure, and renal pathology were evaluated. In vitro, sera from diseased NZM2328 mice were used to stimulate a podocyte cell line, and the cells were analyzed by flow cytometry. Results NLRP3 inflammasomes were activated in podocytes from lupus-prone mice and from patients with LN. Inhibition of NLRP3 with MCC950 ameliorated proteinuria, renal histologic lesions, and podocyte foot process effacement in lupus-prone mice. In vitro, sera from diseased NZM2328 mice activated NLRP3 inflammasomes in the podocyte cell line through the production of reactive oxygen species. Conclusion NLRP3 inflammasomes were activated in podocytes from lupus-prone mice and from LN patients. Activation of NLRP3 is involved in the pathogenesis of podocyte injuries and the development of proteinuria in LN. [ABSTRACT FROM AUTHOR]

Objective Glycogen synthase kinase 3β (GSK-3β) has been demonstrated to be involved in immune and inflammatory responses via multiple signaling pathways, leading to the production of proinflammatory cytokines. The purpose of this study was to investigate the role of GSK-3β in the pathogenesis of lupus nephritis in 2 mouse models. Methods Thiadiazolidinone 8 (TDZD-8), a selective inhibitor of GSK-3β, was administered intraperitoneally to 12-week-old MRL/ lpr mice for 8 weeks or to 22-week-old (NZB × NZW)F1 mice for 12 weeks. The expression of GSK-3β and NLRP3 inflammasome components was analyzed. Proteinuria, biochemical parameters, proinflammatory cytokines, anti-double-stranded DNA (anti-dsDNA) antibody levels, and renal pathology were examined. In vitro, the effect of GSK-3β-directed small interfering RNA (siRNA) on NLRP3 inflammasome activation was evaluated in bone marrow-derived macrophages (BMMs) from the mice and in the J774A.1 macrophage cell line. Results The incidence of severe proteinuria and renal inflammation was significantly attenuated in both models, with a significant reduction in anti-dsDNA antibody production, immune complex deposition in the kidney, and circulating proinflammatory cytokine levels. TDZD-8 inhibited the activation of GSK-3β and caspase 1, with a concomitant decrease in interleukin-1β (IL-1β) synthesis. In vitro, GSK-3β siRNA transfection of mouse BMMs and the J774A.1 cell line with GSK-3β siRNA inhibited the expression of GSK-3β, the activation of caspase 1, and the production of IL-1β. Conclusion These results show that GSK-3β promotes lupus nephritis at least partly by activating the NLRP3/IL-1β pathway. The linking of GSK-3β to the NLRP3/IL-1β pathway is a novel observation in our study. Our results suggest that the GSK-3β/NLRP3/IL-1β pathway may be a potential therapeutic target for lupus in humans. [ABSTRACT FROM AUTHOR]

Objective The NLRP3 inflammasome plays key roles in inflammation and autoimmunity, and purinergic receptor P2X7 has been proposed to be upstream of NLRP3 activation. The aim of the present study, using murine models, was to investigate whether the P2X7/NLRP3 inflammasome pathway contributes to the pathogenesis of lupus nephritis (LN). Methods MRL/ lpr mice were treated with the selective P2X7 antagonist brilliant blue G (BBG) for 8 weeks. Following treatment, the severity of renal lesions, production of anti-double-stranded DNA (anti-dsDNA) antibodies, rate of survival, activation of the NLRP3/ASC/caspase 1 inflammasome pathway, and ratio of Th17 cells to Treg cells were evaluated. P2X7-targeted small interfering RNA (siRNA) was also used for in vivo intervention. Similar evaluations were carried out in NZM2328 mice, a model of LN in which the disease was accelerated by administration of adenovirus-expressing interferon-α (AdIFNα). Results Significant up-regulation of P2X7/NLRP3 inflammasome signaling molecules was detected in the kidneys of MLR/ lpr mice as compared with normal control mice. Blockade of P2X7 activation by BBG suppressed NLRP3/ASC/caspase 1 assembly and the subsequent release of interleukin-1β (IL-1β), resulting in a significant reduction in the severity of nephritis and circulating anti-dsDNA antibodies. The lifespan of the treated mice was significantly prolonged. BBG treatment reduced the serum levels of IL-1β and IL-17 and the Th17:Treg cell ratio. Similar results were obtained by specific siRNA silencing of P2X7 in vivo. The effectiveness of BBG treatment in modulating LN was confirmed in NZM2328 mice with AdIFNα-accelerated disease. Conclusion Activation of the P2X7 signaling pathway accelerates murine LN by activating the NLRP3/ASC/caspase 1 inflammasome, resulting in increased IL-1β production and enhanced Th17 cell polarization. Thus, targeting of the P2X7/NLRP3 pathway should be considered as a novel therapeutic strategy in patients with lupus. [ABSTRACT FROM AUTHOR]