Propionic Acid Receptor Subunit Research Articles

The Effects of Ninjinyoeito on Impaired Spatial Memory and Prefrontal Cortical Synaptic Plasticity through α-Amino-3-hydroxy-5-4-isoxazole Propionic Acid Receptor Subunit in a Rat Model with Cerebral Ischemia and β-Amyloid Injection.

Ninjinyoeito (NYT), a traditional Japanese medicine, is effective for improving physical strength and treating fatigue and anorexia. Recently, a clinical report revealed that NYT ameliorates cognitive dysfunction in Alzheimer's disease (AD) patients, although the mechanisms remain unclear. AD is a neurodegenerative disorder accompanied by a progressive deficit in memory. Current therapeutic agents are largely ineffective in treating cognitive dysfunction in AD patients. In this study, we investigated the effects of NYT on spatial memory impairment in a rat model of dementia. Rats were prepared with transient cerebral ischemia and intraventricular injection of β-amyloid1-42 for 7 days (CI + Aβ). NYT was orally administered for 7 days after cerebral ischemia. We evaluated spatial memory using the Morris water maze and investigated the expression of α-amino-3-hydroxy-5-4-isoxazole propionic acid receptor subunits, the phosphorylation level of glutamate receptor A (GluA)1 at serine sites S831 and S845, and the Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the hippocampus and prefrontal cortex of CI + Aβ rats. In the CI + Aβ rats, NYT treatment shortened the extended time to reach the platform. However, NYT did not restore the decrease in the hippocampal GluA1, GluA2, or CaMKII expression but increased prefrontal cortical phosphorylation levels of S845-GluA1 and CaMKII. Therefore, NYT may alleviate spatial memory impairment by promoting glutamatergic transmission involved in the phosphorylation of S845-GluA1 and CaMKII in the prefrontal cortex of CI + Aβ rats. Our results suggest that NYT is a valuable treatment for AD patients.

Type I interferons impair BDNF‐induced cell signaling and neurotrophic activity in differentiated human SH‐SY5Y neuroblastoma cells and mouse primary cortical neurons

Type I interferons (IFNs) have been shown to act on neurons and to cause neuronal damage through mechanisms not completely defined. Here, we investigated the effects of type I IFNs on brain-derived neurotrophic factor (BDNF)-induced TrkB receptor signaling and neurotrophic activity. In retinoic acid-treated human SH-SY5Y neuroblastoma cells and mouse primary cortical neurons, long-term exposure to IFNs curtailed BDNF-induced activation of phosphatidylinositol 3-kinase, phospholipase Cγ and extracellular-regulated kinases 1 and 2 signaling. Moreover, IFN-β inhibited BDNF-induced cell survival, neurite outgrowth, and expression of neuronal markers, such as neurofilament proteins, growth-associated protein-43 and glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR1. The IFN inhibitory effects were associated with down-regulation of TrkB and inhibition of TrkB autophosphorylation. In SH-SY5Y cells, blockade of either Janus kinase with pyridone 6 or signal transducer and activator of transcription (STAT) 1 with siRNA transfection attenuated IFN-β-induced TrkB down-regulation. Quantitative real time RT-PCR indicated that IFN-β significantly reduced TrkB mRNA levels. Moreover, blockade of protein kinase R counteracted IFN-β-induced inhibition of TrkB expression and signaling. These data indicate that in neuronal cells IFNs negatively regulate BDNF signaling and neurotrophic activity through inhibition of TrkB activation and Janus kinase/Signal transducer and activator of transcription-dependent down-regulation of TrkB.

Early continuous white noise exposure alters l‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptor subunit glutamate receptor 2 and γ‐aminobutyric acid type a receptor subunit β3 protein expression in rat auditory cortex

Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate-level noise delayed the emergence of adult-like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical-period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA(A) receptor subunit beta3 in the auditory cortex after noise rearing. Our results show that continuous moderate-level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA(A)beta3. Furthermore, noise rearing also induced a significant decrease in the level of GABA(A) receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA(A)beta3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing.

N-Methyl-d-aspartate Receptor-dependent Regulation of the Glutamate Transporter Excitatory Amino Acid Carrier 1

The neuronal transporter excitatory amino acid carrier 1 (EAAC1) is enriched in perisynaptic regions, where it may regulate synaptic spillover of glutamate. In this study we examined potential interactions between EAAC1 and ionotropic glutamate receptors. N-Methyl-D-aspartate (NMDA) receptor subunits NR1, NR2A, and NR2B, but not the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR2, were co-immunoprecipitated with EAAC1 from neuron-enriched hippocampal cultures. A similar interaction was observed in C6 glioma and human embryonic kidney cells after co-transfection with Myc epitope-tagged EAAC1 and NMDA receptor subunits. Co-transfection of C6 glioma with the combination of NR1 and NR2 subunits dramatically increased (approximately 3-fold) the amount of Myc-EAAC1 that can be labeled with a membrane-impermeable biotinylating reagent. In hippocampal cultures, brief (5 min), robust (100 microM NMDA, 10 microM glycine) activation of the NMDA receptor decreased biotinylated EAAC1 to approximately 50% of control levels. This effect was inhibited by an NMDA receptor antagonist, intracellular or extracellular calcium chelators, or hypertonic sucrose. Glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with cyclothiazide, and thapsigargin mimicked the effects of NMDA. These studies suggest that NMDA receptors interact with EAAC1, facilitate cell surface expression of EAAC1 under basal conditions, and control internalization of EAAC1 upon activation. This NMDA receptor-dependent regulation of EAAC1 provides a novel mechanism that may shape excitatory signaling during synaptic plasticity and/or excitotoxicity.

Brain-derived Neurotrophic Factor Regulates the Expression and Synaptic Delivery ofα-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor Subunits in Hippocampal Neurons

Brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity in the hippocampus, but the mechanisms involved are not fully understood. The neurotrophin couples synaptic activation to changes in gene expression underlying long term potentiation and short term plasticity. Here we show that BDNF acutely up-regulates GluR1, GluR2, and GluR3 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits in 7-day in vitro cultured hippocampal neurons. The increase in GluR1 and GluR2 protein levels in developing cultures was impaired by K252a, a tropomyosin-related [corrected] kinase (Trk) inhibitor, and by translation (emetine and anisomycin) and transcription (alpha-amanitine and actinomycin D) inhibitors [corrected] The increase in GluR1 and GluR2 protein levels in developing cultures was impaired by K252a, a Trk inhibitor, and by translation (emetine and anisomycin) and transcription (alpha-amanitine and actinomycin D) inhibitors. Accordingly, BDNF increased the mRNA levels for GluR1 and GluR2 subunits. Biotinylation studies showed that stimulation with BDNF for 30 min selectively increased the amount of GluR1 associated with the plasma membrane, and this effect was abrogated by emetine. Under the same conditions, BDNF induced GluR1 phosphorylation on Ser-831 through activation of protein kinase C and Ca(2+)-calmodulin-dependent protein kinase II. Chelation of endogenous extracellular BDNF with TrkB-IgG selectively decreased GluR1 protein levels in 14-day in vitro cultures of hippocampal neurons. Moreover, BDNF promoted synaptic delivery of homomeric GluR1 AMPA receptors in cultured organotypic slices, by a mechanism independent of NMDA receptor activation. Taken together, the results indicate that BDNF up-regulates the protein levels of AMPA receptor subunits in hippocampal neurons and induces the delivery of AMPA receptors to the synapse.

Regions of α-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits that are permissive for the insertion of green fluorescent protein

The green fluorescent protein can be fused to the ends of a mature glutamate receptor subunit to produce functional, fluorescent receptors. However, there are good reasons to search for internal regions of receptor subunits that can tolerate green fluorescent protein insertion. First, internal insertions of green fluorescent protein may produce functional, fluorescent subunits that traffic more correctly. Second, fluorescent proteins inserted near interacting surfaces of subunits could potentially create reagents suitable for fluorescence resonance energy transfer measurements. Finally, internal green fluorescent protein insertions could potentially produce subunits capable of signaling conformational changes through intrinsic changes in fluorescence intensity. To identify regions of receptor subunits that are permissive for green fluorescent protein insertion, we used a series of recombinant transposons to create fluorescent protein insertions in three α-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits. A combined analysis of the relative fluorescence intensity and glutamate-gated ion channel function of 69 different green fluorescent protein fusion proteins identified permissive zones for the creation of bright and fully functional receptor subunits in the C-terminal portion of the amino terminal domain, the intracellular tail of the carboxy terminal domain, and within the pore-forming regions of the channel.

Characterization of the Intracellular Transport of GluR1 and GluR2 α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor Subunits in Hippocampal Neurons

Little is known about the dynamics of the dendritic transport of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) to synapses. Here, using virally expressed green fluorescent protein (GFP)-GluR1 and GFP-GluR2 and confocal photobleach techniques we show near real-time movement of these subunits in living cultured hippocampal neurons. GFP-GluR1 fluorescence was widely distributed throughout the extranuclear compartment with no evidence for discrete intracellular stores. GFP-GluR1 transport was predominantly proximal to distal at rates of 0.2-0.4 mum.s-1. GFP-GluR2 fluorescence was more punctate and localized at or close to the plasma membrane. Overall, GFP-GluR2 movement was less dynamic with distinct mobile and immobile pools. Neither activation nor inhibition of surface-expressed N-methyl-d-aspartate receptors or AMPARs had any significant effect on the rates of GFP-GluR1 or GFP-GluR2 dendritic transport. These results demonstrate that GluR1 is constitutively and rapidly transported throughout the neuron. GluR2, on the other hand, is less mobile, with a majority retained in relatively immobile membrane-associated clusters, with approximately 40% showing synaptic co-localization. Furthermore, the transport of both subunits is activity-independent, suggesting that the regulated delivery of AMPARs to the vicinity of synapses is not a mechanism that is involved in processes such as synaptic plasticity.

A desensitization-inhibiting mutation in the glutamate binding site of rat α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits is dominant in heteromultimeric complexes

Recently, it has been shown that a single leucine-to-tyrosine mutation in the agonist binding domains of the homomerically expressed α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors GluR3 and GluR1 is sufficient to completely block receptor desensitization. In the present study we tested heteromeric subunit combinations of AMPA receptors to demonstrate that the block of desensitization afforded by this mutation is dominant in heteromeric subunit complexes containing the leucine-to-tyrosine mutation in at least one of the subunits. In addition, by comparing mutated, desensitization-deficient forms of unedited GluR1 and GluR1 edited at the Q/R-site of the ion pore we demonstrate that the desensitization properties of AMPA receptors are not linked to the editing state of the ion pore domain and thus are independent of the permeability properties of the ion channel.

Oligomerization and Ligand-binding Properties of the Ectodomain of the α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor Subunit GluRD

The extracellular part of ionotropic glutamate receptor (iGluR) subunits can be divided into a conserved two-lobed ligand-binding domain ("S1S2") and an N-terminal approximately 400-residue segment of unknown function ("X domain") which shows high sequence variation among subunits. To investigate the structure and properties of the N-terminal domain, we have now produced affinity-tagged recombinant fragments which represent the X domain of the GluRD subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptors either alone or covalently linked to the ligand-binding domain ("XS1S2"). These fragments were expressed in insect cells as secreted soluble proteins and were recognized by a conformation-specific anti-GluRD monoclonal antibody. A hydrodynamic analysis of the purified fragments revealed them to be dimers, in contrast to the S1S2 ligand-binding domain which is monomeric. The X domain did not bind radiolabeled AMPA or glutamate nor did its presence affect the ligand binding properties of the S1S2 domain. Our findings demonstrate that the N-terminal domain of AMPA receptor can be expressed as a soluble polypeptide and suggest that subunit interactions in iGluR may involve the extracellular domains.

Subtype-specific Assembly of α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor Subunits Is Mediated by Their N-terminal Domains

Glutamate receptors (GluR) are oligomeric protein complexes formed by the assembly of four or perhaps five subunits. The rules that govern the selectivity of this process are not well understood. Here, we expressed combinations of subunits from two related GluR subfamilies in COS7 cells, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. By co-immunoprecipitation experiments, we assessed the ability of AMPA receptor subunits to assemble into multimeric complexes. Subunits GluR1-4 associated with indistinguishable efficiency with each other, whereas the kainate receptor subunits GluR6 and 7 showed a much lower degree of association with GluR1. Using chimeric receptors and truncation fragments of subunits, we show that this assembly specificity is determined by N-terminal regions of these subunits and that the most N-terminal domain of GluR2 together with a membrane anchor efficiently associates with GluR1.

Simple method for adapting DNA fragments and PCR products to all of the commonly used restriction sites.

BioTechniquesVol. 20, No. 1 BenchmarksOpen AccessSimple Method for Adapting DNA Fragments and PCR Products to All of the Commonly Used Restriction SitesT.C. Tsang, D.T. Harris, E.T. Akporiaye, S.F. Schluter, G.T. Bowden & E.M. HershT.C. Tsang*Address correspondence to Tom C. Tsang, Department of Microbiology and Immunology, Building 90, University of Arizona, Tucson, AZ 85721, USA.Arizona Cancer Center, Tucson, AZ, USASearch for more papers by this author, D.T. HarrisArizona Cancer Center, Tucson, AZ, USASearch for more papers by this author, E.T. AkporiayeArizona Cancer Center, Tucson, AZ, USASearch for more papers by this author, S.F. SchluterArizona Cancer Center, Tucson, AZ, USASearch for more papers by this author, G.T. BowdenArizona Cancer Center, Tucson, AZ, USASearch for more papers by this author & E.M. HershArizona Cancer Center, Tucson, AZ, USASearch for more papers by this authorPublished Online:2 Aug 2018https://doi.org/10.2144/96201bm11AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail "Simple Method for Adapting DNA Fragments and PCR Products to All of the Commonly Used Restriction Sites." , 20(1), pp. 51–52FiguresReferencesRelatedDetailsCited ByHeat-inducible amplifier vector for high-level expression of granulocyte-macrophage colony-stimulating factor9 July 2009 | International Journal of Hyperthermia, Vol. 22, No. 5Novel Amplifier Expression Vectors Producing Higher Levels of IL-2 Led to Slower Tumor Growth and Longer Survival In VivoDNA and Cell Biology, Vol. 24, No. 9Gene silencing studies in the gymnosperm species Pinus radiata21 January 2005 | Plant Cell Reports, Vol. 24, No. 2High-Efficiency Cloning System for Versatile Adaptation of DNA FragmentsP. Luo, T.C. Tsang, C. Takeuchi, J. Dekker, M. Badowski & D.T. Harris17 September 2018 | BioTechniques, Vol. 33, No. 4Isolation of the MIG1 Gene from Candida albicans and Effects of Its Disruption on Catabolite RepressionJournal of Bacteriology, Vol. 182, No. 2Heat-inducible vectors for use in gene therapy9 July 2009 | International Journal of Hyperthermia, Vol. 16, No. 2Subtype-specific Assembly of α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor Subunits Is Mediated by Their N-terminal DomainsJournal of Biological Chemistry, Vol. 274, No. 24Mammalian Expression Vector with Two Multiple Cloning Sites for Expression of Two Foreign GenesT.C. Tsang, D.T. Harris, E.T. Akporiaye, R.S. Chu, J. Brailey, F. Liu, F.H. Vasanwala, S.F. Schluter & E.M. Hersh7 August 2018 | BioTechniques, Vol. 22, No. 1 Vol. 20, No. 1 Follow us on social media for the latest updates Metrics Downloaded 103 times History Published online 2 August 2018 Published in print January 1996 Information© 2018 Author(s)PDF download