Glutamatergic neurons in the paraventricular nucleus of the hypothalamus participate in the regulation of visceral pain induced by pancreatic cancer in mice.

Background: Visceral pain induced by pancreatic cancer seriously affects patients' quality of life, and there is no effective treatment, because the mechanism of its neural circuit is unknown. Therefore, the aim of this study is to explore the main neural circuit mechanism regulating visceral pain induced by pancreatic cancer in mice. Methods: The mouse model of pancreatic cancer visceral pain was established on C57BL/6N mice by pancreatic injection of mPAKPC-luc cells. Abdominal mechanical hyperalgesia and hunch score were performed to assess visceral pain; the pseudorabies virus (PRV) was used to identify the brain regions innervating the pancreas; the c-fos co-labeling method was used to ascertain the types of activated neurons; in vitro electrophysiological patch-clamp technique was used to record the electrophysiological activity of specific neurons; the calcium imaging technique was used to determine the calcium activity of specific neurons; specific neuron destruction and chemogenetics methods were used to explore whether specific neurons were involved in visceral pain induced by pancreatic cancer. Results: The PRV injected into the pancreas was detected in the paraventricular nucleus of the hypothalamus (PVN). Immunofluorescence staining showed that the majority of c-fos were co-labeled with glutamatergic neurons in the PVN. In vitro electrophysiological results showed that the firing frequency of glutamatergic neurons in the PVN was increased. The calcium imaging results showed that the calcium activity of glutamatergic neurons in the PVN was enhanced. Both specific destruction of glutamatergic neurons and chemogenetics inhibition of glutamatergic neurons in the PVN alleviated visceral pain induced by pancreatic cancer. Conclusions: Glutamatergic neurons in the PVN participate in the regulation of visceral pain induced by pancreatic cancer in mice, providing new insights for the discovery of effective targets for the treatment of pancreatic cancer visceral pain.

Pharmacological Validation of ASIC1a as a Druggable Target for Neuroprotection in Cerebral Ischemia Using an Intravenously Available Small Molecule Inhibitor.

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.

Postsynaptic Targeting and Mobility of Membrane Surface-Localized hASIC1a.

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.

Bilirubin enhances the activity of ASIC channels to exacerbate neurotoxicity in neonatal hyperbilirubinemia in mice.

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.

Protein Kinase C Lambda Mediates Acid-Sensing Ion Channel 1a-Dependent Cortical Synaptic Plasticity and Pain Hypersensitivity.

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 λ-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.SIGNIFICANCE STATEMENT 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.

Fear extinction requires ASIC1a-dependent regulation of hippocampal-prefrontal correlates.

Extinction of conditioned fear necessitates the dynamic involvement of hippocampus, medial prefrontal cortex (mPFC), and basolateral amygdala (BLA), but key molecular players that regulate these circuits to achieve fear extinction remain largely unknown. Here, we report that acid-sensing ion channel 1a (ASIC1a) is a crucial molecular regulator of fear extinction, and that this function requires ASIC1a in ventral hippocampus (vHPC), but not dorsal hippocampus, mPFC, or BLA. While genetic disruption or pharmacological inhibition of ASIC1a in vHPC attenuated the extinction of conditioned fear, overexpression of the channel in this area promoted fear extinction. Channelrhodopsin-2-assisted circuit mapping revealed that fear extinction involved an ASIC1a-dependent modification of the long-range hippocampal-prefrontal correlates in a projection-specific manner. Gene expression profiling analysis and validating experiments identified several neuronal activity-regulated and memory-related genes, including Fos, Npas4, and Bdnf, as the potential mediators of ASIC1a regulation of fear extinction. Mechanistically, genetic overexpression of brain-derived neurotrophic factor (BDNF) in vHPC or supplement of BDNF protein in mPFC both rescued the deficiency in fear extinction and the deficits on extinction-driven adaptations of hippocampal-prefrontal correlates caused by the Asic1a gene inactivation in vHPC. Together, these results establish ASIC1a as a critical constituent in fear extinction circuits and thus a promising target for managing adaptive behaviors.

The acid-sensing ion channel ASIC1a mediates striatal synapse remodeling and procedural motor learning.

Acid-sensing ion channel 1a (ASIC1a) is abundant in multiple brain regions, including the striatum, which serves as the input nucleus of the basal ganglia and is critically involved in procedural learning and motor memory. We investigated the functional role of ASIC1a in striatal neurons. We found that ASIC1a was critical for striatum-dependent motor coordination and procedural learning by regulating the synaptic plasticity of striatal medium spiny neurons. Global deletion of Asic1a in mice led to increased dendritic spine density but impaired spine morphology and postsynaptic architecture, which were accompanied by the decreased function of N-methyl-d-aspartate (NMDA) receptors at excitatory synapses. These structural and functional changes caused by the loss of ASIC1a were largely mediated by reduced activation (phosphorylation) of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated protein kinases (ERKs). Consequently, Asic1a null mice exhibited poor performance on multiple motor tasks, which was rescued by striatal-specific expression of either ASIC1a or CaMKII. Together, our data reveal a previously unknown mechanism mediated by ASIC1a that promotes the excitatory synaptic function underlying striatum-related procedural learning and memory.

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.

Molecular mechanism of constitutive endocytosis of Acid-sensing ion channel 1a and its protective function in acidosis-induced neuronal death.

Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the peripheral and CNSs, which critically contribute to a variety of pathophysiological conditions that involve tissue acidosis, such as ischemic stroke and epileptic seizures. However, the trafficking mechanisms of ASICs and the related proteins remain largely unknown. Here, we demonstrate that ASIC1a, the main ASIC subunit in the brain, undergoes constitutive endocytosis in a clathrin- and dynamin-dependent manner in both mouse cortical neurons and heterologous cell cultures. The endocytosis of ASIC1a was inhibited by either the small molecular inhibitor tyrphostin A23 or knockdown of the core subunit of adaptor protein 2 (AP2) µ2 using RNA interference, supporting a clathrin-dependent endocytosis of ASIC1a. In addition, the internalization of ASIC1a was blocked by dominant-negative dynamin1 mutation K44A and the small molecular inhibitor dynasore, suggesting that it is also dynamin-dependent. We show that the membrane-proximal residues (465)LCRRG(469) at the cytoplasmic C terminus of ASIC1a are critical for interaction with the endogenous adaptor protein complex and inhibition of ASIC1a internalization strongly exacerbated acidosis-induced death of cortical neurons from wild-type but not ASIC1a knock-out mice. Together, these results reveal the molecular mechanism of ASIC1a internalization and suggest the importance of endocytic pathway in functional regulation of ASIC1a channels as well as neuronal damages mediated by these channels during neurodegeneration.

Therapeutic effects of inhaling aerosolized surfactant alone or with dexamethasone generated by a novel noninvasive apparatus on acute lung injury in rats.

BACKGROUND: Pulmonary surfactant (PS) administration has been attempted for the treatment of adults with acute lung injury (ALI)/adult respiratory distress syndrome. Aerosolized surfactants inhaled by spontaneous breathing may be an effective method of surfactant-based therapies. Using a noninvasive apparatus, we evaluated the therapeutic effects of aerosolized PS alone or together with dexamethasone (Dex) on a rat model of ALI. METHODS: Severe ALI was induced by intravenous injection of 20% oleic acid (0.2 mL/kg) into adult Sprague-Dawley rats. Animals were divided into eight groups: sham (n = 10); model (injury only, n = 10); normal saline (NS) aerosol driven by compressed air (air-NS, n = 13); PS aerosol driven by compressed air (air-PS, n = 13); NS aerosol driven by O2 (O2-NS, n = 13); PS aerosol driven by O2 (O2-PS, n = 13); Dex aerosol driven by O2 (O2-Dex, n = 13); and PS and Dex aerosol driven by O2 (O2-PS-Dex, n = 13). Blood gases, breathing rate, lung index, total protein, and proinflammatory cytokines (tumor necrosis factor-α, interleukin 1ß, interleukin 6) in the bronchoalveolar lavage fluid (BALF), and lung histology were examined. RESULTS: Animals treated with air-PS for 20 minutes had significantly improved lung function, reduced pulmonary edema, decreased concentration of total protein and proinflammatory cytokines in BALF, ameliorated lung injury, and improved animal survival. In the O2-PS group, the breathing rates and lung injury scores were significantly lower than that of the air-PS group. In the O2-PS-Dex group, lung edema, total protein, and inflammatory cytokines in BALF were significantly reduced in comparison with the O2-PS group. CONCLUSION: 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.

Upregulations of glucocorticoid-induced leucine zipper by hypoxia and glucocorticoid inhibit proinflammatory cytokines under hypoxic conditions in macrophages.

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-1ß, 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-1ß and IL-6 in hypoxia and abrogated the inhibitory effect of Dex on expression of IL-1ß 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.