Small-molecule inhibitors at the PSD-95/nNOS interface attenuate MPP+-induced neuronal injury through Sirt3 mediated inhibition of mitochondrial dysfunction

• Disruption of PSD-95/nNOS interaction attenuates MPP+-induced neuron injury.
• IC87201 and ZL006 inhibit MPP+-induced mitochondrial dysfunction.
• IC87201 and ZL006 preserve mitochondrial Ca2+ homeostasis after MPP+ exposure.
• IC87201 and ZL006 induced protection is mediated by Sirt3 activation.

Post-synaptic density protein 95 (PSD-95) links neuronal nitric oxide synthase (nNOS) with the N-methyl-D-aspartic acid (NMDA) receptor in the central nervous system, and this molecular complex has been implicated in regulating neuronal excitability in several neurological disorders. Here, small-molecule inhibitors of the PSD-95/nNOS interaction, IC87201 and ZL006 were tested for neuroprotective effects in an in vitro Parkinson's disease (PD) model. We now report that IC87201 and ZL006 reduced MPP+-induced neuronal injury and apoptotic cell death in a dose-dependent manner in cultured cortical neurons. These protective effects were associated with suppressed mitochondrial dysfunction, as evidenced by decreased reactive oxygen species (ROS) generation, cytochrome c release, mitochondrial membrane potential (MMP) collapse, and the preserved mitochondrial complex I activity and ATP synthesis. IC87201 and ZL006 also preserved intracellular homeostasis through mitigating mitochondrial Ca2+ uptake and promoting mitochondrial Ca2+ buffering capacity. Moreover, treatment with IC87201 and ZL006 significantly increased the expression of Sirt3 after MPP+ exposure, and knockdown of Sirt3 using specific targeted small interfere RNA (siRNA) partially nullified the protective effects induced by these two inhibitors. These data strongly support the hypothesis that targeting the PSD-95/nNOS interaction produces neuroprotective effects and may represent a novel class of therapeutics for PD as well as other neurological diseases where detrimental NMDA receptor signaling plays a major role.