Inhibiting pro-death NMDA receptor signaling dependent on the NR2 PDZ ligand may not affect synaptic function or synaptic NMDA receptor signaling to gene expression

NMDA receptors (NMDARs) mediate ischemic brain damage, in part through interactions of the PDZ ligand of NR2 subunits with the PDZ domain proteins PSD-95 and neuronal nitric oxide synthase located within the NMDAR signaling complex. We have recently shown that this PDZ ligand-dependent pathway promotes neuronal death via p38 activation. A peptide mimetic of the NR2B PDZ ligand (TAT-NR2B9c) reduces p38-mediated death in vitro and p38-dependent ischemic damage in vivo. In the absence of the PDZ ligand-p38 pathway, such as in TAT-NR2B9c-treated neurons, or in NMDAR-expressing non-neuronal cells, NMDAR-dependent excitotoxicity is mediated largely by JNK and requires greater Ca2+ influx. A major reason for blocking pro-death signaling events downstream of the NMDAR as an anti-excitotoxic strategy is that it may spare physiological synaptic function and signaling. We find that neuroprotective doses of TAT-NR2B9c do not alter the frequency of spontaneous synaptic events within networks of cultured cortical neurons nor is mini-EPSC frequency altered. Furthermore, TAT-NR2B9c does not inhibit the capacity of synaptic NMDAR activity to promote neuroprotective changes in gene expression, including the up-regulation of PACAP via CREB, and suppression of the pro-oxidative FOXO target gene Txnip. Thus, while the NR2 PDZ ligand does not account for all the excitotoxic effects of excessive NMDAR activity, these findings underline the value of the specific targeting of death pathways downstream of the NMDAR.     

Adenosine A2A receptors are required for glutamate mGluR5‐ and dopamine D1 receptor‐evoked ERK1/2 phosphorylation in rat hippocampus: involvement of NMDA receptor

Interaction between mGluR5 and NMDA receptors (NMDAR ) is vital for synaptic plasticity and cognition. We recently demonstrated that stimulation of mGluR5 enhances NMDAR responses in hippocampus by phosphorylating NR2B(Tyr1472) subunit, and this reaction was enabled by adenosine A_2A receptors (A_2AR) (J Neurochem, 135, 2015, 714). In this study, by using in vitro phosphorylation and western blot analysis in hippocampal slices of male Wistar rats, we show that mGluR5 stimulation or mGluR5/NMDAR s co‐stimulation synergistically activate ERK 1/2 signaling leading to c‐Fos expression. Interestingly, both reactions are under the permissive control of endogenous adenosine acting through A_2ARs. Moreover, mGluR5‐mediated ERK 1/2 phosphorylation depends on NMDAR , which however exhibits a metabotropic way of function, since no ion influx through its ion channel is required. Furthermore, our results demonstrate that mGluR5 and mGluR5/NMDAR ‐evoked ERK 1/2 activation correlates well with the mGluR5/NMDAR ‐evoked NR2B(Tyr1472) phosphorylation, since both phenomena coincide temporally, are Src dependent, and are both enabled by A_2ARs. This indicates a functional involvement of NR2B(Tyr1472) phosphorylation in the ERK 1/2 activation. Our biochemical results are supported by electrophysiological data showing that in CA 1 region of hippocampus, the theta burst stimulation (TBS)‐induced long‐term potentiation coincides temporally with an increase in ERK 1/2 activation and both phenomena are dependent on the tripartite A_2A, mGlu5, and NMDAR s. Furthermore, we show that the dopamine D1 receptors evoked ERK 1/2 activation as well as the NR2B(Tyr1472) phosphorylation are also regulated by endogenous adenosine and A_2ARs. In conclusion, our results highlight the A_2ARs as a crucial regulator not only for NMDAR responses, but also for regulating ERK 1/2 signaling and its downstream pathways, leading to gene expression, synaptic plasticity, and memory consolidation.

     

Computational investigation of the changing patterns of subtype specific NMDA receptor activation during physiological glutamatergic neurotransmission

NMDA receptors (NMDARs) are the major mediator of the postsynaptic response during synaptic neurotransmission. The diversity of roles for NMDARs in influencing synaptic plasticity and neuronal survival is often linked to selective activation of multiple NMDAR subtypes (NR1/NR2A-NMDARs, NR1/NR2B-NMDARs, and triheteromeric NR1/NR2A/NR2B-NMDARs). However, the lack of available pharmacological tools to block specific NMDAR populations leads to debates on the potential role for each NMDAR subtype in physiological signaling, including different models of synaptic plasticity. Here, we developed a computational model of glutamatergic signaling at a prototypical dendritic spine to examine the patterns of NMDAR subtype activation at temporal and spatial resolutions that are difficult to obtain experimentally. We demonstrate that NMDAR subtypes have different dynamic ranges of activation, with NR1/NR2A-NMDAR activation sensitive at univesicular glutamate release conditions, and NR2B containing NMDARs contributing at conditions of multivesicular release. We further show that NR1/NR2A-NMDAR signaling dominates in conditions simulating long-term depression (LTD), while the contribution of NR2B containing NMDAR significantly increases for stimulation frequencies that approximate long-term potentiation (LTP). Finally, we show that NR1/NR2A-NMDAR content significantly enhances response magnitude and fidelity at single synapses during chemical LTP and spike timed dependent plasticity induction, pointing out an important developmental switch in synaptic maturation. Together, our model suggests that NMDAR subtypes are differentially activated during different types of physiological glutamatergic signaling, enhancing the ability for individual spines to produce unique responses to these different inputs.     

Differential roles of NR2A- and NR2B-containing NMDA receptors in Ras-ERK signaling and AMPA receptor trafficking

NMDA receptors (NMDARs) control bidirectional synaptic plasticity by regulating postsynaptic AMPA receptors (AMPARs). Here we show that NMDAR activation can have differential effects on AMPAR trafficking, depending on the subunit composition of NMDARs. In mature cultured neurons, NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1, primarily by regulating its surface insertion. In mature neurons, NR2B is coupled to inhibition rather than activation of the Ras-ERK pathway, which drives surface delivery of GluR1. Moreover, the synaptic Ras GTPase activating protein (GAP) SynGAP is selectively associated with NR2B-NMDARs in brain and is required for inhibition of NMDAR-dependent ERK activation. Preferential coupling of NR2B to SynGAP could explain the subtype-specific function of NR2B-NMDARs in inhibition of Ras-ERK, removal of synaptic AMPARs, and weakening of synaptic transmission.     

Superoxide generation via the NR2B-NMDAR/RasGRF1/NOX2 pathway promotes dendritogenesis

N-methyl-D-aspartate receptors (NMDARs) that contain the NR2A and NR2B subunits play a critical role in neuronal plasticity and dendritogenesis. Gain-and-loss-of function studies indicate that NR2B, but not NR2A, promotes dendritic branching. Accumulating evidence indicates that stimulation of NMDARs activates NADPH oxidase (NOX2), thereby generating superoxide. However, the molecular underpinnings of this process are not understood. RasGRF1, a guanine nucleotide exchange factor, is key for several forms of neuronal plasticity and interacts directly with the tail of NR2B. We investigated whether the NR2B-NMDAR/RasGRF1 pathway regulates the activity of NOX2 and whether superoxide production is required for dendritogenesis. We measured superoxide production in developing primary cultures of hippocampal neurons from 3 to 25 days in vitro (DIV) with the probe dihydroethidium (dHE). We found the highest dHE levels at early and intermediate developmental stages (3–15 DIV), when the NR2B-NMDAR expression is abundant. During these early/intermediate developmental stages, but not in mature neurons (>15 DIV), NMDAR activity is required for superoxide production. We also found that disrupting the NR2B-RasGRF1 interaction led to reduced dHE fluorescence intensity and moreover inhibited dendritic branching in hippocampal neurons. Together, our data indicate that superoxide production is induced by the NR2B-NMDARs/RasGRF1/NOX2 pathway and promotes dendritogenesis.     

Essential involvement of the NMDA receptor in ethanol preconditioning-dependent neuroprotection from amyloid-βin vitro

In several epidemiological studies, moderate ethanol consumption has been associated with reduced risks of cognitive decline or Alzheimer's dementia. Of potential relevance is that brain cultures preconditioned with moderate ethanol concentrations are resistant to neurotoxic Alzheimer's amyloid-β (Aβ) peptides. Using rat cerebellar mixed cultures we investigated whether certain membrane receptors were early 'sensors' in moderate ethanol preconditioning (MEP). In a 6-day MEP protocol (30 mM ethanol), neuroprotection from Aβ25–35 was undiminished by antagonism during the first 3 days of either adenosine A₁ or Gα_i/o protein-coupled receptors. However, similar cotreatment with memantine or DL-2-amino-5-phosphono-pentanoic acid (AP-5), antagonists of NMDA receptors (NMDAR), abolished neuroprotection, indicating key early involvement of this ionotropic glutamate receptor. Also in these cultures, directly activating NMDAR using subexcitotoxic NMDA preconditioning prevented Aβ neurotoxicity. By day 2 of MEP, we observed increased levels of NMDAR subunits NR1, NR2B, and NR2C that persisted through day 6. Interestingly, memantine co-exposure blocked elevations in the obligatory NR1 subunit. Furthermore, 2 days of MEP significantly increased two indicators of synaptic NMDAR localization, NR2B phospho-Tyr1472, and post-synaptic density 95 scaffolding protein. The results indicate that ethanol preconditioning-dependent neuroprotection is associated with early increases in NR subunits concomitant with enhancement of synaptic localization and activity of NMDAR.     

Protein kinase C-induced activation of a ceramide/protein phosphatase 1 pathway leading to dephosphorylation of p38 MAPK

Recently we showed that, in human breast cancer cells, activation of protein kinase C by 4beta-phorbol 12-myristate 13-acetate (PMA) produced ceramide formed from the salvage pathway (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). In this study, we investigated intracellular signaling events mediated by this novel activated pathway of ceramide generation. PMA treatment resulted in transient activation of mitogen-activated protein kinases (ERK1/2, JNK1/2, and p38) followed by dephosphorylation/inactivation. Interestingly, fumonisin B1 (FB1), an inhibitor of the salvage pathway, attenuated loss of phosphorylation of p38, suggesting a role for ceramide in p38 dephosphorylation. This was confirmed by knock-down of longevity-assurance homologue 5, which partially suppressed the formation of C(16)-ceramide induced by PMA and increased the phosphorylation of p38. These results demonstrate a role for the salvage pathway in feedback inhibition of p38. To determine which protein phosphatases act in this pathway, specific knock-down of serine/threonine protein phosphatases was performed, and it was observed that knock-down of protein phosphatase 1 (PP1) catalytic subunits significantly increased p38 phosphorylation, suggesting activation of PP1 results in an inhibitory effect on p38. Moreover, PMA recruited PP1 catalytic subunits to mitochondria, and this was significantly suppressed by FB1. In addition, phospho-p38 resided in PMA-stimulated mitochondria. Upon PMA treatment, a mitochondria-enriched/purified fraction exhibited significant increases in C(16)-ceramide, a major ceramide specie, which was suppressed by FB1. Taken together, these data suggest that accumulation of C(16)-ceramide in mitochondria formed from the protein kinase C-dependent salvage pathway results at least in part from the action of longevity-assurance homologue 5, and the generated ceramide modulates the p38 cascade via PP1.     

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.     

Tyrosine dephosphorylation and ethanol inhibition of N-Methyl-D-aspartate receptor function

The inhibitory effect of ethanol on N-methyl-d-aspartate receptors (NMDARs) is well documented in several brain regions. However, the molecular mechanisms by which ethanol affects NMDARs are not well understood. In contrast to the inhibitory effect of ethanol, phosphorylation of the NMDAR potentiates channel currents (Lu, W. Y., Xiong, Z. G., Lei, S., Orser, B. A., Dudek, E., Browning, M. D., and MacDonald, J. F. (1999) Nat. Neurosci. 2, 331-338). We have previously shown that protein kinase C activators induce tyrosine phosphorylation and potentiation of the NMDAR (Grosshans, D. R., Clayton, D. R., Coultrap, S. J., and Browning, M. D. (2002) Nat. Neurosci. 5, 27-33). We therefore hypothesized that the ethanol inhibition of NMDARs might be due to changes in tyrosine phosphorylation of NMDAR subunits. In support of this hypothesis, we found that tyrosine phosphorylation of both NR2A and NR2B subunits was significantly reduced following in situ exposure of hippocampal slices to 100 mm ethanol. Specifically, phosphorylation of tyrosine 1472 on NR2B was reduced 23.5%. These data suggest a possible mechanism by which ethanol may inhibit the NMDAR via activation of a tyrosine phosphatase. Electrophysiological studies demonstrated that ethanol inhibited NMDAR field excitatory postsynaptic potential slope and amplitude to a similar degree as previously reported by our laboratory and others (Schummers, J., Bentz, S., and Browning, M. D. (1997) Alcohol Clin. Exp. Res. 21, 404-408). Inclusion of bpV(phen), a potent phosphotyrosine phosphatase inhibitor, in the recording chamber prior to and during ethanol exposure significantly reduced the inhibitory effect of ethanol on NMDAR field excitatory postsynaptic potentials. Taken together, these data suggest that phosphatase-mediated dephosphorylation of NMDAR subunits may play an important role in mediating the inhibitory effects of ethanol on the N-methyl-D-aspartate receptor.     

The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRF1

The NMDA subtype of glutamate receptors (NMDAR) at excitatory neuronal synapses plays a key role in synaptic plasticity. The extracellular signal-regulated kinase (ERK1,2 or ERK) pathway is an essential component of NMDAR signal transduction controlling the neuroplasticity underlying memory processes, neuronal development, and refinement of synaptic connections. Here we show that NR2B, but not NR2A or NR1 subunits of the NMDAR, interacts in vivo and in vitro with RasGRF1, a Ca(2+)/calmodulin-dependent Ras-guanine-nucleotide-releasing factor. Specific disruption of this interaction in living neurons abrogates NMDAR-dependent ERK activation. Thus, RasGRF1 serves as NMDAR-dependent regulator of the ERK kinase pathway. The specific association of RasGRF1 with the NR2B subunit and study of ERK activation in neurons with varied content of NR2B suggests that NR2B-containing channels are the dominant activators of the NMDA-dependent ERK pathway.     

Sequence determinants on the NR2A and NR2B subunits of NMDA receptor responsible for specificity of phosphorylation by CaMKII

Calcium/calmodulin-dependent protein kinase type II (CaMKII) and NMDA-type glutamate receptor (NMDAR) are neuronal proteins involved in learning and memory. CaMKII binds to the NR2B subunit of NMDAR in more than one mode, a stable association involving a noncatalytic site on CaMKII and an enzyme-substrate mode of interaction by its catalytic site. The latter binding results in phosphorylation of serine-1303 on NR2B. We have investigated this binding by studying the kinetics of phosphorylation of synthetic peptides harboring nested sequences of the phosphorylation site motif. We find that residues 1292-1297 of NR2B enhance the affinity of the catalytic site-mediated binding of CaMKII to the minimal phosphorylation site motif, 1298-1308 of NR2B, as evident from measurements of K(m) values for phosphorylation. However, CaMKII shows decreased affinity towards the closely related NR2A subunit due to an -Ile-Asn- motif present as a natural insertion in the analogous sequence on NR2A.     

Spatial memory formation induces recruitment of NMDA receptor and PSD-95 to synaptic lipid rafts

NMDA receptors (NMDARs) activation in the hippocampus and insular cortex is necessary for spatial memory formation. Recent studies suggest that localization of NMDARs to lipid rafts enhance their signalization, since the kinases that phosphorylate its subunits are present in larger proportion in lipid raft membrane microdomains. We sought to determine the possibility that NMDAR translocation to synaptic lipid rafts occurs during plasticity processes such as memory formation. Our results show that water maze training induces a rapid recruitment of NMDAR subunits (NR1, NR2A, NR2B) and PSD-95 to synaptic lipid rafts and decrease in the post-synaptic density plus an increase of NR2B phosphorylation at tyrosine 1472 in the rat insular cortex. In the hippocampus, spatial training induces selective translocation of NR1 and NR2A subunits to lipid rafts. These results suggest that NMDARs translocate from the soluble fraction of post-synaptic membrane (non-raft PSD) to synaptic lipid raft during spatial memory formation. The recruitment of NMDA receptors and other proteins to lipid rafts could be an important mechanism for increasing the efficiency of synaptic transmission during synaptic plasticity process.     

Ligand Directed Signaling Differences between Rodent and Human κ-Opioid Receptors

KOR activation of Gβγ dependent signaling results in analgesia, whereas the dysphoric effects of KOR agonists are mediated by a different pathway involving G protein receptor kinase and non-visual arrestin. Based on this distinction, a partial KOR agonist that does not efficiently activate arrestin-dependent biased signaling may produce analgesia without dysphoria. No KOR-selective partial agonists are currently available, and preclinical assessment is complicated by sequence differences between rodent (r) and human (h) KOR. In this study, we compared the signaling initiated by the available partial agonists. Pentazocine was significantly more potent at activating p38 MAPK in hKOR than rKOR expressed in HEK293 cells but equally potent at arrestin-independent activation of ERK1/2 in hKOR and rKOR. Similarly, butorphanol increased phospho-p38-ir in hKOR-expressing cells but did not activate p38 in rKOR-HEK293. Like pentazocine, butorphanol was equally efficacious at activating ERK1/2 in rKOR and hKOR. In contrast, levorphanol, nalorphine, and U50,488 did not distinguish between hKOR and rKOR in p38 MAPK activation. Consistent with its low potency at p38 activation, pentazocine did not produce conditioned place aversion in mice. hKOR lacks the Ser-369 phosphorylation site in rKOR required for G protein receptor kinase/arrestin-dependent p38 activation, but mutation of the Ser-358 to asparagine in hKOR blocked p38 activation without affecting the acute arrestin-independent activation of ERK1/2. This study shows that hKOR activates p38 MAPK through a phosphorylation and arrestin-dependent mechanism; however, activation differs between hKOR and rKOR for some ligands. These functional selectivity differences have important implications for preclinical screening of partial KOR agonists.     

Negative regulation of neurogenesis and spatial memory by NR2B-containing NMDA receptors

Several lines of evidence suggest involvement of NMDA receptors (NMDARs) in the regulation of neurogenesis in adults and the formation of spatial memory. Functional properties of NMDARs are strongly influenced by the type of NR2 subunits incorporated. In adult forebrain regions such as the hippocampus and cortex, only NR2A and NR2B subunits are available to form the receptor complex with NR1 subunit. NR2B is predominant NR2 subunit in any of rat or human neural stem cells (NSCs). Thus, we suppose that NR2B-containing NMDAR should be critical in regulating adult neurogenesis, and thereby playing a role in the formation of spatial memory. In the cultured NSCs derived from the embryonic brain of rats, NR2B subunit-specific NMDAR antagonist Ro25-6981 increased cell proliferation, whereas MK-801, non-selective open-channel blocker of NMDARs, inhibited cell proliferation. Blockade of NR2B-containing NMDAR stimulated neurogenesis in the adult hippocampus and facilitated the formation of spatial memory. The enhanced spatial memory dropped back to base level when the NR2B antagonist-induced neurogenesis was neutralized by 3'-azido-deoxythymidine, a telomerase inhibitor. In addition, blockade of NR2B inhibited neuronal nitric oxide synthase (nNOS) enzymatic activity. In null mutant mice lacking nNOS gene (nNOS−/−), the effects of NR2B antagonist on neurogenesis disappeared. Moreover, nitric oxide donor DETA/NONOate attenuated and nNOS inhibitor 7-nitroindazole enhanced the effect of Ro 25-6981 on NSCs proliferation. Our findings suggest that NR2B-containing NMDAR subtypes negatively regulate neurogenesis in the adult hippocampus by activating nNOS activity and thereby hinder the formation of spatial memory.     

Activation of dopamine D1 receptors blocks phencyclidine-induced neurotoxicity by enhancing N-methyl-D-aspartate receptor-mediated synaptic strength

Early postnatal blockade of NMDA receptors by phencyclidine (PCP) causes cortical apoptosis in animals. This is associated with the development of schizophrenia-like behaviors in rats later in life. Recent studies show that the mechanism involves a loss of neurotrophic support from the phosphoinositol-3 kinase/Akt pathway, which is normally maintained by synaptic NMDA receptor activation. Here we report that activation of dopamine D1 receptors (D1R) with dihydrexidine (DHX) prevents PCP-induced neurotoxicity in cortical neurons by enhancing the efficacy of NMDAergic synapses. DHX increases serine phosphorylation of the NR1 subunit through protein kinase A activation and tyrosine phosphorylation of the NR2B subunit via Src kinase. DHX enhances recruitment of NR1 and NR2B, but not NR2A, into synapses. DHX also facilitated the synaptic response in cortical slices and this was blocked by an NR2B antagonist. DHX pre-treatment of rat pups prior to PCP on postnatal days 7, 9 and 11 inhibited PCP-induced caspase-3 activation on PN11 and deficits in pre-pulse inhibition of acoustic startle measured on PN 26-28. In summary, these data demonstrate that PCP-induced deficits in NMDA receptor function, neurotoxicity and subsequent behavioral deficits may be prevented by D1R activation in the cortex and further, it is suggested that D1R activation may be beneficial in treating schizophrenia.