S-Nitrosylation of AMPA receptor GluA1 regulates phosphorylation, single-channel conductance, and endocytosis

Solomon H. Snyder Department of Neuroscience and Departments of Psychiatry and Behavioral Sciences, Pharmacology and Molecular Sciences, and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2012; 110(3). DOI: 10.1073/pnas.1221295110
Source: PubMed


NMDA receptor activation can elicit synaptic plasticity by augmenting conductance of the AMPA receptor GluA1 subsequent to phosphorylation at S831 by Ca(2+)-dependent kinases. NMDA receptor activation also regulates synaptic plasticity by causing endocytosis of AMPA receptor GluA1. We demonstrate a unique signaling cascade for these processes mediated by NMDA receptor-dependent NO formation and GluA1 S-nitrosylation. Thus, S-nitrosylation of GluA1 at C875 enhances S831 phosphorylation, facilitates the associated AMPA receptor conductance increase, and results in endocytosis by increasing receptor binding to the AP2 protein of the endocytotic machinery.

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    • "Glutamate plays a crucial role during development of the central nervous system (CNS) where it regulates neurogenesis, neurite outgrowth, synaptogenesis and apoptosis (Mattson et al., 1988; Monnerie et al., 2003) in addition to the regulation of neuroplasticity in the adult brain (Mattson, 2008; Selvakumar et al., 2013). "
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    ABSTRACT: Glutamate and nitric oxide 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 (500 μM) or NMDA (100-500 μM) combined with glycine suppresses neurite outgrowth. A similar reduction of neurite outgrowth was observed with the nitric oxide (NO) precursor L-arginine and NO donor sodium nitroprusside (SNP) (100 and 300 μM). The NMDA-R antagonists ketamine and MK-801 (10 nM) counteracted the NMDA/glycine-induced reduction in neurite outgrowth and the nNOS inhibitor 1-[2-(trifluoromethyl)phenyl] imidazole (TRIM) (100 nM) 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) (10 μM) also protected against L-arginine-induced decreases in neurite outgrowth. Since the NMDA-R is functionally coupled to neuronal NO synthase (nNOS) via the postsynaptic protein 95 kDa (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) (100-1000 nM) and 4-(3,5-dichloro-2-hydroxy-benzylamino)-2-hydroxybenzoic acid (ZL-006) (10-1000 nM) 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. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Jun 2015 · Neuroscience
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    • "Down-regulation of immediate early genes (IEGs) [12] including arc, zif-268, homer-1, c-fos and inhibition of synapse-associated genes [13], [14], [15] including psd-95, synpo, adam-10 have also been reported in AD brain. In addition, oxidative [16] and nitrosylative [17], [18] damages in different hippocampal proteins also have been implicated in the loss of function and eventual death of hippocampal neurons. Many pharmacological compounds have been tested in the treatment of AD, including cholinesterase inhibitors and memantine, but most of them generate several side effects because of the poor metabolic activities of the elderly population. "
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    ABSTRACT: Increase of the density of dendritic spines and enhancement of synaptic transmission through ionotropic glutamate receptors are important events, leading to synaptic plasticity and eventually hippocampus-dependent spatial learning and memory formation. Here we have undertaken an innovative approach to upregulate hippocampal plasticity. RNS60 is a 0.9% saline solution containing charge-stabilized nanobubbles that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), PNS60 (saline containing a comparable level of oxygen without the TCP modification), or RNS10.3 (TCP-modified normal saline without excess oxygen), stimulated morphological plasticity and synaptic transmission via NMDA- and AMPA-sensitive calcium influx in cultured mouse hippocampal neurons. Using mRNA-based targeted gene array, real-time PCR, immunoblot, and immunofluorescence analyses, we further demonstrate that RNS60 stimulated the expression of many plasticity-associated genes in cultured hippocampal neurons. Activation of type IA, but not type IB, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated upregulation of plasticity-related proteins (NR2A and GluR1) and increase in spine density, neuronal size, and calcium influx by LY294002, a specific inhibitor of PI-3 kinase, suggest that RNS60 upregulates hippocampal plasticity via activation of PI-3 kinase. Finally, in the 5XFAD transgenic model of Alzheimer's disease (AD), RNS60 treatment upregulated expression of plasticity-related proteins PSD95 and NR2A and increased AMPA- and NMDA-dependent hippocampal calcium influx. These results describe a novel property of RNS60 in stimulating hippocampal plasticity, which may help AD and other dementias.
    Full-text · Article · Jul 2014 · PLoS ONE
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    • "Over the last decade hundreds of proteins have been shown to be S-nitrosylated [6], [7]. Functional consequences of S-nitrosylation have been demonstrated for a small number of proteins, including caspases [8], parkin [9], glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [10], tubulin [11], microtubule-associated protein MAP1B [12], histone deacetylase-2 (HDAC2) [13], PSD-95 [14] and AMPA receptors [15]. However, for most of the identified targets, the exact role and relevance of S-nitrosylation remain elusive. "
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    ABSTRACT: Protein S-nitrosylation, the covalent attachment of a nitroso moiety to thiol groups of specific cysteine residues, is one of the major pathways of nitric oxide signaling. Hundreds of proteins are subject to this transient post-translational modification and for some the functional consequences have been identified. Biochemical assays for the analysis of protein S-nitrosylation have been established and can be used to study if and under what conditions a given protein is S-nitrosylated. In contrast, the equally desirable subcellular localization of specific S-nitrosylated protein isoforms has not been achieved to date. In the current study we attempted to specifically localize S-nitrosylated α- and β-tubulin isoforms in primary neurons after fixation. The approach was based on in situ replacement of the labile cysteine nitroso modification with a stable tag and the subsequent use of antibodies which recognize the tag in the context of the tubulin polypeptide sequence flanking the cysteine residue of interest. We established a procedure for tagging S-nitrosylated proteins in cultured primary neurons and obtained polyclonal anti-tag antibodies capable of specifically detecting tagged proteins on immunoblots and in fixed cells. However, the antibodies were not specific for tubulin isoforms. We suggest that different tagging strategies or alternative methods such as fluorescence resonance energy transfer techniques might be more successful.
    Full-text · Article · Jun 2013 · PLoS ONE
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