Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca(2+) permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASIC1a-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca(2+) elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.     

Zinc reverses glycine-dependent inactivation of NMDARs in cultured rat hippocampal neurons

In the presence of glutamate and co-agonists, e.g., glycine, the N-methyl-D-aspartate receptor (NMDAR) plays an important role in physiological and pathophysiological brain processes. Previous studies indicate glycine could inhibit NMDAR responses induced by high concentration of NMDA in hippocampal neurons. The mechanism underlying this inhibitory impact, however, has been unclear. In this study, the whole-cell patch-clamp recording and Ca²⁺ imaging with Fluo-3/AM under laser scanning confocal microscope were used to analyze the possible involvement of NMDAR subunits in this effect. We found that the peak current of NMDARs and Ca²⁺ influx induced by high concentration of NMDA were reduced by treatment of glycine (0.03?C10 ??mol L^?1) in a dose-dependent manner, and that the glycine-dependent inhibition of NMDAR responses, which were induced at 300 ??mol L^?1 NMDA, was reversed by ZnCl₂ through the blocking of the NR2A subunit of NMDARs, but was less influenced by ifenprodil, a NR2B inhibitor. Our results suggest that the glycine-dependent inactivation of NMDARs is potentially modulated by the regulatory subunit NR2A.     

Specificity and actions of an arylaspartate inhibitor of glutamate transport at the Schaffer collateral-CA1 pyramidal cell synapse

In this study we characterized the pharmacological selectivity and physiological actions of a new arylaspartate glutamate transporter blocker, L-threo-ß-benzylaspartate (L-TBA). At concentrations up to 100 µM, L-TBA did not act as an AMPA receptor (AMPAR) or NMDA receptor (NMDAR) agonist or antagonist when applied to outside-out patches from mouse hippocampal CA1 pyramidal neurons. L-TBA had no effect on the amplitude of field excitatory postsynaptic potentials (fEPSPs) recorded at the Schaffer collateral-CA1 pyramidal cell synapse. Excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons were unaffected by L-TBA in the presence of physiological extracellular Mg²⁺ concentrations, but in Mg²⁺-free solution, EPSCs were significantly prolonged as a consequence of increased NMDAR activity. Although L-TBA exhibited approximately four-fold selectivity for neuronal EAAT3 over glial EAAT1/EAAT2 transporter subtypes expressed in Xenopus oocytes, the L-TBA concentration-dependence of the EPSC charge transfer increase in the absence of Mg²⁺ was the same in hippocampal slices from EAAT3 +/+ and EAAT3 −/− mice, suggesting that TBA effects were primarily due to block of glial transporters. Consistent with this, L-TBA blocked synaptically evoked transporter currents in CA1 astrocytes with a potency in accord with its block of heterologously expressed glial transporters. Extracellular recording in the presence of physiological Mg²⁺ revealed that L-TBA prolonged fEPSPs in a frequency-dependent manner by selectively increasing the NMDAR-mediated component of the fEPSP during short bursts of activity. The data indicate that glial glutamate transporters play a dominant role in limiting extrasynaptic transmitter diffusion and binding to NMDARs. Furthermore, NMDAR signaling is primarily limited by voltage-dependent Mg²⁺ block during low-frequency activity, while the relative contribution of transport increases during short bursts of higher frequency signaling.     

CaMKII translocation requires local NMDA receptor-mediated Ca2+ signaling

Excitatory synaptic transmission and plasticity are critically modulated by N-methyl-D-aspartate receptors (NMDARs). Activation of NMDARs elevates intracellular Ca(2+) affecting several downstream signaling pathways that involve Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Importantly, NMDAR activation triggers CaMKII translocation to synaptic sites. NMDAR activation failed to induce Ca(2+) responses in hippocampal neurons lacking the mandatory NMDAR subunit NR1, and no EGFP-CaMKIIalpha translocation was observed. In cells solely expressing Ca(2+)-impermeable NMDARs containing NR1(N598R)-mutant subunits, prolonged NMDA application elevated internal Ca(2+) to the same degree as in wild-type controls, yet failed to translocate CaMKIIalpha. Brief local NMDA application evoked smaller Ca(2+) transients in dendritic spines of mutant compared to wild-type cells. CaMKIIalpha mutants that increase binding to synaptic sites, namely CaMKII-T286D and CaMKII-TT305/306VA, rescued the translocation in NR1(N598R) cells in a glutamate receptor-subtype-specific manner. We conclude that CaMKII translocation requires Ca(2+) entry directly through NMDARs, rather than other Ca(2+) sources activated by NMDARs. Together with the requirement for activated, possibly ligand-bound, NMDARs as CaMKII binding partners, this suggests that synaptic CaMKII accumulation is an input-specific signaling event.     

Local ASIC3 modulates pain and disease progression in a rat model of osteoarthritis

Background

Recent data have suggested a relationship between acute arthritic pain and acid sensing ion channel 3 (ASIC3) on primary afferent fibers innervating joints. The purpose of this study was to clarify the role of ASIC3 in a rat model of osteoarthritis (OA) which is considered a degenerative rather than an inflammatory disease.

Methods

We induced OA via intra-articular mono-iodoacetate (MIA) injection, and evaluated pain-related behaviors including weight bearing measured with an incapacitance tester and paw withdrawal threshold in a von Frey hair test, histology of affected knee joint, and immunohistochemistry of knee joint afferents. We also assessed the effect of ASIC3 selective peptide blocker (APETx2) on pain behavior, disease progression, and ASIC3 expression in knee joint afferents.

Results

OA rats showed not only weight-bearing pain but also mechanical hyperalgesia outside the knee joint (secondary hyperalgesia). ASIC3 expression in knee joint afferents was significantly upregulated approximately twofold at Day 14. Continuous intra-articular injections of APETx2 inhibited weight distribution asymmetry and secondary hyperalgesia by attenuating ASIC3 upregulation in knee joint afferents. Histology of ipsilateral knee joint showed APETx2 worked chondroprotectively if administered in the early, but not late phase.

Conclusions

Local ASIC3 immunoreactive nerve is strongly associated with weight-bearing pain and secondary hyperalgesia in MIA-induced OA model. APETx2 inhibited ASIC3 upregulation in knee joint afferents regardless of the time-point of administration. Furthermore, early administration of APETx2 prevented cartilage damage. APETx2 is a novel, promising drug for OA by relieving pain and inhibiting disease progression.     

A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons

From a systematic screening of animal venoms, we isolated a new toxin (APETx2) from the sea anemone Anthopleura elegantissima, which inhibits ASIC3 homomeric channels and ASIC3-containing heteromeric channels both in heterologous expression systems and in primary cultures of rat sensory neurons. APETx2 is a 42 amino-acid peptide crosslinked by three disulfide bridges, with a structural organization similar to that of other sea anemone toxins that inhibit voltage-sensitive Na+ and K+ channels. APETx2 reversibly inhibits rat ASIC3 (IC50=63 nM), without any effect on ASIC1a, ASIC1b, and ASIC2a. APETx2 directly inhibits the ASIC3 channel by acting at its external side, and it does not modify the channel unitary conductance. APETx2 also inhibits heteromeric ASIC2b+3 current (IC50=117 nM), while it has less affinity for ASIC1b+3 (IC50=0.9 microM), ASIC1a+3 (IC50=2 microM), and no effect on the ASIC2a+3 current. The ASIC3-like current in primary cultured sensory neurons is partly and reversibly inhibited by APETx2 with an IC50 of 216 nM, probably due to the mixed inhibitions of various co-expressed ASIC3-containing channels.     

In silico assessment of interaction of sea anemone toxin APETx2 and acid sensing ion channel 3

Acid sensing ion channels (ASICs) are proton-gated cation channels that are expressed throughout the nervous system and have been implicated in mediating sensory perception of noxious stimuli. Amongst the six ASIC isoforms, ASIC1a, 1b, 2a and 3 form proton-gated homomers, which differ in their activation and inactivation kinetics, expression profiles and pharmacological modulation; protons do not gate ASIC2b and ASIC4. As with many other ion channels, structure-function studies of ASICs have been greatly aided by the discovery of some toxins that act in isoform-specific ways. ASIC3 is predominantly expressed by sensory neurons of the peripheral nervous system where it acts to detect acid as a noxious stimulus and thus plays an important role in nociception. ASIC3 is the only ASIC subunit that is inhibited by the sea anemone (Anthopleura elegantissima)-derived toxin APETx2. However, the molecular mechanism by which APETx2 interacts with ASIC3 remains largely unknown. In this study, we made a homology model of ASIC3 and used extensive protein–protein docking to predict for the first time, the probable sites of APETx2 interaction on ASIC3. Additionally, using computational alanine scanning, we also suggest the ‘hot-spots’ that are likely to be critical for ASIC3–APETx2 interaction.     

NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

Accumulating evidence indicates the involvement of N-methyl-d-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Ro 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.     

NMDA‐type glutamate receptors mediate the acrosome reaction and motility initiation in newt sperm

The N‐methyl d ‐aspartate type glutamate receptor (NMDAR) is a ligand‐gated cation channel that causes Ca²⁺ influx in nerve cells. An NMDAR agonist is effective to the sperm motility in fowls, although the actual role of NMDAR in sperm function is unknown. In the present study, RNA‐seq of the spermatogenic testes suggested the presence of NMDAR in the sperm of the newt Cynops pyrrhogaster. Glutamate of at least 0.7 ± 0.5 mM was detected in the egg‐jelly substances along with acrosome reaction‐inducing substance (ARIS) and sperm motility‐initiating substance (SMIS). In the egg‐jelly extract (JE) that included the ARIS and SMIS, the acrosome reaction was inhibited by a NMDAR antagonists, memantine and MK801. MK801 also inhibited the spontaneous acrosome reaction in Steinberg's salt solution (ST). Furthermore, memantine and MK801 suppressed the progressive motility of the sperm in JE and spontaneous waving of the undulating membrane, which is the tail structure giving thrust for forward motility, in ST. The spontaneous waving of the undulating membrane was promoted when Mg²⁺, which blocks Ca²⁺ influx through gated NMDARs, was removed from the ST. In addition, the ARIS‐induced acrosome reaction was inhibited by a selective antagonist of the transient receptor potential vanilloid 4, whose activation might result in the membrane depolarization to release Mg²⁺ from the NMDAR. These results suggest that NMDAR acts together with other cation channels in the induction of the acrosome reaction and motility of the sperm during the fertilization process of C. pyrrhogaster.     

CaMKII T286 phosphorylation has distinct essential functions in three forms of long-term plasticity

The Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) mediates long-term potentiation or depression (LTP or LTD) after distinct stimuli of hippocampal NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD prevails in juvenile mice, but a mechanistically different form of LTD can be readily induced in adults by instead stimulating metabotropic glutamate receptors (mGluRs). However, the role that CaMKII plays in the mGluR-dependent form of LTD is not clear. Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation (pT286), which induces Ca²⁺-independent autonomous kinase activity. In addition, we compared the role of pT286 among three forms of long-term plasticity (NMDAR-dependent LTP and LTD, and mGluR-dependent LTD) using simultaneous live imaging of endogenous CaMKII together with synaptic marker proteins. We determined that after LTP stimuli, pT286 autophosphorylation accelerated CaMKII movement to excitatory synapses. After NMDAR-LTD stimuli, pT286 was strictly required for any movement to inhibitory synapses. Similar to NMDAR-LTD, we found the mGluR-LTD stimuli did not induce CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, we demonstrate that the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Thus, despite its prominent role in LTP, we conclude that CaMKII T286 autophosphorylation is also required for both major forms of hippocampal LTD, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.     

Red and near-infrared light induces intracellular Ca2+ flux via the activation of glutamate N-methyl-D-aspartate receptors

Red and near-infrared (NIR) light effect on Ca²⁺ ions flux through the influence on N-methyl-D-aspartate receptors (NMDARs) and their functioning in HeLa cells was studied in vitro. Cells were irradiated by 650 and 808 nm laser light at different power densities and doses and the obtained effect was compared with that caused by the pharmacological agents. The laser light was found to elevate Ca²⁺ influx into cell cytoplasm in a dose-dependent manner without changes of the NMDAR functioning. Furthermore, the light of both wavelengths demonstrated the ability to elevate Ca²⁺ influx under the pharmacological blockade of NMDARs and also might partially abolish the blockade enhancing Ca²⁺ influx after selective stimulation of the receptors with NMDA. Simultaneously, the light at moderate doses demonstrated a photobiostimulating effect on cells. Based on our experiments and data reported in the literature, we suggest that the low-power visible and NIR light can instigate a cell membrane depolarization via nonthermal activation, resulting in the fast induction of Ca²⁺ influx into cells. The obtained results also demonstrate that NIR light can be used for nonthermal and nonpharmacological stimulation of NMDARs in cancer cells.     

Protein kinase C promotes N-methyl-D-aspartate (NMDA) receptor trafficking by indirectly triggering calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation

Regulation of neuronal NMDA receptor (NMDAR) is critical in synaptic transmission and plasticity. Protein kinase C (PKC) promotes NMDAR trafficking to the cell surface via interaction with NMDAR-associated proteins (NAPs). Little is known, however, about the NAPs that are critical to PKC-induced NMDAR trafficking. Here, we showed that calcium/calmodulin-dependent protein kinase II (CaMKII) could be a NAP that mediates the potentiation of NMDAR trafficking by PKC. PKC activation promoted the level of autophosphorylated CaMKII and increased association with NMDARs, accompanied by functional NMDAR insertion, at postsynaptic sites. This potentiation, along with PKC-induced long term potentiation of the AMPA receptor-mediated response, was abolished by CaMKII antagonist or by disturbing the interaction between CaMKII and NR2A or NR2B. Further mutual occlusion experiments demonstrated that PKC and CaMKII share a common signaling pathway in the potentiation of NMDAR trafficking and long-term potentiation (LTP) induction. Our results revealed that PKC promotes NMDA receptor trafficking and induces synaptic plasticity through indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs.     

The Role of Hippocampal NMDA Receptors in Long-Term Emotional Responses following Muscarinic Receptor Activation

Extensive evidence indicates the influence of the cholinergic system on emotional processing. Previous findings provided new insights into the underlying mechanisms of long-term anxiety, showing that rats injected with a single systemic dose of pilocarpine—a muscarinic receptor (mAChR) agonist—displayed persistent anxiogenic-like responses when evaluated in different behavioral tests and time-points (24 h up to 3 months later). Herein, we investigated whether the pilocarpine-induced long-term anxiogenesis modulates the HPA axis function and the putative involvement of NMDA receptors (NMDARs) following mAChRs activation. Accordingly, adult male Wistar rats presented anxiogenic-like behavior in the elevated plus-maze (EPM) after 24 h or 1 month of pilocarpine injection (150 mg/kg, i.p.). In these animals, mAChR activation disrupted HPA axis function inducing a long-term increase of corticosterone release associated with a reduced expression of hippocampal GRs, as well as consistently decreased NMDAR subunits expression. Furthermore, in another group of rats injected with memantine–an NMDARs antagonist (4 mg/kg, i.p.)–prior to pilocarpine, we found inhibition of anxiogenic-like behaviors in the EPM but no further alterations in the pilocarpine-induced NMDARs downregulation. Our data provide evidence that behavioral anxiogenesis induced by mAChR activation effectively yields short- and long-term alterations in hippocampal NMDARs expression associated with impairment of hippocampal inhibitory regulation of HPA axis activity. This is a novel mechanism associated with anxiety-like responses in rats, which comprise a putative target to future translational studies.     

Mechanisms for localising calcineurin and CaMKII in dendritic spines

Calcineurin and calmodulin-dependent protein kinase II (CaMKII) are both highly abundant in neurons, and both are activated by calmodulin at similar Ca²⁺ concentrations in the test tube. However, they fulfill opposite functions in dendritic spines, with CaMKII activity driving long-term synaptic potentiation following large influxes of Ca²⁺ through NMDA-type glutamate receptors (NMDARs), and calcineurin responding to smaller influxes of Ca²⁺ through the same receptors to induce long-term depression. In this review, we explore the notion that precise dynamic localisation of the two enzymes at different sites within dendritic spines is fundamental to this behaviour. We describe the structural basis of calcineurin and CaMKII localisation by their interaction with proteins including AKAP79, densin-180, α-actinin, and NMDARs. We then consider how interactions with these proteins likely position calcineurin and CaMKII at different distances from Ca²⁺ microdomains emanating from the mouths of NMDARs in order to drive the divergent responses. We also highlight shortcomings in our current understanding of synaptic localisation of these two important signalling enzymes.     

CaMKII uses GTP as a phosphate donor for both substrate and autophosphorylation

The vast majority of serine/threonine protein kinases have a strong preference for ATP over GTP as a phosphate donor. CK2 (Casein kinase 2) is an exception to this rule and in this study we investigate whether calcium/calmodulin-dependent protein kinase II (CaMKII) has the same extended nucleotide range. Using the Drosophila enzyme, we have shown that CaMKII uses Mg²⁺GTP with a higher K_m and V_max compared to Mg²⁺ATP. Substitution of Mn²⁺ for Mg²⁺ resulted in a much lower K_m for GTP, while nearly abolishing the ability of CaMKII to use ATP. These similar results were obtained with rat αCaMKII, showing the ability to use GTP to be a general property of CaMKII. The V_max difference between Mg²⁺ATP and Mg²⁺GTP was found to be due to the fact that ADP is a potent inhibitor of phosphorylation, while GDP has modest effects. There were no differences found between sites autophosphorylated by ATP and GTP, either by partial proteolysis or mass spectrometry. Phosphorylation of fly head extract revealed that similar proteins are substrates for CaMKII whether using Mg²⁺ATP or Mg²⁺GTP. This new information confirms that CaMKII can use both ATP and GTP, and opens new avenues for the study of regulation of this kinase.     

On the Mechanism of Synaptic Depression Induced by CaMKIIN,an Endogenous Inhibitor of CaMKII

Activity-dependent synaptic plasticity underlies, at least in part, learning and memory processes. NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) is a major synaptic plasticity model. During LTP induction, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is activated, autophosphorylated and persistently translocated to the postsynaptic density, where it binds to the NMDAR. If any of these steps is inhibited, LTP is disrupted. The endogenous CaMKII inhibitor proteins CaMKIINα,β are rapidly upregulated in specific brain regions after learning. We recently showed that transient application of peptides derived from CaMKIINα (CN peptides) persistently depresses synaptic strength and reverses LTP saturation, as it allows further LTP induction in previously saturated pathways. The treatment disrupts basal CaMKII-NMDAR interaction and decreases bound CaMKII fraction in spines. To unravel CaMKIIN function and to further understand CaMKII role in synaptic strength maintenance, here we more deeply investigated the mechanism of synaptic depression induced by CN peptides (CN-depression) in rat hippocampal slices. We showed that CN-depression does not require glutamatergic synaptic activity or Ca²⁺ signaling, thus discarding unspecific triggering of activity-dependent long-term depression (LTD) in slices. Moreover, occlusion experiments revealed that CN-depression and NMDAR-LTD have different expression mechanisms. We showed that CN-depression does not involve complex metabolic pathways including protein synthesis or proteasome-mediated degradation. Remarkably, CN-depression cannot be resolved in neonate rats, for which CaMKII is mostly cytosolic and virtually absent at the postsynaptic densities. Overall, our results support a direct effect of CN peptides on synaptic CaMKII-NMDAR binding and suggest that CaMKIINα,β could be critical plasticity-related proteins that may operate as cell-wide homeostatic regulators preventing saturation of LTP mechanisms or may selectively erase LTP-induced traces in specific groups of synapses.