Browsing by Subject "NMDA receptor"

Now showing 1 - 6 of 6
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    Characterizing Calcium Influx Via Voltage- and Ligand-Gated Calcium Channels in Embryonic Alligator Neurons in Culture
    (De Gruyter, 2013) Ju, Weina; Wu, Jiang; Pritz, Michael B.; Khanna, Rajesh; Neurological Surgery, School of Medicine
    Vertebrate brains share many features in common. Early in development, both the hindbrain and diencephalon are built similarly. Only later in time do differences in morphology occur. Factors that could potentially influence such changes include certain physiological properties of neurons. As an initial step to investigate this problem, embryonic Alligator brain neurons were cultured and calcium responses were characterized. The present report is the first to document culture of Alligator brain neurons in artificial cerebrospinal fluid (ACSF) as well as in standard mammalian tissue culture medium supplemented with growth factors. Alligator brain neuron cultures were viable for at least 1 week with unipolar neurites emerging by 24 hours. Employing Fura-2 AM, robust depolarization-induced calcium influx, was observed in these neurons. Using selective blockers of the voltage-gated calcium channels, the contributions of N-, P/Q-, R-, T-, and L-type channels in these neurons were assessed and their presence documented. Lastly, Alligator brain neurons were challenged with an excitotoxic stimulus (glutamate + glycine) where delayed calcium deregulation could be prevented by a classical NMDA receptor antagonist.
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    Cross-platform validation of neurotransmitter release impairments in schizophrenia patient-derived NRXN1-mutant neurons
    (National Academy of Sciences, 2021) Pak, ChangHui; Danko, Tamas; Mirabella, Vincent R.; Wang, Jinzhao; Liu, Yingfei; Vangipuram, Madhuri; Grieder, Sarah; Zhang, Xianglong; Ward, Thomas; Huang, Yu-Wen Alvin; Jin, Kang; Dexheimer, Philip; Bardes, Eric; Mitelpunkt, Alexis; Ma, Junyi; McLachlan, Michael; Moore, Jennifer C.; Qu, Pingping; Purmann, Carolin; Dage, Jeffrey L.; Swanson, Bradley J.; Urban, Alexander E.; Aronow, Bruce J.; Pang, Zhiping P.; Levinson, Douglas F.; Wernig, Marius; Südhof, Thomas C.; Neurology, School of Medicine
    Heterozygous NRXN1 deletions predispose to schizophrenia and other neurodevelopmental disorders. Engineered heterozygous NRXN1 deletions impair neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. In a multicenter effort to test the generality and robustness of this pivotal observation, we used, at two laboratories, independent analyses of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. Schizophrenia patient-derived neurons with NRXN1 deletions exhibited the same major decrease in neurotransmitter release and an increase in CASK protein as engineered human neurons with NRXN1 deletions. Strikingly, engineered mouse Nrxn1-deficient neurons derived by the same method displayed no such phenotype, suggesting a human-specific role for NRXN1. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons, enabling future drug discovery efforts.
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    Delayed calcium dysregulation in neurons requires both the NMDA receptor and the reverse Na+/Ca2+ exchanger
    (Elsevier, 2012) Brittain, Matthew K.; Brustovetsky, Tatiana; Sheets, Patrick L.; Brittain, Joel M.; Khanna, Rajesh; Cummins, Theodore R.; Brustovetsky, Nickolay; Pharmacology and Toxicology, School of Medicine
    Glutamate-induced delayed calcium dysregulation (DCD) is a causal factor leading to neuronal death. The mechanism of DCD is not clear but Ca2+ influx via N-methyl-d-aspartate receptors (NMDAR) and/or the reverse plasmalemmal Na+/Ca2+ exchanger (NCXrev) could be involved in DCD. However, the extent to which NMDAR and NCX(rev) contribute to glutamate-induced DCD is uncertain. Here, we show that both NMDAR and NCX(rev) are critical for DCD in neurons exposed to excitotoxic glutamate. In rat cultured hippocampal neurons, 25 μM glutamate produced DCD accompanied by sustained increase in cytosolic Na+ ([Na+]c) and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added shortly after glutamate, completely prevented DCD whereas AP-5, a competitive NMDAR inhibitor, failed to protect against DCD. None of the tested inhibitors lowered elevated [Na+]c or restored plasma membrane potential. In the experiments with NCX reversal by gramicidin, MK801 and memantine robustly inhibited NCXrev while AP-5 was much less efficacious. In electrophysiological patch-clamp experiments MK801 and memantine inhibited NCXrev-mediated ion currents whereas AP-5 failed. Thus, MK801 and memantine, in addition to NMDAR, inhibited NCXrev. Inhibition of NCXrev either with KB-R7943, or by collapsing Na+ gradient across the plasma membrane, or by inhibiting Na+/H+ exchanger with 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and thus preventing the increase in [Na+]c failed to preclude DCD. However, NCXrev inhibition combined with NMDAR blockade by AP-5 completely prevented DCD. Overall, our data suggest that both NMDAR and NCXrev are essential for DCD in glutamate-exposed neurons and inhibition of individual mechanism is not sufficient to prevent calcium dysregulation.
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    Disruption of nNOS-NOS1AP protein-protein interactions suppresses neuropathic pain in mice
    (Wolters Kluwer, 2018-05) Lee, Wan-Hung; Li, Li-Li; Chawla, Aarti; Hudmon, Andy; Lai, Yvonne Y.; Courtney, Michael J.; Hohmann, Andrea G.; Biochemistry and Molecular Biology, School of Medicine
    Elevated N-methyl-D-aspartate receptor (NMDAR) activity is linked to central sensitization and chronic pain. However, NMDAR antagonists display limited therapeutic potential because of their adverse side effects. Novel approaches targeting the NR2B-PSD95-nNOS complex to disrupt signaling pathways downstream of NMDARs show efficacy in preclinical pain models. Here, we evaluated the involvement of interactions between neuronal nitric oxide synthase (nNOS) and the nitric oxide synthase 1 adaptor protein (NOS1AP) in pronociceptive signaling and neuropathic pain. TAT-GESV, a peptide inhibitor of the nNOS-NOS1AP complex, disrupted the in vitro binding between nNOS and its downstream protein partner NOS1AP but not its upstream protein partner postsynaptic density 95 kDa (PSD95). Putative inactive peptides (TAT-cp4GESV and TAT-GESVΔ1) failed to do so. Only the active peptide protected primary cortical neurons from glutamate/glycine-induced excitotoxicity. TAT-GESV, administered intrathecally (i.t.), suppressed mechanical and cold allodynia induced by either the chemotherapeutic agent paclitaxel or a traumatic nerve injury induced by partial sciatic nerve ligation. TAT-GESV also blocked the paclitaxel-induced phosphorylation at Ser15 of p53, a substrate of p38 MAPK. Finally, TAT-GESV (i.t.) did not induce NMDAR-mediated motor ataxia in the rotarod test and did not alter basal nociceptive thresholds in the radiant heat tail-flick test. These observations support the hypothesis that antiallodynic efficacy of an nNOS-NOS1AP disruptor may result, at least in part, from blockade of p38 MAPK-mediated downstream effects. Our studies demonstrate, for the first time, that disrupting nNOS-NOS1AP protein-protein interactions attenuates mechanistically distinct forms of neuropathic pain without unwanted motor ataxic effects of NMDAR antagonists.
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    Ifenprodil, a NR2B-selective antagonist of NMDA receptor, inhibits reverse Na+/Ca2+ exchanger in neurons
    (Elsevier, 2012) Brittain, Matthew K.; Brustovetsky, Tatiana; Brittain, Joel M.; Khanna, Rajesh; Cummins, Theodore R.; Brustovetsky, Nickolay; Pharmacology and Toxicology, School of Medicine
    Glutamate-induced delayed calcium dysregulation (DCD) is causally linked to excitotoxic neuronal death. The mechanisms of DCD are not completely understood, but it has been proposed that the excessive influx of external Ca(2+) is essential for DCD. The NMDA-subtype of glutamate receptor (NMDAR) and the plasmalemmal Na(+)/Ca(2+) exchanger operating in the reverse mode (NCX(rev)) have been implicated in DCD. In experiments with "younger" neurons, 6-8 days in vitro (6-8 DIV), in which the NR2A-containing NMDAR expression is low, ifenprodil, an inhibitor of NR2B-containing NMDAR, completely prevented DCD whereas PEAQX, another NMDAR antagonist that preferentially interacts with NR2A-NMDAR, was without effect. With "older" neurons (13-16 DIV), in which NR2A- and NR2B-NMDARs are expressed to a greater extent, both ifenprodil and PEAQX applied separately failed to prevent DCD. However, combined application of ifenprodil and PEAQX completely averted DCD. Ifenprodil and ifenprodil-like NR2B-NMDAR antagonists Ro 25-6981 and Co 101244 but not PEAQX or AP-5 inhibited gramicidin- and Na(+)/NMDG-replacement-induced increases in cytosolic Ca(2+) mediated predominantly by NCX(rev). This suggests that ifenprodil, Ro 25-6981, and Co 101244 inhibit NCX(rev). The ability of ifenprodil to inhibit NCX(rev) correlates with its efficacy in preventing DCD and emphasizes an important role of NCX(rev) in DCD. Overall our data suggest that both NR2A- and NR2B-NMDARs are involved in DCD in "older" neurons, and it is necessary to inhibit both NMDARs and NCX(rev) to prevent glutamate-induced DCD.
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    Regulation of neuronal calcium homeostasis in Huntington's
    (2015-07-28) Pellman, Jessica J.; Brustovetsky, Nickolay; Cummins, Theodore R.; Jerde, Travis J.; Khanna, Rajesh; Vasko, Michael R.
    Huntington’s Disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder. There is no cure for HD and the existing therapies only alleviate HD symptoms without eliminating the cause of this neuropathology. HD is linked to a mutation in the huntingtin gene, which results in an elongation of the poly-glutamine stretch in the huntingtin protein (Htt). A major hypothesis is that mutant Htt (mHtt) leads to aberrant Ca2+ homeostasis in affected neurons. This may be caused by increased Ca2+ influx into the cell via the N-methyl-Daspartate (NMDA)-subtype of glutamate receptors. The contribution of two major Ca2+ removal mechanisms, mitochondria and plasmalemmal Na+/Ca2+ exchangers (NCX), in neuronal injury in HD remains unclear. We investigated Ca2+ uptake capacity in isolated synaptic (neuronal) and nonsynaptic mitochondria from the YAC128 mouse model of HD. We found that both Htt and mHtt bind to brain mitochondria and the amount of mitochondriabound mHtt correlates with increased mitochondrial Ca2+ uptake capacity. Mitochondrial Ca2+ accumulation was not impaired in striatal neurons from YAC128 mice. We also found that expression of the NCX1 isoform is increased with age in striatum from YAC128 mice compared to striatum from wild-type mice. Interestingly, mHtt and Htt bind to the NCX3 isoform but not to NCX1. NCX3 expression remains unchanged. To further investigate Ca2+ homeostasis modulation, we examined the role of collapsin response mediator protein 2 (CRMP2) in wild-type neurons. CRMP2 is viewed as an axon guidance protein, but has been found to be involved in Ca2+ signaling. We found that CRMP2 interacts with NMDA receptors (NMDAR) and disrupting this interaction decreases NMDAR activity. CRMP2 also interacts with and regulates NCX3, resulting in NCX3 internalization and decreased activity. Augmented mitochondrial Ca2+ uptake capacity and an increased expression of NCX1 in the presence of mHtt suggest a compensatory reaction in response to increased Ca2+ influx into the cell. The role of NCX warrants further investigation in HD. The novel interactions of CRMP2 with NMDAR and NCX3 provide additional insight into the complexity of Ca2+ homeostasis regulation in neurons and may also be important in HD neuropathology.