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.

Hypoxia and inflammation often develop concurrently in numerous diseases, and the influence of hypoxia on natural evolution of inflammatory responses is widely accepted. Glucocorticoid-induced leucine zipper (GILZ) is thought to be an important mediator of anti-inflammatory and immune-suppressive actions of glucocorticoid (GC). However, whether GILZ is involved in hypoxic response is still unclear. In this study, we investigated the effects of hypoxic exposure and/or the administration of dexamethasone (Dex), a synthetic GC on GILZ expression both in vitro and in vivo, and further explored the relationship between GILZ and proinflammatory cytokines IL-1β, IL-6, and TNF-α under normoxic and hypoxic conditions. We found that hypoxia not only remarkably upregulated the expression of GILZ, but also significantly enhanced Dex-induced expression of GILZ in macrophages and the spleen of rats. ERK activity is found involved in the upregulation of GILZ induced by hypoxia. Inhibiting the expression of GILZ in RAW264.7 cells using specific GILZ small interfering RNA led to a significant increase in mRNA production and protein secretion of IL-1β and IL-6 in hypoxia and abrogated the inhibitory effect of Dex on expression of IL-1β and IL-6 in hypoxia. We also found that adrenal hormones played pivotal roles in upregulation of GILZ expression in vivo. Altogether, data presented in this study suggest that GILZ has an important role not only in adjusting adaptive responses to hypoxia by negatively regulating the activation of macrophages and the expression of proinflammatory cytokines, but also in mediating the anti-inflammatory action of GC under hypoxic conditions.

Chronic pain is a serious debilitating disease for which effective treatment is still lacking. Acid-sensing ion channel 1a (ASIC1a) has been implicated in nociceptive processing at both peripheral and spinal neurons. However, whether ASIC1a also contributes to pain perception at the supraspinal level remains elusive. Here, we report that ASIC1a in ACC is required for thermal and mechanical hypersensitivity associated with chronic pain. ACC-specific genetic deletion or pharmacological blockade of ASIC1a reduced the probability of cortical LTP induction and attenuated inflammatory thermal hyperalgesia and mechanical allodynia in male mice. Using cell type-specific manipulations, we demonstrate that ASIC1a in excitatory neurons of ACC is a major player in cortical LTP and pain behavior. Mechanistically, we show that ASIC1a tuned pain-related cortical plasticity through protein kinase C -mediated increase of membrane trafficking of AMPAR subunit GluA1 in ACC. Importantly, postapplication of ASIC1a inhibitors in ACC reversed previously established nociceptive hypersensitivity in both chronic inflammatory pain and neuropathic pain models. These results suggest that ASIC1a critically contributes to a higher level of pain processing through synaptic potentiation in ACC, which may serve as a promising analgesic target for treatment of chronic pain.Chronic pain is a debilitating disease that still lacks effective therapy. Ion channels are good candidates for developing new analgesics. Here, we provide several lines of evidence to support an important role of cortically located ASIC1a channel in pain hypersensitivity through promoting long-term synaptic potentiation in the ACC. Our results indicate a promising translational potential of targeting ASIC1a to treat chronic pain.

Motor learning and striatal neuronal function in mice are promoted by the acid-sensing channel ASIC1a.

Neonatal hyperbilirubinemia is a common clinical condition that can lead to brain encephalopathy, particularly when concurrent with acidosis due to infection, ischemia, and hypoxia. The prevailing view is that acidosis increases the permeability of the blood-brain barrier to bilirubin and exacerbates its neurotoxicity. In this study, we found that the concentration of the cell death marker, lactate dehydrogenase (LDH) in cerebrospinal fluid (CSF), is elevated in infants with both hyperbilirubinemia and acidosis and showed stronger correlation with the severity of acidosis rather than increased bilirubin concentration. In mouse neonatal neurons, bilirubin exhibits limited toxicity but robustly potentiates the activity of acid-sensing ion channels (ASICs), resulting in increases in intracellular Ca2+ concentration, spike firings, and cell death. Furthermore, neonatal conditioning with concurrent hyperbilirubinemia and hypoxia-induced acidosis promoted long-term impairments in learning and memory and complex sensorimotor functions in vivo, which are largely attenuated in ASIC1a null mice. These findings suggest that targeting acidosis and ASICs may attenuate neonatal hyperbilirubinemia complications.

Acidosis is a hallmark of ischemic stroke and a promising neuroprotective target for preventing neuronal injury. Previously, genetic manipulations showed that blockade of acid-sensing ion channel 1a (ASIC1a)-mediated acidotoxicity could dramatically alleviate the volume of brain infarct and restore neurological function after cerebral ischemia. However, few pharmacological candidates have been identified to exhibit efficacy on ischemic stroke through inhibition of ASIC1a. In this work, we examined the ability of a toxin-inspired compound 5b (C5b), previously found to effectively inhibit ASIC1a in vitro, to exert protective effects in animal models of ischemic stroke in vivo. We found that C5b exerts significant neuroprotective effects not only in acid-induced neuronal death in vitro but also ischemic brain injury in vivo, suggesting that ASIC1a is a druggable target for therapeutic development. More importantly, C5b is able to cross the blood brain barrier and significantly reduce brain infarct volume when administered intravenously in the ischemic animal model, highlighting its systemic availability for therapies against neurodegeneration due to acidotoxicity. Together, our data demonstrate that C5b is a promising lead compound for neuroprotection through inhibiting ASIC1a, which warrants further translational studies.

Acid-sensing ion channels (ASICs), the main H+ receptors in the central nervous system, sense extracellular pH fluctuations and mediate cation influx. ASIC1a, the major subunit responsible for acid-activated current, is widely expressed in brain neurons, where it plays pivotal roles in diverse functions including synaptic transmission and plasticity. However, the underlying molecular mechanisms for these functions remain mysterious. Using extracellular epitope tagging and a novel antibody recognizing the hASIC1a ectodomain, we examined the membrane targeting and dynamic trafficking of hASIC1a in cultured cortical neurons. Surface hASIC1a was distributed throughout somata and dendrites, clustered in spine heads, and co-localized with postsynaptic markers. By extracellular pHluorin tagging and fluorescence recovery after photobleaching, we detected movement of hASIC1a in synaptic spine heads. Single-particle tracking along with use of the anti-hASIC1a ectodomain antibody revealed long-distance migration and local movement of surface hASIC1a puncta on dendrites. Importantly, enhancing synaptic activity with brain-derived neurotrophic factor accelerated the trafficking and lateral mobility of hASIC1a. With this newly-developed toolbox, our data demonstrate the synaptic location and high dynamics of functionally-relevant hASIC1a on the surface of excitatory synapses, supporting its involvement in synaptic functions.

Inhalation of aerosolized PS generated by the noninvasive apparatus could significantly reduce lung injury, while using oxygen line available in the clinical wards to generate PS aerosol is more convenient and adds further benefits. This method can also be used to deliver Dex and other therapeutic agents to ameliorate lung injury.