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.

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.

Differential regulation of H3K9/H3K14 acetylation by small molecules drives neuron-fate-induction of glioma cell.

Differentiation therapy using small molecules is a promising strategy for improving the prognosis of glioblastoma (GBM). Histone acetylation plays an important role in cell fate determination. Nevertheless, whether histone acetylation in specific sites determines GBM cells fate remains to be explored. Through screening from a 349 small molecule-library, we identified that histone deacetylase inhibitor (HDACi) MS-275 synergized with 8-CPT-cAMP was able to transdifferentiate U87MG GBM cells into neuron-like cells, which were characterized by cell cycle arrest, rich neuron biomarkers, and typical neuron electrophysiology. Intriguingly, acetylation tags of histone 3 at lysine 9 (H3K9ac) were decreased in the promoter of multiple oncogenes and cell cycle genes, while ones of H3K9ac and histone 3 at lysine 14 (H3K14ac) were increased in the promoter of neuron-specific genes. We then compiled a list of genes controlled by H3K9ac and H3K14ac, and proved that it is a good predictive power for pathologic grading and survival prediction. Moreover, cAMP agonist combined with HDACi also induced glioma stem cells (GSCs) to differentiate into neuron-like cells through the regulation of H3K9ac/K14ac, indicating that combined induction has the potential for recurrence-preventive application. Furthermore, the combination of cAMP activator plus HDACi significantly repressed the tumor growth in a subcutaneous GSC-derived tumor model, and temozolomide cooperated with the differentiation-inducing combination to prolong the survival in an orthotopic GSC-derived tumor model. These findings highlight epigenetic reprogramming through H3K9ac and H3K14ac as a novel approach for driving neuron-fate-induction of GBM cells.

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.

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.

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).

ß-Estradiol Protects Against Acidosis-Mediated and Ischemic Neuronal Injury by Promoting ASIC1a (Acid-Sensing Ion Channel 1a) Protein Degradation.

Background and Purpose- Sex differences in the incidence and outcome of stroke have been well documented. The severity of stroke in women is, in general, significantly lower than that in men, which is mediated, at least in part, by the protective effects of ß-estradiol. However, the detailed mechanisms underlying the neuroprotection by ß-estradiol are still elusive. Recent studies have demonstrated that activation of ASIC1a (acid-sensing ion channel 1a) by tissue acidosis, a common feature of brain ischemia, plays an important role in ischemic brain injury. In the present study, we assessed the effects of ß-estradiol on acidosis-mediated and ischemic neuronal injury both in vitro and in vivo and explored the involvement of ASIC1a and underlying mechanism. Methods- Cultured neurons and NS20Y cells were subjected to acidosis-mediated injury in vitro. Cell viability and cytotoxicity were measured by methylthiazolyldiphenyl-tetrazolium bromide and lactate dehydrogenase assays, respectively. Transient (60 minutes) focal ischemia in mice was induced by suture occlusion of the middle cerebral artery in vivo. ASIC currents were recorded using whole-cell patch-clamp technique while intracellular Ca2+ concentration was measured with fluorescence imaging using Fura-2. ASIC1a expression was detected by Western blotting and quantitative real-time polymerase chain reaction. Results- Treatment of neuronal cells with ß-estradiol decreased acidosis-induced cytotoxicity. ASIC currents and acid-induced elevation of intracellular Ca2+ were all attenuated by ß-estradiol treatment. In addition, we showed that ß-estradiol treatment reduced ASIC1a protein expression, which was mediated by increased protein degradation, and that estrogen receptor α was involved. Finally, we showed that the level of ASIC1a protein expression in brain tissues and the degree of neuroprotection by ASIC1a blockade were lower in female mice, which could be attenuated by ovariectomy. Conclusions- ß-estradiol can protect neurons against acidosis-mediated neurotoxicity and ischemic brain injury by suppressing ASIC1a protein expression and channel function. Visual Overview- An online visual overview is available for this article.

Treatment for ischemic stroke: From thrombolysis to thrombectomy and remaining challenges.

Stroke is a leading cause of death and long-term disabilities. Despite decades of extensive efforts in search of brain injury mechanisms and therapeutic interventions, pharmacological treatment is limited to the use of thrombolytic agent tissue plasminogen activator, which has limited therapeutic time window and potential side effect of intracranial hemorrhage. Over the past few years, endovascular thrombectomy with stent-retriever devices combined with advanced imaging modalities has transformed the standard of stroke care, offering an opportunity to improve the outcome in selected patients as late as 24 h after the onset of stroke. This mini-review summarizes the advancement in the treatment of ischemic stroke, from thrombolysis to thrombectomy and remaining challenges in the field.

Acute Ethanol Exposure Promotes Autophagy-Lysosome Pathway-Dependent ASIC1a Protein Degradation and Protects Against Acidosis-Induced Neurotoxicity.

Tissue acidosis is a common feature of brain ischemia which causes neuronal injury. Activation of acid-sensing ion channel 1a (ASIC1a) plays an important role in acidosis-mediated neurotoxicity. Acute ethanol administration has been shown to provide neuroprotective effects during ischemic stroke, but the precise mechanisms have yet to be determined. In this study, we investigated the effect of ethanol on the activity/expression of ASIC1a channels and acidosis-induced neurotoxicity. We showed that acute treatment of neuronal cells with ethanol for more than 3 h could reduce ASIC1a protein expression, ASIC currents, and acid-induced [Ca2+]i elevation. We further demonstrated that ethanol-induced reduction of ASIC1a expression is mediated by autophagy-lysosome pathway (ALP)-dependent protein degradation. Finally, we showed that ethanol protected neuronal cells against acidosis-induced cytotoxicity, which effect was mimicked by autophagy activator rapamycin and abolished by autophagy inhibitor CQ. Together, these results indicate that moderate acute ethanol exposure can promote autophagy-lysosome pathway-dependent ASIC1a protein degradation and protect against acidosis-induced neurotoxicity.

TRPM7 channels play a role in high glucose-induced endoplasmic reticulum stress and neuronal cell apoptosis.

High-glucose (HG) levels and hyperglycemia associated with diabetes are known to cause neuronal damage. The detailed molecular mechanisms, however, remain to be elucidated. Here, we investigated the role of transient receptor potential melastatin 7 (TRPM7) channels in HG-mediated endoplasmic reticulum stress (ERS) and injury of NS20Y neuronal cells. The cells were incubated in the absence or presence of HG for 48 h. We found that mRNA and protein levels of TRPM7 and of ERS-associated proteins, such as C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (GRP78), and inducible nitric-oxide synthase (iNOS), increased in HG-treated cells, along with significantly increased TRPM7-associated currents in these cells. Similar results were obtained in cerebral cortical tissue from an insulin-deficiency model of diabetic mice. Moreover, HG treatment of cells activated ERS-associated proapoptotic caspase activity and induced cellular injury. Interestingly, a NOS inhibitor, l-NAME, suppressed the HG-induced increase of TRPM7 expression and cellular injury. siRNA-mediated TRPM7 knockdown or chemical inhibition of TRPM7 activity also suppressed HG-induced ERS and decreased cleaved caspase-12/caspase-3 levels and cell injury. Of note, TRPM7 overexpression increased ERS and cell injury independently of its kinase activity. Taken together, our findings suggest that TRPM7 channel activities play a key role in HG-associated ERS and cytotoxicity through an apoptosis-inducing signaling cascade involving HG, iNOS, TRPM7, ERS proteins, and caspases.

Cholestane-3ß, 5α, 6ß-triol suppresses neuronal hyperexcitability via binding to voltage-gated sodium channels.

Neuronal hyperexcitability is identified as a critical pathological basis of epileptic seizures. Cholestane-3ß, 5α, 6ß-triol (Triol) is a major metabolic oxysterol of cholesterol. Although its neuroprotective effect on ischemia-induced neuronal injury and negative modulation of voltage-gated sodium (Nav) channels were well established, the physical binding site of triol to sodium channels and its effects on neuronal hyperexcitability have not yet been explored. In this study, we utilized molecular docking and molecular dynamics simulation to investigate the interaction between triol and Nav Channels. Our results demonstrated that triol binds to the indole ring of Trp122 of the Nav Channel in silico with a high biological affinity. We further found that triol negatively modulates the action potentials bursts of hippocampal neurons by cell-attached patch recording. Moreover, triol significantly inhibits low Mg2+-induced hyperexcitability in vitro. In addition, triol attenuates pentylenetetrazole (PTZ)-induced convulsive-form behavioral deficits in vivo. Together, our results suggest that triol suppresses neuronal hyperexcitability via binding to Nav channel, indicating that triol might be an attractive lead compound for the treatment of neuronal hyperexcitability-related neurological disorders, especially epileptic seizures.

Lidocaine suppresses glioma cell proliferation by inhibiting TRPM7 channels.

BACKGROUND: Malignant glioma is the most common brain cancer with devastating prognosis. Recurrence of malignant glioma following surgery is very common with few preventive and therapeutic options. Novel targets and therapeutic agents are constantly sought for better outcome. Our previous study established that inhibition of transient receptor potential melastatin 7 (TRPM7) channels resulted in significant decrease of human glioma cell growth and proliferation. As local anesthetic lidocaine has been shown to inhibit TRPM7 currents, we hypothesize that lidocaine may suppress glioma cell proliferation through TRPM7 channel inhibition. METHODS: TRPM7 currents were recorded in rat C6 glioma cells using the whole cell patch clamp technique. Cell growth and proliferation were assessed under microscopic examination and biochemical assays. RESULTS: Lidocaine inhibits TRPM7-like currents in a dose-dependent and reversible manner. At 1 and 3 mM, it inhibits ~30% and ~50% of TRPM7 currents. At these concentrations, it is effective in inhibiting the proliferation of C6 cells. As expected, the TRPM7 inhibitors gadolinium and 2-Aminoethoxydiphenyl borate have similar effects on TRPM7 currents and proliferation of C6 cells. Similar to its effect on C6 cells, lidocaine inhibits the proliferation of A172 cells, a human glioblastoma cell line. CONCLUSIONS: Lidocaine significantly inhibits the proliferation of glioma cells. The effect of lidocaine is mediated, at least in part, by inhibiting TRPM7 channels.

Neuroprotectant androst-3ß, 5α, 6ß-triol suppresses TNF-α-induced endothelial adhesion molecules expression and neutrophil adhesion to endothelial cells by attenuation of CYLD-NF-κB pathway.

Neuroinflammation is one of key pathologic element in neurological diseases including stroke, traumatic brain injury, Alzheimer' s Disease, Parkinson's Disease, and multiple sclerosis as well. Up-regulation of endothelial adhesion molecules, which facilitate leukocyte adhesion to the endothelium, is the vital process of endothelial cells mediated neuroinflammation. Androst-3ß, 5α, 6ß-triol (Triol) is a synthetic steroid which has been reported to have neuroprotective effects in hypoxia/re-oxygenation-induced neuronal injury model. In the present study, we firstly investigated whether Triol inhibited the TNF-α-induced inflammatory response in rat brain microvascular endothelial cells (RBMECs). Our data showed that Triol decreased TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and the adhesion of neutrophil to RBMECs. We also found that Triol inhibited TNF-α-induced degradation of IκBα and phosphorylation of NF-κBp65 that are required for NF-κB activation. Furthermore, Triol significantly reversed TNF-α-induced down-expression of CYLD, which is a deubiquitinase that negatively regulates activation of NF-κB. These results suggest that Triol displays an anti-inflammatory effect on TNF-α-induced RBMECs via downregulating of CYLD-NF-κB signaling pathways and might have a potential benefit in therapeutic neuroinflammation related diseases.

Region specific contribution of ASIC2 to acidosis-and ischemia-induced neuronal injury.

Acidosis in the brain plays a critical role in neuronal injury in neurological diseases, including brain ischemia. One key mediator of acidosis-induced neuronal injury is the acid-sensing ion channels (ASICs). Current literature has focused on ASIC1a when studying acid signaling. The importance of ASIC2, which is also widely expressed in the brain, has not been appreciated. We found here a region-specific effect of ASIC2 on acid-mediated responses. Deleting ASIC2 reduced acid-activated current in cortical and striatal neurons, but had no significant effect in cerebellar granule neurons. In addition, we demonstrated that ASIC2 was important for ASIC1a expression, and that ASIC2a but not 2b facilitated ASIC1a surface trafficking in the brain. Further, we showed that ASIC2 deletion attenuated acidosis/ischemia-induced neuronal injury in organotypic hippocampal slices but had no effect in organotypic cerebellar slices. Consistent with an injurious role of ASIC2, we showed that ASIC2 deletion significantly protected the mouse brain from ischemic damage in vivo. These data suggest a critical region-specific contribution of ASIC2 to neuronal injury and reveal an important functional difference between ASIC2a and 2b in the brain.

ASIC1 promotes differentiation of neuroblastoma by negatively regulating Notch signaling pathway.

In neurons, up-regulation of Notch activity either inhibits neurite extension or causes retraction of neurites. Conversely, inhibition of Notch1 facilitates neurite extension. Acid-sensing ion channels (ASICs) are a family of proton-gated cation channels, which play critical roles in synaptic plasticity, learning and memory and spine morphogenesis. Our pilot proteomics data from ASIC1a knock out mice implicated that ASIC1a may play a role in regulating Notch signaling, therefore, we explored whether or not ASIC1a regulates neurite growth during neuronal development through Notch signaling. In this study, we determined the effects of ASIC1a on neurite growth in a mouse neuroblastoma cell line, NS20Y cells, by modulating ASIC1a expression. We also determined the relationship between ASIC1a and Notch signaling on neuronal differentiation. Our results showed that down-regulation of ASIC1a in NS20Y cells inhibits CPT-cAMP induced neurite growth, while over expression of ASIC1a promotes its growth. In addition, down-regulation of ASIC1a increased the expression of Notch1 and its target gene Survivin while inhibitor of Notch significantly prevented the neurite extension induced by ASIC1a in NS20Y cells. These data indicate that Notch1 signaling may be required for ASIC1a-mediated neurite growth and neuronal differentiation.

Effect of Redox-Modifying Agents on the Activity of Channelrhodopsin-2.

BACKGROUND: The algal protein Channelrhodopsin-2 (ChR2) has been widely used in recent years in optogenetic technique to investigate the functions of complex neuronal networks through minimally invasive and temporally precise photostimulation of genetically defined neurons. However, as with any other new technique, current optogentic approaches have various limitations. In addition, how ChR2 may behave in response to complex biochemical changes associated with various physiological/pathological conditions is largely unknown. AIM: In this study, we investigated whether a change in redox state of the cell affects the activity of ChR2 channels. METHODS: Whole-cell patch-clamp recordings were used to examine the effect of reducing and oxidizing agents on ChR2 currents activated by blue light. RESULTS: We show that the reducing agent dithiothreitol (DTT) dramatically potentiates the ChR2 currents in a reversible and concentration-dependent manner. Glutathione, an endogenous reducing agent, shows a similar effect on ChR2 currents. The oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) has no effect on ChR2 currents by itself; however, it completely reverses the potentiating effect of DTT. DTT also causes a shift in the current-voltage relationship by 23 ± 4.31 mV, suggesting a change in ion selectivity. CONCLUSION: Taken together, these data suggest that redox modification of ChR2 plays an important role in its sensitivity to the light stimulation. Our findings not only help for a better understanding of how ChR2 may behave in physiological/pathological conditions where changes in redox state are common, but also provide a new direction for further optimization of this important opsin.

Simultaneous Determination of Seven Neuroactive Steroids Associated with Depression in Rat Plasma and Brain by High Performance Liquid Chromatography-Tandem Mass Spectrometry.

Sensitive and specific biomarkers are required for the diagnosis and treatment of depression because the existing diagnostic criteria are subjective and could produce false positives or negatives. Some endogenous neuroactive steroids that have shown either antidepressant effects or concentration changes in individuals with depression could provide potential biomarkers. In this study, a simple and specific method was developed to simultaneously determine seven endogenous neuroactive steroids in biological samples: cortisone, cortisol, dehydroepiandrosterone, estradiol, progesterone, pregnenolone, and testosterone. After liquid-liquid extraction, chromatographic separation was achieved on a C18 column with gradient elution using water-methanol at a flow rate of 300 µL min(-1). Detection and quantitation were performed by tandem mass spectrometry with atmospheric pressure chemical ionization and selected reaction monitoring. Plasma and brain neuroactive steroid levels were then determined in control rats and rats exposed to forced swimming, a classical rodent model of depression. The results showed that the plasma concentrations of testosterone, pregnenolone, and progesterone significantly increased in rats exposed to the forced swimming test. In contrast, brain homogenate levels of cortisol, estradiol, and progesterone decreased, while pregnenolone levels were elevated in this model of depression. In conclusion, a new method to quantify neuroactive steroids was successfully developed and applied to their investigation in rat plasma and brain. The findings of this study indicated that plasma testosterone, pregnenolone, and progesterone levels could provide potential biomarkers for the diagnosis and treatment of depression.

TRPM7 channel inhibition mediates midazolam-induced proliferation loss in human malignant glioma.

The melastatin-like transient receptor potential 7 (TRPM7) has been implicated in proliferation or apoptosis of some cancers, indicating the potential of TRPM7 as an anti-anaplastic target. Here, we identified the characteristic TRPM7 channel currents in human malignant glioma MGR2 cells, which could be blocked by a pharmacologic inhibitor Gd3+. We mined the clinical sample data from Oncomine Database and found that human malignant glioma tissues expressed higher TRPM7 mRNA than normal brain ones. Importantly, we identified a widely used clinical anesthetic midazolam as a TRPM7 inhibitor. Midazolam treatment for seconds suppressed the TRPM7 currents and calcium influx, and treatment for 48 h inhibited the TRPM7 expression. The inhibitory effect on TRPM7 accounts for the proliferation loss and G0/G1 phase cell cycle arrest induced by midazolam. Our data demonstrates that midazolam represses proliferation of human malignant glioma cells through inhibiting TRPM7 currents, which may be further potentiated by suppressing the expression of TRPM7. Our result indicates midazolam as a pharmacologic lead compound with brain-blood barrier permeability for targeting TRPM7 in the glioma.