Iron overload promotes synaptic dysfunction and mediated excitotoxicity via NMDAR–PSD-95–nNOS signaling in dopaminergic neurons

Iron deposition in the substantia nigra (SN) is recognized as a critical factor in the pathogenesis of Parkinson’s disease. However, the precise effects of iron on dopaminergic neuron damage, and the underlying molecular mechanisms remain largely unclear. This study aimed to investigate how excessive iron disrupts dopamine homeostasis and synaptic integrity, with a focus on NMDAR-mediated excitotoxicity. In iron dextran-treated rats, iron disrupted dopamine homeostasis and reduced the expression of NMDAR and AMPAR subunits, and several synaptic plasticity related proteins in SN and striatum. Ferric ammonium citrate (FAC) inhibited spontaneous and evoked firing of dopaminergic neurons in the substantia nigra pars compacta. In the primary ventral midbrain (VM) neurons of rats, FAC elevated intracellular free iron and ROS, reduced mitochondrial membrane potential, and decreased total protein levels of NMDARs and AMPARs. Notably, in vitro study, FAC upregulated the expressions of PSD-95 and nNOS and increased membrane expression of GluN2B and GluN2A, as well as promoted GluN2B–PSD-95–nNOS complex assembly. Tat-NR2B9c, a peptide that disrupts the interaction between GluN2B and PSD-95, protected against iron-induced neurotoxicity in both cultured neurons and rats. This protection was associated with suppression of nNOS upregulation, NO overproduction, restoration of mitochondrial membrane potential and BDNF expression, which were achieved through inhibiting PSD-95–nNOS–GluN2B complex assembly.These results indicated that iron overload disrupted dopamine homeostasis, promoted synaptic deficits and NMDAR-mediated excitotoxicity via NMDAR–PSD-95–nNOS signaling, identifying the disruption of the PSD-95/NMDAR interaction as a novel protective strategy against iron-induced neuronal injury.