The Role Of Neuronal Nitric Oxide Synthase SOMOylation In Synaptic Plasticity

Objective Synaptic plasticity refers to the variability of the structure and function of the synapse. The long-term potentiation (LTP) and long-term depression (LTD) are the two manifestations of synaptic function plasticity, which are the biological basis of learning and memory at the cellular level. NO catalyzed by neuronal nitric oxide synthase (nNOS) is involved in synaptic plasticity, but the role of nNOS and regulatory mechanisms in LTP have not yet been elucidated. In addition, recent studies have shown that the neurological function could be regulated by SUMOylation. nNOS is an important signaling protein in the postsynaptic membrane PSD-95 signaling complex, and SUMO ligase PIAS3 is expressed and highly enriched in the postsynaptic membrane. Therefore, this study is to investigate the role of nNOS SUMOylation in synaptic plasticity.Methods The cell models of synaptic plasticity is build by stimulating the cortical neurons with 50μM bicuculline or 50 mM KC1. The nNOS SUMOylation level and nNOS-PIAS interaction are detected by immunoprecipitation and immunoblotting. Immunofluorescence technique is used to clarify nNOS-SUMOl and nNOS-PIAS3 colocalization in neurons. Molecular biology techniques and PCR-directed mutagenesis is used to identify the SUMOylation site in nNOS. The structure domain deleted mutants of nNOS and PIAS3 are constructed to identify nNOS-PIAS3 interaction basis. Intervention of nNOS-PIAS3 binding by specific small peptides is used to define the role and the mechanism of nNOS in LTP. Patch clamp for field potentials recording is applied to observe the role of small peptides in LTP.Results 1. The covalent binding of nNOS and SUMO1 is detected in rat hippocampus.2. nNOS SUMOylation level increased gradually after the bicuculline stimulation, and is inhibited significantly by NMDA receptor antagonist MK801. The transient elevated nNOS SUMOylation is induced by KC1.3. Compared with wildtype nNOS, nNOS K725R and K739R SUMOylation level is decreased, and nNOS K725/739R SUMOylation presents a lower level obviously.4. The nNOS-PIAS3 interaction level is raised after bicuculline stimulation, consistent with the nNOS SUMOylation trends.5. In COS7 cells, PIAS3 SAP domain(1-86 aa) and nNOS CaM-binding domain (721-756 aa) are the main binding region for nNOS-PIAS3 interaction, and overexpression of nNOS CaM-binding domain restrains the binding between nNOS and PIAS3.6. The small peptide CaMB inhibits nNOS SUMOylation, ERK1/2 phosphorylation, Elkl phosphorylation, Arc and BDNF protein expression.7. The small peptide CaMB can significantly interfere with the LTP induction.Conclusion nNOS is SUMOylated in rat hippocampus. The SUMOylation of nNOS have a pivotal role in LTP. The key SUMOylation site in nNOS is K725 and K739. PIAS3 is the main SUMO ligase involved in nNOS SUMOylation. Sumoylated nNOS activate ERK1/2, promote the Elkl activation, upregulate the downstream plasticity-related protein Arc and BDNF expression, and participate in the LTP induction. The clarification of nNOS SUMOylation regulatory mechanism may provide a theoretical basis for the treatment of learning and memory dysfunction related diseases.