Elevated plasma level of homocysteine (hyper-homocysteinemia) is a major risk factor for neurodegenerative disorders. In spite of fortification of food with folic acid as an attempt to lower homocysteine levels and reduce homocysteine related disorders, the incidence of hyper-homocysteinemia in the elderly population is still quite large. This is mainly due to lowered nutritional absorption and decreased metabolic function with advanced age. Recent studies indicate that homocysteine can penetrate the blood-brain barrier and increased levels of homocysteine in brain is associated with aggravation and acceleration of neuronal damage in ischemic stroke, Alzheimer's and Parkinson's diseases. However, the underlying signaling mechanism(s) by which homocysteine exacerbates neuronal cell death in these disorders are not well understood. One key mechanism that may be fundamental to homocysteine-induced brain damage is through stimulation of NMDAR. In this study we propose to define the intracellular targets and the specific signaling mechanism(s) that link homocysteine mediated NMDA receptor activation to neuronal cell death. Contrary to the presumption that homocysteine-mediated NMDA receptor signaling mechanisms are analogous to other NMDA receptor agonists like glutamate, our findings indicate that the effects of homocysteine on NMDA receptor activation and the downstream signaling events are quite different. Glutamate-mediated stimulation of NR2A containing NMDA receptor subtypes promotes while stimulation of NR2B containing NMDA receptor subtypes inhibits activation of ERK resulting in transient activation of ERK MAPK. In contrast, our findings show that NR2B containing NMDA receptors did not play a role in regulating homocysteine-mediated activation of ERK MAPK resulting in sustained ERK activation. Again glutamate-mediated activation of NR2A containing NMDA receptor is thought to be pro-survival while activation of NR2B containing NMDA receptor is thought to be pro-apoptotic. Our findings, on the other hand, indicate that NR2B containing NMDA receptors have no role in homocysteine- induced neurotoxicity implicating the critical involvement of NR2A containing NMDA receptor in neuronal death. Contrary to the notion that activation of ERK MAPK promotes neuronal cell survival, while p38 MAPK promotes injury, our preliminary findings also indicate a role of ERK MAPK in homocysteine-NMDA receptor mediated neuronal cell death. Experiments proposed in Specific Aim 1 will expand on these initial findings to perform functional studies to determine the role of NR2A containing NMDA receptor in homocysteine induced neuronal cell death. Our preliminary data further indicate that homocysteine-induced activation of p38 MAPK is regulated by ERK MAPK indicating that a unique crosstalk between the two MAPK pathways may play a role in homocysteine-mediated neuronal cell death. The data also indicate that both AMPAR and nNOS regulate activation of p38 and play critical roles in mediating homocysteine induced neuronal cell death. Furthermore we provide evidence for ERK MAPK dependent increased trafficking of AMPA receptor subunits to neuronal surface in response to homocysteine. Experiments in the Specific Aim 2 will evaluate the role of nNOS and AMPA receptors in mediating the crosstalk between ERK and p38 MAPK and subsequent neuronal cell death. The above studies will involve neuron culture experiments of homocysteine toxicity and utilize pharmacological, electrophysiological, Ca2+ imaging, RNAi, fluorescence imaging, electron paramagnetic resonance and biochemical approaches. The proposed studies will potentially establish an important distinction between mechanisms leading to homocysteine & glutamate toxicity. Understanding these novel molecular events triggered by homocysteine may facilitate development of targeted therapeutic approaches to reduce brain damage associated with neurodegenerative disorders in hyper-homocysteinemic individuals.