4. DISCUSSION
4.5. Neuronal protection due to attenuated NMDAR-mediated excitotoxicity?
Another conceivable reason for the protective effect seen in mice lacking PSD-95 is the disruption of NMDAR-mediated excitotoxicity cascades. Hypothetically, due to loss of PSD-95 as a linker protein, key proteins involved in these cascades get uncoupled from NMDAR overactivation and subsequent Ca2+ overload. Excess of Ca2+ has been shown to trigger diverse toxic downstream pathways including activation of the neuronal NO synthase (nNOS), which has been especially implicated in neuronal disorders such as ischemic stroke (Dawson et al., 1992; Huang et al., 1994; Volbracht et al., 2005). Indeed, inhibition of nNOS activity reduced infarct volumes and neurological deficits in mice subjected to cerebral ischemia (Huang et al., 1994).
As stated earlier, the proposed role of PSD-95 in mediating toxic NO production resulted from studies on neuronal cultures, demonstrating that suppressed PSD-95 expression attenuates NMDAR-mediated NO synthesis as well as excitotoxicity (Sattler et al., 1999). Interestingly, PSD-93 has also been implicated in providing protection against ischemic injury. Specifically,
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PSD-93 deletion also prevented NMDAR/nNOS-dependent toxicity in cortical cultures (Xu et al., 2004; Zhang et al., 2010). Furthermore, KO of PSD-93 in a mouse stroke model resulted in reduced infarct size and diminished neurological deficits (Zhang et al., 2014). However, another study showed that loss of PSD-93 did not provide neuroprotection in the hippocampus of neonatal mice subjected to hypoxia-induced ischemia (Jiang et al., 2003), which is similar to the present study not detecting any protection of PSD-93 KO against hypoxia. Collectively, these studies strongly implicate PSD-95 in stroke-induced neurotoxicity pathways, while the effect of PSD-93 KO needs further investigation.
The structural similarity of PSD-95 and PSD-93 (Cho et al., 1992) and the ability of both to directly interact with NMDARs (Kornau et al., 1995; Niethammer et al., 1996) suggests that both paralogs are capable of mediating NMDAR-dependent excitotoxicity. They interact with NMDARs by binding of their N-terminal PDZ1 and PDZ2 domains to the C-terminal cytoplasmatic tail of GluN2A and GluN2B (Kornau et al., 1995; Niethammer et al., 1996).
These first two PDZ repeats are highly conserved throughout all DLG-MAGUK family members, showing 80–90% amino acid sequence identity (Niethammer et al., 1996).
Interaction of PSD-95 or PSD-93 with nNOS has further been shown to be mediated by PDZ2 of the MAGUKs, in turn binding to the PDZ domain of nNOS (Brenman et al., 1996).
4.5.1. Reduced activity of the NMDAR-nNOS pathway in PSD-95 KO mice?
Given the structural similarity of PSD-95 and PSD-93, the binding specificity of the PSD-95 inhibitors can be questioned. These small molecules – including Tat-NR2B9c (NA-1), ZL006 and Tat-N-dimer – are assumed to prevent the interaction of PSD-95 with either GluN2B or nNOS (Sun et al., 2008; Zhou et al., 2010; Bach et al., 2012). They have aroused much attention in the past years with one of them (NA-1) having already been tested in a clinical trial of ischemic stroke (Hill et al., 2012).
In addition to stroke-related studies, the same (or similar) interfering molecules have even been used in other contexts of neuronal disorders. Interestingly, two peptides (i.e. IC87201 and Tat-nNOS) – in vitro interfering with the nNOS-PSD-95 interaction – showed protection in mouse models of NMDA-induced acute and chronic pain (Florio et al., 2009). Furthermore, nNOS-PSD-95 interaction has been proposed as a novel target for fear-related disorders such as posttraumatic stress disorder, since the peptide ZL006 attenuated fear memory in the amygdala (Li et al., 2018). Despite the well-known involvement of nNOS in mechanisms of depression (Wegener & Volke, 2010), the same peptide (ZL006) did not induce antidepressant-like effects in rodent depression models (Tillmann et al., 2017). Notably, in a mechanistical in vitro study, ZL006 and IC87201 were unable to interact with the PDZ domains
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of PSD-95 or nNOS, nor inhibited the PDZ-PDZ binding interface of the two proteins (Bach et al., 2015). This is in sharp contrast to the previously reported in vitro interference capability of IC87201 by Florio et al. (2009). Additional doubt on the proposed mechanism of action was generated by a finding of Cui et al. (2007), that Tat-NR2B9c contains higher potency of affecting the interaction of PSD-95PDZ2 with GluN2A, rather than with GluN2B. This finding seems surprising, since GluN2A is implicated in pro-survival pathways located at the synapse, while extrasynaptic GluN2B mainly mediates excitotoxicity (Hardingham & Bading, 2003). On top of that, despite the assumed interference of Tat-NR2B9c with GluN2B-PSD-95 interaction (Aarts et al., 2002), the peptide is much more effective at disrupting nNOS-PSD-95 by means of the half maximal inhibitory concentrations (IC50, 8 µM for PSD-95PDZ2-GluN2B vs. 0.2 µM for PSD-95PDZ2-nNOS, respectively) (Cui et al., 2007). Hence, these controversial studies on the synthetic peptides do not allow any clarification about their in vivo mechanism of action that mediates the protection against ischemic injury.
By taking into account the high structural similarity of PSD-95 and PSD-93 and the reported ability of Tat-NR2B9c to bind both paralogs (Cui et al., 2007), it is likely that the interaction of PSD-93 with GluN2B or nNOS is also affected. Nevertheless, the present study clearly implicates PSD-95 – rather than PSD-93 – in mediating hypoxia-induced excitotoxicity, as evidenced by substantial protection provided by genetic loss of PSD-95 but not PSD-93.
The here obtained results indicate either higher levels of silent synapses or reduced excitotoxicity such as impaired NO production to account for the protective effects seen in PSD-95 KO mice, while both mechanisms do not have to be mutually exclusive. However, the fact that the DKO mice, in which PSD-95-mediated excitotoxicity is abolished as well, showed almost no protection, clearly points towards a key role of high silent synapse levels in PSD-95 KO mice in providing protection against hypoxia.
A possible approach to further investigate a potential reduction in nNOS activity by PSD-95 nitrosylation of these proteins activates death signaling or inhibits pro-survival cascades.
Reported targets of S-nitrosylation include GAPDH (Hara et al., 2005), the GluR6 subunit of the kainate receptor (Yu et al., 2008) and the src homology 2 domain-containing phosphatase (SHP-2) (Shi et al., 2013). Hence, specific antibodies could be used to detect possible differences in the level of nitration or nitrosylation between WT and PSD-95 KO following the hypoxic event as an indicator for nNOS activity and thus, for excitotoxicity.
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