Peripheral nerve injury decreases the expression of metabolic glutamate receptor 7 in dorsal root ganglion neurons

Group II and III metabolic glutamate receptors (mGluRs) are responsible for the glutamate-mediated postsynaptic excitation of neurons. Previous pharmacological evidences show that activation of mGluR7 could inhibit nociceptive reception. However, the distribution and expression patterns of mGluR7 after peripheral injury remain unclear. Herein we found that mGluR7 was expressed in the rat peptidergic dorsal root ganglion (DRG) neurons and large neurons, but rarely in isolectin B4 positive neurons. Sciatic nerve ligation experiment showed that mGluR7 was anterogradely transported from cell body to the peripheral site. Furthermore, after peripheral nerve injury, mGluR7 expression was down-regulated in both peptidergic and large DRG neurons. Our work suggests that mGluR7 might be involved in the regulation of pathological pain after peripheral nerve injury.     

Analysis of SUMO-1 modification of neuronal proteins containing consensus SUMOylation motifs

SUMOylation is emerging as an important mechanism for modulating protein function in many cell types. A large variety of proteins have been proposed as SUMO targets based on the presence of a consensus SUMOylation core motif (Psi-K-x-D/E). In neurons these include multiple synaptic proteins but it has not been established whether proteins carrying this motif are SUMOylated either in vitro or in vivo. Here we use a bacterial SUMOylation assay to systematically test for SUMO-1 modification of a selection of neuronal proteins containing one or more amino acid sequences predicted as high-probability SUMOylation sites in computer-based searches. Of the 39 proteins analysed only 14 sites were posttranslationally modified by SUMO-1, including the group III metabotropic glutamate receptors and the kainate receptor subunit GluR7. These results identify new candidate proteins that may be involved in the SUMO regulation of synaptic activity and also demonstrate that the presence of the Psi-K-x-D/E motif is not sufficient to indicate that a protein can be SUMOylated in this bacterial system.     

Evidence for involvement of group II/III metabotropic glutamate receptors in NMDA receptor-independent long-term potentiation in area CA1 of rat hippocampus

Previous studies implicated metabotropic glutamate receptors (mGluRs) in N-methyl-D-aspartate (NMDA) receptor-independent long-term potentiation (LTP) in area CA1 of the rat hippocampus. To learn more about the specific roles played by mGluRs in NMDA receptor-independent LTP, we used whole cell recordings to load individual CA1 pyramidal neurons with a G-protein inhibitor [guanosine-5'-O-(2-thiodiphosphate), GDPbetaS]. Although loading postsynaptic CA1 pyramidal neurons with GDPbetaS significantly reduced G-protein dependent postsynaptic potentials, GDPbetaS failed to prevent NMDA receptor- independent LTP, suggesting that postsynaptic G-protein-dependent mGluRs are not required. We also performed a series of extracellular field potential experiments in which we applied group-selective mGluR antagonists. We had previously determined that paired-pulse facilitation (PPF) was decreased during the first 30-45 min of NMDA receptor-independent LTP. To determine if mGluRs might be involved in these PPF changes, we used a twin-pulse stimulation protocol to measure PPF in field potential experiments. NMDA receptor-independent LTP was prevented by a group II mGluR antagonist [(2S)-alpha-ethylglutamic acid] and a group III mGluR antagonist [(RS)-alpha-cyclopropyl-4-phosphonophenylglycine], but was not prevented by other group II and III mGluR antagonists [(RS)-alpha-methylserine-O-phosphate monophenyl ester or (RS)-alpha-methylserine-O-phosphate]. NMDA receptor-independent LTP was not prevented by either of the group I mGluR antagonists we examined, (RS)-1-aminoindan-1,5-dicarboxylic acid and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester. The PPF changes which accompany NMDA receptor-independent LTP were not prevented by any of the group-selective mGluR antagonists we examined, even when the LTP itself was blocked. Finally, we found that tetanic stimulation in the presence of group III mGluR antagonists lead to nonspecific potentiation in control (nontetanized) input pathways. Taken together, our results argue against the involvement of postsynaptic group I mGluRs in NMDA receptor-independent LTP. Group II and/or group III mGluRs are required, but the specific details of the roles played by these mGluRs in NMDA receptor-independent LTP are uncertain. Based on the pattern of results we obtained, we suggest that group II mGluRs are required for induction of NMDA receptor-independent LTP, and that group III mGluRs are involved in determining the input specificity of NMDA receptor-independent LTP by suppressing potentiation of nearby, nontetanized synapses.     

Unique presynaptic and postsynaptic roles of Group II metabotropic glutamate receptors in the modulation of thalamic network activity

The thalamic reticular nucleus (TRN) is a sheet of GABAergic neurons that project to other TRN neurons and to associated thalamocortical relay nuclei. The TRN receives glutamatergic synaptic inputs from cortex as well as reciprocal inputs from the collaterals of thalamocortical neurons. In addition to ionotropic glutamate receptors, metabotropic glutamate receptors (mGluRs) are present in the TRN circuitry. Using whole cell voltage clamp recordings, we pharmacologically characterized unique pre- and postsynaptic functions for Group II mGluRs (mGluR 2 and mGluR 3) within the TRN circuitry in ferrets. mGluR 2 was found on presynaptic cortical axon terminals in the TRN, where it reduced glutamate release, while mGluR 3 acted postsynaptically on TRN cells to increase membrane conductance. Using miniature inhibitory postsynaptic current analysis, we also found that picrotoxin-sensitive intra-TRN GABA-mediated neurotransmission was not affected by administration of a Group II mGluR agonist, indicating that neither mGluR 2 nor 3 acts on presynaptic GABA-containing terminals within the TRN. Because strong corticothalamic activation is implicated in abnormal thalamic rhythms, we used extracellular recordings in the lateral geniculate nucleus to study the effect of Group II mGluR agonists upon these slow oscillations. We induced approximately 3 Hz spike-and-wave discharge activity through corticothalamic stimulation, and found that such activity was reduced in the presence of the Group II mGluR agonist, (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268). These data indicate that Group II mGluR reduce the impact of corticothalamic excitation, and that they may be a useful target in the reduction of absence-like rhythms.     

Subcellular and subsynaptic localization of group I metabotropic glutamate receptors in the nucleus accumbens of cocaine-treated rats

There is significant pharmacological and behavioral evidence that group I metabotropic glutamate receptors (mGluR1a and mGluR5) in the nucleus accumbens play an important role in the neurochemical and pathophysiological mechanisms that underlie addiction to psychostimulants. To further address this issue, we undertook a detailed ultrastructural analysis to characterize changes in the subcellular and subsynaptic localization of mGluR1a and mGluR5 in the core and shell of nucleus accumbens following acute or chronic cocaine administration in rats. After a single cocaine injection (30 mg/kg) and 45 min withdrawal, there was a significant decrease in the proportion of plasma membrane-bound mGluR1a in accumbens shell dendrites. Similarly, the proportion of plasma membrane-bound mGluR1a was decreased in large dendrites of accumbens core neurons following chronic cocaine exposure (i.e. 1-week treatment followed by 3-week withdrawal). However, neither acute nor chronic cocaine treatments induced significant change in the localization of mGluR5 in accumbens core and shell, which is in contrast with the significant reduction of plasma membrane-bound mGluR1a and mGluR5 induced by local intra-accumbens administration of the group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG). In conclusion, these findings demonstrate that cocaine-induced glutamate imbalance has modest effects on the trafficking of group I mGluRs in the nucleus accumbens. These results provide valuable information on the neuroadaptive mechanisms of accumbens group I mGluRs in response to cocaine administration.     

Modulation of synaptic transmission from primary afferents to spinal substantia gelatinosa neurons by group III mGluRs in GAD65-EGFP transgenic mice

Group III metabotropic glutamate receptors (mGluRs) are involved in nociceptive transmission in the spinal cord. However, the cellular mechanism underlying the modulation of synaptic transmission from nociceptive primary afferents to dorsal horn neurons by group III mGluRs has yet to be explored. In this study, we used transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the glutamate decarboxylase (GAD) 65 promoter to identify specific subpopulations of GABAergic inhibitory interneurons. By GABA immunolabeling, we confirmed the majority of GAD65-EGFP-expressing neurons were GABAergic. Because GAD65-EGFP-expressing neurons have not been examined in detail before, we first investigated the physiological properties of GAD65-EGFP- and non-EGFP-expressing neurons in substantia gelatinosa (SG) of the spinal dorsal horn. Membrane properties, such as the resting membrane potential, membrane capacitance, action potential threshold, and action potential height, differed significantly between these two groups of neurons. Most EGFP-expressing neurons displayed a tonic firing pattern (73% of recorded neurons) and received monosynaptic Aδ and/or C primary afferent inputs (85% of recorded neurons). In contrast, we observed a delayed firing pattern in 53% of non-EGFP-expressing neurons. After identifying the physiological properties of EGFP-expressing neurons, we tested the effects of group III mGluRs on synaptic transmission pharmacologically. A group III mGluR agonist, L-AP4, attenuated Aδ fiber-evoked synaptic transmission but did not affect C fiber-evoked synaptic transmission to EGFP-expressing neurons. Similar primary afferent-specific inhibition by L-AP4 was also observed in non-EGFP-expressing neurons. Moreover, Aδ fiber-evoked synaptic transmission was suppressed by a selective mGluR7 agonist, AMN082. These results suggest that modulation of the synaptic transmission from primary afferents to SG neurons by group III mGluR agonist is specific to the type of nociceptive primary afferents but not to the type of target neurons.     

Cellular localization of metabotropic glutamate receptors in cortical tubers and subependymal giant cell tumors of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder associated with cortical malformations (cortical tubers) and the development of glial tumors (subependymal giant-cell tumors, SGCTs). Expression of metabotropic glutamate receptor (mGluR) subtypes is developmentally regulated and several studies suggest an involvement of mGluR-mediated glutamate signaling in the regulation of proliferation and survival of neural stem-progenitor cells, as well as in the control of tumor growth. In the present study, we have investigated the expression and cell-specific distribution of group I (mGluR1, mGluR5), group II (mGluR2/3) and group III (mGluR4 and mGluR8) mGluR subtypes in human TSC specimens of both cortical tubers and SGCTs, using immunocytochemistry. Strong group I mGluR immunoreactivity (IR) was observed in the large majority of TSC specimens in dysplastic neurons and in giant cells within cortical tubers, as well as in tumor cells within SGCTs. In particular mGluR5 appeared to be most frequently expressed, whereas mGluR1alpha was detected in a subpopulation of neurons and giant cells. Cells expressing mGluR1alpha and mGluR5, demonstrate IR for phospho-S6 ribosomal protein (PS6), which is a marker of the mammalian target of rapamycin (mTOR) pathway activation. Group II and particularly group III mGluR IR was less frequently observed than group I mGluRs in dysplastic neurons and giant cells of tubers and tumor cells of SGCTs. Reactive astrocytes were mainly stained with mGluR5 and mGluR2/3. These findings expand our knowledge concerning the cellular phenotype in cortical tubers and in SGCTs and highlight the role of group I mGluRs as important mediators of glutamate signaling in TSC brain lesions. Individual mGluR subtypes may represent potential pharmacological targets for the treatment of the neurological manifestations associated with TSC brain lesions.     

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.     

Regulation of phosphorylation of NMDA receptor NR1 subunits in the rat neostriatum by group I metabotropic glutamate receptors in vivo

Group I metabotropic glutamate receptors (mGluRs) are Gαq-protein-coupled receptors and are densely expressed in medium-sized spiny projection neurons of the neostriatum. Among different subtypes of glutamate receptors, group I mGluRs have been demonstrated to actively interact with the ionotropic glutamate receptor N-methyl-d-aspartate (NMDA) subtypes for regulating various forms of cellular activities and synaptic plasticity. In this study, the possible role of group I mGluRs in regulating serine phosphorylation of NMDA receptor NR1 subunits in the neostriatum was investigated in vivo. We found in chronically cannulated rats that injection of the group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) into the dorsal striatum (caudate putamen) significantly increased phosphorylation of the two serine residues (serine 896 and serine 897) on the intracellular C-terminus of the NR1. The increase in NR1 phosphorylation was dose-dependent and DHPG had no effect on basal levels of NR1 proteins. Intrastriatal infusion of the group I mGluR antagonist N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) significantly attenuated the DHPG-stimulated NR1 phosphorylation. Pretreatment with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) also produced the same effect. These data suggest that group I mGluRs, likely mGluR5 subtypes, possess the ability to upregulate protein phosphorylation of NMDA receptor NR1 subunits in striatal neurons in vivo.     

Characterization of pre- and post-synaptic metabotropic glutamate receptor-mediated inhibitory responses in substantia nigra dopamine neurons

Two inhibitory responses mediated by both pre- and post-synaptic metabotropic glutamate receptors (mGluRs) were investigated in dopamine neurons of the substantia nigra using whole-cell patch recordings. (2R,4R)-APDC, a group II mGluR agonist, and L-2-amino-4-phosphonobutyrate (L-AP4), a group III mGluR agonist, reversibly suppressed the amplitude of excitatory postsynaptic currents (EPSCs). However, (S)-3,5-DHPG, a group I mGluR agonist, exhibited less inhibitory action on the EPSCs. LY341495, a highly potent group II mGluR antagonist, antagonized the broad spectrum mGluR agonist, 1S,3R-ACPD-induced suppression of EPSCs. In acutely dissociated dopamine neurons, glutamate (Glu) in the presence of CNQX and AP-5 evoked an outward current accompanied by an increase in K(+) conductance. (S)-3,5-DHPG, but not (2R,4R)-APDC or L-AP4, also induced an outward current. Glu-induced outward current (I(Glu-out)) was partially inhibited by LY367385, a selective mGluR1 antagonist, but not by MPEP, a selective mGluR5 antagonist. Ryanodine and cyclopiazonic acid blocked the I(Glu-out). In the presence of caffeine, Glu failed to induce a current. Charybdotoxin, but not apamin or iberiotoxin, inhibited the I(Glu-out). Taken together, both group II and III mGluRs are mainly involved in the presynaptic inhibition of Glu release to dopamine neurons, while group I mGluRs, including at least mGluR1, participate in the hyperpolarization of dopamine neurons mediated by the opening of charybdotoxin-sensitive Ca(2+)-activated K(+) channels.     

Group II metabotropic glutamate receptors inhibit glutamate release at thalamocortical synapses in the developing somatosensory cortex

Thalamocortical synapses provide a strong glutamatergic excitation to cortical neurons that is critical for processing sensory information. Unit recordings in vivo indicate that metabotropic glutamate receptors (mGluRs) reduce the effect of thalamocortical input on cortical circuits. However, it is not known whether this reduction is due to a reduction in glutamate release from thalamocortical terminals or from a decrease in cortical neuron excitability. To directly determine whether mGluRs act as autoreceptors on thalamocortical terminals, we examined the effect of mGluR agonists on thalamocortical synapses in slices. Thalamocortical excitatory postsynaptic currents (EPSCs) were recorded in layer IV cortical neurons in developing mouse brain slices. The activation of group II mGluRs with (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) reduced thalamocortical EPSCs in both excitatory and inhibitory neurons, while the stimulation of group I or group III mGluRs had no effect on thalamocortical EPSCs. Consistent with a reduction in glutamate release, DCG IV increased the paired pulse ratio and the coefficient of variation of the EPSCs. The reduction induced by DCG IV was reversed by the group II mGluR antagonist, LY341495, and mimicked by another selective group II agonist, (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid (APDC). The mGluR2 subtype appears to mediate the reduction of thalamocortical EPSCs, since the selective mGluR3 agonist, N-acetylaspartylglutamate (NAAG), had no effect on the EPSCs. Consistent with this, we showed that mGluR2 is expressed in the barrels. Furthermore, blocking group II mGluRs with LY341495 reduced the synaptic depression induced by a short stimulus train, indicating that synaptically released glutamate activates these receptors. These results indicate that group II mGluRs modulate thalamocortical processing by inhibiting glutamate release from thalamocortical synapses. This inhibition provides a feedback mechanism for preventing excessive excitation of cortical neurons that could play a role in the plasticity and refinement of thalamocortical connections during this early developmental period.     

A unilateral 6-hydroxydopamine lesion decreases the expression of metabotropic glutamate receptors in rat substantia nigra

Loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) in Parkinson's disease (PD) leads to augmentation of glutamatergic activity in the subthalamic nucleus (STN). Moreover, antagonizing excitotoxicity has yielded mostly symptomatic improvements in experimental animals of PD. Therefore, we used immunocytochemistry to examine the effect of unilateral 6-hydroxydopamine lesions of SNc on the expression of metabotropic glutamate receptors (mGluR1a, 2/3, 5, 4, 8) and tyrosine hydroxylase (TH). 6-Hydroxydopamine causes a fall in the number of mGluRs and TH in the lesioned lateral substantia nigra. Pharmacological activation of group II or III mGluRs or blockade of group I mGluRs for 1 week significantly increased the expression of the same group receptors. The alteration in the receptor expression may be a compensatory mechanism developed after mGluRs ligands neuroprotective treatment.     

Activation of presynaptic group III metabotropic receptors enhances glutamate release in rat entorhinal cortex

The role of group III metabotropic glutamate receptors (mGluRs) in modulating excitatory synaptic transmission was investigated in the rat entorhinal cortex (EC) in vitro. AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) were recorded in the whole cell configuration of the patch-clamp technique from visually identified neurons in layers V and II. In layer V, bath application of the specific group III mGluR agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 500 microM) resulted in a marked facilitation of both spontaneous and activity-independent "miniature" (s/mEPSC) event frequency. The facilitatory effect of L-AP4 (100 microM) on sEPSC frequency prevailed in the presence of DL-2-amino-5-phosphonopentanoic acid (100 microM) but was abolished by the group III antagonist (RS)-cyclopropyl-4-phosphonophenylglycine (20 microM). These data confirmed that group III mGluRs, and not N-methyl-D-aspartate (NMDA) receptors were involved in the response to L-AP4. Bath application of the specific mGluR4a agonist (1S,3R,4S)-1-aminocyclopentane-1,2, 4-tricarboxylic acid (20 microM) also had a facilitatory effect on sEPSC frequency, suggesting involvement of mGluR4a. In layer II neurons, L-AP4 caused a reduction in sEPSC frequency but did not affect mEPSCs recorded in the presence of tetrodotoxin. These findings suggest that a group III mGluR with mGluR4a-like pharmacology is involved in modulating synaptic transmission in layer V cells of the EC. The effect on mEPSCs suggests that this receptor is located presynaptically and that its activation results in a direct facilitation of glutamate release. This novel facilitatory effect is specific to layer V and, to our knowledge, is the first report of a direct facilitatory action of group III mGluRs on synaptic transmission. In layer II, L-AP4 had an inhibitory effect on glutamate release similar to that reported in other brain regions.     

Protective effect of metabotropic glutamate receptor inhibition on amyotrophic lateral sclerosis-cerebrospinal fluid toxicity in vitro

Conflicting results have been reported concerning the toxicity of cerebrospinal fluid from patients with amyotrophic lateral sclerosis (ALS-CSF) when added to neuronal cultures. The possible toxic factor(s) and the exact mode of action (e.g. requirement of glial cells) have not been identified so far. Glutamate is a potential candidate for this toxic effect, since antagonists of ionotropic glutamate receptors have been shown to attenuate ALS-CSF toxicity. We studied the effects of ALS-CSF on mixed and motoneuron-enriched chick embryonic spinal cord cultures. We found a toxic action of ALS-CSF in both culture types which could not be attenuated by 5 kDa-filtration or 15 min 90 degrees C heating. Nevertheless, the metabotropic glutamate receptor (mGluR) group I antagonist 1-aminoindan-1,5-dicarboxylic acid, but also the group I agonist (s)-3,5-dihydroxyphenylglycine (DHPG) exerted protective effects against ALS-CSF toxicity. In this experimental setting, DHPG may functionally act via a receptor blockade due to sustained activation. No protective effect was seen with the mGluR group III inhibitor (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG). Addition of DHPG did not increase the protective action of the AMPA inhibitor 6-chloro-4-hydroxyquinoline-2-carboxylic acid (6-CKU). Addition of l-glutamate did not mimic these toxic ALS-CSF effects in motoneuron-enriched cultures. Our experiments demonstrate that ALS-CSF toxicity is mediated by a small heat-resistant molecule which may act directly on neurons. Since blockade of group I mGluRs exerts a protective effect, the possibility of targeting these mGluRs pharmacologically in motoneuron disease should be kept in mind.     

The mouse small ubiquitin-like modifier-2 (SUMO-2) inhibits interleukin-12 (IL-12) production in mature dendritic cells by blocking the translocation of the p65 subunit of NFκB into the nucleus

Post-translational modification by small ubiquitin-like modifier (SUMO) is involved in several significant cellular events. In particular, SUMO-1 and SUMO-4 modifications of IκBα have been shown to be actively involved in NFκB regulation. However, among the SUMO family, the specific function of SUMO-2/3 remains relatively unknown. In addition, it is not clear whether SUMO-2/3 follows the same functional role as SUMO-1 and SUMO-4 during the activation of NFκB. In this study, we examined the influence of mouse SUMO-2 during the maturation of dendritic cells (DCs). Our results showed that the ectopic expression of SUMO-2 does not affect the cell surface expression of MHC class II molecule (A(b)) and co-stimulatory molecules (CD80 and CD86), and the efficiency of antigen uptake. However, the ectopic expression of mouse SUMO-2 inhibited IL-12 secretion by blocking the translocation of the p65 subunit of NFκB into the nucleus, which led to the polarization of na?ve CD4(+) T cells to T helper 2 (Th2) shift in vitro. Further analyses showed that SUMO-2 directly modified IκBα. These results indicate that the functional role of SUMO-2/3 in the regulation of NFκB activity was conserved during evolution.     

SUMOylation attenuates sensitivity toward hypoxia- or desferroxamine-induced injury by modulating adaptive responses in salivary epithelial cells

Hypoxic stress activates various signal transduction pathways including posttranslational modification with the ubiquitin-like SUMO protein (SUMOylation). However, the molecular mechanisms by which SUMOylation regulates hypoxic responses remain unclear. Here, we investigated the ability of rat salivary Pa-4 epithelial cells to resist cell injury elicited by 1% O(2)- or hypoxia-mimetic desferroxamine (DFO)-stimulated SUMOylation processes. By using Pa-4 cells stably transduced with lenti-SUMO-1 and a cell-permeant peptide harboring SUMO-binding motif to interfere with SUMO-dependent protein-protein interactions, we demonstrate that SUMOylation augments cell survival against DFO treatment. This appeared to be partly mediated through attenuation of Protein Kinase C (PKC)-delta activation and caspase-3 cleavage, hallmarks of pro-apoptotic signaling. Intriguingly, DFO-induced phosphorylation of DNA damage marker ataxia-telangiectasia-mutated protein S1981 preceded activation of PKCdelta and caspase-3. Constitutive SUMOylation facilitated 1% O(2)- or DFO-induced nuclear factor kappaB transactivation, possibly via activation of genotoxic signaling cascade. In addition, we observed transient preservation of transepithelial electrical resistance during the early stage of hypoxia (1% O(2)) as well as enhanced transepithelial electrical resistance recovery after prolonged hypoxia in SUMO-1-expressing cell monolayers. In conclusion, our results unveil a previously unrecognized mechanism by which SUMOylation and activation of ataxia-telangiectasia-mutated protein, PKCdelta, caspase-3, and nuclear factor kappaB signaling pathways modulate salivary adaptive responses to stress in cells exposed to either 1% O(2) or DFO.     

Distribution and synaptic localisation of the metabotropic glutamate receptor 4 (mGluR4) in the rodent CNS

Group III metabotropic glutamate receptors (mGluRs) are selectively activated by L-2-amino-4-phosphonobutyrate (L-AP4), which produces depression of synaptic transmission. The relative contribution of different group III mGluRs to the effects of L-AP4 remains to be clarified. Here, we assessed the distribution of mGluR4 in the rat and mouse brain using affinity-purified antibodies raised against its entire C-terminal domain. The antibodies reacted specifically with mGluR4 and not with other mGluRs in transfected COS 7 cells. No immunoreactivity was detected in brains of mice with gene-targeted deletion of mGluR4. Pre-embedding immunocytochemistry for light and electron microscopy showed the most intense labelling in the cerebellar cortex, basal ganglia, the sensory relay nuclei of the thalamus, and some hippocampal areas. Immunolabelling was most intense in presynaptic active zones. In the basal ganglia, both the direct and indirect striatal output pathways showed immunolabelled terminals forming mostly type II synapses on dendritic shafts. The localisation of mGluR4 on GABAergic terminals of striatal projection neurones suggests a role as a presynaptic heteroreceptor. In the cerebellar cortex and hippocampus, mGluR4 was also localised in terminals establishing type I synapses, where it probably operates as an autoreceptor. In the hippocampus, mGluR4 labelling was prominent in the dentate molecular layer and CA1-3 strata lacunosum moleculare and oriens. Somatodendritic profiles of some stratum oriens/alveus interneurones were richly decorated with mGluR4-labelled axon terminals making either type I or II synapses. This differential localisation suggests a regulation of synaptic transmission via a target cell-dependent synaptic segregation of mGluR4.Our results demonstrate that, like other group III mGluRs, presynaptic mGluR4 is highly enriched in the active zone of boutons innervating specific classes of neurones. In addition, the question of alternatively spliced mGluR4 isoforms is discussed.