title: Small-molecule inhibitors at the PSD-95/nNOS interface protect against glutamate-induced neuronal atrophy in primary cortical neurons.
abstract: Glutamate and nitric oxide (NO) are important regulators of dendrite and axon development in the central nervous system. Excess glutamatergic stimulation is a feature of many pathological conditions and manifests in neuronal atrophy and shrinkage with eventual neurodegeneration and cell death. Here we demonstrate that treatment of cultured primary cortical rat neurons for 24h with glutamate (500μM) or N-methyl-d-aspartate (NMDA) (100-500μM) combined with glycine suppresses neurite outgrowth. A similar reduction of neurite outgrowth was observed with the NO precursor l-arginine and NO donor sodium nitroprusside (SNP) (100 and 300μM). The NMDA-receptor (NMDA-R) antagonists ketamine and MK-801 (10nM) counteracted the NMDA/glycine-induced reduction in neurite outgrowth and the neuronal NO synthase (nNOS) inhibitor 1-[2-(trifluoromethyl)phenyl] imidazole (TRIM) (100nM) counteracted both the NMDA/glycine and l-arginine-induced decreases in neurite outgrowth. Furthermore, targeting soluble guanylate cyclase (sGC), a downstream target of NO, with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10μM) also protected against l-arginine-induced decreases in neurite outgrowth. Since the NMDA-R is functionally coupled to nNOS via the postsynaptic protein 95kDa (PSD-95), inhibitors of the PSD-95/nNOS interaction were tested for their ability to protect against glutamate-induced suppression in neurite outgrowth. Treatment with the small-molecule inhibitors of the PSD-95/nNOS interface 2-((1H-benzo[d] [1,2,3]triazol-5-ylamino) methyl)-4,6-dichlorophenol (IC87201) (10 and 100nM) and 4-(3,5-dichloro-2-hydroxy-benzylamino)-2-hydroxybenzoic acid (ZL-006) (10 and 100nM) attenuated NMDA/glycine-induced decreases in neurite outgrowth. These data support the hypothesis that targeting the NMDA-R/PSD-95/nNOS interaction downstream of NMDA-R promotes neurotrophic effects by preventing neurite shrinkage in response to excess glutamatergic stimulation. The PSD-95/nNOS interface may be an attractive target for treating deficits in neuronal outgrowth and atrophy associated with excessive glutamatergic neurotransmission in neurodevelopmental and neurodegenerative conditions.
authors.affiliation: Neuropsychopharmacology Research Group, School of Pharmacy & Pharmaceutical Sciences and Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland.
authors.forename: M V
authors.initials: MV
authors.lastname: Doucet
authors.affiliation: Neuropsychopharmacology Research Group, School of Pharmacy & Pharmaceutical Sciences and Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland.
authors.forename: E
authors.initials: E
authors.lastname: O'Toole
authors.affiliation: Neuroimmunology Research Group, School of Medicine and Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland.
authors.forename: T
authors.initials: T
authors.lastname: Connor
authors.affiliation: Neuropsychopharmacology Research Group, School of Pharmacy & Pharmaceutical Sciences and Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland. Electronic address: aharkin@tcd.ie.
authors.forename: A
authors.initials: A
authors.lastname: Harkin
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001284:Atrophy / Q000188:drug therapy; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D002733:Chlorophenols; D000076122:Disks Large Homolog 4 Protein; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D005260:Female; D018698:Glutamic Acid / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D016202:N-Methylaspartate / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects; D009474:Neurons / Q000166:cytology* / Q000187:drug effects*; D018696:Neuroprotective Agents / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase
chemical_list: D002733:Chlorophenols; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D018690:Excitatory Amino Acid Agonists; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D018696:Neuroprotective Agents; D020030:Nitric Oxide Donors; D018160:Receptors, Cytoplasmic and Nuclear; D009599:Nitroprusside; D018698:Glutamic Acid; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D052248:Nitric Oxide Synthase Type I; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; C004762:2,4-dichlorophenol
keywords: NMDA receptor; PSD-95; glutamate; neurite outgrowth; nitric oxide