Role of Cholesterol Metabolic Enzyme CYP46A1 and Its Metabolite 24S-Hydroxycholesterol in Ischemic Stroke.

BACKGROUND: For several decades, it has been recognized that overactivation of the glutamate-gated N-methyl-D-aspartate receptors (NMDARs) and subsequent Ca2+ toxicity play a critical role in ischemic brain injury. 24S-hydroxycholesterol (24S-HC) is a major cholesterol metabolite in the brain, which has been identified as a potent positive allosteric modulator of NMDAR in rat hippocampal neurons. We hypothesize that 24S-HC worsens ischemic brain injury via its potentiation of the NMDAR, and reducing the production of 24S-HC by targeting its synthetic enzyme CYP46A1 provides neuroprotection. METHODS: We tested this hypothesis using electrophysiological, pharmacological, and transgenic approaches and in vitro and in vivo cerebral ischemia models. RESULTS: Our data show that 24S-HC potentiates NMDAR activation in primary cultured mouse cortical neurons in a concentration-dependent manner. At 10 µmol/L, it dramatically increases the steady-state currents by 51% and slightly increases the peak currents by 20%. Furthermore, 24S-HC increases NMDA and oxygen-glucose deprivation-induced cortical neuronal injury. The increased neuronal injury is largely abolished by NMDAR channel blocker MK-801, suggesting an NMDAR-dependent mechanism. Pharmacological inhibition of CYP46A1 by voriconazole or gene knockout of Cyp46a1 dramatically reduces ischemic brain injury. CONCLUSIONS: These results identify a new mechanism and signaling cascade that critically impacts stroke outcome: CYP46A1 → 24S-HC → NMDAR → ischemic brain injury. They offer proof of principle for further development of new strategies for stroke intervention by targeting CYP46A1 or its metabolite 24S-HC.

Construction of platelet count-optical method reflex test rules using Micro-RBC#, Macro-RBC%, "PLT clumps?" flag, and "PLT abnormal histogram" flag on the Mindray BC-6800plus hematology analyzer in clinical practice.

OBJECTIVES: Utilizing RBC or PLT-related parameters to establish rules for the PLT-O reflex test can assist laboratories in quickly identifying specimens with interfered PLT-I that require PLT-O retesting. METHODS: Prospective PLT-I and PLT-O testing was performed on 6857 EDTA-anticoagulated whole blood samples, split randomly into training and validation cohorts at a 2:3 ratio. Reflex and non-reflex groups were distinguished based on the differences between PLT-I and PLT-O results. By comparing RBC and PLT parameter differences and flags in the training set, we pinpointed factors linked to PLT-O reflex testing. Utilizing Lasso regression, then refining through univariate and multivariate logistic regression, candidate parameters were selected. A predictive nomogram was constructed from these parameters and subsequently validated using the validation set. ROC curves were also plotted. RESULTS: Significant differences were observed between the reflex and non-reflex groups for 19 parameters including RBC, MCV, MCH, MCHC, RDW-CV, RDW-SD, Micro-RBC#, Micro-RBC%, Macro-RBC#, Macro-RBC%, MPV, PCT, P-LCC, P-LCR, PLR,"PLT clumps?" flag, "PLT abnormal histogram" flag, "IDA Anemia?" flag, and "RBC abnormal histogram" flag. After further analysis, Micro-RBC#, Macro-RBC%,"PLT clumps?", and "PLT abnormal histogram" flag were identified as candidate parameters to develop a nomogram with an AUC of 0.636 (95 %CI: 0.622-0.650), sensitivity of 42.9 % (95 %CI: 37.8-48.1 %), and specificity of 90.5 % (95 %C1: 89.6-91.3 %). CONCLUSIONS: The established rules may help laboratories improve efficiency and increase accuracy in determining platelet counts as a supplement to ICSH41 guidelines.

Cholestane-3ß,5α,6ß-Triol Inhibits Acid-Sensing Ion Channels and Reduces Acidosis-Mediated Ischemic Brain Injury.

BACKGROUND: Activation of the acid-sensing ion channels (ASICs) by tissue acidosis, a common feature of brain ischemia, contributes to ischemic brain injury, while blockade of ASICs results in protection. Cholestane-3ß,5α,6ß-triol (Triol), a major cholesterol metabolite, has been demonstrated as an endogenous neuroprotectant; however, the mechanism underlying its neuroprotective activity remains elusive. In this study, we tested the hypothesis that inhibition of ASICs is a potential mechanism. METHODS: The whole-cell patch-clamp technique was used to examine the effect of Triol on ASICs heterogeneously expressed in Chinese hamster ovary cells and ASICs endogenously expressed in primary cultured mouse cortical neurons. Acid-induced injury of cultured mouse cortical neurons and middle cerebral artery occlusion-induced ischemic brain injury in wild-type and ASIC1 and ASIC2 knockout mice were studied to examine the protective effect of Triol. RESULTS: Triol inhibits ASICs in a subunit-dependent manner. In Chinese hamster ovary cells, it inhibits homomeric ASIC1a and ASIC3 without affecting ASIC1ß and ASIC2a. In cultured mouse cortical neurons, it inhibits homomeric ASIC1a and heteromeric ASIC1a-containing channels. The inhibition is use-dependent but voltage- and pH-independent. Structure-activity relationship analysis suggests that hydroxyls at the 5 and 6 positions of the A/B ring are critical functional groups. Triol alleviates acidosis-mediated injury of cultured mouse cortical neurons and protects against middle cerebral artery occlusion-induced brain injury in an ASIC1a-dependent manner. CONCLUSIONS: Our study identifies Triol as a novel ASIC inhibitor, which may serve as a new pharmacological tool for studying ASICs and may also be developed as a potential drug for treating stroke.

Inhibition of ASIC1a Improves Behavioral Recovery after Stroke.

Stroke continues to be a leading cause of death and long-term disabilities worldwide, despite extensive research efforts. The failure of multiple clinical trials raises the need for continued study of brain injury mechanisms and novel therapeutic strategies for ischemic stroke. The contribution of acid-sensing ion channel 1a (ASIC1a) to neuronal injury during the acute phase of stroke has been well studied; however, the long-term impact of ASIC1a inhibition on stroke recovery has not been established. The present study sought to bridge part of the translational gap by focusing on long-term behavioral recovery after a 30 min stroke in mice that had ASIC1a knocked out or inhibited by PcTX1. The neurological consequences of stroke in mice were evaluated before and after the stroke using neurological deficit score, open field, and corner turn test over a 28 d period. ASIC1a knock-out and inhibited mice showed improved neurological scores more quickly than wild-type control and vehicle-injected mice after the stroke. ASIC1a knock-out mice also recovered from mobility deficits in the open field test more quickly than wild-type mice, while PcTX1-injected mice did not experience significant mobility deficits at all after the stroke. In contrast to vehicle-injected mice that showed clear-sidedness bias in the corner turn test after stroke, PcTX1-injected mice never experienced significant-sidedness bias at all. This study supports and extends previous work demonstrating ASIC1a as a potential therapeutic target for the treatment of ischemic stroke.

Using immune clusters for classifying Mycobacterium tuberculosis infection.

BACKGROUND: The differential diagnosis between active tuberculosis (ATB) and latent tuberculosis infection (LTBI) is still a challenge worldwide. METHODS: Immune indicators involved in innate, humoral, and cellular immune cells, as well as antigen-specific cells were simultaneously assessed in patients with ATB and LTBI. RESULTS: Of 54 immune indicators, no indicator could distinguish ATB from LTBI, likely due to an obvious heterogeneity of immune indicators noticed in ATB patients. Cluster analysis of ATB patients identified three immune clusters with different severity. Cluster 1 (42.1 %) was a ''Treg/Th1/Tfh unbalance type" cluster, whereas cluster 2 (42.1 %) was an "effector type'' cluster, and cluster 3 was a ''inhibition type'' cluster (15.8 %) which showed the highest severity. A prediction model based on immune indicators was established and showed potential in classifying Mycobacterium tuberculosis infection. CONCLUSIONS: We depicted the immune landscape of patients with ATB and LTBI. Three immune subtypes were identified in ATB patients with different severity.

Mechanism of ShuiJingDan in Treating Acute Gouty Arthritis Flares Based on Network Pharmacology and Molecular Docking.

Purpose: This study examined the underlying mechanisms of SJD's anti-inflammatory and analgesic effects on acute GA flares. Methods: This study used pharmacology network and molecular docking methods. The active ingredients of ShuiJingDan (SJD) were obtained from the Traditional Chinese Medicine Systems Pharmacology Analysis Platform (TCMSP), and the relevant targets of GA were obtained from the Online Mendelian Inheritance in Man (OMIM) database and Therapeutic Target Database (TTD). The core drug group-target-disease Venn diagram was formed by crossing the active ingredients of SJD and the relevant targets. Gene Ontology (GO) analysis was conducted for functional annotation, DAVID was used for Kyoto Encyclopedia of Genes, and Genomes pathway enrichment analysis, and R was used to find the core targets. The accuracy of SJD network pharmacology analysis in GA treatment was verified by molecular docking simulations. Finally, a rat GA model was used to further verify the anti-inflammatory mechanism of SJD in the treatment of GA. Results: SJD mainly acted on target genes including IL1B, PTGS2, CXCL8, EGF, and JUN, as well as signal pathways including NF-κB, Toll-like receptor (TLR), IL-17, and MAPK. The rat experiments showed that SJD could significantly relieve ankle swelling, reduce the local skin temperature, and increased the paw withdrawal threshold. SJD could also reduce synovial inflammation, reduced the concentrations of interleukin-1ß (IL-1ß), IL-8, and COX-2 in the synovial fluid, and suppressed the expression of IL1B, CXCL8, and PTGS2 mRNA in the synovial tissue. Conclusion: SJD has a good anti-inflammatory effect to treat GA attacks, by acting on target genes such as IL-1ß, PTGS2, and CXCL8.

Inhibition of Acid-Sensing Ion Channels by KB-R7943, a Reverse Na+/Ca2+ Exchanger Inhibitor.

KB-R7943, an isothiourea derivative, is widely used as a pharmacological inhibitor of reverse sodium-calcium exchanger (NCX). It has been shown to have neuroprotective and analgesic effects in animal models; however, the detailed molecular mechanisms remain elusive. In the current study, we investigated whether KB-R7943 modulates acid-sensing ion channels (ASICs), a group of proton-gated cation channels implicated in the pathophysiology of various neurological disorders, using the whole-cell patch clamp techniques. Our data show that KB-R7943 irreversibly inhibits homomeric ASIC1a channels heterologously expressed in Chinese Hamster Ovary (CHO) cells in a use- and concentration-dependent manner. It also reversibly inhibits homomeric ASIC2a and ASIC3 channels in CHO cells. Both the transient and sustained current components of ASIC3 are inhibited. Furthermore, KB-R7943 inhibits ASICs in primary cultured peripheral and central neurons. It inhibits the ASIC-like currents in mouse dorsal root ganglion (DRG) neurons and the ASIC1a-like currents in mouse cortical neurons. The inhibition of the ASIC1a-like current is use-dependent and unrelated to its effect on NCX since neither of the other two well-characterized NCX inhibitors, including SEA0400 and SN-6, shows an effect on ASIC. Our data also suggest that the isothiourea group, which is lacking in other structurally related analogs that do not affect ASIC1a-like current, may serve as a critical functional group. In summary, we characterize KB-R7943 as a new ASIC inhibitor. It provides a novel pharmacological tool for the investigation of the functions of ASICs and could serve as a lead compound for developing small-molecule drugs for treating ASIC-related disorders.

Changes in NMDA Receptor Function in Rapid Ischemic Tolerance: A Potential Role for Tri-Heteromeric NMDA Receptors.

In this study, we characterize biophysical changes in NMDA receptor function in response to brief non-injurious ischemic stress (ischemic preconditioning). Electrophysiological studies show NMDA receptor function is reduced following preconditioning in cultured rat cortical neurons. This functional change is not due to changes in the reversal potential of the receptor, but an increase in desensitization. We performed concentration-response analysis of NMDA-evoked currents, and demonstrate that preconditioned neurons show a reduced potency of NMDA to evoke currents, an increase in Mg2+ sensitivity, but no change in glycine sensitivity. Antagonists studies show a reduced inhibition of GluN2B antagonists that have an allosteric mode of action (ifenprodil and R-25-6981), but competitive antagonists at the GluR2A and 2B receptor (NVP-AMM077 and conantokin-G) appear to have similar potency to block currents. Biochemical studies show a reduction in membrane surface GluN2B subunits, and an increased co-immunoprecipitation of GluN2A with GluN2B subunits, suggestive of tri-heteromeric receptor formation. Finally, we show that blocking actin remodeling with jasplakinolide, a mechanism of rapid ischemic tolerance, prevents NMDA receptor functional changes and co-immunoprecipitation of GluN2A and 2B subunits. Together, this study shows that alterations in NMDA receptor function following preconditioning ischemia are associated with neuroprotection in rapid ischemic tolerance.

Ultrasonography-Guided Combination with Elbow Arthrography-Assisted Minimally Invasive Treatment of Radial Neck Fractures in Young Children.

Background: A radius neck fracture in children is a common fracture that not only affects the growth and development of children but also has a certain impact on the function of children's elbow joints. Objective: To probe into the application value of ultrasonography- (US-) guided combination with elbow arthrography in the minimally invasive treatment of radial neck fractures in young children, summarize its clinical effect and provide a minimally invasive, safe, effective, and reliable method for treating radial neck fractures in young children. Methods: Seventy-three patients with type III or IV radial neck fractures were treated from June 2013 to December 2020 and were divided into the Métaizeau group (n = 31, treatment group) and Kirschner wire (k-wire) k-wire group (n = 42, control group). The Métaizeau group was given US-guided combination with elbow arthrography-assisted modified Métaizeau technique, the k-wire group received open reduction and internal fixation with k-wire and compared the surgical effect of the two groups. Results: In comparison with the k-wire group, time of operation, intraoperative bleeding volume, and hospital stay were signally junior to those in the Métaizeau group (P < 0.05). After surgery, in comparison with the k-wire group, the number of degrees to contralateral flexion or forearm rotation was visually lower in the Métaizeau group (P < 0.05), and postoperative complication incidence in the Métaizeau group was visually lower than that in k-wire group (P < 0.05). Conclusion: In the minimally invasive treatment of radial neck fractures, US-guided combination with elbow arthrography in young children has better efficacy and high safety. It can be widely promoted and applied clinically.

pH and proton-sensitive receptors in brain ischemia.

Extracellular proton concentration is at 40 nM when pH is 7.4. In disease conditions such as brain ischemia, proton concentration can reach µM range. To respond to this increase in extracellular proton concentration, the mammalian brain expresses at least three classes of proton receptors. Acid-sensing ion channels (ASICs) are the main neuronal cationic proton receptor. The proton-activated chloride channel (PAC), which is also known as (aka) acid-sensitive outwardly rectifying anion channel (ASOR; TMEM206), mediates acid-induced chloride currents. Besides proton-activated channels, GPR4, GPR65 (aka TDAG8, T-cell death-associated gene 8), and GPR68 (aka OGR1, ovarian cancer G protein-coupled receptor 1) function as proton-sensitive G protein-coupled receptors (GPCRs). Though earlier studies on these GPCRs mainly focus on peripheral cells, we and others have recently provided evidence for their functional importance in brain injury. Specifically, GPR4 shows strong expression in brain endothelium, GPR65 is present in a fraction of microglia, while GPR68 exhibits predominant expression in brain neurons. Here, to get a better view of brain acid signaling and its contribution to ischemic injury, we will review the recent findings regarding the differential contribution of proton-sensitive GPCRs to cerebrovascular function, neuroinflammation, and neuronal injury following acidosis and brain ischemia.

Diagnostic Model for Discrimination Between Tuberculous Meningitis and Bacterial Meningitis.

Background: The differential diagnosis between tuberculous meningitis (TBM) and bacterial meningitis (BM) remains challenging in clinical practice. This study aimed to establish a diagnostic model that could accurately distinguish TBM from BM. Methods: Patients with TBM or BM were recruited between January 2017 and January 2021 at Tongji Hospital (Qiaokou cohort) and Sino-French New City Hospital (Caidian cohort). The detection for indicators involved in cerebrospinal fluid (CSF) and T-SPOT assay were performed simultaneously. Multivariate logistic regression was used to create a diagnostic model. Results: A total of 174 patients (76 TBM and 98 BM) and another 105 cases (39 TBM and 66 BM) were enrolled from Qiaokou cohort and Caidian cohort, respectively. Significantly higher level of CSF lymphocyte proportion while significantly lower levels of CSF chlorine, nucleated cell count, and neutrophil proportion were observed in TBM group when comparing with those in BM group. However, receiver operating characteristic (ROC) curve analysis showed that the areas under the ROC curve (AUCs) produced by these indicators were all under 0.8. Meanwhile, tuberculosis-specific antigen/phytohemagglutinin (TBAg/PHA) ratio yielded an AUC of 0.889 (95% CI, 0.840-0.938) in distinguishing TBM from BM, with a sensitivity of 68.42% (95% CI, 57.30%-77.77%) and a specificity of 92.86% (95% CI, 85.98%-96.50%) when a cutoff value of 0.163 was used. Consequently, we successfully established a diagnostic model based on the combination of TBAg/PHA ratio, CSF chlorine, CSF nucleated cell count, and CSF lymphocyte proportion for discrimination between TBM and BM. The established model showed good performance in differentiating TBM from BM (AUC: 0.949; 95% CI, 0.921-0.978), with 81.58% (95% CI, 71.42%-88.70%) sensitivity and 91.84% (95% CI, 84.71%-95.81%) specificity. The performance of the diagnostic model obtained in Qiaokou cohort was further validated in Caidian cohort. The diagnostic model in Caidian cohort produced an AUC of 0.923 (95% CI, 0.867-0.980) with 79.49% (95% CI, 64.47%-89.22%) sensitivity and 90.91% (95% CI, 81.55%-95.77%) specificity. Conclusions: The diagnostic model established based on the combination of four indicators had excellent utility in the discrimination between TBM and BM.

The Dynamic Immunological Parameter Landscape in Coronavirus Disease 2019 Patients With Different Outcomes.

Objectives: The longitudinal and systematic evaluation of immunity in coronavirus disease 2019 (COVID-19) patients is rarely reported. Methods: Parameters involved in innate, adaptive, and humoral immunity were continuously monitored in COVID-19 patients from onset of illness until 45 days after symptom onset. Results: This study enrolled 27 mild, 47 severe, and 46 deceased COVID-19 patients. Generally, deceased patients demonstrated a gradual increase of neutrophils and IL-6 but a decrease of lymphocytes and platelets after the onset of illness. Specifically, sustained low numbers of CD8+ T cells, NK cells, and dendritic cells were noted in deceased patients, while these cells gradually restored in mild and severe patients. Furthermore, deceased patients displayed a rapid increase of HLA-DR expression on CD4+ T cells in the early phase, but with a low level of overall CD45RO and HLA-DR expressions on CD4+ and CD8+ T cells, respectively. Notably, in the early phase, deceased patients showed a lower level of plasma cells and antigen-specific IgG, but higher expansion of CD16+CD14+ proinflammatory monocytes and HLA-DR-CD14+ monocytic-myeloid-derived suppressor cells (M-MDSCs) than mild or severe patients. Among these immunological parameters, M-MDSCs showed the best performance in predicting COVID-19 mortality, when using a cutoff value of ≥10%. Cluster analysis found a typical immunological pattern in deceased patients on day 9 after onset, which was characterized as the increase of inflammatory markers (M-MDSCs, neutrophils, CD16+CD14+ monocytes, and IL-6) but a decrease of host immunity markers. Conclusions: This study systemically characterizes the kinetics of immunity of COVID-19, highlighting the importance of immunity in patient prognosis.

Molecular Analysis and Antimicrobial Resistance Pattern of Tigecycline-Non-Susceptible K. pneumoniae Isolated from a Tertiary Care Hospital of East Asia.

INTRODUCTION: Tigecycline is one of the last resorts for carbapenem-resistant K. pneumoniae (CRKP) infections. Indeed, tigecycline-non-susceptible K. pneumoniae (TNSKP) strains are increasingly treated with the use of tigecycline. In this study, we attempted to better understand their epidemiological trends and characteristics. K. pneumoniae were collected from 2017 to 2020 at the First Affiliated Hospital of Nanchang University. METHODS: Thirty-four TNSKP strains were selected during the study period, all of which were analyzed using antimicrobial susceptibility testing, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). PCR and DNA sequencing were performed for the detection of ß-lactamase genes and carbapenemase genes, and the mutation analysis of tet(A), tet(X), tet(L), tet(M), rpsJ, ramR, and oqxR, which are related to tigecycline resistance. Virulence gene and capsular genotype testing were conducted to identify whether the TNSKP strains were hypervirulent Klebsiella pneumoniae. RESULTS: An epidemiology analysis showed that Klebsiella pneumoniae carbapenemase-2 (KPC-2) was the predominant carbapenemase in tigecycline non-susceptible carbapenem-resistant K. pneumoniae (TNSCRKP) (96.7%), and the dominant clone type was ST11-K14K64 (82.4%). Among them, 55.9% (19/34) of strains were from each department of ICU, particularly EICU and neurosurgery ICU. In order to further understand the molecular mechanisms of the TNSKP, a polymerase chain reaction of the resistant determinants was carried out. The results detected many tigecycline-resistant genes, such as tet(A) (97.1%), tet(X) (17.6%), rpsJ (97.1%), and ramR (8.8%). CONCLUSION: As the results of this study reveal, we should take effective measures to control the increase in TNSKP.

Immunologic memory to SARS-CoV-2 in convalescent COVID-19 patients at 1 year postinfection.

BACKGROUND: Understanding the complexities of immune memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key to gain insights into the durability of protective immunity against reinfection. OBJECTIVE: We sought to evaluate the immune memory to SARS-CoV-2 in convalescent patients with longer follow-up time. METHODS: SARS-CoV-2-specific humoral and cellular responses were assessed in convalescent patients with coronavirus disease 2019 (COVID-19) at 1 year postinfection. RESULTS: A total of 78 convalescent patients with COVID-19 (26 moderate, 43 severe, and 9 critical) were recruited after 1 year of recovery. The positive rates of both anti-receptor-binding domain and antinucleocapsid antibodies were 100%, whereas we did not observe a statistical difference in antibody levels among different severity groups. Accordingly, the prevalence of neutralizing antibodies (nAbs) reached 93.59% in convalescent patients. Although nAb titers displayed an increasing trend in convalescent patients with increased severity, the difference failed to achieve statistical significance. Notably, there was a significant correlation between nAb titers and anti-receptor-binding domain levels. Interestingly, SARS-CoV-2-specific T cells could be robustly maintained in convalescent patients, and their number was positively correlated with both nAb titers and anti-receptor-binding domain levels. Amplified SARS-CoV-2-specific CD4+ T cells mainly produced a single cytokine, accompanying with increased expression of exhaustion markers including PD-1, Tim-3, TIGIT, CTLA-4, and CD39, while the proportion of multifunctional cells was low. CONCLUSIONS: Robust SARS-CoV-2-specific humoral and cellular responses are maintained in convalescent patients with COVID-19 at 1 year postinfection. However, the dysfunction of SARS-CoV-2-specific CD4+ T cells supports the notion that vaccination is needed in convalescent patients for preventing reinfection.

Acid-sensing ion channel 1 (ASIC1) mediates weak acid-induced migration of human malignant glioma cells.

Glioblastoma is the most aggressive and lethal tumor in the central nervous system in adult and has poor prognosis due to strong proliferation and aggressive invasion capacity. Acidic microenvironment is commonly observed in tumor tissues but the exact role of acidosis in the pathophysiology of glioblastoma and underlying mechanisms remain unclear. Acid-sensing ion channels (ASICs) are proton-gated cation channels activated by low extracellular pH. Recent studies have suggested that ASICs are involved in the pathogenesis of some tumors, such as lung cancer and breast cancer. But the effect of acidosis and activation of ASICs on malignant glioma of the central nervous system has not been reported. In this study, we investigated the expression of ASIC1 in human glioma cell lines (U87MG and A172) and its possible effect on the proliferation and migration of these cells. The results demonstrated that ASIC1 is functionally expressed in U87MG and A172 cells. Treatment with extracellular weak acid (pH 7.0) has no effect on the proliferation but increases the migration of the two cell lines. Application of PcTX1, a specific inhibitor of ASIC1a and ASIC1a/2b channels, or knocking down ASIC1 by siRNA, can abolish the effect of weak acid-induced cell migration. Together, our results indicate that ASIC1 mediates extracellular weak acid induced migration of human malignant glioma cells and may therefore serve as a therapeutic target for malignant glioma in human.

Upregulation of acid sensing ion channel 1a (ASIC1a) by hydrogen peroxide through the JNK pathway.

Oxidative stress is intimately tied to neurodegenerative diseases, including Parkinson's disease and amyotrophic lateral sclerosis, and acute injuries, such as ischemic stroke and traumatic brain injury. Acid sensing ion channel 1a (ASIC1a), a proton-gated ion channel, has been shown to be involved in the pathogenesis of these diseases. However, whether oxidative stress affects the expression of ASIC1a remains elusive. In the current study, we examined the effect of hydrogen peroxide (H2O2), a major reactive oxygen species (ROS), on ASIC1a protein expression and channel function in NS20Y cells and primary cultured mouse cortical neurons. We found that treatment of the cells with H2O2 (20 µM) for 6 h or longer increased ASIC1a protein expression and ASIC currents without causing significant cell injury. H2O2 incubation activated mitogen-activated protein kinases (MAPKs) pathways, including the extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 pathways. We found that neither inhibition of the MEK/ERK pathway by U0126 nor inhibition of the p38 pathway by SB203580 affected H2O2-induced ASIC1a expression, whereas inhibition of the JNK pathway by SP600125 potently decreased ASIC1a expression and abolished the H2O2-mediated increase in ASIC1a expression and ASIC currents. Furthermore, we found that H2O2 pretreatment increased the sensitivity of ASIC currents to the ASIC1a inhibitor PcTx1, providing additional evidence that H2O2 increases the expression of functional ASIC1a channels. Together, our data demonstrate that H2O2 increases ASIC1a expression/activation through the JNK signaling pathway, which may provide insight into the pathogenesis of neurological disorders that involve both ROS and activation of ASIC1a.

Novel insights into ferroptosis: Implications for age-related diseases.

Rapid increase in aging populations is an urgent problem because older adults are more likely to suffer from disabilities and age-related diseases (ARDs), burdening healthcare systems and society in general. ARDs are characterized by the progressive deterioration of tissues and organs over time, eventually leading to tissue and organ failure. To date, there are no effective interventions to prevent the progression of ARDs. Hence, there is an urgent need for new treatment strategies. Ferroptosis, an iron-dependent cell death, is linked to normal development and homeostasis. Accumulating evidence, however, has highlighted crucial roles for ferroptosis in ARDs, including neurodegenerative and cardiovascular diseases. In this review, we a) summarize initiation, regulatory mechanisms, and molecular signaling pathways involved in ferroptosis, b) discuss the direct and indirect involvement of the activation and/or inhibition of ferroptosis in the pathogenesis of some important diseases, and c) highlight therapeutic targets relevant for ARDs.

GPR68 Is a Neuroprotective Proton Receptor in Brain Ischemia.

BACKGROUND AND PURPOSE: Brain acidosis is prevalent in stroke and other neurological diseases. Acidosis can have paradoxical injurious and protective effects. The purpose of this study is to determine whether a proton receptor exists in neurons to counteract acidosis-induced injury. METHODS: We analyzed the expression of proton-sensitive GPCRs (G protein-coupled receptors) in the brain, examined acidosis-induced signaling in vitro, and studied neuronal injury using in vitro and in vivo mouse models. RESULTS: GPR68, a proton-sensitive GPCR, was present in both mouse and human brain, and elicited neuroprotection in acidotic and ischemic conditions. GPR68 exhibited wide expression in brain neurons and mediated acidosis-induced PKC (protein kinase C) activation. PKC inhibition exacerbated pH 6-induced neuronal injury in a GPR68-dependent manner. Consistent with its neuroprotective function, GPR68 overexpression alleviated middle cerebral artery occlusion-induced brain injury. CONCLUSIONS: These data expand our knowledge on neuronal acid signaling to include a neuroprotective metabotropic dimension and offer GPR68 as a novel therapeutic target to alleviate neuronal injuries in ischemia and multiple other neurological diseases.

Protein Kinase C Regulates ASIC1a Protein Expression and Channel Function via NF-kB Signaling Pathway.

Tissue acidosis is a common feature in many pathological conditions. Activation of acid-sensing ion channel 1a (ASIC1a) plays a key role in acidosis-mediated neurotoxicity. Protein kinase C (PKC) activity has been proved to be associated with many physiological processes and pathological conditions; however, whether PKC activation regulates ASIC1a protein expression and channel function remains ill defined. In this study, we demonstrated that treatment with phorbol 12-myristate 13-acetate (PMA, a PKC activator) for 6 h significantly increased ASIC1a protein expression and ASIC currents in NS20Y cells, a neuronal cell line, and in primary cultured mouse cortical neurons. In contrast, treatment with Calphostin C (a nonselective PKC inhibitor) for 6 h or longer decreased ASIC1a protein expression and ASIC currents. Similar to Calphostin C, PKC α and ßI inhibitor Go6976 exposure also reduced ASIC1a protein expression. The reduction in ASIC1a protein expression by PKC inhibition involves a change in ASIC1a protein degradation, which is mediated by ubiquitin-proteasome system (UPS)-dependent degradation pathway. In addition, we showed that PKC regulation of ASIC1a protein expression involves NF-κB signaling pathway. Consistent with their effects on ASIC1a protein expression and channel function, PKC inhibition protected NS20Y cells against acidosis-induced cytotoxicity, while PKC activation potentiated acidosis-induced cells injury. Together, these results indicate that ASIC1a protein expression and channel function are closely regulated by the activity of protein kinase C and its downstream signaling pathway(s).

The TRPM7 Channel in the Nervous and Cardiovascular Systems.

Transient receptor potential melastatin 7 (TRPM7), along with the closely related TRPM6, are unique channels that have dual operations: cation permeability and kinase activity. In contrast to the limited tissue distribution of TRPM6, TRPM7 is widely expressed among tissues and is therefore implicated in a variety of cellular functions physiologically and pathophysiologically. The discovery of TRPM7's unique structure imparting dual ion channel and kinase activities shed light onto novel and peculiar biological functions, such as Mg2+ homeostasis, cellular Ca2+ flickering, and even intranuclear transcriptional regulation by a cleaved kinase domain translocated to nuclei. Interestingly, at a higher level, TRPM7 participates in several biological processes in the nervous and cardiovascular systems, in which excitatory responses in neurons and cardiomyocytes are critical for their function. Here, we review the roles of TRPM7 in cells involved in the nervous and cardiovascular systems and discuss its potential as a future therapeutic target.