Metabotropic glutamate receptor 5 and calcium signaling in retinal amacrine cells

To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)-dependent Ca(2+) elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both G(q) and G(s). Selective activation of mGluR5 stimulated Ca(2+) elevations that varied in waveform from cell to cell. Experiments using high external K(+) revealed that the mGluR5-dependent Ca(2+) elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca(2+) -free external solution demonstrated that the variability in the time course of mGluR5-dependent Ca(2+) elevations is largely due to the influx of extracellular Ca(2+). The sensitivity of the initial phase of the Ca(2+) elevation to thapsigargin indicates that this phase of the response is due to the release of Ca(2+) from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5-mediated Ca(2+) elevations are dependent upon the activation of phospholipase C. We rule out a role for L-type Ca(2+) channels and cAMP-gated channels as pathways for Ca(2+) entry, but provide evidence of transient receptor potential (TRP) channel-like immunoreactivity, suggesting that Ca(2+) influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C-dependent Ca(2+) release and Ca(2+) influx, possibly through TRP channels.     

Permissive role of adenosine A2A receptors on metabotropic glutamate receptor 5 (mGluR5)-mediated effects in the striatum

The metabotropic glutamate receptors 5 (mGlu5Rs) and the adenosine A2A receptors (A2ARs) have been reported to functionally interact in the striatum. The aim of the present work was to verify the hypothesis that the state of activation of A2A Rs could influence mGlu5R-mediated effects in the striatum. In electrophysiological experiments (extracellular recording in rat corticostriatal slices), the ability of the selective mGlu5R agonist CHPG to potentiate the reduction of the field potential amplitude induced by NMDA was prevented not only by the selective mGlu5R antagonist MPEP, but also by the selective A2AR antagonist ZM 241385. Analogously, the application of CHPG potentiated NMDA-induced toxicity (measured by LDH release) in cultured striatal neurons, an effect that was abolished by both MPEP and ZM 241385. Finally, the A2AR agonist CGS 21680 potentiated CHGP effects, an action that was reproduced and abolished, respectively, by forskolin (an activator of the cAMP/protein kinase A, PKA, pathway) and KT 5720 (a PKA inhibitor). The results indicate that A2ARs exert a permissive role on mGlu5R-induced effects in the striatum. Such an interaction may represent an additional target for the development of therapeutic strategies towards striatal disorders.     

The P2X7 receptor as a route for non‐exocytotic glutamate release: dependence on the carboxyl tail

P2X7 receptors trigger Ca²⁺‐dependent exocytotic glutamate release, but also function as a route for non‐exocytotic glutamate release from neurons or astrocytes. To gain an insight into the mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full‐length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18‐amino acid cysteine‐rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non‐competitive way by extracellular Mg²⁺, did not require the recruitment of pore‐forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine‐rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca²⁺ elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca²⁺‐dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non‐exocytotic glutamate efflux.     

Differential regulation of metabotropic glutamate receptor 5-mediated phosphoinositide hydrolysis and extracellular signal-regulated kinase responses by protein kinase C in cultured astrocytes

The metabotropic glutamate receptor 5 (mGluR5) exhibits a rapid loss of receptor responsiveness to prolonged or repeated agonist exposure. This receptor desensitization has been seen in a variety of native and recombinant systems, and is thought to result from receptor-mediated, protein kinase C (PKC)-dependent phosphorylation of the receptor, uncoupling it from the G protein in a negative feedback regulation. We have investigated the rapid PKC-mediated desensitization of mGluR5 in cortical cultured astrocytes by measuring downstream signals from activation of mGluR5. These include activation of phosphoinositide (PI) hydrolysis, intracellular calcium transients, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation. We present evidence that PKC plays an important role in rapid desensitization of PI hydrolysis and calcium signaling, but not in ERK2 phosphorylation. This differential regulation of mGluR5-mediated responses suggests divergent signaling and regulatory pathways which may be important mechanisms for dynamic integration of signal cascades.     

Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long‐QT syndrome

Patient‐specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long‐QT syndromes (LQTS). To study the LQT2‐associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (I_Kr) conducted by the HERG channel and the action potential (AP) duration in iPSC‐derived cardiomyocytes (CMs). Introduction of the same mutation reduced I_Kr and prolonged the AP duration in hESC‐derived CMs. Further characterization of N996I‐HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.     

Comparison of human-induced pluripotent stem cells and mesenchymal stem cell differentiation potential to insulin producing cells in 2D and 3D culture systems in vitro

Diabetes is one of the most common diseases in the world that is chronic, progressive, and costly, and causes many complications. Common drug therapies are not able to cure it, and pancreas transplantation is not responsive to the high number of patients. The production of the insulin producing cells (IPCs) from the stem cells in the laboratory and their transplantation to the patient's body is one of the most promising new approaches. In this study, the differentiation potential of the induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) into IPCs was compared to each other while cultured on poly(lactic-co-glycolic) acid (PLGA)/polyethylene glycol (PEG) nanofibrous scaffold as a 3D substrate and tissue culture polystyrene (TCPS) as a 2D substrate. Although the expression level of the insulin, Glut2 and pdx-1 genes in stem cells cultured on 3D substrate was significantly higher than the stem cells cultured on 2D substrate, the highest expression level of these genes was detected in the iPSCs cultured on PLGA-PEG. Insulin and C-peptide secretions from differentiated cells were also investigated and the results showed that secretions in cultured iPSCs on the PLGA-PEG were significantly higher than cultured iPSCs on the TCPS and cultured MSCs on both PLGA-PEG and TCPS. In addition, insulin protein was also expressed in the cultured iPSCs on the PLGA-PEG significantly higher than cultured MSCs on the PLGA-PEG. It can be concluded that differentiation potential of iPSCs into IPCs is significantly higher than human MSCs at both 2D and 3D culture systems.     

Application of Human‐Induced Pluripotent Stem Cells (hiPSCs) to Study Synaptopathy of Neurodevelopmental Disorders

Synapses are the basic structural and functional units for information processing and storage in the brain. Their diverse properties and functions ultimately underlie the complexity of human behavior. Proper development and maintenance of synapses are essential for normal functioning of the nervous system. Disruption in synaptogenesis and the consequent alteration in synaptic function have been strongly implicated to cause neurodevelopmental disorders such as autism spectrum disorders (ASDs) and schizophrenia (SCZ). The introduction of human‐induced pluripotent stem cells (hiPSCs) provides a new path to elucidate disease mechanisms and potential therapies. In this review, we will discuss the advantages and limitations of using hiPSC‐derived neurons to study synaptic disorders. Many mutations in genes encoding for proteins that regulate synaptogenesis have been identified in patients with ASDs and SCZ. We use Methyl‐CpG binding protein 2 (MECP2), SH3 and multiple ankyrin repeat domains 3 (SHANK3) and Disrupted in schizophrenia 1 (DISC1) as examples to illustrate the promise of using hiPSCs as cellular models to elucidate the mechanisms underlying disease‐related synaptopathy.     

Activation of P2X(7) receptors decreases glutamate uptake and glutamine synthetase activity in RBA-2 astrocytes via distinct mechanisms

Glutamate clearance by astrocytes is critical for controlling excitatory neurotransmission and ATP is an important mediator for neuron-astrocyte interaction. However, the effect of ATP on glutamate clearance has never been examined. Here we report that treatment of RBA-2 cells, a type-2-like astrocyte cell line, with ATP and the P2X(7) receptor selective agonist 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) decreased the Na+-dependent [3H]glutamate uptake within minutes. Mechanistic studies revealed that the decreases were augmented by removal of extracellular Mg2+ or Ca2+, and was restored by P2X7 selective antagonist , periodate-oxidized 2',3'-dialdehyde ATP (oATP), indicating that the decreases were mediated through P2X(7) receptors. Furthermore, stimulation of P2X7 receptors for 2 h inhibited both activity and protein expression of glutamine synthetase (GS), and oATP abolished the inhibition. In addition, removal of extracellular Ca(2+) and inhibition of protein kinase C (PKC) restored the ATP-decreased GS expression but failed to restore the P2X(7)-decreased [3H]glutamate uptake. Therefore, P2X7-mediated intracellular signals play a role in the down-regulation of GS activity/expression. Activation of P2X7 receptors stimulated increases in intracellular Na+ concentration ([Na+](i)) suggesting that the P2X(7)-induced increases in [Na+](i) may affect the local Na+ gradient and decrease the Na+-dependent [3H]glutamate uptake. These findings demonstrate that the P2X7-mediated decreases in glutamate uptake and glutamine synthesis were mediated through distinct mechanisms in these cells.     

Sustained activation of metabotropic glutamate receptor 5 and protein tyrosine phosphatases mediate the expression of (S)-3,5-dihydroxyphenylglycine-induced long-term depression in the hippocampal CA1 region

Previous studies have shown that brief application of group I metabotropic glutamate receptor (mGluR) agonist (S)-3, 5-dihydroxyphenylglycine (DHPG) to hippocampal slices can induce a chemical form of long-term depression (DHPG-LTD) in the hippocampal CA1 region; however, the expression mechanisms of this LTD remain unclear. We show here that the expression of DHPG-LTD can be specifically reversed by application of the broad-spectrum mGluR antagonists, (S)-alpha-methyl-4-carboxyphenylglycine (MCPG) and LY341495, and mGluR5 antagonist, 2-methyl-6-(phenylethyl)pyridine, but not by NMDA receptor antagonist, D-2-amino-5-phosphonopentanoic acid, mGluR1 antagonist, LY367385, group II mGluR antagonist, (2S)-alpha-ethylglutamic acid, or group III mGluR antagonist, (S)-2-amino-2-methyl-4-phosphonobutanic acid (MAP4). In addition, the ability of MCPG to reverse DHPG-LTD was mimicked by the protein tyrosine phosphatase inhibitors, phenylarsine oxide and orthovanadate, but not phospholipase C inhibitor, U73122, protein kinase C inhibitor, bisindolylmaleimide 1, p38 mitogen-activated protein kinase inhibitor, SB203580, or protein phosphatases 1/2 A inhibitor, okadaic acid. Moreover, MCPG reversed the DHPG-LTD without affecting the paired-pulse facilitation. The expression of DHPG-LTD was associated with the reduction of both tyrosine phosphorylation and surface expression of AMPA receptor GluR2 subunits. Together, these results suggest that sustained activation of mGluR5 and in turn triggering a protein tyrosine phosphatase-dependent regulation of postsynaptic expression of AMPA receptors may contribute to the expression of DHPG-LTD.     

ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell‐derived neurons

Induced pluripotent stem cells (iPSCs) hold promise to revolutionize studies of intracellular transport in live human neurons and to shed new light on the role of dysfunctional transport in neurodegenerative disorders. Here, we describe an approach for live imaging of axonal and dendritic transport in iPSC‐derived cortical neurons. We use transfection and transient expression of genetically‐encoded fluorescent markers to characterize the motility of Rab‐positive vesicles, including early, late and recycling endosomes, as well as autophagosomes and mitochondria in iPSC‐derived neurons. Comparing transport parameters of these organelles with data from primary rat hippocampal neurons, we uncover remarkable similarities. In addition, we generated lysosomal‐associated membrane protein 1 (LAMP1)‐enhanced green fluorescent protein (EGFP) knock‐in iPSCs and show that knock‐in neurons can be used to study the transport of endogenously labeled vesicles, as a parallel approach to the transient overexpression of fluorescently labeled organelle markers.     

Antagonism of metabotropic glutamate receptor type 5 attenuates l-DOPA-induced dyskinesia and its molecular and neurochemical correlates in a rat model of Parkinson's disease

Metabotropic glutamate receptor type 5 (mGluR5) modulates dopamine and glutamate neurotransmission at central synapses. In this study, we addressed the role of mGluR5 in l-DOPA-induced dyskinesia, a movement disorder that is due to abnormal activation of both dopamine and glutamate receptors in the basal ganglia. A selective and potent mGluR5 antagonist, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl] pyridine, was tested for its ability to modulate molecular, behavioural and neurochemical correlates of dyskinesia in 6-hydroxydopamine-lesioned rats treated with l-DOPA. The compound significantly attenuated the induction of abnormal involuntary movements (AIMs) by chronic l-DOPA treatment at doses that did not interfere with the rat physiological motor activities. These effects were paralleled by an attenuation of molecular changes that are strongly associated with the dyskinesiogenic action of l-DOPA (i.e. up-regulation of prodynorphin mRNA in striatal neurons). Using in vivo microdialysis, we found a temporal correlation between the expression of l-DOPA-induced AIMs and an increased GABA outflow within the substantia nigra pars reticulata. When co-administered with l-DOPA, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl] pyridine greatly attenuated both the increase in nigral GABA levels and the expression of AIMs. These data demonstrate that mGluR5 antagonism produces strong anti-dyskinetic effects in an animal model of Parkinson's disease through central inhibition of the molecular and neurochemical underpinnings of l-DOPA-induced dyskinesia.     

Fluorescently labeled adrenomedullin allows real‐time monitoring of adrenomedullin receptor trafficking in living cells

The human adrenomedullin (ADM) is a 52 amino acid peptide hormone belonging to the calcitonin family of peptides, which plays a major role in the development and regulation of cardiovascular and lymphatic systems. For potential use in clinical applications, we aimed to investigate the fate of the peptide ligand after binding and activation of the adrenomedullin receptor (AM₁), a heterodimer consisting of the calcitonin receptor‐like receptor (CLR), a G protein‐coupled receptor, associated with the receptor activity‐modifying protein 2 (RAMP2). Full length and N‐terminally shortened ADM peptides were synthesized using Fmoc/tBu solid phase peptide synthesis and site‐specifically labeled with the fluorophore carboxytetramethylrhodamine (Tam) either by amide bond formation or copper(I)‐catalyzed azide alkyne cycloaddition. For the first time, Tam‐labeled ligands allowed the observation of co‐internalization of the whole ligand‐receptor complex in living cells co‐transfected with fluorescent fusion proteins of CLR and RAMP2. Application of a fluorescent probe to track lysosomal compartments revealed that ADM together with the CLR/RAMP2‐complex is routed to the degradative pathway. Moreover, we found that the N‐terminus of ADM is not a crucial component of the peptide sequence in terms of AM₁ internalization behavior. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Involvement of neuropeptide Y and its Y1 and Y5 receptors in maintaining self‐renewal and proliferation of human embryonic stem cells

Neuropeptide Y (NPY) and NPY receptors are widely expressed in various organs and cell types and have been shown to have pleiotropic functions. However, their presence or role in human embryonic stem cells (hESCs) remains unknown. We now show that undifferentiated hESCs primarily express NPY and its Y1 and Y5 receptors. Inhibition of NPY signalling using either the selective NPY Y1 or Y5 receptor antagonist reduces the maintenance of self‐renewal and proliferation of undifferentiated hESCs. We also provide compelling evidence that exogenous NPY supports the long‐term growth of undifferentiated hESCs in the absence of feeder cell factors using only knockout serum replacement media. Further, NPY facilitates the use of chemically defined medium made up of N2/B27 supplement and basic fibroblast growth factor (bFGF) for hESC feeder‐free culture. Our results indicate that both Y1 and Y5 receptors appear to be involved in the NPY‐mediated activation of AKT/protein kinase B and extracellular signal‐regulated kinase 1/2 (ERK1/2) in hESCs. Notably, only Y1 receptor, but not Y5 receptor, is responsible for the NPY‐induced activation of cAMP‐response element binding (CREB) in hESCs. These results provide the first evidence that NPY and its Y1 and Y5 receptors have potential role in maintaining hESC self‐renewal and pluripotency. We demonstrate the underlying importance of NPY signalling and its usefulness in the development of a defined and xeno‐free culture condition for the large‐scale propagation of undifferentiated hESCs.     

Regulation of insulin resistance by targeting the insulin‐like growth factor 1 receptor with microRNA‐122‐5p in hepatic cells

Insulin resistance (IR) is a common etiology of type 2 diabetes (T2D) defined by a state of decreased reactivity to insulin in multiple organs, such as the liver. This study aims to investigate how microRNA‐122‐5p (miR‐122) regulates the hepatic IR in vitro. We first found that the miR‐122 level was upregulated in the liver of rats fed with a high‐fat diet and injected with streptozotocin (T2D rats), while the expression level of insulin‐like growth factor 1 receptor (IGF‐1R), a potential target of miR‐122, was downregulated in the diabetic liver. In vitro, glucosamine‐induced IR was introduced in HepG2 hepatic cells, and the levels of miR‐122 and IGF‐1R were further assessed. An increase of miR‐122 level and a decrease of IGF‐IR level were observed in IR hepatic cells, which was the same as that in the diabetic liver. Results of the luciferase reporter assay validated IGF‐1R as a direct target of miR‐122. Moreover, in IR HepG2 cells, antagonizing miR‐122 with its specific inhibitor enhanced glucose uptake and suppressed the expression of glucose 6‐phosphatase and phosphoenolpyruvate carboxykinase, two key enzymes in regulating gluconeogenesis. Such alterations induced by the miR‐122 inhibitor in IR hepatic cells were impaired when IGF‐1R was simultaneously knocked down. In addition, the PI3K/Akt pathway was deactivated in IR cells, and then reactivated with miR‐122 inhibitor transfection. In conclusion, our study demonstrates that miR‐122 is able to regulate IR in hepatic cells by targeting IGF‐1R.