Propofol post-conditioning induced long-term neuroprotection and reduced internalization of AMPAR GluR2 subunit in a rat model of focal cerebral ischemia/reperfusion

We previously reported that propofol (20 mg/kg/h) post-conditioning provided acute (up to 24 h) neuroprotection in rats with transient middle cerebral artery occlusion. In this study, we extend these data by examining long-term protection and exploring underlying mechanisms involving AMPA receptor GluR2 subunit internalization. Rats were treated with propofol 20 mg/kg/h after 60 min of occlusion (beginning of reperfusion for 4 h). Propofol post-conditioning reduced infarct volume and improved spatial memory deficiencies (up to 28 days) induced by ischemia/reperfusion injury. Additionally, Propofol post-conditioning promoted neurogenesis in the dentate gyrus of hippocampus, as measured by bromodeoxyuridine and neuron-specific nuclear protein immunofluorescence-double staining at day 28 after reperfusion. Finally, propofol post-conditioning increased the surface expression of AMPA receptor GluR2 subunit, thus inhibited the internalization of this part until 28 days after stroke. In conclusion, our data suggest that propofol post-conditioning provides long-term protection against focal cerebral ischemia/reperfusion injury in rats. Furthermore, we found that the inhibition of AMPA receptor GluR2 subunit internalization may contributed to this long-term neuroprotection.     

Human spinal motoneurons express low relative abundance of GluR2 mRNA: an implication for excitotoxicity in ALS

AMPA receptor-mediated neurotoxicity is currently the most plausible hypothesis for the etiology of amyotrophic lateral sclerosis (ALS). The mechanism initiating this type of neuronal death is believed to be exaggerated Ca2+-influx through AMPA receptors, which is critically determined by the presence or absence of the glutamate receptor subunit 2 (GluR2) in the assembly. We have provided the first quantitative measurements of the expression profile of AMPA receptor subunits mRNAs in human single neurons by means of quantitative RT-PCR with a laser microdissector. Among the AMPA subunits, GluR2 shared the vast majority throughout the neuronal subsets and tissues examined. Furthermore, both the expression level and the proportion of GluR2 mRNA in motoneurons were the lowest among all neuronal subsets examined, whereas those in motoneurons of ALS did not differ from the control group, implying that selective reduction of the GluR2 subunit cannot be a mechanism of AMPA receptor-mediated neurotoxicity in ALS. However, the low relative abundance of GluR2 might provide spinal motoneurons with conditions that are easily affected by changes of AMPA receptor properties including deficient GluR2 mRNA editing in ALS.     

Extent of RNA editing of glutamate receptor subunit GluR5 in different brain regions of the rat

Summary 1. The structure and function of glutamate receptor subunits GluR2, GluR5, and GluR6 are changed by RNA editing. This reaction produces a base transition in the second transmembrane spanning region. The triplet CAG (coding for glutamine) is changed to CGG (coding for arginine). This transition has a pronounced effect on calcium fluxes through the respective ion channels, because calcium currents decrease with the rate of editing.2. In the present study the extent of RNA editing of the glutamate receptor subunit GluR5 was studied in different brain regions of control rats using a newly developed analysis system. This system is based on restriction analysis of the polymerase chain reaction (PCR) product, derived from reverse-transcribed mRNA as template, with the enzymeBbv1.Bbv1 recognizes the sequence of the nonedited receptor subunit around the edited base (sequence GCAGC) but not that of the edited subunit (sequence GCGGC; A edited to G).3. Total RNA was isolated from the cerebral cortex, striatum, hippocampus, thalamus, hypothalamus, cerebellum, pons/medulla oblongata, and white matter and reverse transcribed into cDNA. The region across the edited sequence was amplified by PCR using GluR5-specific primers and the cDNA as template. PCR products were cleaned by ethanol precipitation, incubated withBbv1, and electrophoresed on an agarose gel together with standards. Gels were photographed and the extent of GluR5 mRNA editing was quantified using an image analysis system. A calibration curve was obtained using PCR products amplified from plasmids with edited and nonedited GluR5 as inserts.4. In the brain of control rats the extent of RNA editing of the GluR5 subunit amounted to 62±6.0% of total (cortex), 43±5.3% (striatum), 52±5.3% (hippocampus), 91±6.3% (thalamus), 85±10.2% (hypothalamus), 82±6.5% (cerebellum), 88±6.8% (pons/medulla oblongata), and 41±2.7% (white matter).5. The extent of RNA editing varied, thus, considerably in different brain regions, being lowest in the white matter and striatum and highest in the thalamus and pons/medulla oblongate. RNA editing of glutamate receptor subunits may play an important role in the control of calcium fluxes through non-N-methyl-D-aspartate receptor channels in different physiological and/or pathological states of the brain.     

Regulation of the intracellular distribution, cell surface expression, and protein levels of AMPA receptor GluR2 subunits by the monocarboxylate transporter MCT2 in neuronal cells

The neuronal monocarboxylate transporter, MCT2, is not only an energy substrate carrier but it is also purported to be a binding partner for the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit. To unravel a putative role of MCT2 in the regulation of GluR2 subcellular distribution, Neuro2A cells and primary cultures of mouse cortical neurons were co-transfected with plasmids containing sequences to express the fluorescent proteins mStrawberry (mStb)-fused MCT2 and Venus-fused GluR2. Subsequently, their subcellular distribution was visualized by fluorescence microscopy. GluR2 was led to form perinuclear and dendritic clusters together with MCT2 when co-transfected in Neuro2A cells or in neurons, following the original distribution of MCT2. MCT2 co-transfection had no effect on the intracellular distribution of several other post-synaptic proteins, although it partially affected the intracellular distribution of GluR1 similarly to GluR2. Both cell surface and total protein expression levels of GluR2 were significantly reduced by co-expression with MCT2. Finally, partial perinuclear and dendritic co-localization between MCT2 and Rab8, a member of the small GTPase family involved in membrane trafficking of AMPA receptors, was also observed in co-transfected neurons. These results suggest that MCT2 could influence AMPA receptor trafficking within neurons by modulating GluR2 sorting between different subcellular compartments.     

Zebrafish TARP Cacng2 is required for the expression and normal development of AMPA receptors at excitatory synapses

Fast excitatory synaptic transmission in the CNS is mediated by the neurotransmitter glutamate, binding to and activating AMPA receptors (AMPARs). AMPARs are known to interact with auxiliary proteins that modulate their behavior. One such family of proteins is the transmembrane AMPA receptor‐related proteins, known as TARPs. Little is known about the role of TARPs during development, or about their function in non‐mammalian organisms. Here we report the presence of TARPs, specifically the prototypical TARP, stargazin, in developing zebrafish. We find that zebrafish express two forms of stargazin, Cacng2a and Cacng2b from as early as 12‐h post fertilization (hpf). Knockdown of Cacng2a and Cacng2b via splice‐blocking morpholinos resulted in embryos that exhibited deficits in C‐start escape responses, showing reduced C‐bend angles, smaller tail velocities and aberrant C‐bend turning directions. Injection of the morphants with Cacng2a or 2b mRNA rescued the morphological phenotype and the synaptic deficits. To investigate the effect of reduced Cacng2a and 2b levels on synaptic physiology, we performed whole cell patch clamp recordings of AMPA mEPSCs from zebrafish Mauthner cells. Knockdown of Cacng2a results in reduced AMPA currents and lower mEPSC frequencies, whereas knockdown of Cacng2b displayed no significant change in mEPSC amplitude or frequency. Non‐stationary fluctuation analysis confirmed a reduction in the number of active synaptic receptors in the Cacng2a but not in the Cacng2b morphants. Together, these results suggest that Cacng2a is required for normal trafficking and function of synaptic AMPARs, while Cacng2b is largely non‐functional with respect to the development of AMPA synaptic transmission. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 487–506, 2016     

选择性5-HT1A受体拮抗剂WAY-100635增加6-羟多巴胺损毁大鼠杏仁基底外侧核的神经活动

本文采用玻璃微电极细胞外记录法,观察正常大鼠和6-羟多巴胺(6-hydroxydopamine,6-OHDA)损毁黑质致密部大鼠杏仁基底外侧核(basolateral nucleus,BL)神经元电活动的变化,以及体循环给予选择性5-HT1A受体拮抗剂WAY-100635对神经元电活动的影响.结果显示,正常大鼠BL投射神经元和中间神经元的放电频率分别足(O.39±0.04)Hz和(0.83±0.16)Hz,6-OHDA损毁大鼠BL投射神经元和中间神经元的放电频率分别足(0.32±0.04)Hz和(0.53±0.12)Hz,与正常大鼠相比无显著差异.在正常大鼠,所有投射神经元呈现爆发式放电;94%的中间神经元为爆发式放电,6%为不规则放电.在6.OHDA损毁大鼠,85%的投射神经元呈现爆发式放电,15%为不规则放电;86%的中间神经元为爆发式放电,14%为不规则放电,与正常大鼠相比无显著差别.静脉给予0.1 mg/kg体重的WAY-100635不改变正常大鼠和6-OHDA损毁人鼠BL投射神经元和中间神经元的放电频率.然而,0.5 mg/kg体重的WAY-100635却显著降低正常大鼠BL投射神经元的平均放电频率(P<0.01),明显增加6-OHDA损毁大鼠BL投射神经元的平均放电频率(P<0.004).高剂量WAY-100635不影响正常大鼠和6-OHDA损毁大鼠BL中间神经元的平均放电频率.结果表明,黑质多巴胺能损毁后内在和外在的传入调节BL神经元的活动,在正常大鼠和6-OHDA损毁大鼠5-HT1A 受体调节投射神经元的活动,并且在6-OHDA损毁大鼠WAY-100635诱发投射神经元平均放电频率增加.结果提示,5-HT1A 受体在帕金森病情感性症状的产生中起重要作用.     

Role of AMPA receptor trafficking in NMDA receptor-dependent synaptic plasticity in the rat lateral amygdala

Stimulated exocytosis and endocytosis of post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors (AMPARs) have been proposed as primary mechanisms for the expression of hippocampal CA1 long-term potentiation (LTP) and long-term depression (LTD), respectively. LTP and LTD, the two most well characterized forms of synaptic plasticity, are thought to be important for learning and memory in behaving animals. Both LTP and LTD can also be induced in the lateral amygdala (LA), a critical structure involved in fear conditioning. However, the role of AMPAR trafficking in the expression of either LTP or LTD in this structure remains unclear. In this study, we show that NMDA receptor-dependent LTP and LTD can be reliably induced at the synapses of the auditory thalamic inputs to the LA in brain slices. The expression of LTP was prevented by post-synaptic blockade of vesicle-mediated exocytosis with application of a light chain of Clostridium tetanus neurotoxin and was associated with increased cell-surface AMPAR expression. In contrast, the expression of LTD was prevented by post-synaptic application of a glutamate receptor 2-derived interference peptide, which specifically blocks the stimulated clathrin-dependent endocytosis of AMPARs, and was correlated with a reduction in plasma membrane-surface expression of AMPARs. These results strongly suggest that regulated trafficking of post-synaptic AMPARs is also involved in the expression of LTP and LTD in the LA.     

Serotonin 5-HT receptor blockade enhances Ca(2+)/calmodulin-dependent protein kinase II function and membrane expression of AMPA receptor subunits in the rat hippocampus: implications for memory formation

Stimulation of hippocampal 5-HT(1A) receptors impairs memory retention. The highly selective 5-HT(1A) antagonist, WAY-100635, prevents the cognitive deficits induced not only by 5-HT(1A) stimulation but also by cholinergic or NMDA receptor blockade. On this basis, the effects of WAY-100635 on molecular events associated with memory storage were explored. In rat hippocampus, WAY-100635 produced a rapid increase in phosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and in Ca(2+)-independent CaMKII and protein kinase A (PKA) enzyme activity. This increase was followed a few hours later by an enhanced membrane expression of AMPA receptor subunits, especially of the GluR1 subunit phosphorylated at the CaMKII site, pGluR1(Ser831). The same qualitative effects were found with the weaker 5-HT(1A) antagonist NAN-190. The effects of both antagonists were no longer apparent in rats with a previous 5-HT depletion induced by the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA), suggesting that 5-HT(1A) receptor blockade removes the tonic inhibition of 5-HT through 5-HT(1A) receptor stimulation on excitatory hippocampal neurons, with the consequent increase in PKA activity. In addition, administration of WAY-100635 potentiated the learning-specific increase in the hippocampus of phospho-CaMKII, Ca(2+)-independent CaMKII activity, as well as the phosphorylation of either the CaMKII or the PKA site on the AMPA receptor GluR1 subunit. This study suggests that blockade of hippocampal 5-HT(1A) receptors favours molecular events critically involved in memory formation, and provides an in vivo molecular basis for the proposed utility of 5-HT(1A) receptor antagonists in the treatment of cognitive disorders.     

Identification of NADPH oxidase as a key mediator in the post‐ischemia‐induced sequestration and degradation of the GluA2 AMPA receptor subunit

A hallmark of ischemic/reperfusion injury is a change in subunit composition of synaptic 2‐amino‐3‐(3‐hydroxy‐5‐methylisoazol‐4‐yl)propionic acid receptors (AMPARs). This change in AMPAR subunit composition leads to an increase in surface expression of GluA2‐lacking Ca²⁺/Zn²⁺ permeable AMPARs. These GluA2‐lacking AMPARs play a key role in promoting delayed neuronal death following ischemic injury. At present, the mechanism(s) responsible for the ischemia/reperfusion‐induced subunit composition switch and degradation of the GluA2 subunit remain unclear. In this study, we investigated the role of NADPH oxidase, and its importance in mediating endocytosis and subsequent degradation of the GluA2 AMPAR subunit in adult rat hippocampal slices subjected to oxygen–glucose deprivation/reperfusion (OGD/R) injury. In hippocampal slices pre‐treated with the NADPH oxidase inhibitor apocynin attenuated OGD/R‐mediated sequestration of GluA2 and GluA1 as well as prevent the degradation of GluA2. We provide compelling evidence that NADPH oxidase mediated sequestration of GluA1‐ and GluA2‐ involved activation of p38 MAPK. Furthermore, we demonstrate that inhibition of NADPH oxidase blunts the OGD/R‐induced association of GluA2 with protein interacting with C kinase‐1. In summary, this study identifies a novel mechanism that may underlie the ischemia/reperfusion‐induced AMPAR subunit composition switch and a potential therapeutic target.

     

N-Linked Glycosylation of the α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate(AMPA)-Selective Glutamate Receptor Channel α2 Subunit Is Essential for the Acquisition of Ligand-Binding Activity

Abstract: The N-linked glycosylation of the α2 subunit of the mouse α-amino-3-hydroxy-5-methylisoxazole-4-propionate(AMPA)-selective glutamate receptor (GluR) channel was characterized. The receptor subunit protein has five putative N -glycosylation sites. The recombinant receptor proteins were identified by [³⁵S]methionine/[³⁵S]cysteine metabolic labeling, western blot analysis, immunocytochemical detection, and [³H]AMPA binding experiments when expressed in insect Spodoptera frugiperda cells using a baculovirus system. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of ∼102 kDa and a minor species of ∼98 kDa, correspond to glycosylated and unglycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. Immunofluorescence staining of tunicamycin-treated cells expressing only the unglycosylated form differed little from that of tunicamycin-nontreated cells expressing both glycosylated and unglycosylated forms. The lack of AMPA-binding activity of the unglycosylated form expressed in the presence of tunicamycin suggested that N -glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. These results demonstrate that occupancy of at least one N -glycosylation site is required for the formation and maintenance of the GluRα2 subunit protein in an active conformation for ligand binding. Possible roles of N -glycosylation of GluRα2 subunit protein are discussed.     

Transient expression of NMDA receptor subunit NR2B in the developing rat heart

NMDA receptors represent a subtype of the ionotropic glutamate receptor family, comprising three classes of subunits (NR1, NR2A-D, NR3), which exhibit distinct patterns of regional and developmental expression in the CNS. Recently, some NMDA receptor subunits have also been described in adult extraneuronal tissues and keratinocytes. However, their developmental expression patterns are currently unknown. With use of RT-PCR and western blot analysis, the expression of NMDA receptor subunit NR2B was investigated in the developing rat heart. NR2B mRNA and protein were detected in heart tissue of rats from embryonic day 14 until postnatal day 21 but disappeared 10 weeks after birth. In contrast, no NMDA receptor subunit NR1, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit GluR2, or anchoring postsynaptic density protein-95 could be detected in rat heart at any developmental stage. Confocal microscopy of cultured cardiac myocytes (CMs) from neonatal rats revealed distinct NR2B staining mainly of intracellular structures. However, no functional NMDA receptor could be detected on CMs by whole-cell recordings. In conclusion, high concentrations of NR2B protein can be detected in early rat heart development, but its function still remains elusive.     

Adenosine A2A receptors inhibit the conductance of NMDA receptor channels in rat neostriatal neurons

Summary Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70–80%), NMDA-induced inward currents were inhibited by the adenosine AZA receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the AZA receptor selective antagonist 8-(3chlorostyryl) caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP--S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14–24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca²⁺ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, AZA receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca²⁺ pathway rather than the adenylate cyclase/PKA pathway.     

Membrane depolarization regulates AMPA receptor subunit expression in cerebellar granule cells in culture

The physiological responses of AMPA receptors can be modulated through the differential expression of their subunits and by modifying their number at the cell surface. Here we have studied the expression of AMPA receptor subunits (GluR1-4) mRNAs in cerebellar granule cells grown in depolarizing (25 mM K⁺) medium, and we have evaluated the effect of decreasing the [K⁺] in the culture medium for 24 h on both GluR1-4 expression (both mRNA and protein) and their presence at the plasma membrane. The expression of the four AMPAR subunits increases as the [K⁺] decreases, although the increase in GluR2 and GluR3 was only observed in the cell soma but not in the dendrites. Calcium entry through L-type calcium channel and CaMKIV activation are responsible for the reduction in the expression of AMPA receptor subunits in cells cultured in depolarizing conditions. Indeed, prolonged reduction of extracellular [K⁺] or blockage of L-type calcium channels enhanced both the surface insertion of the four AMPAR subunits and the AMPA response measured through intracellular calcium increase. These findings reveal a balanced increase in functional AMPA receptors at the surface of cells that can trigger strong increases in calcium in response to the persistent reduction of calcium entry.     

Cloning and expression of soluble recombinant protein comprising the extracellular domain of the human type I interferon receptor 2c subunit (IFNAR-2c) in E. coli

An optimized procedure was developed for production of the extracellular domain encoding amino acids 1–243 of the human type I interferon receptor 2c subunit (IFNAR-2c) as a fusion protein with glutathione S-transferase (GST-IFNAR2cEC) in E. coli to obtain active, soluble protein. Induction of protein expression at 37 °C resulted in a formation of inclusion body. Co-expression with bacterial chaperones, GroEL and GroES, failed to support the folding of GST-IFNAR2cEc under IPTG induction at 37 °C. Expression induced at 25 °C decreased the inclusion body formation up to 62% and cell disruption by a French press provided higher recovery of the recombinant protein than cell disruption by sonication.     

Activation of GluR6-containing kainate receptors induces ubiquitin-dependent Bcl-2 degradation via denitrosylation in the rat hippocampus after kainate treatment

We previously showed that Bcl-2 (B-cell lymphoma 2) is down-regulated in a kainate (KA)-induced rat epileptic seizure model. The underlying mechanism had remained largely unknown, but we here report for the first time that denitrosylation and ubiquitination are involved. Our results show that the S-nitrosylation levels of Bcl-2 are down-regulated after KA injection and that the GluR6 (glutamate receptor 6) antagonist NS102 can inhibit the denitrosylation of Bcl-2. Moreover, the ubiquitin-dependent degradation of Bcl-2 was found to be promoted after KA treatment, which could be suppressed by the proteasome inhibitor MG132 and the NO donors, sodium nitroprusside and S-nitrosoglutathione. In addition, experiments based on siRNA transfections were performed in the human SH-SY5Y neuroblastoma cell line to verify that the stability of Bcl-2 is causal to neuronal survival. At the same time, it was found that the exogenous NO donor GSNO could protect neurons when Bcl-2 is targeted. Subsequently, these mechanisms were morphologically validated by immunohistochemistry, cresyl violet staining, and in situ TUNEL staining to analyze the expression of Bcl-2 as well as the survival of CA1 and CA3/DG pyramidal neurons. NS102, GSNO, sodium nitroprusside, and MG132 contribute to the survival of CA1 and CA3/DG pyramidal neurons by attenuating Bcl-2 denitrosylation. Taken together, our data reveal that Bcl-2 ubiquitin-dependent degradation is induced by Bcl-2 denitrosylation during neuronal apoptosis after KA treatment.     

ASF/SF2 and SC35 regulate the glutamate receptor subunit 2 alternative flip/flop splicing

The properties of the glutamate receptor subunits 1-4 (GluR1-4) are influenced by the alternative splicing of two homologous and mutually exclusive exons flip and flop. The flip form is most abundant during early development, while the flop form is dominant in adults. From transfections with a GluR2 mini-gene we show that flip is the preferred splice form in all tested cell lines, but coexpression of the SR-proteins ASF/SF2 and SC35 increases the flop to flip splice ratio. The increased flop incorporation depends on ASF/SF2- and SC35-dependent enhancer elements located in the flop exon, which stimulate the splicing between the flop exon and the preceding exon 13.