Orexin-A increases cell surface expression of AMPA receptors in the striatum

Accumulating evidence suggests that orexin signaling is involved in reward and motivation circuit functions. However, the underlying mechanisms are not yet fully understood. Here, we show that orexin-A potentiates AMPAR-mediated synaptic transmission in the striatum, possibly by regulating the surface expression of AMPARs. Primary culture of striatal neurons revealed increased surface expression of AMPARs following orexin-A treatment. The increase in surface-expressed AMPARs induced by orexin-A treatment was dependent on both ERK activation and the presence of extracellular Ca²⁺. In the corticostriatal synapses of rat brain slices, orexin-A bath-application caused a delayed increase in the AMPAR/NMDAR EPSC ratio, suggesting that orexin-A sets in motion a series of events that lead to functional alterations in the striatal circuits. Our findings provide a potential link between the activation of orexin signaling in the striatum in response to addictive substances and neural adaptations in the reward circuitry that may mediate the long-lasting addiction-related behaviors.     

Subunit-dependent and subunit-independent rules of AMPA receptor trafficking during chemical long-term depression in hippocampal neurons

Long-term potentiation (LTP) and long-term depression (LTD) of excitatory neurotransmission are believed to be the neuronal basis of learning and memory. Both processes are primarily mediated by neuronal activity–induced transport of postsynaptic AMPA-type glutamate receptors (AMPARs). While AMPAR subunits and their specific phosphorylation sites mediate differential AMPAR trafficking, LTP and LTD could also occur in a subunit-independent manner. Thus, it remains unclear whether and how certain AMPAR subunits with phosphorylation sites are preferentially recruited to or removed from synapses during LTP and LTD. Using immunoblot and immunocytochemical analysis, we show that phosphomimetic mutations of the membrane-proximal region (MPR) in GluA1 AMPAR subunits affect the subunit-dependent endosomal transport of AMPARs during chemical LTD. AP-2 and AP-3, adaptor protein complexes necessary for clathrin-mediated endocytosis and late endosomal/lysosomal trafficking, respectively, are reported to be recruited to AMPARs by binding to the AMPAR auxiliary subunit, stargazin (STG), in an AMPAR subunit–independent manner. However, the association of AP-3, but not AP-2, with STG was indirectly inhibited by the phosphomimetic mutation in the MPR of GluA1. Thus, although AMPARs containing the phosphomimetic mutation at the MPR of GluA1 were endocytosed by a chemical LTD-inducing stimulus, they were quickly recycled back to the cell surface in hippocampal neurons. These results could explain how the phosphorylation status of GluA1-MPR plays a dominant role in subunit-independent STG-mediated AMPAR trafficking during LTD.     

Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain

The ligand-binding domains of AMPA receptor subunits carry two conserved N-glycosylation sites. In order to gain insight into the functional role of the corresponding N-glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand-binding characteristics, structural stability, cell-surface expression, and channel properties of homomeric GluR-D (GluR4) receptor and its soluble ligand-binding domain (S1S2). GluR-D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand-binding properties were observed between the 'wild-type' S1S2 and non-glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N-glycosylation. Purified glycosylated and non-glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full-length homomeric GluR-D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild-type GluR-D. In outside-out patches, GluR-D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand-binding domain glycans do not play any major role in receptor-ligand interactions, do not impart a stabilizing effect on the ligand-binding domain, and are not critical for the formation and surface localization of homomeric GluR-D AMPA receptors in HEK293 cells.     

Impaired AMPA receptor trafficking by a double knockout of zebrafish olfactomedin1a/b

The olfm1a and olfm1b genes in zebrafish encode conserved secreted glycoproteins. These genes are preferentially expressed in the brain and retina starting from 16 h post‐fertilization until adulthood. Functions of the Olfm1 gene is still unclear. Here, we produced and analyzed a null zebrafish mutant of both olfm1a and olfm1b genes (olfm1 null). olfm1 null fish were born at a normal Mendelian ratio and showed normal body shape and fertility as well as no visible defects from larval stages to adult. Olfm1 proteins were preferentially localized in the synaptosomes of the adult brain. Olfm1 co‐immunoprecipitated with GluR2 and soluble NSF attachment protein receptor complexes indicating participation of Olfm1 in both pre‐ and post‐synaptic events. Phosphorylation of GluR2 was not changed while palmitoylation of GluR2 was decreased in the brain synaptosomal membrane fraction of olfm1 null compared with wt fish. The levels of GluR2, SNAP25, flotillin1, and VAMP2 were markedly reduced in the synaptic microdomain of olfm1 null brain compared with wt. The internalization of GluR2 in retinal cells and the localization of VAMP2 in brain synaptosome were modified by olfm1 null mutation. This indicates that Olfm1 may regulate receptor trafficking from the intracellular compartments to the synaptic membrane microdomain, partly through the alteration of post‐translational GluR2 modifications such as palmitoylation. Olfm1 may be considered a novel regulator of the composition and function of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor complex.

     

Diaphanous-1 affects the nanoscale clustering and lateral diffusion of receptor for advanced glycation endproducts (RAGE)

The interactions between the cytoplasmic protein diaphanous-1 (Diaph1) and the receptor for advanced glycation endproducts (RAGE) drive the negative consequences of RAGE signaling in several disease processes. Reported in this work is how Diaph1 affects the nanoscale clustering and diffusion of RAGE measured using super-resolution stochastic optical reconstruction microscopy (STORM) and single particle tracking (SPT). Altering the Diaph1 binding site has a different impact on RAGE diffusion compared to when Diaph1 expression is reduced in HEK293 cells. In cells with reduced Diaph1 expression (RAGE-Diaph1^?/?), the average RAGE diffusion coefficient is increased by 35%. RAGE diffusion is known to be influenced by the dynamics of the actin cytoskeleton. Actin labeling shows that a reduced Diaph1 expression leads to cells with reduced filopodia density and length. In contrast, when two RAGE amino acids that interact with Diaph1 are mutated (RAGE_RQ/AA), the average RAGE diffusion coefficient is decreased by 16%. Since RAGE diffusion is slowed when the interaction between Diaph1 and RAGE is disrupted, the interaction of the two proteins results in faster RAGE diffusion. In both RAGE_RQ/AA and RAGE-Diaph1^?/? cells the number and size of RAGE clusters are decreased compared to cells expressing RAGE and native concentrations of Diaph1. This work shows that Diaph1 has a role in affecting RAGE clusters and diffusion.     

Differential regulation of dendrite complexity by AMPA receptor subunits GluR1 and GluR2 in motor neurons

Activity-dependent developmental mechanisms in many regions of the central nervous system are thought to be responsible for shaping dendritic architecture and connectivity, although the molecular mechanisms underlying these events remain obscure. Since AMPA glutamate receptors are developmentally regulated in spinal motor neurons, we have investigated the role of activation of AMPA receptors in dendritic outgrowth of spinal motor neurons by overexpression of two subunits, GluR1 and GluR2, and find that dendrite outgrowth is differentially controlled by expression of these subunits. Overexpression of GluR1 was associated with greater numbers of filopodia, and an increase in the length and complexity of dendritic arbor. In contrast, GluR2 expression did not alter dendritic complexity, but was associated with a moderate increase in length of arbor, and decreased numbers of filopodia. Neither GluR1 nor GluR2 had any effect on the motility of filopodia. In addition, GluR1 but not GluR2 expression increased the density of dendritic puncta incorporating a GFP-labeled PSD95, suggesting that GluR1 may mediate its effect in part by augmenting the number of excitatory synapses within motor neuron dendrites. Together these results suggest that in spinal motor neurons, AMPA receptors composed of GluR1 subunits may facilitate neurotrophic mechanisms in these neurons, permitting sustained dendrite outgrowth and synaptogenesis, whereas expression of AMPA receptors containing GluR2 acts to preserve existing dendritic arbor. Thus, the observed downregulation of GluR1 in motor neurons during postnatal development may limit the formation of new dendrite segments and synapses, promoting stabilized synaptic connectivity.     

Modeling of slow glutamate diffusion and AMPA receptor activation in the cerebellar glomerulus

Synaptic conductances are influenced markedly by the geometry of the space surrounding the synapse since the transient glutamate concentration in the synaptic cleft is determined by this geometry. Our paper is an attempt to understand the reasons for slow glutamate diffusion in the cerebellar glomerulus, a structure situated around the enlarged mossy fiber terminal in the cerebellum and surrounded by a glial sheath. For this purpose, analytical expressions for glutamate diffusion in the glomerulus were considered in models with two-, three-, and fractional two-three-dimensional (2D-3D) geometry with an absorbing boundary. The time course of average glutamate concentration in the synaptic cleft of the mossy fiber-granule cell connection was calculated for both direct release of glutamate from the same synaptic unit, and for cumulative spillover of glutamate from neighboring release sites. Several kinetic schemes were examined, and the parameters of the diffusion models were estimated by identifying theoretical activation of AMPA receptors with direct release and spillover components of published experimental AMPA receptor-mediated EPSCs. For model selection, the correspondence of simulated paired-pulse ratio and EPSC increase after prevention of desensitization to experimental values were also taken into consideration. Our results suggest at least a 7- to 10-fold lower apparent diffusion coefficient of glutamate in the porous medium of the glomerulus than in water. The modeling of glutamate diffusion in the 2D-3D geometry gives the best fit of experimental EPSCs. We show that it could be only partly explained by normal diffusion of glutamate in the complex geometry of the glomerulus. We assume that anomalous diffusion of glutamate occurs in the glomerulus. A good match of experimental estimations and theoretical parameters, obtained in the simulations that use an approximation of anomalous diffusion by a solution for fractional Brownian motion, confirms our assumption.     

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.     

Developmental increase in the amount of rapsyn per acetylcholine receptor promotes postsynaptic receptor packing and stability

Neuromuscular synaptic transmission depends upon tight packing of acetylcholine receptors (AChRs) into postsynaptic AChR aggregates, but not all postsynaptic AChRs are aggregated. Here we describe a new confocal Fluorescence Resonance Energy Transfer (FRET) assay for semi-quantitative comparison of the degree to which AChRs are aggregated at synapses. During the first month of postnatal life the mouse tibialis anterior muscle showed increases both in the number of postsynaptic AChRs and the efficiency with which AChR was aggregated (by FRET). There was a concurrent two-fold increase in immunofluorescent labeling for the AChR-associated cytoplasmic protein, rapsyn. When 1-month old muscle was denervated, postsynaptic rapsyn immunostaining was reduced, as was the efficiency of AChR aggregation. In vivo electroporation of rapsyn-EGFP into muscle fibers increased postsynaptic rapsyn levels. Those synapses with higher ratios of rapsyn-EGFP to AChR displayed a slower metabolic turnover of AChR. Conversely, the reduction of postsynaptic rapsyn after denervation was accompanied by an acceleration of AChR turnover. Thus, a developmental increase in the amount of rapsyn targeted to the postsynaptic membrane may drive enhanced postsynaptic AChRs aggregation and AChR stability within the postsynaptic membrane.     

Emerging role of AMPA receptor subunit GluA1 in synaptic plasticity: Implications for Alzheimer's disease

It is well established that GluA1 mediated synaptic plasticity plays a central role in the early development of AD. The complex cellular and molecular mechanisms that enable GluA1‐related synaptic regulation remain to fully understood. Particularly, understanding the mechanisms that disrupt GluA1 related synaptic plasticity is central to the development of disease‐modifying therapies which are sorely needed as the incidence of AD rises. We surmise that the published evidence establishes deficits in synaptic plasticity as a central factor of AD aetiology. We additionally highlight potential therapeutic strategies for the treatment of AD, and we delve into the roles of GluA1 in learning and memory. Particularly, we review the current understanding of the molecular interactions that confer the actions of this ubiquitous excitatory receptor subunit including post‐translational modification and accessory protein recruitment of the GluA1 subunit. These are proposed to regulate receptor trafficking, recycling, channel conductance and synaptic transmission and plasticity.     

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.     

Structure based evolution of a novel series of positive modulators of the AMPA receptor

Starting from compound 1, we utilized biostructural data to successfully evolve an existing series into a new chemotype with a promising overall profile, exemplified by 19.     

in Silico investigation of the structural requirements for the AMPA receptor antagonism by quinoxaline derivatives

Glutamate receptors have been implicated in various neurological disorders and their antagonism offers a suitable approach for the treatment of such disorders. The field of drug design and discovery aims to find best medicines to prevent, treat and cure diseases quickly and efficiently. In this regard, computational tools have helped medicinal chemists modify and optimize molecules to potent drug candidates with better pharmacokinetic profiles, and guiding biologists and pharmacologists to explore new disease genes as well as novel drug targets. In the present study, to understand the structural requirements for AMPA receptor antagonism, molecular docking study was performed on 41 structurally diverse antagonists based on quinoxaline nucleus. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results obtained signify that the molecular docking approach is reliable and produces a good correlation coefficient (r² = 0.6) between experimental and docking predicted AMPA receptor antagonistic activity. The aromatic moiety of quinoxaline core has been proved to be vital for hydrophobic contacts exhibiting - interactions in docked conformations. However, polar moieties such as carboxylic group and 1,2,4-triazole moieties were noted to be sites for hydrophilic interactions in terms of hydrogen bonding with the receptor. These analyses can be exploited to design and develop novel AMPA receptor antagonists for the treatment of different neurological disorders.     

Facilitation of zinc influx via AMPA/kainate receptor activation in the hippocampus

Glutamate and zinc is co-released by excitation of hippocampal mossy fibers and both concentrations are increased in the extracellular compartment. In a novel environment, however, extracellular zinc is persistently decreased in spite of the increase in extracellular glutamate. The mechanism of the decrease in extracellular zinc was studied in the present paper. In rats subjected to the novelty stress under hippocampal perfusion, the differential changes in extracellular glutamate and zinc were abolished in the presence of 1 μM tetrodotoxin (TTX), a sodium channel blocker, which reduced exploratory behavior. When the hippocampus was perfused with corticosterone (50 ng/ml), extracellular zinc was increased. These results suggest that glutamatergic neuron activation elicited by novelty stress is involved in the decrease in extracellular zinc and that glucocorticoid is not a trigger for its decrease. The differential changes in extracellular glutamate and zinc was induced by electrical stimulation to analyze the decrease in extracellular zinc; the differential changes were elicited by delivery of tetanic stimuli (100 Hz for 1 s, 5 min intervals, three times) to the hippocampus instead of the novelty stress, as reported previously. The changes elicited by tetanic stimulation were abolished in the presence of 10 μM CNQX, an AMPA/kainate receptor antagonist. In a hippocampal slice double-labeled with zinc and calcium indicators, furthermore, CNQX inhibited the increase in intracellular zinc levels in mossy fiber synapses after delivery of tetanic stimuli (100 Hz for 5 s) to dentate granule cells. The in vivo and in vitro experiments suggest that AMPA/kainate receptor activation is involved in zinc influx into hippocampal cells, followed by the decrease in extracellular zinc. It is likely that zinc influx is persistently facilitated via stress-induced glutamatergic neuron activation.     

Disease-causing mutation in PKR2 receptor reveals a critical role of positive charges in the second intracellular loop for G-protein coupling and receptor trafficking

Prokineticins are a pair of signal factors involved in many physiological processes by binding to two closely related G-protein-coupled receptors, PKR1 and PKR2. Recently, mutations in prokineticin 2 (PK2) and PKR2 are found to be associated with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, disorders characterized by delayed puberty and infertility. However, little is known how PKRs interact and activate G-proteins to elicit signal transduction. In the present study, we took advantage of one disease-associated mutation (R164Q) located in the second intracellular (IL2) loop of PKR2, to investigate the role of IL2 loop in the cell signaling, G-protein binding and receptor trafficking. R164Q mutant PKR2 showed normal cell surface expression and ligand binding capacity. However, the PKR2 signaling was abolished by R164Q mutation. We demonstrated that R164Q mutation disrupted the interaction of IL2 loop to the Gα(q), Gα(i), and Gα(16)-proteins. A positive-charged amino acid at this position is required for proper function, and the signaling efficacy and potency depend on the net amount of positive charges. We also demonstrated that the interactive partner of Arg-164 may localize in the C-terminal five residues of Gα(q)-protein. A series of mutation analysis indicated that the basic amino acids at the C terminus of IL2 loop may function cooperatively in GPCRs. Furthermore, R164Q mutation also results in minimal ligand-induced endocytosis of PKR2. As many GPCRs share structural homology in the C terminus of IL2 loop, our findings may have general application in understanding structure and function of GPCRs.     

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.

     

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4)

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.     

Two forms of long-term depression in a polysynaptic pathway in the leech CNS: one NMDA receptor-dependent and the other cannabinoid-dependent

Although long-term depression (LTD) is a well-studied form of synaptic plasticity, it is clear that multiple cellular mechanisms are involved in its induction. In the leech, LTD is observed in a polysynaptic connection between touch mechanosensory neurons (T cells) and the S interneuron following low frequency stimulation. LTD elicited by 450 s low frequency stimulation was blocked by N-methyl-d-aspartic acid (NMDA) receptor antagonists. However, LTD elicited by 900 s low frequency stimulation was insensitive to NMDA receptor antagonists and was instead dependent on cannabinoid signaling. This LTD was blocked by both a cannabinoid receptor antagonist and by inhibition of diacylglycerol lipase, which is necessary for the synthesis of the cannabinoid transmitter 2-arachidonyl glycerol (2-AG). Bath application of 2-AG or the cannabinoid receptor agonist CP55 940 also induced LTD at this synapse. These results indicate that two forms of LTD coexist at the leech T-to-S polysynaptic pathway: one that is NMDA receptor-dependent and another that is cannabinoid-dependent and that activation of either form of LTD is dependent on the level of activity in this circuit. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.