Role of fragile X messenger ribonucleoprotein 1 in the pathophysiology of brain disorders: a glia perspective.

Fragile X messenger ribonucleoprotein 1 (FMRP) is a widely expressed RNA binding protein involved in several steps of mRNA metabolism. Mutations in the FMR1 gene encoding FMRP are responsible for fragile X syndrome (FXS), a leading genetic cause of intellectual disability and autism spectrum disorder, and fragile X-associated tremor-ataxia syndrome (FXTAS), a neurodegenerative disorder in aging men. Although FMRP is mainly expressed in neurons, it is also present in glial cells and its deficiency or altered expression can affect functions of glial cells with implications for the pathophysiology of brain disorders. The present review focuses on recent advances on the role of glial subtypes, astrocytes, oligodendrocytes and microglia, in the pathophysiology of FXS and FXTAS, and describes how the absence or reduced expression of FMRP in these cells can impact on glial and neuronal functions. We will also briefly address the role of FMRP in radial glial cells and its effects on neural development, and gliomas and will speculate on the role of glial FMRP in other brain disorders.

Fragile X mental retardation protein (FMRP) and metabotropic glutamate receptor subtype 5 (mGlu5) control stress granule formation in astrocytes.

Fragile X syndrome (FXS) is a common form of intellectual disability and autism caused by the lack of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in RNA transport and protein synthesis. Upon cellular stress, global protein synthesis is blocked and mRNAs are recruited into stress granules (SGs), together with RNA-binding proteins including FMRP. Activation of group-I metabotropic glutamate (mGlu) receptors stimulates FMRP-mediated mRNA transport and protein synthesis, but their role in SGs formation is unexplored. To this aim, we pre-treated wild type (WT) and Fmr1 knockout (KO) cultured astrocytes with the group-I-mGlu receptor agonist (S)-3,5-Dihydroxyphenylglycine (DHPG) and exposed them to sodium arsenite (NaAsO2), a widely used inducer of SGs formation. In WT cultures the activation of group-I mGlu receptors reduced SGs formation and recruitment of FMRP into SGs, and also attenuated phosphorylation of eIF2α, a key event crucially involved in SGs formation and inhibition of protein synthesis. In contrast, Fmr1 KO astrocytes, which exhibited a lower number of SGs than WT astrocytes, did not respond to agonist stimulation. Interestingly, the mGlu5 receptor negative allosteric modulator (NAM) 2-methyl-6-(phenylethynyl)pyridine (MPEP) antagonized DHPG-mediated SGs reduction in WT and reversed SGs formation in Fmr1 KO cultures. Our findings reveal a novel function of mGlu5 receptor as modulator of SGs formation and open new perspectives for understanding cellular response to stress in FXS pathophysiology.

Endothelin-1 Induces Degeneration of Cultured Motor Neurons Through a Mechanism Mediated by Nitric Oxide and PI3K/Akt Pathway.

Endothelin-1 (ET-1) is a vasoactive peptide produced by activated astrocytes and microglia and is implicated in initiating and sustaining reactive gliosis in neurodegenerative diseases. We have previously suggested that ET-1 can play a role in the pathophysiology of amyotrophic lateral sclerosis (ALS). Indeed, we reported that this peptide is abundantly expressed in reactive astrocytes in the spinal cord of SOD1-G93A mice and ALS patients and exerts a toxic effect on motor neurons (MNs) in an in vitro model of mixed spinal cord cultures enriched with reactive astrocytes. Here, we explored the possible mechanisms underlying the toxic effect of ET-1 on cultured MNs. We show that ET-1 toxicity is not directly caused by oxidative stress or activation of cyclooxygenase-2 but requires the synthesis of nitric oxide and is mediated by a reduced activation of the phosphoinositide 3-kinase pathway. Furthermore, we observed that ET-1 is also toxic for microglia, although its effect on MNs is independent of the presence of this type of glial cells. Our study confirms that ET-1 may contribute to MN death and corroborates the view that the modulation of ET-1 signaling might be a therapeutic strategy to slow down MN degeneration in ALS.

Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in early and late developmental periods. In particular, the RNA-binding and cytoskeleton remodeling functions of FMRP may be differently modulated during development.

5-HT(1A) and 5-HT(7) receptors differently modulate AMPA receptor-mediated hippocampal synaptic transmission.

We have studied the effects of 5-HT(1A) and 5-HT(7) serotonin receptor activation in hippocampal CA3-CA1 synaptic transmission using patch clamp on mouse brain slices. Application of either 5-HT or 8-OH DPAT, a mixed 5-HT(1A)/5-HT(7) receptor agonist, inhibited AMPA receptor-mediated excitatory post synaptic currents (EPSCs); this effect was mimicked by the 5-HT(1A) receptor agonist 8-OH PIPAT and blocked by the 5-HT(1A) antagonist NAN-190. 8-OH DPAT increased paired-pulse facilitation and reduced the frequency of mEPSCs, indicating a presynaptic reduction of glutamate release probability. In another group of neurons, 8-OH DPAT enhanced EPSC amplitude but did not alter paired-pulse facilitation, suggesting a postsynaptic action; this effect persisted in the presence of NAN-190 and was blocked by the 5-HT(7) receptor antagonist SB-269970. To confirm that EPSC enhancement was mediated by 5-HT(7) receptors, we used the compound LP-44, which is considered a selective 5-HT(7) agonist. However, LP-44 reduced EPSC amplitude in most cells and instead increased EPSC amplitude in a subset of neurons, similarly to 8-OH DPAT. These effects were respectively antagonized by NAN-190 and by SB-269970, indicating that under our experimental condition LP-44 behaved as a mixed agonist. 8-OH DPAT also modulated the current evoked by exogenously applied AMPA, inducing either a reduction or an increase of amplitude in distinct neurons; these effects were respectively blocked by 5-HT(1A) and 5-HT(7) receptor antagonists, indicating that both receptors exert a postsynaptic action. Our results show that 5-HT(1A) receptors inhibit CA3-CA1 synaptic transmission acting both pre- and postsynaptically, whereas 5-HT(7) receptors enhance CA3-CA1 synaptic transmission acting exclusively at a postsynaptic site. We suggest that a selective pharmacological targeting of either subtype may be envisaged in pathological loss of hippocampal-dependent cognitive functions. In this respect, we underline the need for new selective agonists of 5-HT(7) receptors.

Expression of multidrug resistance type 1 gene (MDR1) P-glycoprotein in intractable epilepsy with different aetiologies: a double-labelling and electron microscopy study.

The objective of this study was to analyse the clinical characteristics, pathological features and expression patterns of multiple drug resistance type 1 (MDR1) and glial fibrillary acidic protein (GFAP) in intractable epilepsy patients with variable aetiologies and to analyse the relationships between the clinical and pathological findings. Twenty-six patients (15 males, 11 females, age range 4-25 years, mean age 22.92 years, SD 11.19 years) with intractable epilepsy were included in this study; the clinical characteristics were considered, and the pathological changes as well as expression of MDR1 and GFAP in surgically removed brain tissues of each subject were examined under light and electron microscopy. All patients presented a long-lasting, refractory epilepsy, mostly of the partial type, due to different causes, such as trauma, vascular injuries, encephalitis, cortical dysplasia, cavernous angioma and Sturge-Weber disease. Neuronal degenerative damage, reactive proliferation of astrocytes, as well as overexpression of GFAP and MDR1, appeared as common pathological features in all cases. The detection of MDR1 by electron microscopy allowed us to precisely define its cellular location in reactive astrocytes and to exclude the presence of the antigen in other cellular types. In all cases, pathological features, at both light and electron microscopy, were similar, independent of the different clinical presentation and aetiology.

Expression of pannexin1 in the CNS of adult mouse: cellular localization and effect of 4-aminopyridine-induced seizures.

The expression pattern of pannexin1, a gene coding for a protein that forms gap junction channels, was studied as both mRNA and protein in the CNS of adult mouse. Pannexin1 was widely expressed in the CNS by neuronal cell types but not glial cells, except for Bergmann glial cells of the cerebellar cortex. Cells positive to Ca-binding proteins, principally parvalbumin, but also calbindin and calretinin, as well as glutamate decarboxylase 67 kDa isoform, were pannexin1-positive. Pannexin1 labeling was found in cells which are known to exhibit spontaneous and synchronous discharge, such as neurons of the inferior olivary complex and the reticular thalamic nucleus, and also in neurons whose electrical activity is not coupled with neighboring cells, such as motoneurons of the spinal cord. The analysis of cellular localization showed puncta that surrounded cell bodies (e.g. the pyramidal cells of hippocampus) or restricted areas inside the cell bodies (e.g. the spinal motoneurons). In Bergmann glial cells the staining was present as fine grains that covered a large part of the cellular surface. Pannexin1 stained cells that previous studies have reported as expressing connexin36, another protein forming gap junction channels. Thus, it was possible that these two proteins could be integrated in the same functions. Since connexin36 expression levels change after seizures, we examined the expression of both pannexin1 and connexin36 in cerebral cortex, hippocampus, cerebellum and brain stem at different time intervals (2, 4 and 8 h) after i.p. injection of 4-aminopyridine, which resulted in systemic seizures. The only modification of the expression levels observed in this study concerned the progressive decrement of the connexin36 in the hippocampus, while pannexin1 expression was unchanged. This finding suggested that pannexin1 and connexin36 are involved in different functional roles or that they are expressed in different cell types and that only those expressing the Cx36 are induced to apoptosis by epileptic seizures.

Object recognition impairment in Fmr1 knockout mice is reversed by amphetamine: involvement of dopamine in the medial prefrontal cortex.

Fragile X syndrome is an X-linked form of mental retardation including, among others, symptoms such as stereotypic behaviour, hyperactivity, hyperarousal, and cognitive deficits. We hypothesized that hyperactivity and/or compromised attentional, cognitive functions may lead to impaired performance in cognitive tasks in Fmr1 knockout mice, the most widely used animal model of fragile X syndrome, and suggested that psychostimulant treatment may improve performance by acting on one or both components. Since hyperactivity and cognitive functions have been suggested to depend on striatal and prefrontal cortex dopaminergic dysfunction, we assessed whether amphetamine produced beneficial, positive effects by acting on dopaminergic corticostriatal systems. Our results show that Fmr1 knockout mice are not able to discriminate between a familiar object and a novel one in the object recognition test, thus showing a clear-cut cognitive impairment that, to date, has been difficult to demonstrate in other cognitive tasks. Amphetamine improved performance of Fmr1 knockout mice, leading to enhanced ability to discriminate novel versus familiar objects, without significantly affecting locomotor activity. In agreement with behavioural data, amphetamine produced a greater increase in dopamine release in the prefrontal cortex of Fmr1 knockout compared with the wild-type mice, while a weak striatal dopaminergic response was observed in Fmr1 knockout mice. Our data support the view that the psychostimulant ameliorates performance in Fmr1 knockout mice by improving merely cognitive functions through its action on prefrontal cortical dopamine, irrespective of its action on motor hyperactivity. These results indicate that prefrontal cortical dopamine plays a major role in cognitive impairments characterizing Fmr1 knockout mice, thus pointing to an important aetiological factor in the fragile X syndrome.

An increased expression of the mGlu5 receptor protein following LTP induction at the perforant path-dentate gyrus synapse in freely moving rats.

The involvement of metabotropic glutamate (mGlu) receptors in the induction of long-term potentiation (LTP) in vivo has been consistently documented. We have investigated whether LTP induction in the dentate gyrus of rats leads to changes in expression of mGlu2/3 or -5 receptor subtypes in the hippocampus. LTP was induced at the medial perforant path-dentate gyrus synapses, and mGlu receptor expression was examined by Western blot or in situ hybridization. An up-regulation of mGlu5 receptors was observed in the hippocampus both 24 and 48 h following LTP induction. This effect was restricted to the dentate gyrus and CA1 region, whereas no changes in mGlu5 receptor protein (but an increase in mRNA levels) were observed in the CA3 region. The increased expression of mGlu5 receptors was directly related to the induction of LTP, because it was not observed when tetanic stimulation was carried out in animals treated with the NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5). Western blot analysis also showed a reduced expression of mGlu2/3 receptors in the whole hippocampus 24 h after LTP induction, indicating that the increased expression of mGlu5 receptors was specific. These data suggest that an up-regulation of mGlu5 receptors is a component of the plastic changes that follow the induction of LTP at the perforant path-dentate gyrus synapse.

L-Acetylcarnitine induces analgesia by selectively up-regulating mGlu2 metabotropic glutamate receptors.

L-Acetylcarnitine (LAC, 100 mg/kg, s.c.), a drug commonly used for the treatment of painful neuropathies, substantially reduced mechanical allodynia in rats subjected to monolateral chronic constriction injury (CCI) of the sciatic nerve and also attenuated acute thermal pain in intact rats. In both cases, induction of analgesia required repeated injections of LAC, suggesting that the drug induces plastic changes within the nociceptive pathway. In both CCI- and sham-operated rats, a 24-day treatment with LAC increased the expression of metabotropic glutamate (mGlu) receptors 2 and 3 in the lumbar segment of the spinal cord, without changing the expression of mGlu1a or -5 receptors. A similar up-regulation of mGlu2/3 receptors was detected in the dorsal horns and dorsal root ganglia of intact rats treated with LAC for 5-7 days, a time sufficient for the induction of thermal analgesia. Immunohistochemical analysis showed that LAC treatment enhanced mGlu2/3 immunoreactivity in the inner part of lamina II and in laminae III and IV of the spinal cord. An increased mGlu2/3 receptor expression was also observed in the cerebral cortex but not in the hippocampus or cerebellum of LAC-treated animals. Reverse transcription-polymerase chain reaction combined with Northern blot analysis showed that repeated LAC injections selectively induced mGlu2 mRNA in the dorsal horns and cerebral cortex (but not in the hippocampus). mGlu3 mRNA levels did not change in any brain region of LAC-treated animals. To examine whether the selective up-regulation of mGlu2 receptors had any role in LAC-induced analgesia, we have used the novel compound LY 341495, which is a potent and systemically active mGlu2/3 receptor antagonist. LAC-induced analgesia was largely reduced 45 to 75 min after a single injection of LY 341495 (1 mg/kg, i.p.) in both CCI rats tested for mechanical allodynia and intact rats tested for thermal pain. We conclude that LAC produces analgesia against chronic pain produced not only by peripheral nerve injury but also by acute pain in intact animals and that LAC-induced analgesia is associated with and causally related to a selective up-regulation of mGlu2 receptors. This offers the first example of a selective induction of mGlu2 receptors and discloses a novel mechanism for drug-induced analgesia.

Immunohistochemical localization of group I and II metabotropic glutamate receptors in control and amyotrophic lateral sclerosis human spinal cord: upregulation in reactive astrocytes.

Excitotoxicity, which is mediated by the excessive activation of glutamate receptors, has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). There is substantial information about the distribution and function of ionotropic glutamate receptors in the spinal cord, although the role of metabotropic glutamate receptors (mGluRs) is poorly understood in this region of the brain, particularly under pathological conditions. We used immunocytochemistry to study the general distribution of group I and group II mGluR immunoreactivity in the human spinal cord, as well as the cell-specific expression of these receptors. We also investigated whether mGluR expression was altered in the spinal cord of patients with sporadic and familial ALS. Immunocytochemical analysis of control human spinal cord demonstrated that mGluR1alpha and mGluR5 (group I mGluRs) were highly represented in neuronal cells throughout the spinal cord. mGluR1alpha showed the highest relative level of expression in ventral horn neurons (laminae VIII and IX), whereas intense mGluR5 immunoreactivity was observed within the dorsal horn (superficial laminae I and II). Group II mGluRs (mGluR2/3) immunoreactivity was mainly concentrated in the inner part of the lamina II. With respect to specific neuronal populations, mGluR2/3 and mGluR5 appeared to be most frequently expressed in calbindin-containing and calretinin-containing cells, respectively. In control spinal cord only sparse astrocytes showed a weak to moderate mGluR immunoreactivity. Regional differences in immunoreactivity were apparent in ALS compared to control. In particular, mGluR expression was increased in reactive glial cells in both gray (ventral horn) and white matter of ALS spinal cord. Upregulation of mGluRs in reactive astrocytes may represent a critical mechanism for modulation of glial function and changes in glial-neuronal communication in the course of neurodegenerative diseases.

Endogenous activation of group-I metabotropic glutamate receptors is required for differentiation and survival of cerebellar Purkinje cells.

We have applied subtype-selective antagonists of metabotropic glutamate (mGlu) receptors mGlu1 or mGlu5 [7-(hydroxy-imino) cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) or 2-methyl-6-(phenylethynyl)pyridine (MPEP)] to mixed rat cerebellar cultures containing both Purkinje and granule cells. The action of these two drugs on neuronal survival was cell specific. Although CPCCOEt (1, 10, 30 microm) reduced the survival of Purkinje cells, MPEP (3 or 30 microm) selectively reduced the survival of granule cells. Both effects required an early exposure of cultures to antagonists [from 3 to 6 d in vitro (DIV) for CPCCOEt, and from 3 to 6 or 6 to 9 DIV for MPEP]. Addition of MPEP from 6 to 9, 9 to 13, or 13 to 17 DIV also induced profound morphological changes in the dendritic tree and dendritic spines of Purkinje cells, suggesting that endogenous activation of mGlu5 receptors is required for the age-dependent refinement of Purkinje cell phenotype. In in vivo studies, an early blockade of mGlu1 receptors induced in rats by local injections of LY367385 (20 nmol/2 microl), local injections of mGlu1 antisense oligonucleotides (12 nmol/2 microl), or systemic administration of CPCCOEt (5 mg/kg, s.c.) from postnatal day (P) 3 to P9 reduced the number and dramatically altered the morphology of cerebellar Purkinje cells. In contrast, mGlu5 receptor blockade induced by local injections of antisense oligonucleotides reduced the number of granule cells but also produced substantial morphological changes in the dendritic tree of Purkinje cells. These results provide the first evidence that the development of cerebellar neurons is under the control of mGlu1 and mGlu5 receptors, i.e., the two mGlu receptor subtypes coupled to polyphosphoinositide hydrolysis.

Selective blockade of type-1 metabotropic glutamate receptors induces neuroprotection by enhancing gabaergic transmission.

Selective antagonists of mGlu1 (LY367385 and CPCCOEt) and mGlu5 (MPEP) metabotropic glutamate receptors were neuroprotective against NMDA toxicity when either applied to mixed cortical cultures or locally infused into the caudate nucleus. Neuroprotection produced by LY367385 or CPCCOEt was occluded by GABA and was abolished by a cocktail of GABA(A) and GABA(B) receptor antagonists. In contrast, GABAergic drugs did not influence the action of MPEP. In microdialysis studies, LY367385 and CPCCOEt substantially enhanced GABA release in the corpus striatum of freely moving animals, whereas MPEP had no effect on GABA but abolished the stimulation of glutamate release induced by NMDA. A role for mGlu1 receptors in modulating GABAergic transmission was supported by electrophysiological studies carried out in cortico-striatal slices. In this particular model, the mixed mGlu1/5 receptor agonist, DHPG, reduced bicuculline-sensitive inhibitory postsynaptic currents presumably via a presynaptic mechanism. The action of DHPG was antagonized by LY367385, but not by MPEP. Taken together, these results indicate that selective blockade of mGlu1 receptors produces neuroprotection by enhancing GABAergic transmission.

Increased expression of neuronal nitric oxide synthase spliced variants in reactive astrocytes of amyotrophic lateral sclerosis human spinal cord.

The expression of three different neuronal nitric oxide synthase (nNOS) spliced variants, named nNOSalpha, nNOSbeta, and nNOSgamma, was investigated in the spinal cord of control and both familiar and sporadic amyotrophic lateral sclerosis (FALS and SALS) patients. Western blot analysis showed a consistent increase in nNOS expression in six SALS patients compared with controls when antibodies recognizing both nNOSalpha and nNOSbeta, or nNOSalpha, nNOSbeta, and nNOSgamma were used, whereas no change was observed when a selective anti-nNOSalpha antibody was used. Immunoreactivity signal for nNOSalpha-beta-gamma and nNOSalpha-beta was equally present in ventral horn neurons of control and ALS spinal cord but was dramatically increased in reactive astrocytes of the ventral horn and white matter in both FALS and SALS. nNOSalpha signal was equally expressed in motor neurons of normal and ALS spinal cord but was not evident in astrocytes. This finding indicates that nNOSbeta and nNOSgamma spliced variants are upregulated in reactive astrocytes in ALS. This may contribute to the peroxynitrite-mediated oxidative damage involved in the pathogenesis of both FALS and SALS.

Selective activation of mGlu4 metabotropic glutamate receptors is protective against excitotoxic neuronal death.

Activation of group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, and mGluR8) has been established to be neuroprotective in vitro and in vivo. To disclose the identity of the receptor subtype(s) that exert(s) the protective effect, we have used group III agonists in combination with mGluR4 subtype-deficient mice (-/-). In cortical cultures prepared from wild-type (+/+) mice and exposed to a toxic pulse of NMDA, the selective group III agonist (+)-4-phosphonophenylglycine [(+)-PPG] reversed excitotoxicity with an EC(50) value of 4.9 microm, whereas its enantiomer (-)-PPG was inactive. This correlated closely with the potency of (+)-PPG in activating recombinant mGluR4a. In cortical neurons from -/- mice, (+)-PPG showed no protection against the NMDA insult up to 300 microm, whereas group I/II mGluR ligands still retained their protective activity. Classical group III agonists (l-2-amino-4-phosphonobutyrate and l-serine-O-phosphate) were also substantially neuroprotective against NMDA toxicity in +/+ and heterozygous (+/-) cultures but were inactive in -/- cultures. Interestingly, -/- cultures were more vulnerable to low concentrations of NMDA and showed higher extracellular glutamate levels compared with +/+ cultures. We have also examined neurodegeneration induced by intrastriatal infusion of NMDA in wild-type or mGluR4-deficient mice. Low doses of (R,S)-PPG (10 nmol/0.5 microl) substantially reduced NMDA toxicity in +/+ mice but were ineffective in -/- mice. Higher doses of (R,S)-PPG were neuroprotective in both strains of animals. Finally, microdialysis studies showed that intrastriatal infusion of NMDA increased extracellular glutamate levels to a greater extent in -/- than in +/+ mice, supporting the hypothesis that the mGluR4 subtype is necessary for the maintenance of the homeostasis of extracellular glutamate levels.

Group-I metabotropic glutamate receptors: hypotheses to explain their dual role in neurotoxicity and neuroprotection.

The role of group-I metabotropic glutamate receptors (mGlu1 and 5) in neurodegeneration is still controversial. While antagonists of these receptors are consistently neuroprotective, agonists have been found to either amplify or attenuate excitotoxic neuronal death. At least three variables affect responses to agonists: (i) the presence of the NR2C subunit in the NMDA receptor complex; (ii) the existence of an activity-dependent functional switch of group-I mGlu receptors, similar to that described for the regulation of glutamate release; and (iii) the presence of astrocytes expressing mGlu5 receptors. Thus, a number of factors, including the heteromeric composition of NMDA receptors, the exposure time to drugs or to ambient glutamate, and the function of astrocytes clearing extracellular glutamate and producing neurotoxic or neuroprotective factors, must be taken into account when examining the role of group-I mGlu receptors in neurodegeneration/neuroprotection.

An enhanced expression of the immediate early gene, Egr-1, is associated with neuronal apoptosis in culture.

Cultured cerebellar granule cells grown in medium containing 10 mM K+ (K10) underwent apoptosis after four to five days in vitro, unless they were rescued by the addition of insulin-like growth factor-I. The few GABAergic neurons present in the cultures were more resistant to apoptotic degeneration, as indicated by double fluorescent staining with the chromatin dye Hoechst 33258 and with glutamate decarboxylase-67 antibodies. As compared with sister cultures grown in 25 mM K+, K10 cultures showed an increased expression of the Egr-1 protein and a reduced expression of the Fos protein. The increase in Egr-1 was more substantial in granule cells than in GABAergic neurons, and was not observed in K10 cultures chronically exposed to insulin-like growth factor-I. To examine the temporal relationship between the increase in Egr-1 and the development of programmed cell death, we induced apoptosis in K25 cultures at six days in vitro by replacing their medium with serum-free K10 medium. A substantial, but transient, increase in Egr- expression was observed in granule cells 6 h after switching the medium, a time that preceded the appearance of the phenotypical markers of apoptotic death. An early reduction in the Fos protein was observed after switching the medium from K25 into serum-free K10, but also after switching the medium into serum-free K25, a condition which was not associated with the development of apoptosis nor with the increase in Egr-1. We suggest that a transient induction of Egr-1 contributes to the chain of events leading to the execution phase of neuronal apoptosis in culture.

Neuroprotective activity of the potent and selective mGlu1a metabotropic glutamate receptor antagonist, (+)-2-methyl-4 carboxyphenylglycine (LY367385): comparison with LY357366, a broader spectrum antagonist with equal affinity for mGlu1a and mGlu5 receptors.

(+)-2-Methyl-4-carboxyphenylglycine (LY367385), a potent and selective antagonist of mGlu1a metabotropic glutamate receptors, was neuroprotective in the following in vitro and in vivo models of excitotoxic death: (i) mixed cultures of murine cortical cells transiently exposed to N-methyl-D-aspartate (NMDA); (ii) rats monolaterally infused with NMDA into the caudate nucleus; and (iii) gerbils subjected to transient global ischemia. We have compared the activity of LY367385 with that of the novel compound (+/-)-alpha-thioxantylmethyl-4-carboxyphenylglycine (LY367366), which antagonizes both mGlu1a and -5 receptors at low micromolar concentrations, but also recruits other subtypes at higher concentrations. Although LY367366 was neuroprotective, it was in general less efficacious than LY357385, suggesting that inhibition of mGlu1 receptors is sufficient to confer significant neuroprotection. We conclude that endogenous activation of mGlu1a receptors (or perhaps other mGlu1 receptor splice variants) contributes to the development of neuronal degeneration of excitotoxic origin.

AMPA receptor subunits are differentially expressed in parvalbumin- and calretinin-positive neurons of the rat hippocampus.

Recent studies suggest a functional diversity of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)- and calretinin (CR)-positive cells, which represent different populations of non-principal cells. To this end, non-radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double-immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV-containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR-A and GluR-D mRNAs, moderate levels of GluR-C and low levels of GluR-B mRNAs in all the examined regions of hippocampus. The vast majority of CR-containing cells have a much lower expression of GluR-A, -C and -D mRNA than PV-positive neurons, although similarly featuring low levels of GluR-B mRNA. Only a subpopulation of CR-containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR-A and -D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR-B subunit expression, PV- and CR-containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.

Deafferentation up-regulates the expression of the mGlu1a metabotropic glutamate receptor protein in the olfactory bulb.

Chemical lesion of olfactory neuroepitheium induced an up-regulation of the mGlu1a metabotropic glutamate receptor protein in the olfactory bulb, as shown by Western blot analysis. At 2 days after the lesion, the increase in the receptor protein was associated with an increase in mGlu1a mRNA levels; in contrast, at longer times after the lesion (16 days), mRNA levels were reduced in spite of the high expression of the receptor protein, perhaps as a result of product-inhibition of mGlu1 gene expression. Immunohistochemical analysis showed that the increase in mGlu1a induced by olfactory denervation was confined to the glomeruli, which occupy the external portion of the olfactory bulb. Within these structures, mGlu1a receptors are mainly localized on the distal dendrites of mitral cells, which are innervated by the glutamatergic axons of the olfactory nerve. These results demonstrate that the expression of mGlu1a receptors is up-regulated in response to glutamatergic deafferentation, supporting a role for this particular receptor subtype in the physiology of synaptic transmission.