4. DISCUSSION
4.3. Knockout of PSD-95 protects against hypoxia
The interaction of DLG-MAGUKs with NMDARs is thought to mediate the activation of several downstream signaling pathways in response to Ca2+ influx, thus affecting processes such as synaptic plasticity (Xu et al., 2008). Importantly, both PSD-95 and PSD-93 have been implicated in NMDAR-mediated excitotoxicity, which contributes to neuronal dysfunction and cell damage in diverse neuropathologies including ischemic stroke, Alzheimer’s disease and acute brain injury (Dawson & Dawson, 1998; Lai et al., 2014; Parsons & Raymond, 2014).
However, the specific role of PSD-95 and PSD-93 in this context is elusive and, notably, studies on PSD-95 KO mice are still lacking. In order to shed some light on possibly similar or opposing functions of PSD-95 and PSD-93, I induced excitotoxicity in acute brain slices of the respective KO and DKO mice by hypoxia, leading to spreading depression (HSD).
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Importantly, only the KO of PSD-95 but not PSD-93 provided protection against hypoxia. This finding indicates that only PSD-95 is involved in NMDAR-mediated excitotoxicity following metabolic compromise. Furthermore, simultaneous KO of both PSD-95 and PSD-93 showed partly reduced susceptibility to hypoxia, most likely due to the absence of PSD-95 and not PSD-93. The protective effect of PSD-95 KO was demonstrated by multiple parameters. First of all, the onset of HSD was delayed in absence of PSD-95, as indicated by a delayed HSD-accompanied DC shift (Figure 7A). Second, KO of PSD-95 resulted in a markedly attenuated IOS change, as displayed by reduced tissue light reflectance (Figure 8D). Third, PSD-95 KO mice exhibited a trend towards decelerated HSD wave propagation (Figure 9) and slightly delayed synaptic failure (Figure 11). Finally, the most obvious evidence for reduced susceptibility to hypoxia in PSD-95 deficient mice was their substantially improved recovery of synaptic function (Figure 12A and B). By contrast, single KO of PSD-93 had no effect in all of these parameters. Interestingly, the protective effects in PSD-95 KO mice were consistently abolished by additional KO of PSD-93 in DKO mice, except for improved synaptic recovery.
The only parameters which were not altered and therefore did not indicate protection by the absence of PSD-95, were the amplitude and duration of the HSD-associated DC shift (Figure 7B/C) and the size of affected hippocampal area (Figure 9). Whereas a reduced HSD-invaded area would have been expected, the amplitude and duration of the DC shift depend on the synchronization of underlying neuronal and glial depolarizations, and therefore not necessarily provide evidence for the vulnerability to hypoxia. Specifically, while an improved synchronization could shorten the DC shift, the amplitude would be enhanced. Consistent with the lack of differences in the invaded area between genotypes observed in the present study, no morphological alterations in hippocampal structures of PSD-93 KO and PSD-95 KO mice have been detected so far (Migaud et al., 1998; McGee et al., 2001). Detailed morphological investigations in PSD-93/95 DKO mice are still lacking, but the here presented unchanged DC parameters do not indicate alterations.
The delayed occurrence of HSD (Figure 7A) seen in PSD-95 KO mice might have been influenced by the decreased neuronal excitability (Figure 4A), since neuronal excitability is an important parameter defining the vulnerability of brain tissue to HSD generation (Aitken et al., 1991; Müller, 2000; Müller & Somjen, 2000b). However, HSD onset was unchanged in DKO mice, which also presented reduced strength of basal synaptic transmission (Figure 4A).
Therefore, the protective effect seen in PSD-95 mice is more likely to be genotype-specific, rather than due to reduced excitability.
The here demonstrated protection due to loss of PSD-95 against ischemia-like insults is consistent with several previous studies. Sattler et al. (1999) reported protective effects of antisense RNA-mediated KD of PSD-95 in cortical cultures against NMDAR-dependent excitotoxicity as indicated by reduced cell death after NMDA-treatment. These authors addressed delayed neuronal death, while my results indicate short-term protective effects
Discussion
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such as delayed HSD onset and improved synaptic recovery. Sattler et al. (1999) furthermore linked the interaction of PSD-95 with NMDARs to toxic NO production, in which PSD-95 serves to couple nNOS-activity to NMDAR-mediated Ca2+ influx. Consequently, following studies used small interfering peptides, which were synthesized to disrupt the NMDAR-PSD-95-nNOS interaction and tested them in stroke models. In support of the present data, these so-called “PSD-95 inhibitors” provided protection against ischemic stroke in rodents (Aarts et al., 2002; Sun et al., 2008; Zhou et al., 2010), macaques (Cook et al., 2012a), as well as humans tested in phase II clinical trials, which suffered from fewer infarcts following treatment (Hill et al., 2012). Specifically, the peptides reduced neuronal deficits following MCAO (Aarts et al., 2002; Sun et al., 2008; Zhou et al., 2010), which is comparable to the present study showing improved reinstatement of neuronal function upon hypoxia in PSD-95 KO mice (Figure 12A and B).
In contrast to the unchanged size of the HSD-invaded hippocampal area in PSD-95 KO mice seen here (Figure 9), previous studies, testing PSD-95 inhibitors in MCAO models, reported reduced infarct volumes (Aarts et al., 2002; Sun et al., 2008; Zhou et al., 2010; Cook et al., 2012a). However, using a different lesion model which typically does not result in a penumbra region like MCAO, i.e. photothrombotic stroke, another study did not detect reduced lesion sizes in PSD-95 KO mice (Greifzu et al., 2016). This is consistent with the present data, indicating similarities between impairments induced by photothrombotic stroke and the here addressed hypoxia-induced dysfunctions. Nevertheless, various other parameters including the reduced IOS change and improved functional recovery in PSD-95 KO mice are pointing towards diminished brain damage and an enhanced ability to recover from hypoxia such as in a penumbra region following MCAO, if appropriate treatment is performed (Ferrer & Planas, 2003). The extent and severity of delayed neuronal damage and/or cell death can, however, not be investigated here, due to the given time-limit of acute brain slices.
There are two main possibilities, which could account for the protective effect in absence of PSD-95. On the one hand, loss of PSD-95 might result in reduced activation of excitotoxicity pathways such as toxic NO production (Sattler et al., 1999). On the other hand, high levels of silent synapses in PSD-95 KO mice (D. Favaro et al., in press; Huang et al., 2015a) may
“reinstate” a juvenile, more plastic state, possibly promoting synaptic reorganization following hypoxia. Both possibilities will be further discussed in the following sections (4.4 and 4.5).
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