Regulation of p38MAPK in excitotoxicity, a common mechanism of neuronal diseases.

Michael Courtney, University of Eastern Finland, Kuopio, Finland

Excitotoxicity is a multistep mechanism of neurodegeneration common to acute and chronic disorders of the nervous system. It is initiated by excess neurotransmitter glutamate and calcium entry through NMDA receptors (NRs), causing neurons to die. Interaction of calcium-activated neuronal nitric oxide synthase (nNOS) with NRs via PSD95 contributes to excitotoxicity, apparently via activation of p38MAPK cell death pathways. Direct inhibition of calcium influx and nNOS have not emerged as therapeutic approaches, but protein-protein interactions within the NR-PSD95-nNOS ternary complex have become increasingly attractive targets. A peptide competing with the interaction of NR and the PDZ domains of PSD95 was the first successful neuroprotectant in stroke trials, while nNOS-derived peptides and small-molecule inhibitors devised to disrupt PSD95-nNOS interaction show in vitro and in vivo efficacy in models of excitotoxicity, pain and depression. Nevertheless, PSD-95 is a critical player at synapses and nNOS function should also be retained. For this reason we sought targets downstream of the NR-PSD95-nNOS ternary complex, upstream of p38MAPK, which might allow more selective neuroprotection. We identified a novel mediator of excitotoxic cell death, the nNOS ligand NOS1AP encoded by a gene associated with sudden cardiac death, diabetes, and schizophrenia. In resting cortical neurons, interaction of nNOS with NOS1AP is weak. This rapidly increases in parallel with p38MAPK activation in response to NMDA-evoked increase of intracellular [Ca2+]. NOS1AP forms a complex with p38MAPK activator, MKK3, and RNAi methods show that both NOS1AP and MKK3 are required for NMDA-evoked activation of p38. NMDA also induced interaction between nNOS and NOS1AP in organotypic hippocampal slice cultures. The ligand-binding pocket of nNOS that binds to NOS1AP is known to have unusual sequence specificity, which facilitated the development of a cell-permeable peptide TAT-GESV that disrupts nNOS-NOS1AP interaction. TAT-GESV interacts with the nNOS-ligand binding pocket and thus we find it selectively competes with NOS1AP but not with PSD95, nor does it detectably interact with PSD95-PDZ domains. Using this tool we were able to obtain neuroprotection in several models of excitotoxic neurodegeneration. In conclusion, the recruitment of NOS1AP by nNOS is acts as a mediator between activation of the NR-PSD95-nNOS complex signalling and downstream p38-dependent neurodegenerative signalling and may become a valuable basis for design of selective therapeutics for conditions involving aberrant NMDA receptor signalling.