High sensitivity of cerebellar neurons to homocysteine is determined by expression of GluN2C and GluN2D subunits of NMDA receptors

© 2018 Elsevier Inc. Homocysteine (HCY) induced neurotoxicity largely depends on interaction of this endogenous amino acid with glutamate NMDA receptors (NMDARs). This receptor type is composed by GluN1 and different GluN2 (A, B, C or D) subunits. However, the receptor activity of HCY in brain regions which differ in relative contribution of GluN2 subunits was not tested so far. In the current study, we explored the action of HCY on cerebellar neurons which natively express GluN2C and GluN2D subunits of NMDARs and compared this with the action of HCY on cortical neurons which are mainly composed by GluN2A and GluN2B subunits. To validate obtained results, we also studied the responses to HCY in recombinant GluN1/2C and GluN1/2D NMDARs expressed in HEK293T cells. Responses to HCY were compared to membrane currents evoked by glutamate or by the specific agonist NMDA. First, we found that on HEK cells expressing GluN1/2C or GluN1/2D NMDARs, HCY was full agonist producing membrane currents similar in amplitude to currents induced by glutamate. The EC50 values for these particular receptor subtype activation were 80 μM and 31 μM, respectively. Then, we found that HCY similarly to NMDA, evoked large slightly desensitizing membrane currents in native NMDARs of cerebellar and cortical neurons. In cortical neurons, the ratio of the respective currents (IHCY/INMDA) was 0.16 and did not significantly change during in vitro maturation. In sharp contrast, in cerebellar neurons, the ratio of currents evoked by HCY and NMDA was dramatically increased from 0.31 to 0.72 from 7 to 21 day in culture. We show that least 75% of HCY-induced currents in cerebellum were mediated by GluN2C- or GluN2D-containing NMDARs. Thus, our data revealed a large population of cerebellar NMDA receptors highly sensitive to HCY which suggest potential vulnerability of this brain region to pathological conditions associated with enhanced levels of this neurotoxic amino acid

GluN2A subunit-containing NMDA receptors are the preferential neuronal targets of homocysteine

© 2016 Sibarov, Abushik, Giniatullin and Antonov.Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+- independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 and 61.8 ± 8.9 mM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to post-synaptic responses mediated by GluN2A-containing NMDA receptors. On the other hand, HCY toxicity may be limited by desensitization typical for HCY-induced activation of GluN2B-containing extrasynaptic receptors. Our findings, therefore, provide an evidence for the physiological relevance of endogenous HCY, which may represent an effective endogenous modulator of the central excitatory neurotransmission

Homocysteine-induced membrane currents, calcium responses and changes in mitochondrial potential in rat cortical neurons

© 2015, Pleiades Publishing, Ltd. Homocysteine, a sulfur-containing amino acid, exerts neurotoxic effects and is involved in the pathogenesis of many neurodegenerative disorders. In contrast to well-studied glutamate excitotoxicity, the mechanism of homocysteine neurotoxicity is not clearly understood. Using wholecell patch-clamp, calcium imaging (fluo-3) and measurements of mitochondrial membrane potential (rhodamine 123), we studied in vitro in cultured rat cortical neurons transmembrane currents, calcium signals and changes in mitochondrial membrane potential induced by homocysteine versus responses induced by NMDA and glutamate. L-homocysteine (50 μM) induced inward currents that were completely blocked by the selective antagonist of NMDA receptors, AP-5. In contrast to NMDA-induced currents, homocysteine-induced currents exhibited a smaller steady-state amplitude. Comparison of calcium responses to homocysteine, NMDA or glutamate demonstrated that in all cortical neurons homocysteine elicited fast oscillatory-type calcium responses, whereas NMDA or glutamate induced a “classical” sustained elevation of intracellular calcium. In contrast to NMDA, homocysteine did not cause a drop in mitochondrial membrane potential at the early stages of its action. However, after its long-term effect, as in cases of NMDA and glutamate, changes in mitochondrial membrane potential arose comparable with its complete drop caused by protonophore FCCP-induced uncoupling of the respiratory chain. Our data suggest that in cultured rat cortical neurons homocysteine at the initial stages of its action induces in vitro neurotoxic effects due to the activation of NMDA-type ionotropic glutamate receptors followed by a massive calcium influx through the channels of these receptors. The long-term effect of homocysteine may lead to mitochondrial dysfuction manifested as a drop in mitochondrial membrane potential

Functional properties of human NMDA receptors associated with epilepsy-related mutations of GluN2A subunit

© 2017 Sibarov, Bruneau, Antonov, Szepetowski, Burnashev and Giniatullin. Genetic variants of the glutamate activated N-methyl-D-aspartate (NMDA) receptor (NMDAR) subunit GluN2A are associated with the hyperexcitable states manifested by epileptic seizures and interictal discharges in patients with disorders of the epilepsy-aphasia spectrum (EAS). The variants found in sporadic cases and families are of different types and include microdeletions encompassing the corresponding GRIN2A gene as well as nonsense, splice-site and missense GRIN2A defects. They are located at different functional domains of GluN2A and no clear genotype-phenotype correlation has emerged yet. Moreover, GluN2A variants may be associated with phenotypic pleiotropy. Deciphering the consequences of pathogenic GRIN2A variants would surely help in better understanding of the underlying mechanisms. This emphasizes the need for functional studies to unravel the basic functional properties of each specific NMDAR variant. In the present study, we have used patch-clamp recordings to evaluate kinetic changes of mutant NMDARs reconstituted after co-transfection of cultured cells with the appropriate expression vectors. Three previously identified missense variants found in patients or families with disorders of the EAS and situated in the N-terminal domain (p.Ile184Ser) or in the ligand-binding domain (p.Arg518His and p.Ala716Thr) of GluN2A were studied in both the homozygous and heterozygous conditions. Relative surface expression and current amplitude were significantly reduced for NMDARs composed of mutant p.Ile184Ser and p.Arg518His, but not p.Ala716His, as compared with wild-type (WT) NMDARs. Amplitude of whole-cell currents was still drastically decreased when WT and mutant p.Arg518His-GluN2A subunits were co-expressed, suggesting a dominant-negative mechanism. Activation times were significantly decreased in both homozygous and heterozygous conditions for the two p.Ile184Ser and p.Arg518His variants, but not for p.Ala716His. Deactivation also significantly increased for p.Ile184Ser variant in the homozygous but not the heterozygous state while it was increased for p.Arg518His in both states. Our data indicate that p.Ile184Ser and p.Arg518His GluN2A variants both impacted on NMDAR function, albeit differently, whereas p.Ala716His did not significantly influence NMDAR kinetics, hence partly questioning its direct and strong pathogenic role. This study brings new insights into the functional impact that GRIN2A variants might have on NMDAR kinetics, and provides a mechanistic explanation for the neurological manifestations seen in the corresponding human spectrum of disorders

Pro-nociceptive migraine mediator CGRP provides neuroprotection of sensory, cortical and cerebellar neurons via multi-kinase signaling

© 2016, © International Headache Society 2016. Background: Blocking the pro-nociceptive action of CGRP is one of the most promising approaches for migraine prophylaxis. The aim of this study was to explore a role for CGRP as a neuroprotective agent for central and peripheral neurons. Methods: The viability of isolated rat trigeminal, cortical and cerebellar neurons was tested by fluorescence vital assay. Engagement of Nrf2 target genes was analyzed by qPCR. The neuroprotective efficacy of CGRP in vivo was tested in mice using a permanent cerebral ischemia model. Results: CGRP prevented apoptosis induced by the amino acid homocysteine in all three distinct neuronal populations. Using a set of specific kinase inhibitors, we show the role of multi-kinase signaling pathways involving PKA and CaMKII in neuronal survival. Forskolin triggered a very similar signaling cascade, suggesting that cAMP is the main upstream trigger for multi-kinase neuroprotection. The specific CGRP antagonist BIBN4096 reduced cellular viability, lending further support to the proposed neuroprotective function of CGRP. Importantly, CGRP was neuroprotective against permanent ischemia in mice. Conclusion: Our data show an unexpected ‘positive’ role for the endogenous pro-nociceptive migraine mediator CGRP, suggesting more careful examination of migraine prophylaxis strategy based on CGRP antagonism although it should be noted that homocysteine induced apoptosis in primary neuronal cell culture might not necessarily reproduce all the features of cell loss in the living organism

High sensitivity of cerebellar neurons to homocysteine is determined by expression of GluN2C and GluN2D subunits of NMDA receptors

© 2018 Elsevier Inc. Homocysteine (HCY) induced neurotoxicity largely depends on interaction of this endogenous amino acid with glutamate NMDA receptors (NMDARs). This receptor type is composed by GluN1 and different GluN2 (A, B, C or D) subunits. However, the receptor activity of HCY in brain regions which differ in relative contribution of GluN2 subunits was not tested so far. In the current study, we explored the action of HCY on cerebellar neurons which natively express GluN2C and GluN2D subunits of NMDARs and compared this with the action of HCY on cortical neurons which are mainly composed by GluN2A and GluN2B subunits. To validate obtained results, we also studied the responses to HCY in recombinant GluN1/2C and GluN1/2D NMDARs expressed in HEK293T cells. Responses to HCY were compared to membrane currents evoked by glutamate or by the specific agonist NMDA. First, we found that on HEK cells expressing GluN1/2C or GluN1/2D NMDARs, HCY was full agonist producing membrane currents similar in amplitude to currents induced by glutamate. The EC50 values for these particular receptor subtype activation were 80 μM and 31 μM, respectively. Then, we found that HCY similarly to NMDA, evoked large slightly desensitizing membrane currents in native NMDARs of cerebellar and cortical neurons. In cortical neurons, the ratio of the respective currents (IHCY/INMDA) was 0.16 and did not significantly change during in vitro maturation. In sharp contrast, in cerebellar neurons, the ratio of currents evoked by HCY and NMDA was dramatically increased from 0.31 to 0.72 from 7 to 21 day in culture. We show that least 75% of HCY-induced currents in cerebellum were mediated by GluN2C- or GluN2D-containing NMDARs. Thus, our data revealed a large population of cerebellar NMDA receptors highly sensitive to HCY which suggest potential vulnerability of this brain region to pathological conditions associated with enhanced levels of this neurotoxic amino acid

GluN2A subunit-containing NMDA receptors are the preferential neuronal targets of homocysteine

© 2016 Sibarov, Abushik, Giniatullin and Antonov.Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+- independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 and 61.8 ± 8.9 mM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to post-synaptic responses mediated by GluN2A-containing NMDA receptors. On the other hand, HCY toxicity may be limited by desensitization typical for HCY-induced activation of GluN2B-containing extrasynaptic receptors. Our findings, therefore, provide an evidence for the physiological relevance of endogenous HCY, which may represent an effective endogenous modulator of the central excitatory neurotransmission

GluN2A subunit-containing NMDA receptors are the preferential neuronal targets of homocysteine

© 2016 Sibarov, Abushik, Giniatullin and Antonov.Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+- independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 and 61.8 ± 8.9 mM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to post-synaptic responses mediated by GluN2A-containing NMDA receptors. On the other hand, HCY toxicity may be limited by desensitization typical for HCY-induced activation of GluN2B-containing extrasynaptic receptors. Our findings, therefore, provide an evidence for the physiological relevance of endogenous HCY, which may represent an effective endogenous modulator of the central excitatory neurotransmission

GluN2A subunit-containing NMDA receptors are the preferential neuronal targets of homocysteine

© 2016 Sibarov, Abushik, Giniatullin and Antonov.Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+- independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 and 61.8 ± 8.9 mM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to post-synaptic responses mediated by GluN2A-containing NMDA receptors. On the other hand, HCY toxicity may be limited by desensitization typical for HCY-induced activation of GluN2B-containing extrasynaptic receptors. Our findings, therefore, provide an evidence for the physiological relevance of endogenous HCY, which may represent an effective endogenous modulator of the central excitatory neurotransmission

Homocysteine-induced membrane currents, calcium responses and changes in mitochondrial potential in rat cortical neurons

© 2015, Pleiades Publishing, Ltd. Homocysteine, a sulfur-containing amino acid, exerts neurotoxic effects and is involved in the pathogenesis of many neurodegenerative disorders. In contrast to well-studied glutamate excitotoxicity, the mechanism of homocysteine neurotoxicity is not clearly understood. Using wholecell patch-clamp, calcium imaging (fluo-3) and measurements of mitochondrial membrane potential (rhodamine 123), we studied in vitro in cultured rat cortical neurons transmembrane currents, calcium signals and changes in mitochondrial membrane potential induced by homocysteine versus responses induced by NMDA and glutamate. L-homocysteine (50 μM) induced inward currents that were completely blocked by the selective antagonist of NMDA receptors, AP-5. In contrast to NMDA-induced currents, homocysteine-induced currents exhibited a smaller steady-state amplitude. Comparison of calcium responses to homocysteine, NMDA or glutamate demonstrated that in all cortical neurons homocysteine elicited fast oscillatory-type calcium responses, whereas NMDA or glutamate induced a “classical” sustained elevation of intracellular calcium. In contrast to NMDA, homocysteine did not cause a drop in mitochondrial membrane potential at the early stages of its action. However, after its long-term effect, as in cases of NMDA and glutamate, changes in mitochondrial membrane potential arose comparable with its complete drop caused by protonophore FCCP-induced uncoupling of the respiratory chain. Our data suggest that in cultured rat cortical neurons homocysteine at the initial stages of its action induces in vitro neurotoxic effects due to the activation of NMDA-type ionotropic glutamate receptors followed by a massive calcium influx through the channels of these receptors. The long-term effect of homocysteine may lead to mitochondrial dysfuction manifested as a drop in mitochondrial membrane potential