PDZ Protein Interactions Underlying NMDA Receptor-Mediated Excitotoxicity and Neuroprotection by PSD-95 Inhibitors

Abstract

In neuronal synapses, PDZ domains [postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] of PSD-95 proteins interact with C termini of NMDA receptor [NMDAR (NR)]subunits, linking them to downstream neurotoxic signaling molecules. Perturbing NMDAR/ PSD-95 interactions with a Tat peptide comprising the nine C-terminal residues of the NR2B subunit (Tat-NR2B9c) reduces neurons' vulnerability to excitotoxicity and ischemia. However, NR subunit C termini may bind many of Ͼ240 cellular PDZs, any of which could mediate neurotoxic signaling independently of PSD-95. Here, we performed a proteomic and biochemical analysis of the interactions of all known human PDZs with synaptic signaling proteins including NR1, NR2A-NR2D, and neuronal nitric oxide synthase (nNOS). Tat-NR2B9c, whose interactions define PDZs involved in neurotoxic signaling, was also used. NR2A-NR2D subunits and Tat-NR2B9c had similar, highly specific, PDZ protein interactions, of which the strongest were with the PSD-95 family members (PSD-95, PSD-93, SAP97, and SAP102) and Tax interaction protein 1 (TIP1). The PSD-95 PDZ2 domain bound NR2A-NR2C subunits most strongly (EC 50 , ϳ1 M), and fusing the NR2B C terminus to Tat enhanced its affinity for PSD-95 PDZ2 by Ͼ100-fold (EC 50 , ϳ7 nM). IC 50 values for Tat-NR2B9c inhibiting NR2A-NR2C/PSD-95 interactions (ϳ1-10 M) and nNOS/PSD-95 interactions (200 nM) confirmed the feasibility of such inhibition. To determine which of the PDZ interactions of Tat-NR2B9c mediate neuroprotection, one of PSD-95, PSD-93, SAP97, SAP102, TIP1, or nNOS expression was inhibited in cortical neurons exposed to NMDA toxicity. Only neurons lacking PSD-95 or nNOS but not PSD-93, SAP97, SAP102, or TIP1 exhibited reduced excitotoxic vulnerability. Thus, despite the ubiquitousness of PDZ domaincontaining proteins, PSD-95 and nNOS above any other PDZ proteins are keys in effecting NMDAR-dependent excitotoxicity. Consequently, PSD-95 inhibition may constitute a highly specific strategy for treating excitotoxic disorders.

Interaction between connexin35 and zonula occludens-1 and its potential role in the regulation of electrical synapses

“…At chemical synapses, several PDZ proteins such as PSD95/SAP90, GRIP, and INAD mediate protein-protein interactions essential in the formation of effective functional units. As an example, PSD95 is important not only for clustering and localization of NMDA receptors in postsynaptic densities (6,9), but also for linking NMDA receptors with signaling molecules (5,8), such as neuronal NOS (48), and for regulation of channel surface expression (3). AMPA receptors are dynamically regulated by activity (4, 7), a process that involves several PDZ-containing proteins.…”

The Role of PSD-95 and Cypin in Morphological Changes in Dendrites Following Sublethal NMDA Exposure

“…As shown in Figure 8 D-F, overexpressing cypin or knocking down PSD-95 significantly protects neurons from NMDA-induced excitotoxicity, while knocking down cypin or overexpressing PSD-95 potentiates neuronal death. Furthermore, changes in cell viability are not due to aberrant subcellular localization of PSD-95 that may interfere with the NMDA receptor signaling, further influencing neuron survival rates (Sattler et al, 1999;Aarts et al, 2002;Cui et al, 2007). As shown in Figure 8G, the majority of PSD-95 clusters are synaptic in all conditions.…”

Knock-down of postsynaptic density protein 95 expression by antisense oligonucleotides protects against apoptosis-like cell death induced by oxygen-glucose deprivation in vitro

“…In PSDs, binding of the cytosolic C-terminals of NR2 subunits to the PDZ domains of PSD-95 is the major component [10,11] . The interaction between NMDAR NR2 subunits and PSD-95 is important for specific localization of NMDARs in the PSD and for the coupling of NMDARs to cytoplasmic signaling pathways.…”