Xiang-Ping Chu

Neuroprotection in Ischemia: Blocking Calcium-Permeable Acid-Sensing Ion Channels

Ca2+ toxicity remains the central focus of ischemic brain injury. The mechanism by which toxic Ca2+ loading of cells occurs in the ischemic brain has become less clear as multiple human trials of glutamate antagonists have failed to show effective neuroprotection in stroke. Acidosis is a common feature of ischemia and is assumed to play a critical role in brain injury; however, the mechanism(s) remain ill defined. Here, we show that acidosis activates Ca2+ -permeable acid-sensing ion channels (ASICs), inducing glutamate receptor-independent, Ca2+ -dependent, neuronal injury inhibited by ASIC blockers. Cells lacking endogenous ASICs are resistant to acid injury, while transfection of Ca2+ -permeable ASIC1a establishes sensitivity. In focal ischemia, intracerebroventricular injection of ASIC1a blockers or knockout of the ASIC1a gene protects the brain from ischemic injury and does so more potently than glutamate antagonism. Thus, acidosis injures the brain via membrane receptor-based mechanisms with resultant toxicity of [Ca2+]i, disclosing new potential therapeutic targets for stroke.

Effect of ischaemic preconditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation and hypoxia-tolerant states.

BACKGROUND Molecular mechanisms of neuroprotection that lead to ischaemic tolerance are incompletely understood. Identification of genes involved in the process would provide insight into cell survival and therapeutic approaches for stroke. We developed a mouse model of neuroprotection in stroke and did gene expression profiling to identify potential neuroprotective genes and their associated pathways. METHODS Eight mice per condition were subjected to occlusion of the middle cerebral artery for 15 min (preconditioning), 60 min (injurious ischaemia), or preconditioning followed 72 h later by injurious ischaemia. RNA was extracted from the cortical regions of the ischaemic and non-ischaemic hemispheres. Three pools per condition were generated, and RNA was hybridised to oligonucleotide microarrays for comparison of ischaemic and non-ischaemic hemispheres. Real-time PCR and western blots were used to validate results. Follow-up experiments were done to address the biological relevance of findings. FINDINGS Microarray analysis revealed changes in gene expression with little overlap among the conditions of injurious ischaemia, ischaemic preconditioning, or both. Injurious ischaemia induced upregulation of gene expression; 49 (86%) of 57 genes regulated showed increased expression in the ischaemic hemisphere. By contrast, preconditioning followed by injurious ischaemia resulted in pronounced downregulation; 47 (77%) of 61 regulated genes showed lower expression. Preconditioning resulted in transcriptional changes involved in suppression of metabolic pathways and immune responses, reduction of ion-channel activity, and decreased blood coagulation. INTERPRETATION Preconditioning reprogrammes the response to ischaemic injury. Similar changes reported by others support an evolutionarily conserved endogenous response to decreased blood flow and oxygen limitation such as seen during hibernation.

Modulation of D2R-NR2B Interactions in Response to Cocaine

Dopamine-glutamate interactions in the neostriatum determine psychostimulant action, but the underlying molecular mechanisms remain elusive. Here we found that dopamine stimulation by cocaine enhances a heteroreceptor complex formation between dopamine D2 receptors (D2R) and NMDA receptor NR2B subunits in the neostriatum in vivo. The D2R-NR2B interaction is direct and occurs in the confined postsynaptic density microdomain of excitatory synapses. The enhanced D2R-NR2B interaction disrupts the association of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) with NR2B, reduces NR2B phosphorylation at a CaMKII-sensitive site (Ser1303), and inhibits NMDA receptor-mediated currents in medium-sized striatal neurons. Furthermore, the regulated D2R-NR2B interaction is critical for constructing behavioral responsiveness to cocaine. Our findings here uncover a direct and dynamic D2R-NR2B interaction in striatal neurons in vivo. This type of dopamine-glutamate integration at the receptor level may be responsible for synergistically inhibiting the D2R-mediated circuits in the basal ganglia and fulfilling the stimulative effect of psychostimulants.

Subunit-Dependent High-Affinity Zinc Inhibition of Acid-Sensing Ion Channels

Acid-sensing ion channels (ASICs), a novel class of ligand-gated cation channels activated by protons, are highly expressed in peripheral sensory and central neurons. Activation of ASICs may play an important role in physiological processes such as nociception, mechanosensation, and learning-memory, and in the pathology of neurological conditions such as brain ischemia. Modulation of the activities of ASICs is expected to have a significant influence on the roles that these channels can play in both physiological and/or pathological processes. Here we show that the divalent cation Zn2+, an endogenous trace element, dose-dependently inhibits ASIC currents in cultured mouse cortical neurons at nanomolar concentrations. With ASICs expressed in Chinese hamster ovary cells, Zn2+ inhibits currents mediated by homomeric ASIC1a and heteromeric ASIC1a-ASIC2a channels, without affecting currents mediated by homomeric ASIC1β, ASIC2a, or ASIC3. Consistent with ASIC1a-specific modulation, high-affinity Zn2+ inhibition is absent in neurons from ASIC1a knock-out mice. Current-clamp recordings and Ca2+-imaging experiments demonstrated that Zn2+ inhibits acid-induced membrane depolarization and the increase of intracellular Ca2+. Mutation of lysine-133 in the extracellular domain of the ASIC1a subunit abolishes the high-affinity Zn2+ inhibition. Our studies suggest that Zn2+ may play an important role in a negative feedback system for preventing overexcitation of neurons during normal synaptic transmission and ASIC1a-mediated excitotoxicity in pathological conditions.

Transient receptor potential melastatin 7-like current in human head and neck carcinoma cells: role in cell proliferation.

Ion channels are involved in normal physiologic processes and in the pathology of various diseases. In this study, we investigated the presence and potential function of transient receptor potential melastatin 7 (TRPM7) channels in the growth and proliferation of FaDu and SCC25 cells, two common human head and neck squamous carcinoma cell lines, using a combination of patch-clamp recording, Western blotting, immunocytochemistry, small interfering RNA (siRNA), fluorescent Ca(2+) imaging, and cell counting techniques. Although voltage-gated K(+) currents were recorded in all cells, none of FaDu cells express voltage-gated Na(+) or Ca(2+) currents. Perfusion of cells with NMDA or acidic solution did not activate inward currents, indicating a lack of NMDA receptor and acid-sensing channels. Lowering extracellular Ca(2+), however, induced a large nondesensitizing current reminiscent of Ca(2+)-sensing cation current or TRPM7 current previously described in other cells. This Ca(2+)-sensing current can be inhibited by Gd(3+), 2-aminoethoxydiphenyl borate (2-APB), or intracellular Mg(2+), consistent with the TRPM7 current being activated. Immunocytochemistry, Western blot, and reverse transcription-PCR detected the expression of TRPM7 protein and mRNA in these cells. Transfection of FaDu cells with TRPM7 siRNA significantly reduced the expression of TRPM7 mRNA and protein as well as the amplitude of the Ca(2+)-sensing current. Furthermore, we found that Ca(2+) is critical for the growth and proliferation of FaDu cells. Blockade of TRPM7 channels by Gd(3+) and 2-APB or suppression of TRPM7 expression by siRNA inhibited the growth and proliferation of these cells. Similar to FaDu cells, SCC25 cells also express TRPM7-like channels. Suppressing the function of these channels inhibited the proliferation of SCC25 cells.

Stability of surface NMDA receptors controls synaptic and behavioral adaptations to amphetamine

Plastic changes in glutamatergic synapses that lead to endurance of drug craving and addiction are poorly understood. We examined the turnover and trafficking of NMDA receptors and found that chronic exposure to the psychostimulant amphetamine (AMPH) induced selective downregulation of NMDA receptor NR2B subunits in the confined surface membrane pool of rat striatal neurons at synaptic sites. This downregulation was a long-lived event and was a result of the destabilization of surface-expressed NR2B caused by accelerated ubiquitination and degradation of crucial NR2B-anchoring proteins by the ubiquitin-proteasome system. The biochemical loss of synaptic NR2B further translated to the modulation of synaptic plasticity in the form of long-term depression at cortico-accumbal glutamatergic synapses. Behaviorally, genetic disruption of NR2B induced and restoration of NR2B loss prevented behavioral sensitization to AMPH. Our data identify NR2B as an important regulator in the remodeling of excitatory synapses and persistent psychomotor plasticity in response to AMPH.

Extracellular Spermine Exacerbates Ischemic Neuronal Injury through Sensitization of ASIC1a Channels to Extracellular Acidosis

Ischemic brain injury is a major problem associated with stroke. It has been increasingly recognized that acid-sensing ion channels (ASICs) contribute significantly to ischemic neuronal damage, but the underlying mechanism has remained elusive. Here, we show that extracellular spermine, one of the endogenous polyamines, exacerbates ischemic neuronal injury through sensitization of ASIC1a channels to extracellular acidosis. Pharmacological blockade of ASIC1a or deletion of the ASIC1 gene greatly reduces the enhancing effect of spermine in ischemic neuronal damage both in cultures of dissociated neurons and in a mouse model of focal ischemia. Mechanistically, spermine profoundly reduces desensitization of ASIC1a by slowing down desensitization in the open state, shifting steady-state desensitization to more acidic pH, and accelerating recovery between repeated periods of acid stimulation. Spermine-mediated potentiation of ASIC1a activity is occluded by PcTX1 (psalmotoxin 1), a specific ASIC1a inhibitor binding to its extracellular domain. Functionally, the enhanced channel activity is accompanied by increased acid-induced neuronal membrane depolarization and cytoplasmic Ca2+ overload, which may partially explain the exacerbated neuronal damage caused by spermine. More importantly, blocking endogenous spermine synthesis significantly attenuates ischemic brain injury mediated by ASIC1a but not that by NMDA receptors. Thus, extracellular spermine contributes significantly to ischemic neuronal injury through enhancing ASIC1a activity. Our data suggest new neuroprotective strategies for stroke patients via inhibition of polyamine synthesis and subsequent spermine–ASIC interaction.

ASIC1a-Specific Modulation of Acid-Sensing Ion Channels in Mouse Cortical Neurons by Redox Reagents

Acid-sensing ion channel (ASIC)-1a, the major ASIC subunit with Ca2+ permeability, is highly expressed in the neurons of CNS. Activation of these channels with resultant intracellular Ca2+ accumulation plays a critical role in normal synaptic plasticity, learning/memory, and in acidosis-mediated glutamate receptor-independent neuronal injury. Here we demonstrate that the activities of ASICs in CNS neurons are tightly regulated by the redox state of the channels and that the modulation is ASIC1a subunit dependent. In cultured mouse cortical neurons, application of the reducing agents dramatically potentiated, whereas the oxidizing agents inhibited the ASIC currents. However, in neurons from the ASIC1 knock-out mice, neither oxidizing agents nor reducing reagents had any effect on the acid-activated current. In Chinese Hamster Ovary cells, redox-modifying agents only affected the current mediated by homomeric ASIC1a, but not homomeric ASIC1b, ASIC2a, or ASIC3. In current-clamp recordings and Ca2+-imaging experiments, the reducing agents increased but the oxidizing agents decreased acid-induced membrane depolarization and the intracellular Ca2+ accumulation. Site-directed mutagenesis studies identified involvement of cysteine 61 and lysine 133, located in the extracellular domain of the ASIC1a subunit, in the modulation of ASICs by oxidizing and reducing agents, respectively. Our results suggest that redox state of the ASIC1a subunit is an important factor in determining the overall physiological function and the pathological role of ASICs in the CNS.

Modulation of Acid-sensing Ion Channel Currents, Acid-induced Increase of Intracellular Ca2+, and Acidosis-mediated Neuronal Injury by Intracellular pH

Acid-sensing ion channels (ASICs), activated by lowering extracellular pH (pHo), play an important role in normal synaptic transmission in brain and in the pathology of brain ischemia. Like pHo, intracellular pH (pHi) changes dramatically in both physiological and pathological conditions. Although it is known that a drop in pHo activates the ASICs, it is not clear whether alterations of pHi have an effect on these channels. Here we demonstrate that the overall activities of ASICs, including channel activation, inactivation, and recovery from desensitization, are tightly regulated by pHi. In cultured mouse cortical neurons, bath perfusion of the intracellular alkalizing agent quinine increased the amplitude of the ASIC current by ∼50%. In contrast, intracellular acidification by withdrawal of NH4Cl or perfusion of propionate inhibited the current. Increasing pH buffering capacity in the pipette solution with 40 mm HEPES attenuated the effects of quinine and NH4Cl. The effects of intracellular alkalizing/acidifying agents were mimicked by using intracellular solutions with pH directly buffered at high/low values. Increasing pHi induced a shift in H+ dose-response curve toward less acidic pH but a shift in the steady state inactivation curve toward more acidic pH. In addition, alkalizing pHi induced an increase in the recovery rate of ASICs from desensitization. Consistent with its effect on the ASIC current, changing pHi has a significant influence on the acid-induced increase of intracellular Ca2+, membrane depolarization, and acidosis-mediated neuronal injury. Our findings suggest that changes in pHi may play an important role in determining the overall function of ASICs in both physiological and pathological conditions.

N-Glycosylation of Acid-Sensing Ion Channel 1a Regulates Its Trafficking and Acidosis-Induced Spine Remodeling

Acid-sensing ion channel-1a (ASIC1a) is a potential therapeutic target for multiple neurological diseases. We studied here ASIC1a glycosylation and trafficking, two poorly understood processes pivotal in determining the functional outcome of an ion channel. We found that most ASIC1a in the mouse brain was fully glycosylated. Inhibiting glycosylation with tunicamycin reduced ASIC1a surface trafficking, dendritic targeting, and acid-activated current density. N-glycosylation of the two glycosylation sites, Asn393 and Asn366, has differential effects on ASIC1a biogenesis. Maturation of Asn393 increased ASIC1a surface and dendritic trafficking, pH sensitivity, and current density. In contrast, glycosylation of Asn366 was dispensable for ASIC1a function and may be a rate-limiting step in ASIC1a biogenesis. In addition, we revealed that acidosis reduced the density and length of dendritic spines in a time- and ASIC1a-dependent manner. ASIC1a N366Q, which showed increased glycosylation and dendritic targeting, potentiated acidosis-induced spine loss. Conversely, ASIC1a N393Q, which had diminished dendritic targeting and inhibited ASIC1a current dominant-negatively, had the opposite effect. These data tie N-glycosylation of ASIC1a with its trafficking. More importantly, by revealing a site-specific effect of acidosis on dendritic spines, our findings suggest that these processes have an important role in regulating synaptic plasticity and determining long-term consequences in diseases that generate acidosis.