Xiang-ming Zha

Acid-sensing ion channel 1a is a postsynaptic proton receptor that affects the density of dendritic spines

Extracellular proton concentrations in the brain may be an important signal for neuron function. Proton concentrations change both acutely when synaptic vesicles release their acidic contents into the synaptic cleft and chronically during ischemia and seizures. However, the brain receptors that detect protons and their physiologic importance remain uncertain. Using organotypic hippocampal slices and biolistic transfection, we found the acid-sensing ion channel 1a (ASIC1a), localized in dendritic spines where it functioned as a proton receptor. ASIC1a also affected the density of spines, the postsynaptic site of most excitatory synapses. Decreasing ASIC1a reduced the number of spines, whereas overexpressing ASIC1a had the opposite effect. Ca2+-mediated Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling was probably responsible, because acid evoked an ASIC1a-dependent elevation of spine intracellular Ca2+ concentration, and reducing or increasing ASIC1a levels caused parallel changes in CaMKII phosphorylation in vivo. Moreover, inhibiting CaMKII prevented ASIC1a from increasing spine density. These data indicate that ASIC1a functions as a postsynaptic proton receptor that influences intracellular Ca2+ concentration and CaMKII phosphorylation and thereby the density of dendritic spines. The results provide insight into how protons influence brain function and how they may contribute to pathophysiology.

Targeting ASIC1a Reduces Innate Fear and Alters Neuronal Activity in the Fear Circuit

BACKGROUND The molecular mechanisms underlying innate fear are poorly understood. Previous studies indicated that the acid sensing ion channel ASIC1a influences fear behavior in conditioning paradigms. However, these differences may have resulted from an ASIC1a effect on learning, memory, or the expression of fear. METHODS To test the hypothesis that ASIC1a influences the expression of fear or anxiety independent of classical conditioning, we examined the effects of disrupting the mouse ASIC1a gene on unconditioned fear in the open field test, unconditioned acoustic startle, and fear evoked by the predator odor trimethylthiazoline (TMT). In addition, we tested the effects of acutely inhibiting ASIC1a with PcTx, an ASIC1a antagonist in tarantula venom. Our immunohistochemistry suggested ASIC1a is expressed in the bed nucleus of the stria terminalis, medial amygdala, and periaqueductal gray, which are thought to play important roles in the generation and expression of innate fear. Therefore, we also tested whether ASIC1a disruption altered c-fos expression in these structures following TMT exposure. RESULTS We found that the loss of ASIC1a reduced fear in the open field test, reduced acoustic startle, and inhibited the fear response to TMT. Similarly, intracerebroventricular administration of PcTx reduced TMT-evoked freezing in ASIC1a(+/+) mice but not ASIC1a(-/-) mice. In addition, loss of ASIC1a altered TMT-evoked c-fos expression in the medial amydala and dorsal periaqueductal gray. CONCLUSIONS These findings suggest that ASIC1a modulates activity in the circuits underlying innate fear. Furthermore, the data indicate that targeting the ASIC1a gene or acutely inhibiting ASIC1a suppresses fear and anxiety independent of conditioning.

Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala

Significance Synaptic transmission involves the release of neurotransmitters that activate receptors on postsynaptic cells. The results reveal that protons fulfill the criteria for a neurotransmitter and that they activate postsynaptic acid-sensing ion channels. This activity facilitates synaptic plasticity, a requirement for learning and memory in the amygdala. Stimulating presynaptic terminals can increase the proton concentration in synapses. Potential receptors for protons are acid-sensing ion channels (ASICs), Na+- and Ca2+-permeable channels that are activated by extracellular acidosis. Those observations suggest that protons might be a neurotransmitter. We found that presynaptic stimulation transiently reduced extracellular pH in the amygdala. The protons activated ASICs in lateral amygdala pyramidal neurons, generating excitatory postsynaptic currents. Moreover, both protons and ASICs were required for synaptic plasticity in lateral amygdala neurons. The results identify protons as a neurotransmitter, and they establish ASICs as the postsynaptic receptor. They also indicate that protons and ASICs are a neurotransmitter/receptor pair critical for amygdala-dependent learning and memory.

Ca2+/calmodulin-dependent protein kinases II and IV both promote survival but differ in their effects on axon growth in spiral ganglion neurons

Spiral ganglion neuron (SGN) survival in vitro can be maintained by neurotrophins, permeant cAMP analogs, and depolarization in an additive manner, with depolarization being the most efficacious. Therefore, we used cultured SGNs to determine the mechanism by which depolarization promotes neuronal survival. Our data implicate Ca2+/calmodulin‐dependent protein kinase (CaMK) activity by showing that it is induced by depolarization, that CaMK activity is necessary for at least part of the survival‐promoting effect of depolarization, and that CaMKII or CamKIV activity suffices to support neuronal survival in the absence of other trophic stimuli. First, that depolarization of SGNs activates CaMKs is evidenced by observation of increased CaMKII phosphorylation and of CaMK‐dependent CREB phosphorylation. Second, the requirement for CaMKs is shown by a reduction of SGN survival under depolarizing conditions in the presence of CaMK inhibitors. Third, transfection of COOH‐terminal‐truncated (lacking regulatory domain), constitutively active CaMKII or CaMKIV, but not of normal, full‐length CAMKs, promotes SGN survival in the absence of other trophic stimuli, indicating that CaMK activity is sufficient to promote survival. The survival‐promoting effect of truncated CaMKs is additive with that of depolarization, neurotrophins, or cyclic AMP. Although both CaMKII and CaMKIV activities converge in promoting survival, their actions on axon growth are markedly different: Transfection of truncated CaMKII, but not of truncated CaMKIV, into SGNs prevents axon outgrowth.

BDNF synthesis in spiral ganglion neurons is constitutive and CREB-dependent

Brain-derived neurotrophic factor (BDNF), which supports spiral ganglion neuron (SGN) survival in vivo and in vitro, is synthesized by SGNs. The BDNF gene generates multiple different transcripts, each from its own promoter region. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we find that SGNs express only the downstream transcripts III and IV in vivo and in vitro. Using RT-PCR assays of BDNF transcripts and transfection of BDNF promoter-reporter constructs, we tested the hypothesis, originally derived from studies of cortical neurons, that depolarization induces BDNF expression via a signaling pathway that includes Ca2+/calmodulin-dependent kinases (CaMKs) and the transcription factor, Ca2+/cyclic AMP response element binding protein (CREB). In contrast, we found that in SGNs in vivo BDNF expression is constitutive and is not increased by electrical activation. Similarly, BDNF expression in vitro is not increased by stimuli that activate CREB, including depolarization, cAMP, or transfection of activated CaMK mutants. However, transfection of dominant-negative CREB mutants did abrogate gene expression driven by BDNF promoters III and IV, indicating that CREB is necessary for constitutive BDNF expression. Thus, BDNF synthesis within SGNs makes possible an autocrine or paracrine mechanism that can contribute to support SGN survival but SGNs are distinctive in that this mechanism is constitutive and not activity-regulated.

Oxidant regulated inter-subunit disulfide bond formation between ASIC1a subunits

The acid-sensing ion channel-1a (ASIC1a) is composed of 3 subunits and is activated by a decrease in extracellular pH. It plays an important role in diseases associated with a reduced pH and production of oxidants. Previous work showed that oxidants reduce ASIC1a currents. However, the effects on channel structure and composition are unknown. We found that ASIC1a formed inter-subunit disulfide bonds and the oxidant H2O2 increased this link between subunits. Cys-495 in the ASIC1a C terminus was particularly important for inter-subunit disulfide bond formation, although other C-terminal cysteines contributed. Inter-subunit disulfide bonds also produced some ASIC1a complexes larger than trimers. Inter-subunit disulfide bond formation reduced the proportion of ASIC1a located on the cell surface and contributed to the H2O2-induced decrease in H+-gated current. These results indicate that channel function is controlled by disulfide bond formation between intracellular residues on distinct ASIC1a subunits. They also suggest a mechanism by which the redox state can dynamically regulate membrane protein activity by forming intracellular bridges.

Regulation of hippocampal synapse remodeling by epileptiform activity

We examined the regulation of dendritic spines and synapses by epileptiform activity (EA) in rat hippocampal slice cultures. EA, which was induced by a GABA(A) receptor inhibitor, gabazine, reduced pyramidal neuron spine density by approximately 50% after 48 h and also caused an increase in the average length of remaining spines. To directly determine the effects of EA on synapses, we used fluorescent protein-tagged PSD95, which marks postsynaptic densities. EA induced a net loss of synapses on spines but not shafts; conversely, activity blockade (TTX) induced a loss of shaft synapses. Time-lapse confocal imaging in live tissue slices revealed that EA (1) shifts the balance of synapse gain and loss in dendrites leading to a net loss of spine synapses and (2) induces the formation of new filopodia-like dendritic structures having abnormally slow motility. These results identify EA-induced changes in the density and distribution of synaptic structures on dendrites.

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.

Role of Ca2+/calmodulin-dependent protein kinase II in dendritic spine remodeling during epileptiform activity in vitro

Epileptiform activity (EA) in vivo and in vitro induces a loss of dendritic spines and synapses. Because CaMKII has been implicated in synaptogenesis and synaptic plasticity, we investigated the role of CaMKII in the effects of EA on spines, using rat hippocampal slice cultures. To visualize dendrites and postsynaptic densities (PSDs) in pyramidal neurons in the slices, we used biolistic transfection to express either free GFP or a PSD95‐YFP construct that specifically labels PSDs. This allowed us to distinguish two classes of dendritic protrusions: spines that contain PSDs, and filopodia that lack PSDs and that are, on average, longer than spines. By these criteria, 48 hr of EA caused a decrease specifically in the number of spines. Immunoblots showed that EA increased CaMKII activity in the slices. Inhibition of CaMKII by expression of AIP, a specific peptide inhibitor of CaMKII, reduced spine number under basal conditions and failed to prevent EA‐induced spine loss. However, under EA conditions, AIP increased the number of filopodia and the number of PSDs on the dendritic shaft. These data show at least two roles for CaMKII activity in maintenance and remodeling of dendritic spines under basal or EA conditions. First, CaMKII activity promotes the maintenance of spines and spine PSDs. Second, CaMKII activity suppresses EA‐induced formation of filopodia and suppresses an increase in shaft PSDs, apparently by promoting translocation of PSDs from dendritic shafts to spines and/or selectively stabilizing spine rather than shaft PSDs.

Synaptic activity and F-actin coordinately regulate CaMKIIα localization to dendritic postsynaptic sites in developing hippocampal slices

We examined the timing and mechanisms of CaMKIIalpha recruitment to nascent synapses of developing rat hippocampal pyramidal neurons in slice culture. Time-lapse confocal imaging shows that GFP-CaMKIIalpha in transfected neurons accumulates in spines as they are forming, and loss of CaMKIIalpha coincides with spine destabilization. Immunolabeling shows that endogenous CaMKIIalpha is concentrated at postsynaptic sites in spines under ambient slice culture conditions, and this is not disrupted by short-term (3 h) synaptic activity blockade or Latrunculin-induced F-actin depolymerization. However, the combination of activity blockade and F-actin depolymerization significantly reduces synaptic CaMKIIalpha. Conversely, postsynaptic activation induces synaptic recruitment of CaMKIIalpha even in the presence of F-actin depolymerizing drugs. Thus, synaptic-activity-dependent mechanisms and (synaptic activity-independent) F-actin-based mechanisms are individually sufficient and act in parallel to localize CaMKIIalpha to the dendritic spine compartment. Moreover, the timing of CaMKIIalpha recruitment to developing spines suggests a role for CaMKIIalpha in spine assembly and maintenance.