Homocysteine-induced sustained GluN2A NMDA receptor stimulation leads to mitochondrial ROS generation and neurotoxicity

Homocysteine, a sulfur-containing amino acid derived from methionine metabolism, is a known agonist of N-methyl-D-aspartate receptor (NMDAR) and is involved in neurotoxicity. Our previous findings showed that neuronal exposure to elevated homocysteine levels leads to sustained low-level increase in intracellular Ca2+, which is dependent on GluN2A subunit-containing NMDAR (GluN2A-NMDAR) stimulation. These studies further showed a role of ERK MAPK in homocysteine-GluN2A-NMDAR mediated neuronal death. However, the intracellular mechanisms associated with such sustained GluN2A-NMDAR stimulation and subsequent Ca2+ influx have remained unexplored. Using live cell imaging with Fluo3-AM and biochemical approaches, we show that homocysteine-GluN2A NMDAR induced initial Ca2+ influx triggers sequential phosphorylation and subsequent activation of Pyk2 and Src family kinases (SFK), which in turn phosphorylates GluN2A-Tyr1325 residue of GluN2A-NMDARs to maintain channel activity. The continuity of this cycle of events leads to sustained Ca2+ influx through GluN2A-NMDAR. Our findings also show that lack of activation of the regulatory tyrosine phosphatase STEP, which can limit Pyk2 and SFK activity further contributes to the maintenance of this cycle. Additional studies using live cell imaging of neurons expressing a redox sensitive green fluorescent protein (RoGFP) targeted to the mitochondrial matrix show that treatment with homocysteine leads to a progressive increase in mitochondrial reactive oxygen species (ROS) generation, which is dependent on GluN2A-NMDAR mediated sustained ERK MAPK activation. This later finding demonstrates a novel role of GluN2A-NMDAR in homocysteine-induced mitochondrial ROS generation and highlights the role of ERK MAPK as the intermediary signaling pathway between GluN2A-NMDAR stimulation and mitochondrial ROS generation.