The Journal of Neuroscience, May 8, 2013 • 33(19):8185–8201 • 8185 Neurobiology of Disease The nNOS-p38MAPK Pathway Is Mediated by NOS1AP during Neuronal Death Li-Li Li,1 Vanessa Ginet,2,3 Xiaonan Liu,1 Olga Vergun,1 Minna Tuittila,1,4 Marc Mathieu,5 Christophe Bonny,5 Julien Puyal,3 Anita C. Truttmann,2 and Michael J. Courtney1 1Molecular Signalling Laboratory, Department of Neurobiology, A.I. Virtanen Institute, University of Eastern Finland, FI-70211 Kuopio, Finland, 2Division of Neonatology, Department of Paediatrics and Paediatric Surgery and University Hospital Centre and University of Lausanne, CH-1015 Lausanne, Switzerland, 3Department of Fundamental Neurosciences, University of Lausanne, CH-1015 Lausanne, Switzerland, 4Turku Centre for Biotechnology, BioCity, University of Turku and Åbo Akademi University, FI-20521 Turku, Finland, and 5Xigen SA, 1003 Lausanne, Switzerland Neuronal nitric oxide synthase (nNOS) and p38MAPK are strongly implicated in excitotoxicity, a mechanism common to many neuro- degenerative conditions, but the intermediary mechanism is unclear. NOS1AP is encoded by a gene recently associated with sudden cardiac death, diabetes-associated complications, and schizophrenia (Arking et al., 2006; Becker et al., 2008; Brzustowicz, 2008; Lehtinen et al., 2008). Here we find it interacts with p38MAPK-activating kinase MKK3. Excitotoxic stimulus induces recruitment of NOS1AP to nNOS in rat cortical neuron culture. Excitotoxic activation of p38MAPK and subsequent neuronal death are reduced by competing with the nNOS:NOS1AP interaction and by knockdown with NOS1AP-targeting siRNAs. We designed a cell-permeable peptide that competes for the unique PDZ domain of nNOS that interacts with NOS1AP. This peptide inhibits NMDA-induced recruitment of NOS1AP to nNOS and in vivo in rat, doubles surviving tissue in a severe model of neonatal hypoxia-ischemia, a major cause of neonatal death and pediatric disability. The highly unusual sequence specificity of the nNOS:NOS1AP interaction and involvement in excitotoxic signaling may provide future opportunities for generation of neuroprotectants with high specificity. Introduction targetable, and the focus has shifted to downstream events that Excitotoxicity is a multistep mechanism of neurodegeneration mediate neuronal death. Recruitment of neuronal nitric oxide that is common to many acute and chronic diseases of the ner- synthase (nNOS) to activated NRs via PSD-95 contributes to vous system for which no therapeutic approaches are available. excitotoxicity (Ferriero et al., 1996; Cao et al., 2005; Ishii et al., Although the mechanisms are not fully understood, initiation of 2006; Zhou et al., 2010). Thus an NR C-terminus mimetic excitotoxicity is relatively clear. Excess neurotransmitter gluta- (“NR2B9C”) that competes with NR for binding the PSD-95– mate leads to calcium entry through NMDA-subtype glutamate nNOS complex is neuroprotective (Aarts et al., 2002). Similarly, receptors (NRs), which causes neurons to die. However, NRs are competitors of nNOS binding to PSD-95 to nNOS are neuropro- also required for normal brain function and neuronal survival. tective (Cao et al., 2005; Zhou et al., 2010). There is now reason to Multiple clinical trials have revealed them as therapeutically un- be optimistic about the potential therapeutic value of such ap- proaches as promising results have recently been reported from a clinical trial using NR2B9C in patients with iatrogenic stroke Received Sept. 21, 2012; revised March 24, 2013; accepted March 26, 2013. (Hill et al., 2012). However, PSD-95 is also critical for neuronal Author contributions: L.-L.L., A.C.T., and M.J.C. designed research; L.-L.L., V.G., X.L., O.V., M.T., J.P., and M.J.C. function. For example PSD-95 knockdown alone greatly disrupts performedresearch;M.T.,M.M.,C.B.,andM.J.C.contributedunpublishedreagents/analytictools;L.-L.L.,V.G.,O.V., J.P., and M.J.C. analyzed data; L.-L.L. and M.J.C. wrote the paper. synaptic AMPA receptor function (Elias et al., 2006; Elias and This work was funded by the Academy of Finland (Projects 203520, 110445, 127717, 134893, 135073), the Nicoll, 2007), while NO may even have beneficial effects in excito- EuropeanUnionprogramsFP6(STRESSPROTECT)andFP7(MEMOLOAD),theUniversityofEasternFinland,theSigrid toxic conditions (Iadecola, 1997; Atochin et al., 2003). Therefore Juse´lius foundation, and the Doctoral Programme of Molecular Medicine. We thank Bert Mayer (Karl-Franzens- protein-interaction targets downstream of nNOS must be found. Universita¨t, Graz, Austria), Jim Woodgett (Samuel Lunenfeld Research Institute, Toronto, Canada), and Atsushi Miyawaki (Riken Brain Institute, Saitama, Japan) for providing plasmids; Aila Seppa¨nen and Veera Pevgonen for A major obstacle to progress has been the absence of a mech- technical support; and the following people for helpful discussion and pilot experiments before this study: Jiong Cao, anism linking nNOS to downstream excitotoxic events, such as Valdislav Komarovski, Jenni Viholainen, Lotta Parviainen, Anu Ma¨ki-Hokkonen, Jouni Toivola, Kaisa Kosonen, and the activation of p38 stress-activated protein kinase (p38MAPK). members of the STRESSPROTECT consortium. The Biocentre Finland imaging facility MUIC at the University of Eastern This kinase is known to be involved in excitotoxicity but also Finland was used for this work. The authors declare no competing financial interests. contributes to multiple other functions and is itself not a viable CorrespondenceshouldbeaddressedtoMichaelJ.Courtney,A.I.VirtanenInstitute,UniversityofEasternFinland, therapeutic target. Here we show that a binding pocket on nNOS, FIN-70211 Kuopio, Finland. E-mail: [email protected]. which is distinct from the PSD-95-binding loop and does not M. Mathieu’s present address: Debiopharm Laboratory, HES-SO Valais, Route du Rawyl 64, CH-1950 Sion, mediate interaction with PSD-95, is unexpectedly required for Switzerland. C. Bonny’s present address: Division of Medical Genetics, University Hospital, CH-1015 Lausanne, Switzerland. excitotoxic signaling and neuronal death. We find that NMDA DOI:10.1523/JNEUROSCI.4578-12.2013 stimulation recruits NOS1AP to nNOS. A synthetic cell- Copyright © 2013 the authors 0270-6474/13/338185-17$15.00/0 permeable peptide ligand of the binding pocket inhibits this re- 8186 • J. Neurosci., May 8, 2013 • 33(19):8185–8201 Li et al. • NOS1AP Mediates the nNOS-p38MAPK Pathway cruitment and reduces the activation of p38MAPK, which has as controls. pVenus-NOS1AP N terminus and nNOS1–155Y77H/D78E previously been shown to be a nNOS-dependent step contribut- mutants were generated by PCR-based methods from pVenus-NOS1AP ing to excitotoxic death (Cao et al., 2005; Soriano et al., 2008). and pEGFP-nNOS-155, respectively. For recombinant protein expres- ␣␣ Supporting a role for NOS1AP in excitotoxicity we find that sion in bacteria, coding sequences for MKK3b, nNOS 1–300, 1–155wt, siRNA-mediated NOS1AP knockdown reduces excitotoxic death 1–155Y77H/D78E mutant, and 1–99 were transferred in-frame to pGEX-6P3 (APBiotech). pLuc-ASK1 was generated from pcDNA3-HA- and p38MAPK activation. NO can activate multiple MAP3Ks ASK1 (a generous gift from Jim Woodgett, Samuel Lunenfeld Research capable of binding NR-associated molecules such as PSD-95 and Institute, Toronto, Canada) and luciferase vector. The PSD-95-PDZ do- DAB2IP/AIP (Savinainen et al., 2001; Yoshimura et al., 2004; main and pVenus-C1 plasmids were previously described (Cao et al., Zhang et al., 2004; Follstaedt et al., 2008; Hu et al., 2012). Our 2005; Semenova et al., 2007). results suggest that p38MAPK activation by NR requires Recombinant protein preparation. GST-tagged nNOS␣␣1–300wt, NOS1AP because it is able to recruit and supply p38-activating nNOS␣␣1–99wt, nNOS␣␣1–155, and Y77H/D78E; MKK3b; and free MKK3, thereby facilitating the activation of p38MAPK by up- GST were prepared from BL21(DE3) Escherichia coli strain as described stream signals. Based on these results, we developed a cell- previously (Courtney and Coffey, 1999). permeable peptide competing with the nNOS–NOS1AP interaction Preparation of immobilized peptides. Synthetic peptides were coupled via amino groups to affigel10 beads according the manufacturer’s in- that is neuroprotective in an in vivo model of neonatal cerebral ␮ hypoxia-ischemia. structions (Bio-Rad, 2 mg per 500 l beads). Control empty beads were prepared by blocking fresh beads without peptide. Cell culture and transfection. Cerebellar granule neuron cultures, pre- Materials and Methods pared and transfected by calcium phosphate as described previously (Cao Materials. Peptides were synthesized as described previously (Yang et al., et al., 2005), stimulated with glutamate, were used only for Figure 1, because these experiments were based on published data in this model 2011) or obtained from GENECUST or GenicBio: “-GESV,” NH2- using nNOS␣␣1–300 to inhibit glutamate-induced excitotoxicity (Cao YAGQWGESV-COOH; “-GASA,” NH2-YAGQWGASA-COOH; “-ESDV” et al., 2005). Cortical neuron cultures, prepared as described previously or “NR2B9,” NH2-KLSSIESDV-COOH; “-EADA,” NH2-KLSSIEADA- (Semenova et al., 2007) and cultured in NeurobasalA/B27 medium (Life COOH; “L-TAT,” NH2-GRKKRRQRRR-COOH; “L-TAT-NR2B9C,” NH2-GRKKRRQRRR-KLSSIESDV-COOH; “L-TAT-GESV,” NH2-GRK- Technologies) and transfected with Lipofectamine 2000 (Life Technolo- KRRQRRR-YAGQWGESV-COOH; “L-TAT-GESVamide,” NH2-GRK- gies), stimulated with NMDA, were used for the remaining figures be- KRRQRRR-YAGQWGESV-CONH2; “L-TAT-GASA,”