Metabotropic glutamate receptor-mediated signaling in neuroglia

Abstract

Metabotropic glutamate (mGlu) receptors are G-protein-coupled receptors, which include eight subtypes that have been classified into three groups (I–III) based upon sequence homology, signal transduction mechanism and pharmacological profile. Although most studied with regard to neuronal function and modulation, mGlu receptors are also expressed by neuroglia-including astrocytes, microglia and oligodendrocytes. Activation of mGlu receptors on neuroglia under both physiologic and pathophysiologic conditions mediates numerous actions that are essential for intrinsic glial cell function, as well as for glial–neuronal interactions. Astrocyte mGlu receptors play important physiological roles in regulating neurotransmission and maintaining neuronal homeostasis. However, mGlu receptors on astrocytes and microglia also serve to modulate cell death and neurological function in a variety of pathophysiological conditions such as acute and chronic neurodegenerative disorders. The latter effects are complex and bi-directional, depending on which mGlu receptor sub-types are activated.

Neuroglia comprise over 70% of the total cell population in the brain. Originally neuroglia were classified into three types: astrocytes, oligodendrocytes and microglia, but recently this classification has been expanded to include a newly characterized NG2-glial cell or syanantocyte (1). Microglia serve as the intrinsic defense system of the brain and are the primary mediators of the brain’s innate immune response to infection, injury and disease. Under physiologic conditions microglia are spread throughout the brain parenchyma in a resting status and constantly survey their microenvironment for noxious agents and injurious processes (2, 3). They respond to extracellular signals and are responsible for clearing cellular debris and toxic substances by phagocytosis, thereby maintaining normal cellular homeostasis in the CNS (4). Astrocytes are engaged in key aspects of neuronal function such as trophic support, neuronal differentiation, neurite outgrowth, and synaptic efficacy. Their activities are essential for maintaining CNS homeostasis, regulating the local concentration of ions and neuroactive substances (5, 6). The protoplasmic astrocytes create a structural parcellation of the gray matter; astrocyte micro-domains, characterized by elaborated arborization of processes, establish independent structural units (7). Within these structural micro-domains astroglial membranes cover synapses and establish contacts with neuronal membranes (8)and blood vessels, thereby integrating the neural circuitry with local blood flow and participating in the neurovascular unit (9).

Oligodendrocytes play a key role in neurotransmission as neuronal isolators. They produce myelin, which facilitates the propagation of action potentials. In addition, oligodendrocytes express neurotransmitter receptors and are able to sense neuronal activity (10). The function of NG2-glial cells is currently being elucidated. These cells receive synaptic contacts from neurons and may regulate neuronal physiology (11); they are highly reactive and may also participate in gliogenesis, myelination and synaptic plasticity (12).

Glutamate is the major excitatory amino acid transmitter in the brain. It is released from presynaptic vesicles and activates postsynaptic ligand-gated ion channel (iGlu) receptors (NMDA, AMPA and kainate receptors) to ensure fast synaptic transmission (13). Glutamate also activates metabotropic glutamate (mGlu) receptors, which in a feedback loop modulate its release and postsynaptic response, as well as the activity at other synapses (14). The mGlu receptors belong to the G-protein-coupled receptors (GPCRs) superfamily. Eight mGlu receptor subtypes have been identified and based upon sequence homology, signal transduction mechanism and pharmacological profile have been classified into three groups (I–III) (figure 1). Group I include mGlu1 and mGlu5 receptors, which couple to G_q and activate phospholipase C (PLC). Group I receptors are primarily localized postsynaptically, and upon activation they increase excitability. Group II (mGlu2, mGlu3) and group III (mGlu4, mGlu6, mGlu7 and mGlu8) receptors couple to G_i/G_o and inhibit adenylyl cyclase (AC); in contrast to group I receptors, group II/III receptors are generally presynaptic and their activation reduces glutamate release decreasing excitability. The mGlu receptors modulate synaptic transmission and are involved in activity-dependent changes in synaptic transmission such as long-term potentiation and long-term depression (14). As such, mGlu receptors are recognized as promising therapeutic targets (15). In fact, a number of mGlu receptor ligands are now under clinical development for the treatment of a variety of disorders such as Fragile-X syndrome, schizophrenia, Parkinson’s disease and L-DOPA-induced dyskinesias, generalized anxiety disorder, and chronic pain (16).

Figure 1.

Classification of the eight subtypes of mGlu receptors and their pharmacological modulators.

mGlu receptor expression and signal transduction pathways in neuroglia

mGlu receptor subtypes are expressed in well-defined patterns throughout the brain. Considerable research has focused on characterizing and defining the functional roles of neuronal mGlu receptors in modulating synaptic plasticity and in neurological disorders (16). In neurons, mGlu1 and mGlu5 are expressed in post-synaptic densities, whereas mGlu2, mGlu3, mGlu7, and mGlu8 receptors are primarily expressed pre-synaptically where they regulate neurotransmitter release (17). In addition, mGlu receptors are expressed in neuroglia (Figure 2), where their activation exerts numerous effects that are crucial for glial cell function and glial–neuronal interactions under both physiologic and pathophysiologic conditions.

Figure 2.

Characteristics of mGlu receptors in neuroglia.

Astrocyte mGlu receptors

Astrocytes express many different mGlu receptor subtypes. In vitro and in vivo studies indicate that mGlu5 receptors are highly expressed in brain astrocytes (18), although the expression of mGlu5 appears to decrease during development (19). Although mGlu1 receptors are not detected on cultured cortical astrocytes (20–22), they are expressed on astrocytes prepared from the spinal cord (23), as well as spinal cord samples from patients with amyotrophic lateral sclerosis (ALS)(24). The expression of mGlu5 receptors is modulated by extracellular signals: cultured astrocytes grown in conventional serum-containing medium show low expression, whereas expression is up-regulated when cells are cultured in medium containing growth factors such as basic fibroblast growth factor, epidermal growth factor, or transforming growth factor-α(20, 22). It has been suggested that the expression profile induced by the growth medium mimics the activation of astrocytes during reactive astrogliosis. Accordingly, immunohistochemical analysis demonstrates mGlu5 receptor expression in reactive astrocytes surrounding a lesion site or induced by epileptic seizures to be higher than in non-activated astrocytes (25–28). In humans, mGlu5 receptors are up-regulated in reactive astrocytes under pathological conditions such as multiple sclerosis (MS) (29)and in ALS (24).

Among the group II receptors, astrocytes express mGlu3 receptors in vitro and in vivo, whereas mGlu2 receptors are not expressed (30–32). Although mGlu3 mRNA is expressed in cultured astrocytes (33), detection of mGlu3 receptor protein has been hampered by a lack of mGlu3 receptor-specific antibodies (34, 35). Similar to mGlu5 receptor expression, mGlu3 receptor expression is up-regulated in cultured astrocytes by media containing basic fibroblast growth factor and epidermal growth factor (34). Group III mGlu receptors have received the least amount of attention to date, with studies showing inconsistent results. The expression of mGlu4 receptors in astrocytes is controversial; some studies have detected the receptor in primary cultures of rat and mouse cortical astrocytes (36), whereas others have not (33, 35). Neither mGlu6 nor mGlu7 receptors have been detected in cultured astrocytes (35); although mGluR8 mRNA is not expressed in cortical astrocytes grown in conventional medium, it is up-regulated in astrocytes grown in astrocyte-defined media(37). In humans, mGlu4 are not found in resting astrocytes, but are detectable in reactive astrocytes of MS lesions (38).

Activation of mGlu5 receptors in astrocytes stimulates phosphoinositide (PI) hydrolysis (22)and generates oscillatory increases in intracellular calcium (33, 39). This results in the release of neurotransmitters such as glutamate, which in turn modulates neuronal excitability and promotes synchronized activation of groups of neurons (40). Activation of group I receptors by DHPG stimulates MAP kinase pathways (41)and selective activation of mGlu5 receptors stimulates phopholipase D (PLD) signaling in cultured cortical and hippocampal astrocytes (42). Another group I agonist, quisqualic acid, increases the open probability of two types of Ca²⁺-activated K⁺-channels in hippocampal astrocytes through a PLC-mediated mechanism (43). The mGlu2 and mGlu3 receptors are negatively coupled to adenylate cyclase and activation of group II mGlu receptors by the selective agonist LY379268 reduces forskolin-stimulated cAMP formation in the absence of extracellular calcium, but enhances cAMP formation in the presence of calcium (44). This dual regulation of cAMP formation is unique to cultured astrocytes. In addition, activation of group II mGlu receptors amplifies the stimulation of cAMP formation mediated by β2-adrenergic receptors, leading to adenosine release in cultured astrocytes (45). Activation of group II mGlu receptors also stimulates the MAP/ERK kinase and PI-3-kinase signaling pathways (34, 46). Activation of MAP kinase and PI-3-kinase pathways increases formation of transforming growth factor-β (TGF-β), which is neuroprotective (47), and also protects against astrocytic damage caused by oxygen/glucose deprivation in culture (48).

Microglial mGlu receptors

Expression of both mGlu5 receptor mRNA and protein has been reported in cultured cortical microglia as well as in a microglial cell line (33, 49, 50). Although one report indicates that mGlu5 receptors are not expressed in resting microglia of intact brain (30), following spinal cord or brain injury there is significant expression of mGlu5 receptors in activated microglia that surround the injury site (51, 52). In contrast, mGlu1 receptors are not expressed in cultured microglia (33, 49); however, in humans mGlu1α receptor immunreactivity is co-localized with a subset of cells of the microglial/macrophage lineage in MS lesions (29). Cultured microglia also express mRNA and protein for mGlu2 and mGlu3 receptors (53). Although mGlu3 receptor mRNA has not been detected in microglia from intact rat brain (30), mGlu2/3 receptor immunoreactivity has been observed in microglia/macrophage cells in autopsied brain samples from patients with MS (29). With regard to the group III mGlu receptors, microglia express mGlu4, mGlu6, and mGlu8 -but not mGlu7 receptors (54). Analysis of human tissue samples revealed that mGlu8, but not mGlu4 receptors, are expressed in MS lesions, particularly in cells of the microglia/macrophage lineage with an amoeboid morphology (38). With respect to lesion stage, it was found that the mGlu8 receptor expression was strongly co-localized to actively demyelinating lesions.

Data on the mGlu receptor signal transduction pathways in microglia is limited to group I and III mGlu receptors. Selective activation of mGlu5 receptors by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) in microglia stimulates PI hydrolysis (49, 50)and activates a G_αq signal transduction pathway in microglia that requires PLC, PKC and calcium release (49). In contrast, selective activation of group III mGlu receptors (mGlu4, mGlu6, and mGlu8) using the specific group III agonists L-AP4 or (RS)-PPG inhibits forskolin-induced cAMP production (54).

Oligodendrocyte and NG2-glial cell mGlu receptors

It is not clear whether group I mGlu receptors are expressed in oligodendrocytes in vivo. However, oligodendrocytes have been shown to express mGlu3 receptors, but not mGlu2 receptors, in mouse brain (55). In vitro studies show that cultured oligodendrocytes prepared from neonatal rats express mGlu1α, mGlu2/3, mGlu4, and mGlu5 receptors, and that the expression of these receptor subtypes is developmentally regulated -being highly elevated in early and late oligodendrocyte precursors, but low or absent in both immature and mature oligodendrocytes (56). O4-and O1-positive and A2B5-positive early precursors from adult human brain express both mGlu3 and mGlu5 receptors (57, 58). Unfortunately, little is known with regard to mGlu receptor mediated signal transduction pathways in oligodendrocyte cells. The non-selective mGlu agonist, (1S,3R)-ACPD, does not evoke a current in white matter oligodendrocytes in brain slice (59), although in some cultured oligodendrocyte lineage cells it raises intracellular Ca²⁺ levels (60). The non-selective group I mGlu receptor agonist DHPG attenuates oligodendrocyte excitotoxic cell death by reducing downstream oxidative stress after AMPA/Kainate receptor over-activation in a PKCα mediated manner (56). Finally, NG2-glial cells in the hippocampus express functional group I mGlu receptors (61), but given the recent emergence of NG2-glial cells there is limited information on the function of mGlu receptors in these neuroglial cells.

Physiological role of mGlu receptors in neuroglia

Recently the concept of neurotransmission has evolved from a neuron-centric paradigm to include the tripartite synapse (62), composed of three reciprocally interacting compartments: the pre-synaptic nerve terminal, the post-synaptic membrane, and the astrocyte -which regulates synaptic efficacy and plasticity (63). Although astrocytes do not display electrical excitability and are unable to fire all-or-none action potentials like neurons (64), they can regulate neuronal excitability by controlling the levels of extracellular glutamate at synapses and by responding to neurotransmitters with the release of gliotransmitters (transmitters released by glial cells), which in turn modulate neurotransmitter release (65). Importantly, mGlu receptors are implicated in both these processes (figure 3).

Figure 3. Schematic representation of mGlu receptor localization in the tripartite synapse in the CNS.

Glutamate released from the pre-synaptic element leads to the activation of post-synaptic receptors. In parallel, mGlu receptors on astrocytes are activated, causing Ca²⁺ elevation and consequent vesicle-based glutamate exocytosis. Excess glutamate released by astrocytes activates both pre-and post-synaptic receptors. The activation of pre-synaptic mGlu receptors leads to the inhibition of further glutamate release. Blue, green and red colored receptors denote group I, II and III mGlu receptors respectively.

Astrocytes respond to neurotransmitter release from pre-synaptic terminals by increasing intracellular Ca²⁺ and releasing gliotransmitters such as glutamate (66), d-serine (a co-agonist of NMDA receptors) (67), and ATP; the latter, when hydrolyzed to adenosine, plays a role in A1 receptor-mediated pre-synaptic inhibition of excitatory synaptic transmission (63). The glutamate released from astrocytes in response to mGlu5 receptor activation induces a slow inward current in neurons mediated by extra-synaptic NR2B-containing NMDA receptors, which promotes synchronized activation of groups of neurons in the hippocampus and facilitates a slow form of long distance information processing (68, 69). Of recent interest, mGlu5 receptor-mediated glutamate release in astrocytes appears to control neuronal excitability in the nucleus accumbens, a region implicated in substance addiction (70).

In addition to modulating the release of gliotransmitters and neurotransmission, mGlu receptors also alter the expression and function of glutamate transporter proteins in astrocytes. Glutamate in the synaptic cleft is rapidly cleared from the extracellular space by high-affinity glutamate transporters present on neurons and astrocytes, thereby maintaining glutamate concentrations below excitotoxic levels. The glutamate transporters GLAST and GLT1 are almost exclusively expressed by astrocytes, and GLT1 accounts for more than 80% of glutamate uptake in the brain. In vitro studies demonstrate that GLT1 and GLAST are minimally expressed in cultured astrocytes grown in serum-containing medium, but their expression becomes strongly induced when astrocytes are cultured in the presence of neurons or in a defined medium containing growth factors (34, 71). Acute stimulation of mGlu5 receptors increases activity of GLT1 and leads to rapid glutamate uptake in these cells through a mechanism that involves PLC and PKC; this process may help restrict the glutamate signal to the synaptic cleft (71). Similarly, activation of group II and group III mGlu in astrocytes enhances glutamate uptake and is neuroprotective against 1-methyl-4-phenylpyridinium toxicity(MPTP)in midbrain mixed neuron-glial cultures (72).

The physiological role of microglial mGlu receptors under non-pathological conditions is currently unknown. However, in oligodendrocytes mGlu receptors play a key role in oligodendrocyte development. During lineage progression oligodendrocytes precursor cells exhibit a series of distinct developmental morphologies, characterized by the sequential expression of stage-specific oligodendrocyte markers: A2B5 (early precursors), O4 (later precursors), O1 (immature oligodendrocyte), and MBP (mature oligodendrocyte). Notably, group 1 (mGlu 1 and 5), group 2 (mGlu 2/3) and group 3 (mGlu 4a) mGlu receptors are highly expressed in early (A2B5-positive) and late (O4-positive) precursor cells but are dramatically down-regulated in immature (O1-positive) and mature (MBP-positive) oligodendrocytes (56).

Pathophysiological role of neuroglial mGlu receptors

mGlu receptors in neurotoxicity and neuroprotection

Considerable work has been done to clarify the role of mGlu receptors in excitotoxic neuronal cell death cascades. In general, activation of group II and group III mGlu receptors generally provide neuroprotection, whereas activation of mGlu1 and mGlu5 receptors may either accentuate or attenuate cell death depending on the experimental paradigm used (16). Notably, mGlu receptor-mediated neuroprotective mechanisms appear to be dependent on complex glial-neuronal interactions that are modulated by the subtype of mGlu receptor expressed on the glial cell. Neuroprotection mediated by group II mGlu receptors involves such mechanisms. Astrocytes express mGlu3 receptors (but not mGlu2 receptors) and their activation protects neurons from oxygen/glucose deprivation injury (73)and NMDA-mediated excitotoxicity (74). Media collected from cultured astrocytes stimulated with mGlu3 receptor agonists protect neurons against NMDA excitotoxicity (46, 75)whereas neuroprotection is lost when media collected from mGlu3 receptor knockout astrocytes is added to the co-culture system (74). Selective activation of mGlu3 receptors by LY379268 protects cultured astrocytes against apoptotic death induced by oxygen/glucose deprivation (48)and prevents nitric oxide-induced apoptotic cell death (76). Notably, activation of mGlu2/3 receptors in astrocytes stimulates the MAP/ERK kinase and PI-3-kinase signaling pathways (34, 46), leading to increased formation of TGFβ, which is neuroprotective (47). In microglia, selective activation of mGlu2 and mGlu3 receptors by DCGIV and L-CCG-I respectively, induces mitochondrial depolarization and cellular apoptosis, promoting a neurotoxic microglial phenotype and neuronal cell death in co-culture conditions (53). These effects are mediated by the secretion of tumor necrosis factor-α (TNFα) and Fas ligand from activated microglia (77). In addition, chromogranin A (CGA)-and amyloid-β (Aβ_25–35)-induced microglial activation is modulated by group II mGlu receptors because inhibition by the antagonist MCCG reduced toxin-induced microglial reactivity and related neurotoxicity. Recently it was demonstrated that activation of microglial mGlu2 receptors exacerbated neuronal cell death whilst activation of mGlu3 receptors was neuroprotective in a model of myelin-induced microglial neurotoxicity (78). These data highlight the role that group II mGlu receptor subtypes expressed on neuroglia play in neurotoxicity and neuroprotection and demonstrate that mGlu3 receptors may be important therapeutic targets for neuroprotection under conditions of excessive glutamate excitotoxicity and microglial-mediated neuroinflammation.

Group III mGlu receptors in neuroglia also play a role in neuroprotection. In vitro studies demonstrate that agonists of group III mGlu receptors attenuate microglial activation when stimulated by lipopolysaccharide (LPS), CGA or Aβ_25–35, and protect neurons against microglial-mediated neurotoxicity in these models (54). The neuroprotective effects of group III mGlu receptor agonists has been confirmed in a model of myelin-induced microglial neurotoxicity (78). Interestingly, activation of group III mGlu receptors stimulates neurotrophic factor expression and production in microglia during the early stages of glutamate excitotoxicity (79)which may result in neuroprotection. Activation of NMDA and group III mGlu receptors in microglia trigger intracellular Ca²⁺ release from the endoplasmic reticulum which stimulates PKC signaling to induce the expression of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) (79). Selective activation of group III mGlu receptors on neuroglia may have important implications for the treatment of MS because mGlu8 receptors are expressed in MS lesions, in particular in cells of the microglia/macrophage lineage with an amoeboid morphology (38). In addition, in cultured astrocytes exposed to pro-inflammatory cytokines, activation of mGlu4 receptors reduce the production of RANTES (36), a key chemokine which is implicated in the pathophysiology of MS. These studies suggest a potential use of group III mGlu receptor agonists as therapeutic agents in neuroinflammatory disorders such as MS(36).

Group I mGlu receptors expressed on neuroglia are involved in a complex cross talk between activated astrocytes and microglia that can result in the activation of neurotoxic or neuroprotective signaling cascades depending on which mGlu receptor is stimulated (figure 4). The expression level of mGlu5 receptors are increased in reactive astrocytes and a change in the ratio of mGlu receptors under pathological conditions may influence the levels of extracellular glutamate in neurological disorders associated with reactive astrogliosis. Elegant in vivo two-photon microscopy experiments have shown that an enhanced release of glutamate induced by the activation of astrocyte mGlu5 receptors contribute to glutamate-mediated neuronal cell death after status epilepticus (80), and that the mGlu5 receptor antagonist, MPEP, attenuates astrocyte Ca²⁺ signals and is neuroprotective in this model. Coincidently, mGlu receptor-mediated functional responses are impaired in neurological disorders associated with reactive astrogliosis, and the mGlu5 receptor-dependent up-regulation of glutamate transport is significantly impaired in activated astrocytes prepared from a G93A SOD1 model of ALS (81). Interestingly, mGlu5 receptors are down-regulated in cultured astrocytes treated with the pro-inflammatory cytokines, interleukin-1β (IL-1β) and TNFα, or with conditioned media collected from LPS-stimulated activated microglia (82, 83). These data suggest a complex interaction between astrocytes and microglia during pathological conditions associated with neuroinflammation, which further influenced the glutamate-dependent response of astrocytes (82). mGlu5 receptor expression is distinctly up-regulated in activated microglia that surround the injury site following brain injury (52). In cultured microglia, selective activation of mGlu5 receptors by CHPG or DHPG/CPCCOEt (group I mGlu receptor agonist and mGlu1 receptor antagonist combination) markedly attenuates microglial activation induced by LPS or interferon-gamma (49, 50, 84, 85), as well as reducing the levels of inducible nitric-oxide synthase, NO, reactive oxygen species and TNFα. Thus, mGlu5 receptors can modulate neuroinflammatory responses, possibly through the inhibition of the enzyme activity of NADPH oxidase (50). Interestingly, activation of group I mGlu receptors attenuates excitotoxic damage of oligodendrocyte precursor cells, but is not protective against excitotoxicity in mature oligodendrocytes (56). Further, activation of mGlu5 receptors has been shown to completely protect oligodendrocyte precursors and partially protect mature oligodendrocytes against staurosporine-induced apoptosis (86). The different neuroprotective outcomes conferred by group I mGlu receptor agonists in these cells may reflect the development pattern of mGlu receptor expression across the oligodendroglial lineage.

Figure 4. Complex crosstalk between mGlu receptors expressed on activated astrocytes and microglia.

There is a complex interplay between activated astrocytes and microglia that influences responses mediated by mGlu receptors under pathological conditions characterized by neuroinflammation and neurodegeneration. Solid arrows represent causative or activating effects, whereas dashed lines represent inhibitory effects. Blue, green and red colored receptors denote group I, II and III mGlu receptors respectively.

The effective glutamate concentration at the cell membrane may determine whether mGlu receptor signaling results in neuroprotection or neurotoxicity. As discussed, mGlu receptor subtypes are expressed in neuroglia at distinct expression levels under normal and pathological conditions and may have varying affinities for glutamate. Data from cloned mGlu receptors indicate that mGlu2 and mGlur3 receptors respond to much lower glutamate concentrations than group III mGlu receptors (87). Therefore, the final outcome of a given concentration of glutamate on neuroglia will depend on the combined effects of signaling through all mGlu receptors expressed in the microenvironment. This may explain why glutamate does not always attenuate microglial activation. For example, in some cases microglial activation mediated through mGlu2 signaling may overcome the inhibitor effect on microglia initiated by signaling through mGlu5 or group III mGlu receptors (49–51, 54, 88).

In summary, these data indicate that there is a complex interplay between astrocytes, microglia, oligodendrocytes and neurons that influences responses mediated by mGlu receptors under pathological conditions characterized by neuroinflammation and neurodegeneration. Drugs that modulate mGlu receptors are currently proposed as potential therapeutic agents for several neurodegenerative disorders (84). Although their effects on neurons often have justified the interest it is now clear that their modulation of neuroglial mGlu receptors need to be carefully considered.

mGlu receptors in CNS injury

Microglia are the resident immune cells of the CNS. In the healthy adult brain the highly motile ramified processes of resting microglia constantly monitor and probe the brain microenvironment and maintain normal CNS homeostasis by phagocytic clearance of cellular debris (2, 3). However, in response to CNS injury microglia are readily activated and undergo dramatic changes in cell morphology and behavior. Upon activation microglia contract their processes and transform from a ramified to an ameboid morphology resembling that of blood-borne macrophages, followed by proliferation and migration towards the site of injury (3, 89). This convergence upon the site of injury is in response to ATP and other signals released by injured neurons (89). However, when microglia become over-activated or reactive they can induce detrimental neurotoxic effects by releasing multiple cytotoxic substances, including pro-inflammatory mediators (e.g. TNFα, interferon-γ) and oxidative stress factors (e.g. superoxide free radicals, nitric oxide) (90). Importantly, the release of pro-inflammatory cytokines and other soluble factors by activated microglia can significantly influence the subsequent activation of astrocytes and glial scar formation under pathological conditions including CNS injury (91).

Astrocyte activation or astrogliosis is characterized by the increase of intermediate filaments (vimentin and GFAP), increased cell proliferation and an accompanying cellular hypertrophy (92). Reactive astrocytes have multiple roles that can be either harmful or beneficial after CNS injury. Upon activation, astrocytes upregulate a number of neurotrophic factors (GDNF and BDNF) that support and protect against injury-induced cell death (93). In addition, astrocytes play a crucial role in regulating extracellular glutamate levels, which can reduce glutamate excitotoxicity to neurons and other cells (94). Notably, impaired astrocyte performance exacerbates neuronal dysfunction following CNS injury and transgenic ablation of reactive astrocytes increases neuronal cell death and promotes worse outcome after brain and spinal cord injury (95, 96); this may in part reflect the loss of ability to limit the influx of inflammatory cells. Astrocytes provide support and guidance for axonal growth following CNS injury; however, prolonged astogliosis inhibits axon regeneration, hinders functional recovery and secrets excessive neurotoxic substances. Following injury, hypertrophic astrocytes surround the lesion site and deposit an inhibitory extracellular matrix including chondroitin sulfate proteoglycans that contributes to the glial scar. This dense physical and chemical barrier inhibits axonal regeneration and prevents functional connections required for axonal growth and repair (97). Therefore, limiting prolonged astrogliosis may be important for axon regeneration after CNS injury; for example, improved axonal growth and repair has been reported in transgenic mice deficient in both vimentin and GFAP (98, 99).

Following CNS injury, the extracellular concentration of excitatory amino acids, including glutamate, is increased(100–102). The expression of mGlu receptors is also altered by CNS trauma; group II mGlu receptors are reduced after spinal cord injury (SCI) and traumatic brain injury (TBI) (28, 103, 104), whereas mGlu1 receptors are increased rostral and caudal to the injury site following SCI and mGlu5 receptors are increased at the lesion site following TBI (52, 103). Recently, multipotential treatments have become more attractive therapeutic strategies for CNS injuries because the mechanism of such injuries is multi-factorial; therefore, therapies that serve to modulate multiple pathophysiological pathways may prove more effective than those directed at a single target(105). Neurons, astrocytes, microglia, oligodendrocytes, endothelial cells and circulating immune cells all play roles in response to acute and sub-acute injury, as well as chronic neurodegeneration. Therefore, treatments that target multiple cell types and associated pathways may prove most beneficial.

Inhibition of mGlu1 receptors has protective effects following CNS injury. After spinal cord contusion injury, injections of the group I mGlu receptor antagonist AIDA into the lesion site improved functional locomotor recovery. Protection was mediated by the mGlu1α receptor because similar beneficial effects were demonstrated by administration of the mGlu1α receptor specific antagonist, LY367385, but not the mGlu5 receptor specific antagonist, MPEP (106). Furthermore, treatment with the mGlu1α receptor antagonist also produced significant white and gray matter sparing. Similarly, mGlu1 receptor antagonists have also proved protective in TBI models. AIDA administration following lateral fluid percussion in rats significantly improved neurological function outcomes and reduced the lesion size following injury (107). Furthermore, the mGlu1 receptor antagonist YM-202074 is neuroprotective following cerebral ischemia (108); following middle cerebral artery occlusion (MCAO) in rats, YM-202074 administration within 2 hours of the onset of ischemia significantly reduced infarct volumes in the brain and improved neurological scores.

As discussed above, mGlu5 receptors are expressed in both neuroglia and neurons and its actions in different cells suggest strong possibilities for synergistic therapeutic potential. For example, mGlu5 receptor agonists have not only shown considerable anti-apoptotic properties in neuronal cultures (109–112), but also strong anti-inflammatory effects in microglial cultures (49, 50). Group I mGlu receptors can activate PKC which up-regulate inward rectifier potassium channels and reduce microglial activation (113). In a co-culture system of neurons and astrocytes, administration of the mGlu5 receptor agonist CHPG significantly reduced NMDA-mediated currents after stretch-injury (114); interestingly, in the absence of astrocytes, CHPG did not modulate the stretch-injury induced NMDA responses. Although a number of publications indicate that treatment with the selective mGlu5 receptor agonist CHPG is neuroprotective in vitro and in vivo, treatment with them Glu5 receptor antagonist MPEP may also be beneficial. For example, in vitro studies demonstrated that administration of MPEP significantly reduced neuronal death following glutamate or NMDA exposure (115). These confusing actions of mGlu5 receptor agonism/antagonism were highlighted in in vivo neuroprotective studies following MCAO-induced focal ischemia (116). In these studies both the mGlu5 receptor agonist CHPG and antagonist MPEP reduced the infarct volume when applied at 250 nmol concentrations. Later studies shed more light and demonstrated that the neuroprotective actions of the mGlu5 receptor antagonists did not reflect actions at the mGlu5 receptor. Instead, MPEP acted to directly inhibit NMDA receptor signaling as application of the antagonist in mGlu5 receptor knockout cultures produced similar neuroprotective effects as wild-type cultures (117). In contrast, selective activation of mGlu5 receptors by CHPG has powerful neuroprotective effects in vivo. Intrathecal administration of CHPG for 7 days following a moderately severe spinal cord contusion at T9 resulted in functional motor recovery, reduced lesion volume as well as sparing of white matter through 28 days post-injury (51). Notably, CHPG administration attenuated microglial activation and release of pro-inflammatory mediators in the injured spinal cord following injury.

In vivo, the group II mGlu receptor agonist LY379268 reduced neuronal loss in the hippocampus following global ischemia and application of LY379268 up to 2 hours after occlusion was neuroprotective in a focal ischemia model (118). These effects may reflect not only reduced neuronal death directly, but also the impact of stimulating astrocyte production of neuroprotective factors such as TGFβ (47). In other models of CNS injury, such as SCI, TBI or excitotoxic injections, group II and III mGlu receptors provide neuroprotection. For example, following SCI, group II and III mGlu receptor agonists improve some measures of functional recovery, such as reduced allodynia (119). Additionally, administration of the mGlu4 receptor agonist (RS)-PPG reduces NMDA-induced neuronal death (120). Administration of LY379268 30 minutes after controlled cortical impact injury in mice resulted in significant improvements in both motor and cognitive function (121). Similarly, treatment with LY354740 significantly improved neurological outcome scores at 2 weeks post-injury after lateral fluid percussion injury in rats (122).

mGlu receptors in neurological disorders

Drugs acting at mGlu receptors are currently proposed as potential therapeutic agents for the treatment of a variety of neurological disorders such as Fragile-X syndrome, schizophrenia, Parkinson’s disease and L-DOPA-induced dyskinesias, and chronic pain (16). Novel therapeutic agents with either positive or negative modulation of mGlu receptors are under investigation. A growing body of evidence indicates that systemic treatment with mGlu5 receptor negative allosteric modulators (NAMs) are highly effective in relieving motor symptoms and L-DOPA-induced dyskinesias in rodent and primate models of Parkinsonism (123–125). Pharmacological blockade of mGlu5 receptors is also analgesic in models of inflammatory and neuropathic pain, and inhibits the development of tolerance to the analgesic activity of morphine (126, 127). Based on promising pre-clinical data, a number of mGlu5 receptor NAMs are under clinical development for the treatment of Parkinson’s disease and neuropathic pain. Positive allosteric modulators (PAMs) of mGlu5 receptors show promise for the treatment of schizophrenia. Amplification of mGlu5 receptor function appears to be important in schizophrenia and a number of PAMs are active in pre-clinical models and demonstrate efficacy on cognitive dysfunction associated with schizophrenia (128). However, the role that modulation of neuroglial mGlu receptors pathways in these neurological disorders has received limited attention. Astrocyte mGlu3 receptors may be promising therapeutic targets for Alzheimer’s disease (AD) because TGFβ protects neurons against Aβ-toxicity and type-2 TGFβ receptors are defective in the AD brain (129), but further in-depth analysis will be required to fully elucidate the role of mGlu receptors on astrocyte, microglia and oligodendrocytes in these complex neurological disorders.

Conclusions

In past few years the traditional neuro-centric view of CNS physiology and pathology has been replaced by a more complex and nuanced neuronal-neuroglia paradigm that defines increasingly important roles for microglia, astrocytes and oligodendrocytes. In this regard it has become clear that mGlu receptors play critical physiological and pathophysiological roles in neuroglia as well as in neurons. Recent studies have underscored the diverse and important functions of mGlu receptors as modulators of neuroglial function under both physiological conditions and in a variety of neurological and psychiatric disorders. Thus, neuroglia mGlu receptors represent a potential therapeutic target for multiple disorders of the nervous system.