Alpha-secretase inhibition impairs Group I metabotropic glutamate receptor-mediated protein synthesis, long-term potentiation and long-term depression.

Group I metabotropic glutamate receptors (Gp1-mGluRs) exert a host of effects on cellular functions, including enhancement of protein synthesis and the associated facilitation of long-term potentiation (LTP) and induction of long-term depression (LTD). However, the complete cascades of events mediating these events are not fully understood. Gp1-mGluRs trigger α-secretase cleavage of amyloid precursor protein, producing soluble amyloid precursor protein-α (sAPPα), a known regulator of LTP. However, the α-cleavage of APP has not previously been linked to Gp1-mGluR's actions. Using rat hippocampal slices, we found that the α-secretase inhibitor tumour necrosis factor-alpha protease inhibitor-1, which inhibits both disintegrin and metalloprotease 10 (ADAM10) and 17 (ADAM17) activity, blocked or reduced the ability of the Gp1-mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) to stimulate protein synthesis, metaplastically prime future LTP and elicit sub-maximal LTD. In contrast, the specific ADAM10 antagonist GI254023X did not affect the regulation of plasticity, suggesting that ADAM17 but not ADAM10 is involved in mediating these effects of DHPG. However, neither drug affected LTD that was strongly induced by either high-concentration DHPG or paired-pulse synaptic stimulation. Our data suggest that moderate Gp1-mGluR activation triggers α-secretase sheddase activity targeting APP or other membrane-bound proteins as part of a more complex signalling cascade than previously envisioned. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Regulation of mGluR1 on the Expression of PKC and NMDAR in Aluminum-Exposed PC12 Cells.

Aluminum demonstrates clear neurotoxicity and can cause Alzheimer's disease (AD)-like symptoms, including cognitive impairment. One toxic effect of aluminum is a decrease in synaptic plasticity, but the specific mechanism remains unclear. In this study, PC12 cells were treated with Al(mal)3 to construct a toxic cell model. (S)-3,5-Dihydroxyphenylglycine (DHPG), α-methyl-4-carboxyphenylglycine (MCPG), and mGluR1-siRNA were used to interfere with the expression of metabotropic glutamate receptor subtype 1 (mGluR1). Polymerase chain reaction and western blotting were used to investigate the expression of mGluR1, protein kinase C (PKC), and N-methyl-D-aspartate receptor (NMDAR) subunits. ELISA was used to detect PKC enzyme activity. In PC12 cells, mRNA and protein expressions of PKC and NMDAR subunits were inhibited by Al(mal)3. Aluminum may further regulate the expression of NMDAR1 and NMDAR2B through mGluR1 to regulate PKC enzyme activity, thereby affecting learning and memory functions. Furthermore, the results implied that the mGluR1-PKC-NMDAR signaling pathway may predominately involve positive regulation. These findings provide new targets for studying the neurotoxic mechanism of aluminum.

Tumor necrosis factor-alpha aggravates gliosis and inflammation of activated retinal Müller cells.

Tumor necrosis factor-alpha (TNF-α), a major inflammatory factor released from activated retinal glial cells, is implicated in the pathogenesis of glaucoma. In this study, we investigated whether and how TNF-α may affect functional conditions of activated retinal Müller cells. Our results showed that in the group I metabotropic glutamate receptor (mGluR I) agonist DHPG-activated cultured Müller cells, TNF-α treatment aggravated cell gliosis, as evidenced by significantly increased expression of glial fibrillary acidic protein (GFAP). TNF-α treatment of the DHPG-activated Müller cells decreased cell proliferation and induced cell apoptosis. In normal Müller cells, TNF-α treatment increased the mRNA levels of leukocyte inhibitory factor (LIF), intercellular cell adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and chemokine C-C-motif ligand 2 (CCL2), which could be significantly attenuated when Müller cells were pre-activated. However, TNF-α-induced elevation in mRNA levels of inflammatory factors, such as TNF-α, inducible nitric oxide synthase (iNOS), and interleukin-6 (IL-6), in normal Müller cells still kept higher levels when Müller cells were pre-activated. Furthermore, the TNF-α-induced changes of cytokines were partially mediated by NF-κB signaling pathway. Our results suggest that TNF-α may promote gliosis and inflammatory response of activated Müller cells, thus aggravating RGC injury in glaucoma.

Neuroprotective effects of mGluR5 activation through the PI3K/Akt pathway and the molecular switch of AMPA receptors.

Previous studies have demonstrated that antagonists of mGluR1, but not mGluR5, are neuroprotective in models of cerebral ischemia. To investigate the individual roles of mGlu1 and mGlu5 receptors in in vitro model of cerebral ischemia we used low doses of the non-selective group I agonist DHPG and mGlu1 and mGlu5 selective positive allosteric modulators (PAMs). In hippocampal slices subjected to 30 min oxygen-glucose deprivation (OGD), DHPG (1 µM) and the mGluR5 PAM (VU0092273) significantly reduced OGD-induced CA1 injury monitored by propidium iodide staining of the slices and quantitative analysis of CA1 neurons. In contrast, the mGluR1 PAM (VU0483605) showed no neuroprotection. These protective effects of DHPG and VU0092273 were prevented by inhibition of PI3K/Akt pathway by LY294002. The mGluR5 PAM (VU0092273) also prevented GluA2 down-regulation triggered by ischemic injury, via PI3K/Akt pathway, revealing a further contribution to its neuroprotective effects by reducing the excitotoxic effects of increased Ca2+ influx through GluA2-lacking AMPA receptors. Furthermore, immunohistochemical assays confirmed the neuroprotective effect of VU0092273 and revealed activation of glia, indicating the involvement reactive astrogliosis in the mechanisms of neuroprotection. Our data suggest that selective activation/potentiation of mGluR5 signalling represents a promising strategy for the development of new interventions to reduce or prevent ischemia-induced neuronal death.

Effect of different olive oil-derived antioxidants (hydroxytyrosol and 3,4-dihydroxyphenylglycol) on the quality of frozen-thawed ram sperm.

The aim of the present study was to evaluate the effect of the addition of different concentrations of two olive oil-derived antioxidants, hydroxytyrosol (3,4-dihydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), on ovine semen during the freezing-thawing process. Sperm was collected, pooled and diluted with commercial extenders and then divided into aliquots supplemented with different concentrations (10 µg/ml, 30 µg/ml, 50 µg/ml and 70 µg/ml) of HT, DHPG and a mixture (MIX) of both antioxidants. A control group, without antioxidant, was also prepared. Sperm motility, viability, acrosome integrity, mitochondrial membrane potential and lipid peroxidation (LPO) were assessed. The results showed that frozen-thawed ram spermatozoa exhibited lower values for motility, membrane integrity, acrosome and mitochondrial membrane potential than fresh samples (P ≤ 0.01). However, when antioxidants were added, thawed spermatozoa exhibited relatively low LPO, recording values similar to fresh spermatozoa; by contrast, the control group of frozen-thawed spermatozoa without antioxidants exhibited significantly higher LPO (P ≤ 0.01). The addition of a HT+DHPG mixture (MIX) had a negative impact on sperm membrane and acrosome integrity, suggesting that a pure antioxidant supplementation has the potential to offer superior results. In conclusion, HT and DHPG exhibited a positive effect on the frozen-thawed spermatozoa inasmuch as they reduced the LPO. These olive oil-derived antioxidants have the potential to improve frozen-thawed sperm quality, although further studies should be carried out to analyse the antioxidant effect at different times after thawing.

Effect of edible pectin-fish gelatin films containing the olive antioxidants hydroxytyrosol and 3,4-dihydroxyphenylglycol on beef meat during refrigerated storage.

The objective of this research was to evaluate the effect of the addition of two antioxidants naturally present in olives, hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG), to a pectin-fish gelatin edible film on the preservation of raw beef meat during refrigerated storage. A new composite film that included beeswax was also prepared, resulting in a reduction in the film's oxygen permeability. Results showed that the meat samples wrapped with film containing antioxidants reduced the formation of oxidation products in the form of thiobarbituric acid reaction substances (TBARS) compared with control film without antioxidants. HT added at 0.5% to the film with beeswax suppressed the lipid oxidation of beef meat during 7 days of storage at 4 °C, possibly by the combined effect of acting as an oxygen barrier and the specific antioxidant activity. The interference of plasticizer agents (glycerol and sorbitol) incorporated to the film on the TBARS method was showed for the first time.

Enhancement of dendritic persistent Na+ currents by mGluR5 leads to an advancement of spike timing with an increase in temporal precision.

Timing and temporal precision of action potential generation are thought to be important for encoding of information in the brain. The ability of single neurons to transform their input into output action potential is primarily determined by intrinsic excitability. Particularly, plastic changes in intrinsic excitability represent the cellular substrate for spatial memory formation in CA1 pyramidal neurons (CA1-PNs). Here, we report that synaptically activated mGluR5-signaling can modulate the intrinsic excitability of CA1-PNs. Specifically, high-frequency stimulation at CA3-CA1 synapses increased firing rate and advanced spike onset with an improvement of temporal precision. These changes are mediated by mGluR5 activation that induces cADPR/RyR-dependent Ca2+ release in the dendrites of CA1-PNs, which in turn causes an increase in persistent Na+ currents (INa,P) in the dendrites. When group I mGluRs in CA1-PNs are globally activated pharmacologically, afterdepolarization (ADP) generation as well as increased firing rate are observed. These effects are abolished by inhibiting mGluR5/cADPR/RyR-dependent Ca2+ release. However, the increase in firing rate, but not the generation of ADP is affected by inhibiting INa,P. The differences between local and global activation of mGluR5-signaling in CA1-PNs indicates that mGluR5-dependent modulation of intrinsic excitability is highly compartmentalized and a variety of ion channels are recruited upon their differential subcellular localizations. As mGluR5 activation is induced by physiologically plausible brief high-frequency stimulation at CA3-CA1 synapses, our results suggest that mGluR5-induced enhancement of dendritic INa,P in CA1-PNs may provide important implications for our understanding about place field formation in the hippocampus.

mGluR5 mediates post-radiotherapy fatigue development in cancer patients.

Cancer-related fatigue (CRF) is a common burden in cancer patients and little is known about its underlying mechanism. The primary aim of this study was to identify gene signatures predictive of post-radiotherapy fatigue in prostate cancer patients. We employed Fisher Linear Discriminant Analysis (LDA) to identify predictive genes using whole genome microarray data from 36 men with prostate cancer. Ingenuity Pathway Analysis was used to determine functional networks of the predictive genes. Functional validation was performed using a T lymphocyte cell line, Jurkat E6.1. Cells were pretreated with metabotropic glutamate receptor 5 (mGluR5) agonist (DHPG), antagonist (MPEP), or control (PBS) for 20 min before irradiation at 8 Gy in a Mark-1 γ-irradiator. NF-κB activation was assessed using a NF-κB/Jurkat/GFP Transcriptional Reporter Cell Line. LDA achieved 83.3% accuracy in predicting post-radiotherapy fatigue. "Glutamate receptor signaling" was the most significant (p = 0.0002) pathway among the predictive genes. Functional validation using Jurkat cells revealed clustering of mGluR5 receptors as well as increased regulated on activation, normal T cell expressed and secreted (RANTES) production post irradiation in cells pretreated with DHPG, whereas inhibition of mGluR5 activity with MPEP decreased RANTES concentration after irradiation. DHPG pretreatment amplified irradiation-induced NF-κB activation suggesting a role of mGluR5 in modulating T cell activation after irradiation. These results suggest that mGluR5 signaling in T cells may play a key role in the development of chronic inflammation resulting in fatigue and contribute to individual differences in immune responses to radiation. Moreover, modulating mGluR5 provides a novel therapeutic option to treat CRF.

GPR30 activation improves memory and facilitates DHPG-induced LTD in the hippocampal CA3 of middle-aged mice.

Reduced estrogen levels and decreased expression of related receptors are typical cerebral features of aging. The G protein-coupled estrogen receptor 1 (GPER1, also known as GPR30) is considered a novel therapeutic target for neurodegenerative diseases. In this study, we demonstrated that hippocampal GPR30 expression was reduced in middle-aged mice compared with young adult mice. GPR30 agonist G1 improved both fear and spatial memory in both male and female middle-aged mice, but not in young adult mice, which were blocked by the GPR30 antagonist G15. Interestingly, a group I metabotropic glutamate receptor (mGluR) agonist, 3,5-dihydroxyphenylglycine (DHPG)-induced long-term depression (LTD) in mossy fiber-cornu ammonis 3 (MF-CA3) synapses but not Schaffer collateral-CA1 (SC-CA1) synapses was facilitated in brain slices from G1-treated middle-aged mice. Long-term potentiation (LTP) in SC-CA1 synapses was not affected in slices from G1-treated mice. The effects of GPR30 activation on memory and DHPG-LTD in MF-CA3 synapses were further confirmed by viral expression of GPR30 in the CA3. The regulation of hippocampal synaptic plasticity by G1 treatment might be related to brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signaling, as G15 also blocked G1-induced activation of the BDNF-TrkB pathway. Moreover, we found that DHPG triggered GluA internalization in slices from G1-treated mice but not control mice. Pharmacological experiments showed that G1-mediated facilitation of DHPG-induced LTD in MF-CA3 synapses was dependent on protein kinase B (Akt), mammalian target of rapamycin (mTor), and TrkB signaling. In conclusion, our results indicate that GPR30 activation improves memory in middle-aged mice, likely through facilitating synaptic plasticity in the CA3. This study provides novel evidence that GPR30 activation can improve memory in middle-aged animals.

Functional partnership between mGlu3 and mGlu5 metabotropic glutamate receptors in the central nervous system.

mGlu5 receptors are involved in mechanisms of activity-dependent synaptic plasticity, and are targeted by drugs developed for the treatment of CNS disorders. We report that mGlu3 receptors, which are traditionally linked to the control of neurotransmitter release, support mGlu5 receptor signaling in neurons and largely contribute to the robust mGlu5 receptor-mediated polyphosphoinositide hydrolysis in the early postnatal life. In cortical pyramidal neurons, mGlu3 receptor activation potentiated mGlu5 receptor-mediated somatic Ca2+ mobilization, and mGlu3 receptor-mediated long-term depression in the prefrontal cortex required the endogenous activation of mGlu5 receptors. The interaction between mGlu3 and mGlu5 receptors was also relevant to mechanisms of neuronal toxicity, with mGlu3 receptors shaping the influence of mGlu5 receptors on excitotoxic neuronal death. These findings shed new light into the complex role played by mGlu receptors in physiology and pathology, and suggest reconsideration of some of the current dogmas in the mGlu receptor field.

Neuron-astrocyte signaling is preserved in the aging brain.

Astrocytes play crucial roles in brain homeostasis and are emerging as regulatory elements of neuronal and synaptic physiology by responding to neurotransmitters with Ca2+ elevations and releasing gliotransmitters that activate neuronal receptors. Aging involves neuronal and astrocytic alterations, being considered risk factor for neurodegenerative diseases. Most evidence of the astrocyte-neuron signaling is derived from studies with young animals; however, the features of astrocyte-neuron signaling in adult and aging brain remain largely unknown. We have investigated the existence and properties of astrocyte-neuron signaling in physiologically and pathologically aging mouse hippocampal and cortical slices at different lifetime points (0.5 to 20 month-old animals). We found that astrocytes preserved their ability to express spontaneous and neurotransmitter-dependent intracellular Ca2+ signals from juvenile to aging brains. Likewise, resting levels of gliotransmission, assessed by neuronal NMDAR activation by glutamate released from astrocytes, were largely preserved with similar properties in all tested age groups, but DHPG-induced gliotransmission was reduced in aged mice. In contrast, gliotransmission was enhanced in the APP/PS1 mouse model of Alzheimer's disease, indicating a dysregulation of astrocyte-neuron signaling in pathological conditions. Disruption of the astrocytic IP3 R2 mediated-signaling, which is required for neurotransmitter-induced astrocyte Ca2+ signals and gliotransmission, boosted the progression of amyloid plaque deposits and synaptic plasticity impairments in APP/PS1 mice at early stages of the disease. Therefore, astrocyte-neuron interaction is a fundamental signaling, largely conserved in the adult and aging brain of healthy animals, but it is altered in Alzheimer's disease, suggesting that dysfunctions of astrocyte Ca2+ physiology may contribute to this neurodegenerative disease. GLIA 2017 GLIA 2017;65:569-580.

Protein kinase Cɛ activity regulates mGluR5 surface expression in the rat nucleus accumbens.

Type 5 metabotropic glutamate receptors (mGluR5) activate protein kinase C (PKC) via coupling to Gαq/11 protein signaling. We have previously demonstrated that the epsilon isoform of PKC (PKCɛ) is a critical downstream target of mGluR5 in regulating behavioral and biochemical responses to alcohol. Recent evidence suggests that PKC-mediated phosphorylation of mGluR5 can lead to receptor desensitization and internalization. We therefore sought to examine the specific involvement of PKCɛ in the regulation of mGluR5 surface expression in the nucleus accumbens (NAc), a key regulator of alcohol-associated behaviors. Coronal brain sections from male Wistar rats were analyzed for either colocalization of mGluR5 and PKCɛ via immunohistochemistry or changes in mGluR5 surface expression and PKCɛ phosphorylation following local application of PKCɛ translocation activator or inhibitor peptides and/or an orthosteric mGluR5 agonist. We observed colocalization of mGluR5 and PKCɛ in the NAc. We also showed that intra-NAc infusion of the PKCɛ translocation inhibitor ɛV1-2 increased mGluR5 surface expression under baseline conditions. Stimulation of mGluR5 with an orthosteric agonist DHPG, dose dependently increased ERK1/2 and PKCɛ phosphorylation as well as mGluR5 internalization in acute NAc slices. Finally, we observed that activation of PKCɛ translocation with Tat-ΨɛRACK peptide mediates agonist-independent mGluR5 internalization, whereas PKCɛ translocation inhibitor ɛV1-2 prevents agonist-dependent internalization of mGluR5 in NAc slice preparations. These findings suggest that the subcellular localization of mGluR5 in the NAc is regulated by PKCɛ under basal and stimulation conditions, which may influence the role of mGluR5-PKCɛ signaling in alcohol-related behaviors. © 2016 Wiley Periodicals, Inc.

Activated Müller Cells Involved in ATP-Induced Upregulation of P2X7 Receptor Expression and Retinal Ganglion Cell Death.

P2X7 receptor (P2X7R), an ATP-gated ion channel, plays an important role in glaucomatous retinal ganglion cell (RGC) apoptotic death, in which activated retinal Müller glial cells may be involved by releasing ATP. In the present study, we investigated whether and how activated Müller cells may induce changes in P2X7R expression in RGCs by using immunohistochemistry and Western blot techniques. Intravitreal injection of DHPG, a group I metabotropic glutamate receptor (mGluR I) agonist, induced upregulation of GFAP expression, suggestive of Müller cell activation (gliosis), as we previously reported. Accompanying Müller cell activation, P2X7R protein expression was upregulated, especially in the cells of ganglion cell layer (GCL), which was reversed by coinjection of brilliant blue G (BBG), a P2X7R blocker. In addition, intravitreal injection of ATP also induced upregulation of P2X7R protein expression. Similar results were observed in cultured retinal neurons by ATP treatment. Moreover, both DHPG and ATP intravitreal injection induced a reduction in the number of fluorogold retrogradely labeled RGCs, and the DHPG effect was partially rescued by coinjection of BBG. All these results suggest that activated Müller cells may release ATP and, in turn, induce upregulation of P2X7R expression in the cells of GCL, thus contributing to RGC death.

Metabotropic glutamate receptor 1 promotes cementoblast proliferation via MAP kinase signaling pathways.

PURPOSE/AIM: Glutamate is one of the signaling molecules responsible for transmission in the central nervous system. Periodontal ligament (PDL) cells were recently reported to express metabotropic glutamate receptors (mGluRs). However, the functions of mGluR signaling in PDL cells or PDL-related cells remain largely unknown. The aim of this study was to investigate the expression and function of mGluRs in PDL-related cells. MATERIALS AND METHODS: OCCM-30 cells, immortalized murine cementoblasts, were stimulated with l-glutamate or mGluRs antagonists. The cells' proliferative response was evaluated using a colorimetric assay and gene expression was assessed using real-time polymerase chain reaction. The nuclear translocation of cyclin D1 was evaluated by immunohistochemistry. RESULTS: l-Glutamate promoted the proliferation of OCCM-30 cells, which expressed mGluR1, but not mGluR5. Dihydroxyphenylglycine (DHPG), an agonist of group I mGluRs (mGluR1 and mGluR5), also promoted cell proliferation, and this was inhibited by LY456236, an mGluR1 antagonist. DHPG increased the expression of cyclin D1, a key regulator of cell proliferation, and its nuclear translocation. DHPG also increased the expression of Bcl2A1, an antiapoptotic oncogene and simultaneously reduced the expression of Bax, a pro-apoptotic marker. Furthermore, the DHPG-induced proliferation of OCCM-30 cells was reduced by pretreatment with SB203580, SP600125, and PD98059, inhibitors of p38, JNK, and ERK1/2, respectively. CONCLUSIONS: These findings indicate that activation of mGluR1 expressed by OCCM-30 cells induces cell proliferation in a manner that is dependent on mitogen-activated protein kinase pathways and that cyclin D1 and Bcl2A1/Bax may be involved. Our results provide useful information for elucidating the mechanisms underlying cementum homeostasis and regeneration.

Effects of dietary virgin olive oil polyphenols: hydroxytyrosyl acetate and 3, 4-dihydroxyphenylglycol on DSS-induced acute colitis in mice.

Hydroxytyrosol, a polyphenolic compound from extra virgin olive oil (EVOO) has exhibited an improvement in a model of DSS-induced colitis. However, other phenolic compounds present such as hydroxytyrosyl acetate (HTy-Ac) and 3,4-dihydroxyphenylglycol (DHPG) need to be explored to complete the understanding of the overall effects of EVOO on inflammatory colon mucosa. This study was designed to evaluate the effect of both HTy-Ac and DHPG dietary supplementation in the inflammatory response associated to colitis model. Six-week-old mice were randomized in four dietary groups: sham and control groups received standard diet, and other two groups were fed with HTy-Ac and DHPG, respectively, at 0.1%. After 30 days, all groups except sham received 3% DSS in drinking water for 5 days followed by a regime of 5 days of water. Acute inflammation was evaluated by Disease Activity Index (DAI), histology and myeloperoxidase (MPO) activity. Colonic expression of iNOS, COX-2, MAPKs, NF-kB and FOXP3 were determined by western blotting. Only HTy-Ac-supplemented group showed a significant DAI reduction as well as an improvement of histological damage and MPO. COX-2 and iNOS protein expression were also significantly reduced. In addition, this dietary group down-regulated JNK phosphorylation and prevented the DSS-induced nuclear translocation level of p65. However, no significant differences were observed in the FOXP3 expression. These results demonstrated, for the first time, that HTy-Ac exerts an antiinflammatory effect on acute ulcerative colitis. We concluded that HTy-Ac supplement might provide a basis for developing a new dietary strategy for the prevention of ulcerative colitis.

Pannexin-1-mediated ATP release from area CA3 drives mGlu5-dependent neuronal oscillations.

The activation of Group I metabotropic glutamate receptors (GI mGluRs) in the hippocampus results in the appearance of persistent bursts of synchronised neuronal activity. In response to other stimuli, such activity is known to cause the release of the purines ATP and its neuroactive metabolite, adenosine. We have thus investigated the potential release and role of the purines during GI mGluR-induced oscillations in rat hippocampal areas CA3 and CA1 using pharmacological techniques and microelectrode biosensors for ATP and adenosine. The GI mGluR agonist DHPG induced both persistent oscillations in neuronal activity and the release of adenosine in areas CA1 and CA3. In contrast, the DHPG-induced release of ATP was only observed in area CA3. Whilst adenosine acting at adenosine A1 receptors suppressed DHPG-induced burst activity, the activation of mGlu5 and P2Y1 ATP receptors were necessary for the induction of DHPG-induced oscillations. Selective inhibition of pannexin-1 hemichannels with a low concentration of carbenoxolone (10 µM) or probenecid (1 mM) did not affect adenosine release in area CA3, but prevented both ATP release in area CA3 and DHPG-induced bursting. These data reveal key aspects of GI mGluR-dependent neuronal activity that are subject to bidirectional regulation by ATP and adenosine in the initiation and pacing of burst firing, respectively, and which have implications for the role of GI mGluRs in seizure activity and neurodevelopmental disorders.

Stimulation-evoked Ca2+ signals in astrocytic processes at hippocampal CA3-CA1 synapses of adult mice are modulated by glutamate and ATP.

To date, it has been difficult to reveal physiological Ca(2+) events occurring within the fine astrocytic processes of mature animals. The objective of the study was to explore whether neuronal activity evokes astrocytic Ca(2+) signals at glutamatergic synapses of adult mice. We stimulated the Schaffer collateral/commissural fibers in acute hippocampal slices from adult mice transduced with the genetically encoded Ca(2+) indicator GCaMP5E driven by the glial fibrillary acidic protein promoter. Two-photon imaging revealed global stimulation-evoked astrocytic Ca(2+) signals with distinct latencies, rise rates, and amplitudes in fine processes and somata. Specifically, the Ca(2+) signals in the processes were faster and of higher amplitude than those in the somata. A combination of P2 purinergic and group I/II metabotropic glutamate receptor (mGluR) antagonists reduced the amplitude of the Ca(2+) transients by 30-40% in both astrocytic compartments. Blockage of the mGluRs alone only modestly reduced the magnitude of the stimulation-evoked Ca(2+) signals in processes and failed to affect the somatic Ca(2+) response. Local application of group I or I/II mGluR agonists or adenosine triphosphate (ATP) elicited global astrocytic Ca(2+) signals that mimicked the stimulation-evoked astrocytic Ca(2+) responses. We conclude that stimulation-evoked Ca(2+) signals in astrocytic processes at CA3-CA1 synapses of adult mice (1) differ from those in astrocytic somata and (2) are modulated by glutamate and ATP.

Metabotropic glutamate receptor 1 recycles to the cell surface in protein phosphatase 2A-dependent manner in non-neuronal and neuronal cell lines.

Trafficking of G protein-coupled receptors plays a crucial role in controlling the precise signalling of the receptor as well as its proper regulation. Metabotropic glutamate receptor 1 (mGluR1), a G protein-coupled receptor, is a member of the group I mGluR family. mGluR1 plays a critical role in neuronal circuit formation and also in multiple types of synaptic plasticity. This receptor has also been reported to be involved in various neuropsychiatric diseases. Other than the central nervous system, mGluR1 plays crucial roles in various non-neuronal cells like hepatocytes, skin cells, etc. Although it has been reported that mGluR1 gets endocytosed on ligand application, the events after the internalization of the receptor has not been studied. We show here that mGluR1 internalizes on ligand application. Subsequent to endocytosis, majority of the receptors localize at the recycling compartment and no significant presence of the receptor was noticed in the lysosome. Furthermore, mGluR1 returned to the cell membrane subsequent to ligand-mediated internalization. We also show here that the recycling of mGluR1 is dependent on the activity of protein phosphatase 2A. Thus, our data suggest that the ligand-mediated internalized receptors recycle back to the cell surface in protein phosphatase 2A-dependent manner.

Differential deregulation of astrocytic calcium signalling by amyloid-ß, TNFα, IL-1ß and LPS.

In Alzheimer's disease (AD), astrocytes undergo complex morphological and functional changes that include early atrophy, reactive activation and Ca(2+) deregulation. Recently, we proposed a mechanism by which nanomolar Aß42 deregulates mGluR5 and InsP3 receptors, the key elements of astrocytic Ca(2+) signalling toolkit. To evaluate the specificity of these changes, we have now investigated whether the effects of Aß42 on Ca(2+) signalling machinery can be reproduced by pro-inflammatory agents (TNFα, IL-1ß, LPS). Here we report that Aß42 (100nM, 72h) significantly increased mRNA levels of mGluR5, InsP3R1 and InsP3R2, whereas pro-inflammatory agents reduced expression of these specific mRNAs. Furthermore, DHPG-induced Ca(2+) signals and store operated Ca(2+) entry (SOCE) were augmented in Aß42-treated cells due to up-regulation of a set of Ca(2+) signalling-related genes including TRPC1 and TRPC4. Opposite changes were observed when astrocytes were treated with TNFα, IL-1ß and LPS. Last, the effects observed on SOCE by treating wild-type astrocytes with Aß42 were also identified in untreated astrocytes from 3×Tg-AD animals, suggesting a link to the AD pathology. Our results demonstrate that effects of Aß42 on astrocytic Ca(2+) signalling differ from and may contrast to the effects of pro-inflammatory agents.

Changes in mGlu5 receptor-dependent synaptic plasticity and coupling to homer proteins in the hippocampus of Ube3A hemizygous mice modeling angelman syndrome.

Angelman syndrome (AS) is caused by the loss of Ube3A, an ubiquitin ligase that commits specific proteins to proteasomal degradation. How this defect causes autism and other pathological phenotypes associated with AS is unknown. Long-term depression (LTD) of excitatory synaptic transmission mediated by type 5 metabotropic glutamate (mGlu5) receptors was enhanced in hippocampal slices of Ube3A(m-/p+) mice, which model AS. No changes were found in NMDA-dependent LTD induced by low-frequency stimulation. mGlu5 receptor-dependent LTD in AS mice was sensitive to the protein synthesis inhibitor anisomycin, and relied on the same signaling pathways as in wild-type mice, e.g., the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycine pathway, and protein tyrosine phosphatase. Neither the stimulation of MAPK and PI3K nor the increase in Arc (activity-regulated cytoskeleton-associated protein) levels in response to mGlu5 receptor activation were abnormal in hippocampal slices from AS mice compared with wild-type mice. mGlu5 receptor expression and mGlu1/5 receptor-mediated polyphosphoinositide hydrolysis were also unchanged in the hippocampus of AS mice. In contrast, AS mice showed a reduced expression of the short Homer protein isoform Homer 1a, and an increased coupling of mGlu5 receptors to Homer 1b/c proteins in the hippocampus. These findings support the link between Homer proteins and monogenic autism, and lay the groundwork for the use of mGlu5 receptor antagonists in AS.