PDZ protein interactions underlying NMDA receptor-mediated excitotoxicity and neuroprotection by PSD-95 inhibitors

Hong Cui, Amy Hayashi, Hong Shuo Sun, Michael P. Belmares, Carolyn Cobey, Thuymy Phan, Johannes Schweizer, Michael W. Salter, Tian Wang Yu, R. Andrew Tasker, David Garman, Joshua D. Rabinowitz, Peter S. Lu, Michael Tymianski

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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 >240 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, ∼1 μM), and fusing the NR2B C terminus to Tat enhanced its affinity for PSD-95 PDZ2 by >100-fold (EC50, ∼7 nM). IC 50 values for Tat-NR2B9c inhibiting NR2A-NR2C/PSD-95 interactions (∼1-10 μM) 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 domain-containing 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.

Original languageEnglish (US)
Pages (from-to)9901-9915
Number of pages15
JournalJournal of Neuroscience
Volume27
Issue number37
DOIs
StatePublished - Sep 12 2007

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)

Keywords

  • Excitotoxicity
  • NMDA receptors
  • Nitric oxide synthase
  • PDZ domains
  • PSD-95
  • RNA interference

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    Cui, H., Hayashi, A., Sun, H. S., Belmares, M. P., Cobey, C., Phan, T., Schweizer, J., Salter, M. W., Yu, T. W., Tasker, R. A., Garman, D., Rabinowitz, J. D., Lu, P. S., & Tymianski, M. (2007). PDZ protein interactions underlying NMDA receptor-mediated excitotoxicity and neuroprotection by PSD-95 inhibitors. Journal of Neuroscience, 27(37), 9901-9915. https://doi.org/10.1523/JNEUROSCI.1464-07.2007