We conducted

experiments to assess its impact on palmitoylation. NMDA treatment of granule cells reduces palmitoylation of PSD-95 by ∼70% (Figure 2H). Vorinostat in vitro This action reflects NMDA augmentation of NO formation, as it is largely reversed by treatment with L-VNIO. Moreover, we observe reciprocal changes in PSD-95 nitrosylation, suggesting that nitrosylation is responsible for the effects of NMDA-mediated NO formation (Figure 3I). To further explore the importance of endogenous NO in regulating PSD-95 palmitoylation, we made use of nNOS-deleted mice (Figure 3J). We confirm the marked reduction of PSD-95 palmitoylation in response to NMDA treatment. Levels of PSD-95 palmitoylation are significantly augmented in nNOS-deleted granule cells both in the absence and presence of NMDA treatment (Figure 3J). NMDA does signaling pathway elicit some decrease in PSD-95 palmitoylation in nNOS knockout mice, which may reflect the retention of alternatively spliced, catalytically active nNOS (Eliasson

et al., 1997) or compensatory mechanisms involving eNOS or iNOS (Huang and Fishman, 1996). Thus, diverse experimental approaches establish that in mammalian brain, endogenous NO under basal conditions and in response to NMDAR-mediated neurotransmission regulates the palmitoylation of PSD-95. One of the principal roles of PSD-95 palmitoylation is to regulate synaptic clustering of the protein. Thus, synaptic clustering of PSD-95 is abolished with C3 and C5 mutation, which prevents palmitoylation (Craven et al., 1999). Moreover, inhibition of palmitoylation with 2-bromopalmitate (2-BP) reduces PSD-95 synaptic clusters (El-Husseini et al., 2002). Bredt, Nicoll, and Fukata established that glutamate neurotransmission, acting via ionotropic receptors, diminishes PSD-95 clustering both by inhibiting palmitoylation (Noritake

et al., 2009) and by stimulating depalmitoylation (el-Husseini and Bredt, 2002). We wondered whether this process might involve influences of NO acting by displacing Cediranib (AZD2171) palmitate from PSD-95. Accordingly, we examined the effect of NMDA treatment upon PSD-95 clustering in cerebellar granule neurons and evaluated the influence of the nNOS inhibitor VNIO (Figure 4). PSD-95 is synaptic in localization as it is closely apposed to clusters of synapsin, a presynaptic marker, with statistically significant colocalization (Pearson correlation coefficient, 0.45) (Figure 4A). NMDA treatment reduces PSD-95 synaptic clusters by ∼40%, while VNIO significantly reverses this action. In contrast, synapsin clusters are unchanged. The partial reversal by VNIO may indicate that the NMDA effects involve other signaling systems in addition to NO, possibly associated with calcium entering cells through NMDA channels. Double labeling is specific, as demonstrated by omission of primary antibodies (Figure 5C). As previously reported, 2-BP reduces PSD-95 clustering (Figures 5A and 5B).