, 1999) or inhibition of binding between PSD-95 and

the NMDA receptor with exogenous peptides (Aarts et al., 2002) reduces NO-mediated excitotoxicity, emphasizing the role of PSD-95 in transducing signals from the NMDA receptor to nNOS. PSD-95 also regulates AMPA receptors through its interaction with stargazin (Chen et al., 2000). This binding is required for recruitment of AMPA receptors to the synapse (Schnell et al., 2002). Consistent with this observation, mice deficient in PSD-95 have decreased AMPA receptor-mediated neurotransmission (Béïque et al., 2006). Furthermore, appropriate interactions between PSD-95 and A kinase-anchoring protein (AKAP) are required for NMDA-mediated AMPA receptor endocytosis (Bhattacharyya et al., 2009). PSD-95 function SKI-606 cell line is regulated by dynamic cycling of palmitoylation and depalmitoylation (El-Husseini et al.,

2002). Glutamate receptor activation enhances depalmitoylation of PSD-95 (El-Husseini et al., 2002), while blockade of synaptic activity enhances PSD-95 palmitoylation through regulated translocation of the dendritic palmitoyl acyltransferase (PAT) DHHC2 (Noritake et al., 2009). Palmitoylation influences synaptic dynamics by augmenting clustering of PSD-95 at dendritic spines (Craven et al., 1999). Palmitoylation of PSD-95 takes place at cysteines 3 and 5 (Topinka and Bredt, 1998). Nitric oxide signals in large part by S-nitrosylating (hereafter referred to as “nitrosylating”) cysteines PD0332991 in vivo in a variety of proteins (Hess et al., 2005). Hess et al. (1993) showed that NO donors can inhibit the palmitoylation of several proteins in dorsal root ganglia neurons and suggested that a NO-mediated posttranslational modification might compete with palmitoylation. Because of its close physical proximity to both the NMDA receptor and nNOS, we wondered whether PSD-95

might be a target for nitrosylation and whether there might be some interaction between putative nitrosylation and palmitoylation of PSD-95. In the present study we show that PSD-95 is physiologically nitrosylated at cysteines 3 and 5 in a reciprocal relationship with palmitoylation. This process impacts the physiologic clustering of PSD-95 at synapses. We examined the possibility that PSD-95 can be nitrosylated by exposing HEK293 cells containing overexpressed PSD-95 to the NO donor cysteine-NO (Cys-NO) (Figure 1A). The NO donor elicits nitrosylation those of PSD-95, monitored by the biotin-switch assay, in a concentration-dependent fashion. To determine whether PSD-95 is physiologically nitrosylated in mammalian brain, we monitored endogenous PSD-95 in mouse brain from wild-type and nNOS-deleted animals (Figure 1B). We observe ascorbate-dependent basal nitrosylation of endogenous PSD-95 that is abolished in nNOS knockout mice. Levels of nitrosylated PSD-95 are comparable to those of the NR2A subunit of the NMDAR (Figure 1C). PSD-95 is palmitoylated at cysteines 3 and 5 (Topinka and Bredt, 1998).