Genetic exploration of roles of acid-sensing ion channel subtypes in neurosensory mechanotransduction including proprioception.

Although acid-sensing ion channels (ASICs) are proton-gated ion channels responsible for sensing tissue acidosis, accumulating evidence has shown that ASICs are also involved in neurosensory mechanotransduction. However, in contrast to Piezo ion channels, evidence of ASICs as mechanically gated ion channels has not been found using conventional mechanoclamp approaches. Instead, ASICs are involved in the tether model of mechanotransduction, with the channels gated via tethering elements of extracellular matrix and intracellular cytoskeletons. Methods using substrate deformation-driven neurite stretch and micropipette-guided ultrasound were developed to reveal the roles of ASIC3 and ASIC1a, respectively. Here we summarize the evidence supporting the roles of ASICs in neurosensory mechanotransduction in knockout mouse models of ASIC subtypes and provide insight to further probe their roles in proprioception.

ASIC1a contributes to the epithelial-mesenchymal transformation of breast cancer by activating the Ca2+ /ß-catenin pathway.

Breast cancer is the most common cancer in the world, with metastasis being one of the leading causes of death among patients. The acidic environment of breast cancer tissue promotes tumor cell invasion and migration by inducing epithelial-mesenchymal transformation (EMT) in tumor cells, but the exact mechanisms are not yet fully understood. This study investigated the expression of acid-sensitive ion channel 1a (ASIC1a) in breast cancer tissue samples and explored the mechanisms by which ASIC1a mediates the promotion of EMT in breast cancer cells in an acidic microenvironment through in vivo and in vitro experiments. The results showed that first, the expression of ASIC1a was significantly upregulated in breast cancer tissue and was correlated with the TNM (tumor node metastasis) staging of breast cancer. Furthermore, ASIC1a expression was higher in tumors with lymph node metastasis than in those without. Second, the acidic microenvironment promoted [Ca2+ ]i influx via ASIC1a activation and regulated the expression of ß-catenin, Vimentin, and E-cadherin, thus promoting EMT in breast cancer cells. Inhibition of ASIC1a activation with PcTx-1 could suppress EMT in breast cancer cells. Finally, in vivo studies also showed that inhibition of ASIC1a could reduce breast cancer metastasis, invasion, and EMT. This study suggests that ASIC1a expression is associated with breast cancer staging and metastasis. Therefore, ASIC1a may become a new breast cancer biomarker, and the elucidation of the mechanism by which ASIC1a promotes EMT in breast cancer under acidic microenvironments provides evidence for the use of ASIC1a as a molecular target for breast cancer treatment.

Modulation of TRPV1 and TRPA1 Channels Function by Sea Anemones' Peptides Enhances the Viability of SH-SY5Y Cell Model of Parkinson's Disease.

Cellular dysfunction during Parkinson's disease leads to neuroinflammation in various brain regions, inducing neuronal death and contributing to the progression of the disease. Different ion channels may influence the process of neurodegeneration. The peptides Ms 9a-1 and APHC3 can modulate the function of TRPA1 and TRPV1 channels, and we evaluated their cytoprotective effects in differentiated to dopaminergic neuron-like SH-SY5Y cells. We used the stable neuroblastoma cell lines SH-SY5Y, producing wild-type alpha-synuclein and its mutant A53T, which are prone to accumulation of thioflavin-S-positive aggregates. We analyzed the viability of cells, as well as the mRNA expression levels of TRPA1, TRPV1, ASIC1a channels, alpha-synuclein, and tyrosine hydroxylase after differentiation of these cell lines using RT-PCR. Overexpression of alpha-synuclein showed a neuroprotective effect and was accompanied by a reduction of tyrosine hydroxylase expression. A mutant alpha-synuclein A53T significantly increased the expression of the pro-apoptotic protein BAX and made cells more susceptible to apoptosis. Generally, overexpression of alpha-synuclein could be a model for the early stages of PD, while expression of mutant alpha-synuclein A53T mimics a genetic variant of PD. The peptides Ms 9a-1 and APHC3 significantly reduced the susceptibility to apoptosis of all cell lines but differentially influenced the expression of the genes of interest. Therefore, these modulators of TRPA1 and TRPV1 have the potential for the development of new therapeutic agents for neurodegenerative disease treatment.

New Drug Targets and Preclinical Modelling Recommendations for Treating Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is the leading cause of morbidity and mortality worldwide and the primary underlying risk factor for heart failure. Despite decades of research and clinical trials, there are no drugs currently available to prevent organ damage from acute ischaemic injuries of the heart. In order to address the increasing global burden of heart failure, drug, gene, and cell-based regeneration technologies are advancing into clinical testing. In this review we highlight the burden of disease associated with AMI and the therapeutic landscape based on market analyses. New studies revealing the role of acid-sensitive cardiac ion channels and other proton-gated ion channels in cardiac ischaemia are providing renewed interest in pre- and post-conditioning agents with novel mechanisms of action that may also have implications for gene- and cell-based therapeutics. Furthermore, we present guidelines that couple new cell technologies and data resources with traditional animal modelling pipelines to help de-risk drug candidates aimed at treating AMI. We propose that improved preclinical pipelines and increased investment in drug target identification for AMI is critical to stem the increasing global health burden of heart failure.

Auditory independent low-intensity ultrasound stimulation of mouse brain is associated with neuronal ERK phosphorylation and an increase of Tbr2 marked neuroprogenitors.

Transcranial ultrasound stimulation is an emerging technique for the development of a non-invasive neuromodulation device for the treatment of various types of neurodegenerations and brain damages. However, there are very few studies that have quantified the optimal ultrasound dosage and the long-term associated effects of transcranial ultrasound treatments of brain diseases. In this study, we used a simple ex vivo hippocampal tissues stimulated by different dosages of ultrasound in combination with different chemical treatments to quantify the required energy for a measurable effect. After determining the most desirable ex vivo stimulation conditions, it was then replicated for the in vivo mouse brains. It was discovered that transcranial ultrasound promoted the increase of Tbr2-expressing neural progenitors in an ASIC1a-dependent manner. Furthermore, such effect was observable at least a week after the initial ultrasound treatments and was not abolished by auditory toxicity.

Estrogen antagonizes ASIC1a-induced chondrocyte mitochondrial stress in rheumatoid arthritis.

BACKGROUND: Destruction of articular cartilage and bone is the main cause of joint dysfunction in rheumatoid arthritis (RA). Acid-sensing ion channel 1a (ASIC1a) is a key molecule that mediates the destruction of RA articular cartilage. Estrogen has been proven to have a protective effect against articular cartilage damage, however, the underlying mechanisms remain unclear. METHODS: We treated rat articular chondrocytes with an acidic environment, analyzed the expression levels of mitochondrial stress protein HSP10, ClpP, LONP1 by q-PCR and immunofluorescence staining. Transmission electron microscopy was used to analyze the mitochondrial morphological changes. Laser confocal microscopy was used to analyze the Ca2+, mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) level. Moreover, ASIC1a specific inhibitor Psalmotoxin 1 (Pctx-1) and Ethylene Glycol Tetraacetic Acid (EGTA) were used to observe whether acid stimulation damage mitochondrial function through Ca2+ influx mediated by ASIC1a and whether pretreatment with estrogen could counteract these phenomena. Furthermore, the ovariectomized (OVX) adjuvant arthritis (AA) rat model was treated with estrogen to explore the effect of estrogen on disease progression. RESULTS: Our results indicated that HSP10, ClpP, LONP1 protein and mRNA expression and mitochondrial ROS level were elevated in acid-stimulated chondrocytes. Moreover, acid stimulation decreased mitochondrial membrane potential and damaged mitochondrial structure of chondrocytes. Furthermore, ASIC1a specific inhibitor PcTx-1 and EGTA inhibited acid-induced mitochondrial abnormalities. In addition, estrogen could protect acid-stimulated induced mitochondrial stress by regulating the activity of ASIC1a in rat chondrocytes and protects cartilage damage in OVX AA rat. CONCLUSIONS: Extracellular acidification induces mitochondrial stress by activating ASIC1a, leading to the damage of rat articular chondrocytes. Estrogen antagonizes acidosis-induced joint damage by inhibiting ASIC1a activity. Our study provides new insights into the protective effect and mechanism of action of estrogen in RA.

ASIC1a regulates miR-350/SPRY2 by N6 -methyladenosine to promote liver fibrosis.

As a reversible scar repair reaction, liver fibrosis can be blocked or even reversed by proper intervention during its formation. Our work suggests that acid-sensitive ion channel 1a (ASIC1a) participates in liver fibrosis and presents a novel mechanism involving m6 A modification and miR-350/SPRY2. We demonstrated that the expression of ASIC1a was significantly increased in liver tissue of patients with liver fibrosis and animal models of liver fibrosis, as well as PDGF-BB-induced activated HSC-T6. After downregulating the expression of ASIC1a, the degree of liver fibrosis is reduced and HSC activation was inhibited, the level of m6 A modification and miR-350 expression were also reduced. The results of dual luciferase reporter assay showed that miR-350 can bind to the target gene SPRY2 and inhibit its expression. We also found that METTL3 can regulate the extent of m6 A modification of pri-miR-350 by binding to DGCR8. In addition, silencing or blocking the expression of ASIC1a can reduce the expression of PI3K/AKT and ERK signaling pathway-related proteins in activated HSCs. Taken together, we demonstrated that ASIC1a regulates the processing of miR-350 through METTL3-dependent m6 A modification, and mature miR-350 targets SPRY2 and further promotes liver fibrosis through the PI3K/KT and ERK pathways.

Selection of an ASIC1a-blocking combinatorial antibody that protects cells from ischemic death.

Acid-sensing ion channels (ASICs) have emerged as important, albeit challenging therapeutic targets for pain, stroke, etc. One approach to developing therapeutic agents could involve the generation of functional antibodies against these channels. To select such antibodies, we used channels assembled in nanodiscs, such that the target ASIC1a has a configuration as close as possible to its natural state in the plasma membrane. This methodology allowed selection of functional antibodies that inhibit acid-induced opening of the channel in a dose-dependent way. In addition to regulation of pH, these antibodies block the transport of cations, including calcium, thereby preventing acid-induced cell death in vitro and in vivo. As proof of concept for the use of these antibodies to modulate ion channels in vivo, we showed that they potently protect brain cells from death after an ischemic stroke. Thus, the methodology described here should be general, thereby allowing selection of antibodies to other important ASICs, such as those involved in pain, neurodegeneration, and other conditions.

Evidence for role of acid-sensing ion channel 1a in chronic rhinosinusitis with nasal polyps.

PURPOSE: A variety of inflammatory cells are infiltrated histologically in sinonasal mucosa of chronic rhinosinusitis with nasal polyps (CRSwNP), especially CRSwNP with asthma. Acid-sensing ion channel 1a (ASIC1a) is essential in the process of sensing acidification and triggering inflammation. Whereas, its role and mechanism in CRSwNP remain uncertain. The present study aimed to explore the roles and mechanism of ASIC1a in the pathogenesis of CRSwNP. METHODS: Nasal secretions from control subjects, patients with CRSwNP with or without asthma were collected for measuring pH values. Western blotting, real-time PCR and immunohistochemistry (IHC) were employed to assess ASIC1a expression in nasal tissue samples from included subjects. The co-localization of ASIC1a with inflammatory cells was evaluated by immunofluorescence staining. Then, dispersed nasal polyp cells (DNPCs) were cultured under acidified condition (pH 6.0), with or without ASIC1a inhibitor amiloride. Western blotting, real-time PCR, LDH activity kit, and ELISA were performed to assess the effects and mechanisms of stimulators on the cells. RESULTS: The pH values were significantly lower in the nasal secretions from patients with CRSwNP with asthma. Significant upregulation of ASIC1a protein, mRNA levels, and positive cells was found in CRSwNP with asthma. ASIC1a was detected in a variety of inflammatory cells. In cultured DNPCs, significant alterations of ASIC1a levels, LDH activity, HIF-1α levels, and inflammatory cytokines were found under acidified condition (pH 6.0), but were prevented by amiloride. CONCLUSION: Upregulation of ASIC1a might be essential in the process of sensing acidification and triggering inflammatory response via enhancing HIF-1α expression and LDH activity to activate inflammatory cells in the pathogenesis of CRSwNP, especially in CRSwNP with asthma.

ASIC1a channels regulate mitochondrial ion signaling and energy homeostasis in neurons.

Acid-sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~pH 6, whereas its function during physiological brain activity, linked to much milder pH changes, is still poorly understood. Here, by performing live cell imaging utilizing Na+ and Ca2+ sensitive and spatially specific fluorescent dyes, we investigated the role of ASIC1a in cytosolic Na+ and Ca2+ signals elicited by a mild extracellular drop from pH 7.4 to 7.0 and how these affect mitochondrial Na+ and Ca2+ signaling or metabolic activity. We show that in mouse primary cortical neurons, this small extracellular pH change triggers cytosolic Na+ and Ca2+ waves that propagate to mitochondria. Inhibiting ASIC1a with Psalmotoxin 1 or ASIC1a gene knockout blocked not only the cytosolic but also the mitochondrial Na+ and Ca2+ signals. Moreover, physiological activation of ASIC1a by this pH shift enhances mitochondrial respiration and evokes mitochondrial Na+ signaling even in digitonin-permeabilized neurons. Altogether our results indicate that ASIC1a is critical in linking physiological extracellular pH stimuli to mitochondrial ion signaling and metabolic activity and thus is an important metabolic sensor.

PI3-kinase/Akt pathway-regulated membrane transportation of acid-sensing ion channel 1a/Calcium ion influx/endoplasmic reticulum stress activation on PDGF-induced HSC Activation.

Acid-sensing ion channel 1a (ASIC1a) allows Na+ and Ca2+ flow into cells. It is expressed during inflammation, in tumour and ischaemic tissue, in the central nervous system and non-neuronal injury environments. Endoplasmic reticulum stress (ERS) is caused by the accumulation of misfolded proteins that interferes with intracellular calcium homoeostasis. Our recent reports showed ASIC1a and ERS are involved in liver fibrosis progression, particularly in hepatic stellate cell (HSC) activation. In this study, we investigated the roles of ASIC1a and ERS in activated HSC. We found that ASIC1a and ERS-related proteins were up-regulated in carbon tetrachloride (CCl4 )-induced fibrotic mouse liver tissues, and in patient liver tissues with hepatocellular carcinoma with severe liver fibrosis. The results show silencing ASIC1a reduced the expression of ERS-related biomarkers GRP78, Caspase12 and IREI-XBP1. And, ERS inhibition by 4-PBA down-regulated the high expression of ASIC1a induced by PDGF, suggesting an interactive relationship. In PDGF-induced HSCs, ASIC1a was activated and migrated to the cell membrane, leading to extracellular calcium influx and ERS, which was mediated by PI3K/AKT pathway. Our work shows PDGF-activated ASIC1a via the PI3K/AKT pathway, induced ERS and promoted liver fibrosis progression.

Acid-Sensing Ion Channels Activated by Evoked Released Protons Modulate Synaptic Transmission at the Mouse Calyx of Held Synapse.

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases. We found that these channels can be activated in neurons of the medial nucleus of the trapezoid body (MNTB) of the auditory system in the CNS. A drop in extracellular pH induces transient inward ASIC currents (IASICs) in postsynaptic MNTB neurons from wild-type mice. The inhibition of IASICs by psalmotoxin-1 (PcTx1) and the absence of these currents in knock-out mice for ASIC-1a subunit (ASIC1a-/-) suggest that homomeric ASIC-1as are mediating these currents in MNTB neurons. Furthermore, we detect ASIC1a-dependent currents during synaptic transmission, suggesting an acidification of the synaptic cleft due to the corelease of neurotransmitter and H+ from synaptic vesicles. These currents are capable of eliciting action potentials in the absence of glutamatergic currents. A significant characteristic of these homomeric ASIC-1as is their permeability to Ca2+ Activation of ASIC-1a in MNTB neurons by exogenous H+ induces an increase in intracellular Ca2+ Furthermore, the activation of postsynaptic ASIC-1as during high-frequency stimulation (HFS) of the presynaptic nerve terminal leads to a PcTx1-sensitive increase in intracellular Ca2+ in MNTB neurons, which is independent of glutamate receptors and is absent in neurons from ASIC1a-/- mice. During HFS, the lack of functional ASICs in synaptic transmission results in an enhanced short-term depression of glutamatergic EPSCs. These results strongly support the hypothesis of protons as neurotransmitters and demonstrate that presynaptic released protons modulate synaptic transmission by activating ASIC-1as at the calyx of Held-MNTB synapse.SIGNIFICANCE STATEMENT The manuscript demonstrates that postsynaptic neurons of the medial nucleus of the trapezoid body at the mouse calyx of Held synapse express functional homomeric Acid-sensing ion channel-1a (ASIC-1as) that can be activated by protons (coreleased with neurotransmitter from acidified synaptic vesicles). These ASIC-1as contribute to the generation of postsynaptic currents and, more relevant, to calcium influx, which could be involved in the modulation of presynaptic transmitter release. Inhibition or deletion of ASIC-1a leads to enhanced short-term depression, demonstrating that they are concerned with short-term plasticity of the synapse. ASICs represent a widespread communication system with unique properties. We expect that our experiments will have an impact in the neurobiology field and will spread in areas related to neuronal plasticity.

Necrostatin-1 ameliorates adjuvant arthritis rat articular chondrocyte injury via inhibiting ASIC1a-mediated necroptosis.

Necroptosis, a necrotic cell death pathway regulated by receptor interacting protein (RIP) 1 and 3, plays a key role in pathophysiological processes, including rheumatoid arthritis (RA). However, whether necroptosis is involved in RA articular cartilage damage processes remain unclear. The aim of present study was to investigate the dynamic changes in arthritic chondrocyte necroptosis and the effect of RIP1 inhibitor necrostatin-1 (Nec-1) and acid-sensing ion channels (ASICs) inhibitor amiloride on arthritic cartilage injury and acid-induced chondrocyte necroptosis. Our results demonstrated that the expression of RIP1, RIP3 and mixed lineage kinase domain-like protein phosphorylation (p-MLKL) were increased in adjuvant arthritis (AA) rat articular cartilage in vivo and acid-induced chondrocytes in vitro. High co-expression of ASIC1a and RIP1 showed in AA rat articular cartilage. Moreover, Nec-1 and amiloride could reduce articular cartilage damage and necroinflammation in AA rats. In addition, acid-induced increase in necroptosis markers RIP1/RIP3 were inhibited by Nec-1, ASIC1a-specific blocker psalmotoxin-1 (PcTx-1) or ASIC1a-short hairpin RNA respectively, which revealed that necroptosis is triggered in acid-induced chondrocytes and mediated by ASIC1a. These findings indicated that blocking ASIC1a-mediated chondrocyte necroptosis may provide potential therapeutic strategies for RA treatment.

Interleukin-1ß and tumor necrosis factor-α augment acidosis-induced rat articular chondrocyte apoptosis via nuclear factor-kappaB-dependent upregulation of ASIC1a channel.

The acute-phase proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) demonstrate high-level expression and pleiotropic biological effects, and contribute to the progression and persistence of rheumatoid arthritis (RA). Acid hydrarthrosis is also an important pathological characteristic of RA, and the acid-sensing ion channel 1a (ASIC1a) plays a critical role in acidosis-induced chondrocyte cytotoxicity. However, the roles of IL-1ß and TNF-α in acid-induced apoptosis of chondrocytes remain unclear. Rat adjuvant arthritis and primary articular chondrocytes were used as in vivo and in vitro model systems, respectively. ASIC1a expression in articular cartilage was increased and highly colocalized with nuclear factor (NF)-κB expression in vivo. IL-1ß and TNF-α could upregulate ASIC1a expression. These cytokines activated mitogen-activated protein kinase and NF-κB pathways in chondrocytes, while the respective inhibitors of these signaling pathways could partially reverse the ASIC1a upregulation induced by IL-1ß and TNF-α. Dual luciferase and gel-shift assays and chromatin immunoprecipitation-polymerase chain reaction demonstrated that IL-1ß and TNF-α enhanced ASIC1a promoter activity in chondrocytes by increasing NF-κB DNA-binding activities, which was in turn prevented by the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate. IL-1ß and TNF-α also decreased cell viability but enhanced LDH release, intracellular Ca2+ concentration elevation, loss of mitochondrial membrane potential, cleaved PARP and cleaved caspase-3/9 expression, and apoptosis in acid-stimulated chondrocytes, which effects could be abrogated by the specific ASIC1a inhibitor psalmotoxin-1 (PcTX-1), ASIC1a-short hairpin RNA or calcium chelating agent BAPTA-AM. These results indicate that IL-1ß and TNF-α can augment acidosis-induced cytotoxicity through NF-κB-dependent up-regulation of ASIC1a channel expression in primary articular chondrocytes.

Effects of autophagy on acid-sensing ion channel 1a-mediated apoptosis in rat articular chondrocytes.

Rheumatoid arthritis (RA) is a degenerative joint disease that is caused by multiple pathogenic factors. However, the precise etiology of RA is still unknown. Our previous studies demonstrated that acid-sensing ion channel 1a (ASIC1a)-mediated articular chondrocyte apoptosis played a key role in the progression of RA. In this study, we aim to explore whether ASIC1a mediates autophagy or not and the effect of autophagy on ASIC1a-mediated apoptosis. Primary articular chondrocytes, extracted from rat knee joints, were exposed to different concentrations of concentrated hydrochloric acid for different time intervals in vitro. The results indicated that extracellular acid treatment induced autophagy of rat articular chondrocytes. Moreover, inhibition of ASIC1a with either psalmotoxin 1 or ASIC1a short hairpin RNA reduced the autophagy flux. The results suggested that ASIC1a mediated acid-induced autophagy. Pretreatment with autophagy antagonist 3-methyladenine decreased the autophagy, but increased the apoptosis mediated by ASIC1a. Furthermore, knockdown of Beclin 1 by small interfering RNA attenuated autophagy but potentiated ASIC1a-mediated apoptosis of rat articular chondrocytes. Taken together, these findings suggested that both inhibition and silencing of autophagy could enhance ASIC1a-mediated apoptosis in rat articular chondrocytes, and therefore, autophagy is likely to be a new mechanism involved in ASIC1a-mediated apoptosis of articular chondrocytes during the pathogenesis of RA.

Acid-sensing ion channel 1a mediates acid-induced inhibition of matrix metabolism of rat articular chondrocytes via the MAPK signaling pathway.

The acid-sensing ion channel 1a (ASIC1a), which is activated by extracellular acid, contributes to the pathogenesis of rheumatoid arthritis. However, it remains unclear whether ASIC1a mediates acid-induced matrix metabolism in rat articular chondrocytes via activation of the MAPK signaling pathway. In the current study, we found that extracellular acidification (pH 6.0) inhibited proliferation and induced apoptosis of articular chondrocytes in a dose-dependent manner, while the expression of phosphorylated ERK1/2 and P38 MAPK increased, but, this effect was blocked by the Ca2+ chelator BAPTA-AM and the ASIC1a-specific blocker PcTx-1. In addition, extracellular acidification increased the expression of c-fos, GAG, HYP, and TIM1/2. These effects were inhibited by the Ca2+ chelator BAPTA-AM, ERK1/2 inhibitor PD98059, and ASIC1a-specific blocker PcTx-1, but not the P38 MAPK inhibitor SB203580. Finally, extracellular acidification increased the expression of c-jun and MMP-2/9, and these effects were blocked by the Ca2+ chelator BAPTA-AM, P38 MAPK inhibitor SB203580, and ASIC1a-specific blocker PcTx-1, but not the ERK1/2 inhibitor PD98059. In conclusion, ASIC1a inhibits the expression of MMP-2/9, GAG, HYP, and TIMP-1/2 by the Ca2+-dependent P38 MAPK/c-jun and ERK/c-fos signaling pathways.

Role of ASIC1a in Aß-induced synaptic alterations in the hippocampus.

Acid-sensing ion channels (ASICs) are widely expressed in the mammalian central nervous system where they play a key role in synaptic transmission and in specific forms of memory. On the other hand, ASICs can be persistently active under pathological conditions contributing to neuronal damage in ischemic stroke, brain trauma, epilepsy and Parkinson's disease. However, to date no experimental evidence has linked ASICs to Alzheimer's disease (AD). Aim of the present work was to investigate, in CA1 pyramidal neurons, the possible involvement of ASIC1a in the Aß-mediated effect on metabotropic glutamate (mGlu) receptor dependent transmission. We found that, in slices pretreated with Aß, the pharmacological blockade of ASIC1a restored the increased intrinsic excitability following group I mGlu receptor activation. This suggests that, under certain conditions, ASIC1a might further contribute to the Aß-related depolarizing response. We have recently demonstrated that ASIC1a is also involved long-term depression (LTD) induced either by low-frequency stimulation or by application of the group I mGlu receptor agonist DHPG. Here, we have shown that psalmotoxin-1, a selective blocker of ASIC1a, rescued the DHPG-LTD facilitation associated with genetic and non-genetic models of AD. Overall, these results suggest that a functional coupling between ASIC1a and mGlu receptors occurs and might contribute to the synaptic alterations associated with AD.

ASIC1a Promotes Acid-Induced Autophagy in Rat Articular Chondrocytes through the AMPK/FoxO3a Pathway.

Acid-sensing ion channel 1a (ASIC1a) is a member of the extracellular H⁺-activated cation channels family. Our previous studies suggested that ASIC1a contributed to acid-induced rat articular chondrocytes autophagy. However, its potential mechanisms remain unclear. The present study demonstrated the effect of ASIC1a on rat articular chondrocytes autophagy and explored the underlying molecular mechanisms. The results demonstrated that ASIC1a contributed to acid-induced autophagy in rat articular chondrocytes, and which was associated with an increase in (Ca2+)i, as indicated that acid-induced increases in mRNA and protein expression of LC3B-II and other autophagy-related markers were inhibited by ASIC1a-specific blocker, PcTx1 and calcium chelating agent, BAPTA-AM. Furthermore, the results showed that extracellular acid increased level of Forkhead box O (FoxO) 3a, but was reversed by inhibition of ASIC1a and Ca2+ influx. Moreover, gene ablation of FoxO3a prevented acid-induced increases in mRNA and protein expression of LC3B-II, Beclin1 and the formation of autophagosome. Finally, it also showed that ASIC1a activated adenine nucleotide (AMP)-activated protein kinase (AMPK). In addition, suppression of AMPK by Compound C and its small interfering RNA (siRNA) prevented acid-induced upregulation of total and nuclear FoxO3a and increases in mRNA and protein expression of LC3B-II, Beclin1, and ATG5. Taken together, these findings suggested that AMPK/FoxO3a axis plays an important role in ASIC1a-mediated autophagy in rat articular chondrocytes, which may provide novel mechanistic insight into ASIC1a effects on autophagy.

Neuroprotective Effects of Paeoniflorin on 6-OHDA-Lesioned Rat Model of Parkinson's Disease.

Paeoniflorin (PF) is the main active component extracted from the roots of Paeonialactiflora, a traditional Chinese medicine used for the treatment of neurodegenerative disorders, especially Parkinson's disease (PD). The degeneration of dopaminergic (DA-) neurons in PD may be caused by pathological activation of acid-sensing ion channels (ASICs). Thus, we designed a series of experiments to evaluate the therapeutic effects of PF and to test whether its effects are related to its inhibitory effect on ASIC1a. We found that systemic administration of PF or ASICs blockers (psalmotoxin-1 and amiloride) improved behavioral symptoms, delayed DA-neuronal loss and attenuated the reduction of dopamine (DA) and its metabolites in a rat model of 6-hydroxydopamine (6-OHDA)-induced PD. In addition, our data showed that PF, like ASICs blockers, regulated the expression of ASIC1a, decreased the level of α-synuclein (α-SYN), and improved autophagic dysfunction. Further experiments showed that ASIC1a knockdown down-regulated the α-SYN level and alleviated the autophagic injury in the 6-OHDA-treated ASIC1a-silenced PC12 cells. In summary, these findings indicate that PF enhanced the autophagic degradation of α-SYN and, thus, protected DA-neurons against the neurotoxicity caused by 6-OHDA. These findings also provide experimental evidence that PF may be a neuroprotectant for PD by acting on ASIC1a and that ASIC1a may be involved in the pathogenesis of PD.

Therapeutic Effect Analysis of Sinomenine on Rat Cerebral Ischemia-Reperfusion Injury.

OBJECTIVE: The objective of this study is to investigate the therapeutic effect of sinomenine (SIN) on rat cerebral ischemia-reperfusion (IR) injury and the molecular mechanism. METHODS: One hundred thirty-five rats were equally randomized into sham-operated group, middle cerebral artery occlusion (MCAO) group, and SIN group, and reversible rat MCAO model was made according to the Longa method for the MCAO and SIN groups. Then, 15 rats from each group were decapitated at 6, 12, and 24 hours after reperfusion to obtain brain tissue samples. Rats in the SIN group were injected with sinomenine by tail vein (90 mg/kg) 1 hour before ischemia; rats in the MCAO and sham-operated groups were administrated with the same volume of saline. Neurological severity score (NSS), infarction volume, ischemic brain water content, and blood-brain barrier (BBB) permeability were determined at corresponding time points. Acid-sensing ion channel (ASIC) 1a mRNA level was determined by quantitative real-time polymerase chain reaction; ischemic brain contents of lactic acid (LD), lactic dehydrogenase (LDH), ATPase, and inflammatory factors were determined by spectrophotometric method. RESULTS: At 12 hours after reperfusion and since then, NSS in the SIN group decreased obviously; infarction volume, brain water content, and BBB permeability in the SIN group were lower than those in the MCAO group (P < .05). IR injury resulted in the upregulation of the contents of ASIC1a mRNA, LD, LDH, and inflammatory factors and the downregulation of the contents of ATPase, while SIN could reverse the upregulation/downregulation effect induced by IR injury (P < .05). CONCLUSION: Through its anti-inflammation effect, which alleviates acidosis, improves energy metabolism, and inhibits ASIC1a level, SIN protects ischemic rat brain against cerebral IR injury.