The effect of regulating MCU expression on experimental ischemic brain injury.

Mitochondrial calcium uniporter (MCU) is a critical channel for Ca2+ influx into mitochondria. The present study aimed to determine if MCU knockdown has beneficial effects on ischemic brain injury and to explore the underlying mechanisms. The present study demonstrated that MCU knockdown but not total knockout (KO) attenuated ischemia infarction volume and primary cortical neuronal cells' ischemic damage. MCU knockdown maintained mitochondrial ultrastructure, alleviated calcium overload, and reduced mitochondrial apoptosis. Moreover, MCU knockdown regulated the changes of MICU1 and MICU2 after cerebral infarction, while no changes were observed in other mitochondrial calcium handling proteins. Based on metabolomics, MCU knockdown reversed middle cerebral artery occlusion (MCAO)-induced up-regulated phosphoenolpyruvate and down-regulated GDP to protect energy metabolism after cerebral infarction. Furthermore, a total of 87 and 245 differentially expressed genes (DEGs) were detected by transcriptome sequencing among WT mice, MCU KO mice and MCU knockdown mice in the MCAO model, respectively. Then, NR4A1 was identified as one of the DEGs in different MCU expressions in vivo ischemia stroke model via transcriptomic screening and genetic validation. Furthermore, MCU knockdown downregulated the ischemia-induced upregulation of NR4A1 expression. Together, this is the further evidence that the MCU knockdown exerts a protective role after cerebral infarction by promoting calcium homeostasis, inhibiting mitochondrial apoptosis and protecting energy metabolism.

BLK polymorphisms and expression level in neuromyelitis optica spectrum disorder.

AIM: This study aimed to determine the correlation between B-lymphoid tyrosine kinase (BLK) polymorphism, mRNA gene expression of BLK, and NMOSD in a Chinese Han population. BACKGROUND: B-lymphoid tyrosine kinase gene expressed mainly in B cells plays a key role in various autoimmune disorders. However, no studies have investigated the association of BLK polymorphisms with neuromyelitis optica spectrum disorder (NMOSD). METHODS: Han Chinese population of 310 subjects were recruited to analyze three single nucleotide polymorphisms (rs13277113, rs4840568, and rs2248932) under allele, genotype, and haplotype frequencies, followed by clinical characteristics stratified analysis. Real-time PCR was used to analyze mRNA expression levels of BLK in the peripheral blood mononuclear cells of 64 subjects. RESULTS: Patients with NMOSD showed lower frequencies of the minor allele G of rs2248932 than healthy controls (odds ratio (OR) =0.57, 95% confidence intervals (CI) 0.39-0.83, p = 0.003). The association between minor allele G of rs2248932 and reduced NMOSD susceptibility was found by applying genetic models of inheritance (codominant, dominant, and recessive) and haplotypes analysis. Subsequently, by stratification analysis for AQP4-positivity, the minor allele G frequencies of rs2248932 in AQP4-positive subgroup were significantly lower than in the healthy controls (OR =0.46, 95% CI 0.30-0.72, p = 0.001). Notably, the genotype GG of rs2248932 was more frequent in AQP4-negative subgroup (n = 14) than in AQP4-positive subgroup (n = 93) (p = 0.003, OR =0.05, 95% CI =0.01-0.57). BLK mRNA expression levels in the NMOSD patients (n = 36) were lower than in healthy controls (n = 28) (p < 0.05). However, the acute non-treatment (n = 7), who were untreated patients in the acute phase from the NMOSD group, showed BLK mRNA expression levels 1.8-fold higher than healthy controls (n = 8) (p < 0.05). CONCLUSION: This study evaluated that the minor allele G of rs2248932 in BLK is associated with reduced susceptibility to NMOSD and protected the risk of AQP4-positive. BLK mRNA expression in NMOSD was lower as compared to healthy controls while significantly increased in acute-untreated patients.

Nicotinamide adenine dinucleotide treatment alleviates the symptoms of experimental autoimmune encephalomyelitis by activating autophagy and inhibiting the NLRP3 inflammasome.

Nicotinamide adenine dinucleotide (NAD + ) is an essential cofactor in numerous metabolic pathways, and so may support protective and reparative processes against central nervous system diseases such as multiple sclerosis (MS). Here, we investigated the therapeutic potential of NAD + administration in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS and the contributions of autophagic regulation and NLRP3 inflammasome activity. EAE was induced in female C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG) p35-55 and disease severity analyzed by neurological function score and histological scores of spinal cord sections stained with hematoxylin-eosin or luxol fast blue. Outcomes were compared among control mice and EAE groups receiving daily post-immunization vehicle injections, NAD + injections, injection of the autophagy inhibitor 3-methyladenine (3-MA), or co-injection of NAD + and 3-MA. Expression levels of autophagy-related proteins (Beclin1, LC3-II/I, and p62/SQSTM1) were assessed by Western blotting, the activated microglial cells were evaluated by immunohistochemistry, while mRNA expression levels of NOD-like receptor family pyrin domain containing 3 (NLRP3), interleukin (IL)-1ß, IL-2, IL-17, IL-18, interferon-γ (IFN-γ) and IL-10 were detected by real-time PCR. The proportions of Th1 and Th17 cells in spleen were evaluated using flow cytometry. Treatment with NAD + alleviated demyelination, nerve injury, microglial activation and motor function abnormalities of EAE mice. In addition, NAD + increased the expressions of the autophagy-related proteins LC3-II/I and Beclin 1, and reduced the expression of p62. Treatment with NAD + also inhibited the expressions of NLRP3 and modulated the differentiation of Th1 and Th17 cells, reduced the expressions of the pro-inflammatory factors IL-1ß, IL-2, IL-18, IFN-γ and IL-17, and increased the expression of anti-inflammatory IL-10. Conversely, 3-MA aggravated spinal cord inflammation and demyelination, and delayed spontaneous remission from EAE. Furthermore, the beneficial effects of NAD + were abolished by 3-MA cotreatment. Our results indicate that NAD + suppresses the NLRP3 inflammasome at least in part through the activation of autophagy to relieve the symptoms of EAE. Therefore, regulation of autophagy by NAD + treatment may be an effective therapeutic strategy for MS.

Amlexanox attenuates experimental autoimmune encephalomyelitis by inhibiting dendritic cell maturation and reprogramming effector and regulatory T cell responses.

BACKGROUND: Amlexanox (ALX), a TBK1 inhibitor, can modulate immune responses and has anti-inflammatory properties. To investigate its role in regulating the progression of experimental autoimmune encephalomyelitis (EAE), we studied the effect of ALX on the maturation of dendritic cells (DCs) and the responses of effector and regulatory T cells (Tregs). METHODS: In vitro, bone marrow-derived DCs (BMDCs) were cultured and treated with ALX. Their proliferation, maturation, and their stimulatory function to induce T cells responses were detected. In vivo, the development of EAE from different groups was recorded. At the peak stage of disease, HE, LFB, and electronic microscope (EM) were used to evaluate inflammation and demyelination. Maturation of splenic DC and Th1/Th17/Treg response in the CNS and peripheral were also detected. To further explore the mechanism underlying the action of ALX in DC maturation, the activation of TBK1, IRF3, and AKT was analyzed. RESULTS: Our data indicated that ALX significantly inhibited the proliferation and maturation of BMDCs, characterized by the reduced MHCII, a co-stimulatory molecule, IL12, and IL-23 expression, along with morphological alterations. Co-culture of ALX-treated BMDCs inhibited allogeneic T cell proliferation and MOG-specific T cell response. In EAE mice, ALX significantly attenuated the EAE development by decreasing inflammatory infiltration and demyelination in the spinal cords, accompanied by reduced frequency of splenic pathogenic Th1 and Th17 cells and increased Tregs. Moreover, ALX treatment decreased Th1 and Th17 cytokines, but increased Treg cytokines in the CNS and spleen. Notably, ALX treatment reduced the frequency and expression of CD80 and CD86 on splenic DCs and lowered IL-12 and IL-23 secretion, further supporting an impaired maturation of splenic DCs. In addition, ALX potently reduced the phosphorylation of IRF3 and AKT in BMDC and splenic DCs, both of which are substrates of TBK1 and associated with DC maturation. CONCLUSIONS: ALX, a TBK1 inhibitor, mitigated EAE development by inhibiting DC maturation and subsequent pathogenic Th1 and Th17 responses while increasing Treg responses through attenuating the TBK1/AKT and TBK1/IRF3 signaling.