Regulation of gliomagenesis and stemness through acid sensor ASIC1a.

Glioblastoma multiforme (GBM) is the most prevalent and aggressive type of adult gliomas. Despite intensive therapy including surgery, radiation, and chemotherapy, invariable tumor recurrence occurs, which suggests that glioblastoma stem cells (GSCs) render these tumors persistent. Recently, the induction of GSC differentiation has emerged as an alternative method to treat GBM, and most of the current studies aim to convert GSCs to neurons by a combination of transcriptional factors. As the tumor microenvironment is typically acidic due to increased glycolysis and consequently leads to an increased production of lactic acid in tumor cells, in the present study, the role of acid­sensing ion channel 1a (ASIC1a), an acid sensor, was explored as a tumor suppressor in gliomagenesis and stemness. The bioinformatics data from The Cancer Genome Atlas revealed that ASIC1 expression levels in GBM tumor tissues were lower than those in normal brain, and glioma patients with high ASIC1 expression had longer survival than those with low ASIC1 expression. Our immunohistochemistry data from tissue microarray revealed that ASIC1a expression was negatively associated with glioma grading. Functional studies revealed that the downregulation of ASIC1a promoted glioma cell proliferation and invasion, while upregulation of ASIC1a inhibited their proliferation and invasion. Furthermore, ASIC1a suppressed growth and proliferation of glioma cells through G1/S arrest and apoptosis induction. Mechanistically, ASIC1a negatively modulated glioma stemness via inhibition of the Notch signaling pathway and GSC markers CD133 and aldehyde dehydrogenase 1. ASIC1a is a tumor suppressor in gliomagenesis and stemness and may serve as a promising prognostic biomarker and target for GBM patients.

NO in the dPAG modulates panic-like responses and ASIC1a expression in the prefrontal cortex and hippocampus in mice.

Panic disorder (PD) is a multifactorial neuropsychiatric disorder. Our previous study has demonstrated that the nitric oxide (NO) pathway and the acid-sensing ion channel 1a (ASIC1a) level in the dorsal midbrain periaqueductal gray (dPAG) are involved in the modulation of panic-like responses. In addition, the prefrontal cortex (PFC) and the hippocampus also play a role in panic-like responses. However, no studies have investigated the protein level of ASIC1a in the PFC and hippocampus in a mouse model of panic-like disorders after alteration of the NO pathway in the dPAG. We investigated the production of a panic attack with intra-dPAG injections of SNAP, an NO donor, and 7-NI, an nNOS inhibitor. Moreover, we measured ASIC1a protein levels in the PFC and hippocampus. The rat exposure test (RET) is frequently used as an animal model of panic. In our study, C57BL/6 mice received an intra-dPAG injection of SNAP or 7-NI before RET; neurobehavioral tests were then conducted, followed by mechanistic evaluation through western blot analysis in the PFC and hippocampus. An intra-dPAG infusion of SNAP significantly increased the panic-like effect, whereas treatment with 7-NI decreased fear behavior. Mice treated with SNAP/7-NI showed significantly increased/decreased ASIC1a expression in the PFC, and a decreasing/increasing trend in the hippocampus. The present study suggests that the NO pathway in the dPAG plays a key role in panic-like responses in mice confronted by a rat, further, NO intra-dPAG injection also modulates the ASIC1a expression levels in the PFC and hippocampus.

TRPM8, ASIC1, and ASIC3 localization and expression in the human oropharynx.

BACKGROUND: Oropharyngeal dysphagia (OD) is a prevalent disease with poor prognosis among older people and has no pharmacological treatment. Polymodal sensory receptors like the TRP or ASIC family receptors are potential targets to treat OD. TRPM8 agonists and acidic solutions can improve the swallow response in patients with OD, but little is known about the expression of TRPM8, ASIC1, and ASIC3 in the human oropharynx. The aim of this study was to assess the expression and localization of TRPM8, ASIC1, and ASIC3 in human samples of the oropharynx to lay the basis for new pharmacological treatments for OD. METHODS: Pathology-free samples from oropharyngeal regions innervated by cranial nerves V, IX, and X were obtained during major ENT surgery and processed to obtain mRNA (20 patients) or to be used in immunohistochemical assays (12 patients). TRPM8, ASIC1, and ASIC3 expression and localization were studied with RT-qPCR and fluorescent immunohistochemistry. KEY RESULTS: ASIC3 was expressed in the 3 regions studied with similar levels and was localized on sensory fibers innervating the mucosa below the basal lamina of all studied regions. TRPM8 was also co-localized on the sensory fibers innervating the mucosa below the basal lamina of all studied regions. In contrast, ASIC1 was only found in the nerves innervating the tongue muscular fibers. CONCLUSIONS & INFERENCES: TRPM8 and ASIC3 are found on submucosal sensory nerves in the human oropharynx. Our study lays the basis to use oropharyngeal TRPM8 and ASIC3 receptors as therapeutic targets to develop new active pharmacological treatments for OD patients.

Cellular Localization of Acid-Sensing Ion Channel 1 in Rat Nucleus Tractus Solitarii.

By determining its cellular localization in the nucleus tractus solitarii (NTS), we sought anatomical support for a putative physiological role for acid-sensing ion channel Type 1 (ASIC1) in chemosensitivity. Further, we sought to determine the effect of a lesion that produces gliosis in the area. In rats, we studied ASIC1 expression in control tissue with that in tissue with gliosis, which is associated with acidosis, after saporin lesions. We hypothesized that saporin would increase ASIC1 expression in areas of gliosis. Using fluorescent immunohistochemistry and confocal microscopy, we found that cells and processes containing ASIC1-immunoreactivity (IR) were present in the NTS, the dorsal motor nucleus of vagus, and the area postrema. In control tissue, ASIC1-IR predominantly colocalized with IR for the astrocyte marker, glial fibrillary acidic protein (GFAP), or the microglial marker, integrin αM (OX42). The subpostremal NTS was the only NTS region where neurons, identified by protein gene product 9.5 (PGP9.5), contained ASIC1-IR. ASIC1-IR increased significantly (157 ± 8.6% of control, p < 0.001) in the NTS seven days after microinjection of saporin. As we reported previously, GFAP-IR was decreased in the center of the saporin injection site, but GFAP-IR was increased in the surrounding areas where OX42-IR, indicative of activated microglia, was also increased. The over-expressed ASIC1-IR colocalized with GFAP-IR and OX42-IR in those reactive astrocytes and microglia. Our results support the hypothesis that ASIC1 would be increased in activated microglia and in reactive astrocytes after injection of saporin into the NTS.

Resveratrol attenuates bone cancer pain through regulating the expression levels of ASIC3 and activating cell autophagy.

Bone cancer pain (BCP) is one of the most common pains in patients with malignant cancers. The mechanism underlying BCP is largely unknown. Our previous studies and the increasing evidence both have shown that acid-sensing ion channels 3 (ASIC3) is an important protein in the pathological pain state in some pain models. We hypothesized that the expression change of ASIC3 might be one of the factors related to BCP. In this study, we established the BCP model through intrathecally injecting rat mammary gland carcinoma cells (MRMT-1) into the left tibia of Sprague-Dawley female rats, and found that the BCP rats showed bone destruction, increased mechanical pain sensitivities and up-regulated ASIC3 protein expression levels in L4-L6 dorsal root ganglion. Then, resveratrol, which was intraperitoneally injected into the BCP rats on post-operative Day 21, dose-dependently increased the paw withdrawal threshold of BCP rats, reversed the pain behavior, and had an antinociceptive effect on BCP rats. In ASIC3-transfected SH-SY5Y cells, the ASIC3 protein expression levels were regulated by resveratrol in a dose- and time-dependent manner. Meanwhile, resveratrol also had an antinociceptive effect in ASIC3-mediated pain rat model. Furthermore, resveratrol also enhanced the phosphorylation of AMPK, SIRT1, and LC3-II levels in ASIC3-transfected SH-SY5Y cells, indicating that resveratrol could activate the AMPK-SIRT1-autophagy signal pathway in ASIC3-transfected SH-SY5Y cells. In BCP rats, SIRT1 and LC3-II were also down-regulated. These findings provide new evidence for the use of resveratrol as a therapeutic treatment during BCP states.

Distribution of ASIC4 transcripts in the adult wild-type mouse brain.

Acid-sensing ion channel 4 (ASIC4) belongs to the ASIC gene family of neuronal proton-gated cation channels, and is the least understood subtype among the members. Previous studies of ASIC4 expression in the mammalian central nervous system have shown that ASIC4 is abundantly expressed in the spinal cord and in various brain regions, such as the cerebral cortex, the hippocampus, and the cerebellum. However, the detailed distribution of ASIC4 transcripts in mammalian brains still remains to be elucidated. In the present study, radioactive in situ hybridization histochemistry with an ASIC4-specific cRNA probe was performed on wild-type mouse brains, followed by X-gal staining experiments with Asic4-lacZ reporter mice Asic4tm1a(KOMP)Mbp. It was found that ASIC4 mRNAs were widely expressed throughout the wild-type brain, but preferentially concentrated in the olfactory bulb, the piriform cortex, the caudate putamen, the preoptic area, the paraventricular nucleus, the medial habenular nucleus, the pretectal area, the lateral geniculate nucleus, the amygdaloid complex, the superior colliculus, the interpeduncular nucleus, and the granule cell layer of the ventral hippocampus, and these results were in agreement with the X-gal-positive reactions observed in the mutant brain. In addition, X-gal staining combined with immunohistochemistry identified intense signals for ASIC4 transcriptional activity in most of the choline acetyltransferase (ChAT)-positive principal neurons located in the basal forebrain cholinergic nuclei. Our data provide useful information to speculate possible roles of ASIC4 in diverse brain functions.

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.

Site-specific fluorescence spectrum detection and characterization of hASIC1a channels upon toxin mambalgin-1 binding in live mammalian cells.

The synthesis of fluorescent unnatural amino-acid Anap was optimized and the Anap was incorporated into four sites in an acid-pocket or a transmembrane region of human acid-sensing ion channel-1a (hASIC1a). Combinational Anap fluorescence spectra and patch-clamp electrophysiology data illustrated site-specific conformational responses upon toxin mambalgin-1 binding. This combinational approach can be used to analyse conformational properties of many different eukaryotic proteins in their functional states, in a site-specific manner in live mammalian cells.

ASIC2 is present in human mechanosensory neurons of the dorsal root ganglia and in mechanoreceptors of the glabrous skin.

Mechanosensory neurons lead to the central nervous system touch, vibration and pressure sensation. They project to the periphery and form different kinds of mechanoreceptors. The manner in which they sense mechanical signals is still not fully understood, but electrophysiological experiments have suggested that this may occur through the activation of ion channels that gate in response to mechanical stimuli. The acid-sensing ion channels (ASICs), especially ASIC2, may function as mechanosensors or are required for mechanosensation, and they are expressed in both mechanosensory neurons and mechanoreceptors. Here, we have used double immunohistochemistry for ASIC2 together with neuronal and glial markers associated with laser confocal microscopy and image analysis, to investigate the distribution of ASIC2 in human lumbar dorsal root ganglia, as well as in mechanoreceptors of the hand and foot glabrous skin. In lumbar dorsal root ganglia, ASIC2 immunoreactive neurons were almost all intermediate or large sized (mean diameter ≥20-70 µm), and no ASIC2 was detected in the satellite glial. ASIC2-positive axons were observed in Merkel cell-neurite complexes, Meissner and Pacinian corpuscles, all of them regarded as low-threshold mechanoreceptors. Moreover, a variable percent of Meissner (8 %) and Pacinian corpuscles (27 %) also displayed ASIC2 immunoreactivity in the Schwann-related cells. These results demonstrate the distribution of ASIC2 in the human cutaneous mechanosensory system and suggest the involvement of ASIC2 in mechanosensation.

Acid-sensing ion channel 1 and nitric oxide synthase are in adjacent layers in the wall of rat and human cerebral arteries.

Extracellular acidification activates a family of proteins known as acid-sensing ion channels (ASICs). One ASIC subtype, ASIC type 1 (ASIC1), may play an important role in synaptic plasticity, memory, fear conditioning and ischemic brain injury. ASIC1 is found primarily in neurons, but one report showed its expression in isolated mouse cerebrovascular cells. In this study, we sought to determine if ASIC1 is present in intact rat and human major cerebral arteries. A potential physiological significance of such a finding is suggested by studies showing that nitric oxide (NO), which acts as a powerful vasodilator, may modulate proton-gated currents in cultured cells expressing ASIC1s. Because both constitutive NO synthesizing enzymes, neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS), are expressed in cerebral arteries we also studied the anatomical relationship between ASIC1 and nNOS or eNOS in both rat and human cerebral arteries. Western blot analysis demonstrated ASIC1 in cerebral arteries from both species. Immunofluorescent histochemistry and confocal microscopy also showed that ASIC1-immunoreactivity (IR), colocalized with the smooth muscle marker alpha-smooth muscle actin (SMA), was present in the anterior cerebral artery (ACA), middle cerebral artery (MCA), posterior cerebral artery (PCA) and basilar artery (BA) of rat and human. Expression of ASIC1 in cerebral arteries is consistent with a role for ASIC1 in modulating cerebrovascular tone both in rat and human. Potential interactions between smooth muscle ASIC1 and nNOS or eNOS were supported by the presence of nNOS-IR in the neighboring adventitial layer and the presence of nNOS-IR and eNOS-IR in the adjacent endothelial layer of the cerebral arteries.

A comparative study of gelatin sponge scaffolds and PLGA scaffolds transplanted to completely transected spinal cord of rat.

This study sought to investigate whether gelatin sponge (GS) scaffold would produce less acidic medium in injured spinal cord, as compared with poly(lactic-co-glycolic acid) (PLGA) scaffold, to determine which of the two scaffolds as the biomaterial is more suitable for transplantation into spinal cord. GS scaffold or PLGA scaffold was transplanted into a transected spinal cord in this study. Two months after transplantation of scaffolds, acid sensing ion channel 1a (ASIC1a) positive cells expressing microtubule associated protein 2 (Map2) were observed as well as expressing adenomatous polyposis coli (APC) in spinal cord. GFAP positive cells were distributed at the rostral and caudal of the injury/graft area in the GS and PLGA groups. Western blot showed ASIC1a and GFAP expression of injured spinal cord was downregulated in the GS group. The number of CD68 positive cells was fewer and NF nerve fibers were more in the GS group. Nissl staining and cell counting showed that the number of survival neurons was comparable between the GS and PLGA groups in the pyramidal layer of sensorimotor cortex and the red nucleus of midbrain. However, in the Clarke's nucleus at L1 spinal segment, the surviving neurons in the GS group were more numerous than that in the PLGA group. H&E staining showed that the tissue cavities in the GS group were smaller in size than that in the PLGA group. The results suggest that GS scaffold is more suitable for transplantation to promote the recovery of spinal cord injury compared with PLGA scaffold.