GluN2A Subunit-Containing NMDA Receptors Are the Preferential Neuronal Targets of Homocysteine

Abstract

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 μM, 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.

Astaxanthin Protects PC12 Cells against Homocysteine- and Glutamate-Induced Neurotoxicity

“…Previously, Lecleric et al evidenced the occurrence of NMDA receptor in PC12 cells and concluded that PC12 cells express predominantly the splice variant NMDAR1-4a and smaller amounts of NMDAR1-1a, NMDAR1-2a, and NMDAR1-3a [35]. More recently, Sibarov et al implicated that GluN2A subunit-containing NMDA receptors as the preferential neural targets of Hcy [36]. Moreover, Hcy has been confirmed not only to be Ca 2+ -and NMDA receptor-dependent, but also Ca 2+ -independent, mainly mediated by the "synaptic type" GluN1/2 NMDAR [36].…”

“…More recently, Sibarov et al implicated that GluN2A subunit-containing NMDA receptors as the preferential neural targets of Hcy [36]. Moreover, Hcy has been confirmed not only to be Ca 2+ -and NMDA receptor-dependent, but also Ca 2+ -independent, mainly mediated by the "synaptic type" GluN1/2 NMDAR [36]. Suggestively, the degree of binding by different ligands like Glu and Hcy for these receptor subunits and the outcome responses may be synergistically additive on one hand, but competitively expelling on the other hand.…”

“…Calcium ion dysregulation is relevant to the initiation of model in reality has pertinently reflected such a possibility raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium slightly less effective against that by Hcy (10 mM), and was m therapy (133%) (Figure 3a), implicating insufficient mole nu and (Glu + Hcy); suggestively, a higher dose of ATX may be In addition, Hcy has been implicated to indirectly i activating ionotropic and metabotropic receptors [41], which the reasons to elicit different outcomes by such a combina [2,36,41].…”

“…In addition, Hcy has been implicated to indirectly increase intracellular calcium level activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may c the reasons to elicit different outcomes by such a combination of pharmacological actions of [2,36,41]. Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via allev mitochondrial dysfunction and signal crosstalk [37].…”

Metabolism of amino acid neurotransmitters: the synaptic disorder underlying inherited metabolic diseases

“…Evidence is increasing that disturbed biosynthesis or breakdown of amino acid neurotransmitters and hence pre-and/or postsynaptic dysfunction play an important role in many other inherited metabolic diseases. For instance, significant dysfunction of glutamatergic and/or GABAergic metabolism and signaling is known or thought to be involved in urea cycle disorders (Braissant 2010), isolated sulfite oxidase and molybdenum cofactor deficiency (Kumar et al 2017), homocystinurias (Poddar et al 2017;Sibarov et al 2016), branched-chain 2-ketoacid dehydrogenase kinase (Novarino et al 2012), maple syrup urine disease (Zinnanti et al 2009), GABA transaminase deficiency (Koenig et al 2017), glutamine synthetase deficiency (Häberle et al 2005), pyridoxine-dependent epilepsy (Mills et al 2006), guanidinoacetate methyltransferase deficiency (Schulze et al 2016), mucopolysaccharidosis IIIA (Dwyer et al 2017), and disorders of energy metabolism such as pyruvate carboxylase deficiency (Ortez et al 2013). Although amino acid disorders are among the earliest identified inherited metabolic diseases, the delineation of the synaptic disorder underlying the metabolic diseases has long been neglected and remains more fully examined.…”