Molecular Determinants of Agonist Discrimination by NMDA Receptor Subunits: Analysis of the Glutamate Binding Site on the NR2B Subunit

AbstractNMDA receptors require both L-glutamate and the coagonist glycine for efficient channel activation. The glycine binding site of these heteromeric receptor proteins is formed by regions of the NMDAR1 (NR1) subunit that display sequence similarity to bacterial amino acid binding proteins. Here, we demonstrate that the glutamate binding site is located on the homologous regions of the NR2B subunit. Mutation of residues within the N-terminal domain and the loop region between membrane segments M3 and M4 significantly reduced the efficacy of glutamate, but not glycine, in channel gating. Some of the mutations also decreased inhibition by the glutamate antagonists, D-AP5 and R-CPP. Homology-based molecular modeling of the glutamate and glycine binding domains indicates that the NR2 and NR1 subunits use similar residues to ligate the agonists' α-aminocarboxylic acid groups, whereas differences in side chain interactions and size of aromatic residues determine ligand selectivity

Activity-Dependent Shedding of the NMDA Receptor Glycine Binding Site by Matrix Metalloproteinase 3: A PUTATIVE Mechanism of Postsynaptic Plasticity

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS

Loss of Extrasynaptic Inhibitory Glycine Receptors in the Hippocampus of an AD Mouse Model Is Restored by Treatment with Artesunate

Alzheimer’s disease (AD) is characterized by synaptic failure and neuronal loss. Recently, we demonstrated that artemisinins restored the levels of key proteins of inhibitory GABAergic synapses in the hippocampus of APP/PS1 mice, a model of cerebral amyloidosis. In the present study, we analyzed the protein levels and subcellular localization of α2 and α3 subunits of GlyRs, indicated as the most abundant receptor subtypes in the mature hippocampus, in early and late stages of AD pathogenesis, and upon treatment with two different doses of artesunate (ARS). Immunofluorescence microscopy and Western blot analysis demonstrated that the protein levels of both α2 and α3 GlyRs are considerably reduced in the CA1 and the dentate gyrus of 12-month-old APP/PS1 mice when compared to WT mice. Notably, treatment with low-dose ARS affected GlyR expression in a subunit-specific way; the protein levels of α3 GlyR subunits were rescued to about WT levels, whereas that of α2 GlyRs were not affected significantly. Moreover, double labeling with a presynaptic marker indicated that the changes in GlyR α3 expression levels primarily involve extracellular GlyRs. Correspondingly, low concentrations of artesunate (≤1 µM) also increased the extrasynaptic GlyR cluster density in hAPPswe-transfected primary hippocampal neurons, whereas the number of GlyR clusters overlapping presynaptic VIAAT immunoreactivities remained unchanged. Thus, here we provide evidence that the protein levels and subcellular localization of α2 and α3 subunits of GlyRs show regional and temporal alterations in the hippocampus of APP/PS1 mice that can be modulated by the application of artesunate

Cyclin-dependent kinase 5 is involved in the phosphorylation of gephyrin and clustering of GABAA receptors at inhibitory synapses of hippocampal neurons.

CDK5 has been implicated in neural functions including growth, neuronal migration, synaptic transmission and plasticity of excitatory chemical synapses. Here we report robust effects of CDK5 on phosphorylation of the postsynaptic scaffold protein gephyrin and clustering of inhibitory GABAA receptors in hippocampal neurons. shRNA-mediated knockdown of CDK5 and pharmacological inhibition of cyclin-dependent kinases reduced phosphorylated gephyrin clusters and postsynaptic γ2-containing GABAA receptors. Phosphorylation of S270 is antagonized by PP1/PP2a phosphatase and site-directed mutagenesis and in vitro phosphorylation experiments indicate that S270 is a putative CDK5 phosphorylation site of gephyrin. Our data suggest that CDK5 plays an essential role for the stability of gephyrin-dependent GABAA receptor clusters in hippocampal neurons

Transient neuromotor phenotype in transgenic spastic mice expressing low levels of glycine receptor beta-subunit: an animal model of startle disease

Startle disease or hereditary hyperekplexia has been shown to result from mutations in the alpha1-subunit gene of the inhibitory glycine receptor (GlyR). In hyperekplexia patients, neuromotor symptoms generally become apparent at birth, improve with age, and often disappear in adulthood. Loss-of-function mutations of GlyR alpha or beta-subunits in mice show rather severe neuromotor phenotypes. Here, we generated mutant mice with a transient neuromotor deficiency by introducing a GlyR beta transgene into the spastic mouse (spa/spa), a recessive mutant carrying a transposon insertion within the GlyR beta-subunit gene. In spa/spa TG456 mice, one of three strains generated with this construct, which expressed very low levels of GlyR beta transgene-dependent mRNA and protein, the spastic phenotype was found to depend upon the transgene copy number. Notably, mice carrying two copies of the transgene showed an age-dependent sensitivity to tremor induction, which peaked at approximately 3-4 weeks postnatally. This closely resembles the development of symptoms in human hyperekplexia patients, where motor coordination significantly improves after adolescence. The spa/spa TG456 line thus may serve as an animal model of human startle disease

CDK5 knockdown virus infection results in reduced CDK5 expression and reduced numbers of phospho-gephyrin clusters in cultured hippocampal neurons.

<p>Hippocampal neurons (div14) were stained with anti-GFP antibodies to detect infected neurons (upper panel), with anti-CDK5 antibody to quantify CDK5 expression levels (middle panel) and with the phosphospecific anti-gephyrin mAb7a antibody (lower panel). (A) Non-infected cells; (A') CDK5-knockdown; (A'') control shRNA (CDK5-mismatch). Neurons were infected with the indicated viruses at div6. The scale bar represents 15 µm. (B) Quantification of CDK5 fluorescence intensities of neurons infected with three different CDK5 knockdown viruses (kd1, kd2 and kd3). CDK5 knockdown cells were compared to non-infected neurons. n = 3, mean ± SD (C) Quantification of mAb7a cluster numbers of hippocampal neurons infected with three different CDK5 knockdown viruses (kd1, kd2, kd3) compared to non-infected and control shRNA (mismatch). 30 cells from n = 4 independent cultures (for each of non-infected, mismatch, kd2, kd1) or 30 cells from n = 3 independent cultures (kd3), mean ± SE, ANOVA with post-hoc test, ***P<0.001.</p

Binding of gephyrin to microtubules is regulated by its phosphorylation at Ser270

Gephyrin is a multifunctional scaffolding protein anchoring glycine- and subtypes of GABA type A- receptors at inhibitory postsynaptic membrane specializations by binding to the microtubule (MT) and/or the actin cytoskeleton. However, the conditions under which gephyrin can bind to MTs and its regulation are currently unknown. Here, we demonstrate that during the purification of MTs from rat brain by sedimentation of polymerized tubulin using high-speed centrifugation a fraction of gephyrin was bound to MTs, whereas gephyrin phosphorylated at the CDK5-dependent site Ser270 was detached from MTs and remained in the soluble protein fraction. Moreover, after collybistin fostered phosphorylation at Ser270 the binding of a recombinant gephyrin to MTs was strongly reduced in co-sedimentation assays. Correspondingly, upon substitution of wild-type gephyrin with recombinant gephyrin carrying alanine mutations at putative CDK5 phosphorylation sites the binding of gephyrin to MTs was increased. Furthermore, the analysis of cultured HEK293T and U2OS cells by immunofluorescence-microscopy disclosed a dispersed and punctuated endogenous gephyrin immunoreactivity co-localizing with MTs which was evidently not phosphorylated at Ser270. Thus, our study provides additional evidence for the binding of gephyrin to MTs in brain tissue and in in vitro cell systems. More importantly, our findings indicate that gephyrin-MT binding is restricted to a specific gephyrin fraction and depicts phosphorylation of gephyrin as a regulatory mechanism of this process by showing that soluble gephyrin detached from MTs can be detected specifically with the mAb7a antibody, which recognizes the Ser270 phosphorylated- version of gephyrin.publishe

Gephyrin and GABA_A receptor γ2 puncta are reduced upon CDK5/2/1 inhibition.

<p>(A) Hippocampal neurons were double-immunolabeled for gephyrin with antibody mAb7a (red, upper panel) and with anti-VIAAT antibody (green, middle panel). Superposition of both immunolabelings (lower panel, merge). Neurons were fixed and immunolabeled at div16. (A) Control cells, non-treated; (A') cultures treated with aminopurvalanol A (5 µM) for 2 days; (A'') cultures treated with aminopurvalanol A (5 µM) for 3 days. Note a higher number of VIAAT-opposed gephyrin puncta (yellow, merge) under A, compared to A' and A''. The scale bar represents 15 µm. (B) Hippocampal neurons were double-immunolabeled for GABA_A receptors with anti-γ2-subunit antibody (red, upper panel) and with anti-VIAAT antibodies (green, middle panel). Superposition of both immunolabelings (lower panel, merge). Neurons were fixed and immunolabeled at div16. (A) Control cells, non-treated; (A') cultures treated with aminopurvalanol A (5 µM) for 2 days. (A'') cultures treated with aminopurvalanol A (5 µM) for 3 days. Note a higher number of VIAAT-opposed γ2-subunit puncta (yellow, merge) under B, compared to B' and B''. (C) Quantification of the number of gephyrin (mAb7a) puncta. Quantification was done with about 10 cells from 4 independent cultures. Mean ± S.E.; ANOVA with post-hoc test: * P<0.05. (D) Quantification of the number of GABA_A receptors with anti-γ2-subunit antibody. Mean ± SE. Quantification was done with 49 cells from n = 4 independent cultures (control), 34 cells from n = 3 independent cultures (2 days) and 18 cells from n = 3 independent cultures (3 days). ANOVA with post-hoc test: ** P<0.01.</p