|Ischemic stroke is one of the leading causes of long-term disability and death worldwide. Despite the importance of medical treatment, the underlying molecular mechanisms are not fully understood. A critical factor of ischemic stroke is hypoxia, leading to hypoxia-induced spreading depression (HSD) in the affected brain tissue. HSD is characterized by strong cell depolarization and disrupted ion homeostasis, which shuts down neuronal function and can cause severe cell damage. Importantly, two DLG-MAGUKs (Discs large membrane-associated guanylate kinases), namely PSD-95 and PSD-93, have been implicated in NMDA receptor (NMDAR)-mediated excitotoxicity processes contributing to stroke pathology. Both proteins are important players in the core organization of the postsynaptic density and exhibit opposing roles in experience-dependent synapse maturation during early development and in synaptic plasticity in the adult brain. The present study aimed at further investigating the role of PSD-95 and PSD-93 in synaptic transmission and excitotoxicity processes using a model of transient hypoxia in acute hippocampal slices of adult PSD-95 knockout (KO), PSD-93 KO and PSD-93/95 double knockout (DKO) mice.
The strength of basal synaptic transmission was substantially reduced in PSD-95 KO and PSD-93/95 DKO, but not in PSD-93 KO mice. Diminished synaptic function of PSD-95 KO mice is consistent with the previous finding that they maintain high levels of silent synapses into adulthood. Silent synapses lack functional AMPARs and therefore do not transmit at resting potential. Normally, such silent synapses undergo experience-dependent maturation into AMPAR-transmitting synapses during early development, thereby consolidating pre-existing wiring patterns. In contrast to PSD-95 KO, silent synapses in PSD-93 KO mice mature faster during development than in wildtype (WT) mice. Despite silent synapse number in younger DKO mice has been reported to be unchanged, the present results strongly indicate impaired excitatory transmission in adult DKO mice.
Differential effects of MAGUK KO were further seen in HSD. Importantly, the absence of PSD-95 provided protection against hypoxia-induced metabolic compromise of neuronal function. Indeed, PSD-95 KO mice exhibited reduced HSD effects as demonstrated by multiple parameters including delayed onset of HSD, attenuated change of intrinsic optical signals and improved recovery of synaptic function. By contrast, single KO of PSD-93 had no effect on the susceptibility to hypoxia. Interestingly, the protective effects in PSD-95 KO mice were consistently abolished by additional KO of PSD-93, except for improved synaptic recovery. Given its interaction with NMDARs, PSD-95 might couple the massive hypoxia-induced calcium influx to downstream signaling cascades involved in excitotoxicity processes such as toxic nitric oxide production. Toxic signal transduction mainly occurs at extrasynaptic sites involving GluN2B-containing NMDARs. Notably, further biochemical analysis of brain homogenates revealed an impaired switch from GluN2B- to GluN2A-NMDARs in PSD-95 KO mice, which normally occurs at synaptic sites during early development. Thus, silent synapses might mainly contain synaptic GluN2B-NMDARs, rather than extrasynaptic ones mediating excitotoxicity, therefore providing protection. Another important aspect of high silent synapse levels in PSD-95 KO mice is the increased potential for synaptic plasticity, maintaining the brain in a partly premature state. Since stroke is highly influenced by aging, which is associated with low silent synapse levels, increased levels of silent synapses may represent an important substrate for the reduced hypoxia-susceptibility in PSD-95 KO mice. Silent synapses provide opportunities of reorganization and refinement of neuronal networks and might therefore be particularly beneficial for synaptic recovery, which is consistent with the improved recovery in the absence of PSD-95.
Taken together, my data reveals that the KO of PSD-95 provides protection against hypoxia, which is likely due to high levels of silent synapses.