Francesco Fazio

Metabotropic glutamate receptor-4 modulates adaptive immunity and restrains neuroinflammation

High amounts of glutamate are found in the brains of people with multiple sclerosis, an inflammatory disease marked by progressive demyelination. Glutamate might affect neuroinflammation via effects on immune cells. Knockout mice lacking metabotropic glutamate receptor-4 (mGluR4) were markedly vulnerable to experimental autoimmune encephalomyelitis (EAE, a mouse model of multiple sclerosis) and developed responses dominated by interleukin-17–producing T helper (TH17) cells. In dendritic cells (DCs) from those mice, defective mGluR4 signaling—which would normally decrease intracellular cAMP formation—biased TH cell commitment to the TH17 phenotype. In wild-type mice, mGluR4 was constitutively expressed in all peripheral DCs, and this expression increased after cell activation. Treatment of wild-type mice with a selective mGluR4 enhancer increased EAE resistance via regulatory T (Treg) cells. The high amounts of glutamate in neuroinflammation might reflect a counterregulatory mechanism that is protective in nature and might be harnessed therapeutically for restricting immunopathology in multiple sclerosis.

Cinnabarinic Acid, an Endogenous Metabolite of the Kynurenine Pathway, Activates Type-4 Metabotropic Glutamate Receptors

Cinnabarinic acid is an endogenous metabolite of the kynurenine pathway that meets the structural requirements to interact with glutamate receptors. We found that cinnabarinic acid acts as a partial agonist of type 4 metabotropic glutamate (mGlu4) receptors, with no activity at other mGlu receptor subtypes. We also tested the activity of cinnabarinic acid on native mGlu4 receptors by examining 1) the inhibition of cAMP formation in cultured cerebellar granule cells; 2) protection against excitotoxic neuronal death in mixed cultures of cortical cells; and 3) protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice after local infusion into the external globus pallidus. In all these models, cinnabarinic acid behaved similarly to conventional mGlu4 receptor agonists, and, at least in cultured neurons, the action of low concentrations of cinnabarinic acid was largely attenuated by genetic deletion of mGlu4 receptors. However, high concentrations of cinnabarinic acid were still active in the absence of mGlu4 receptors, suggesting that the compound may have off-target effects. Mutagenesis and molecular modeling experiments showed that cinnabarinic acid acts as an orthosteric agonist interacting with residues of the glutamate binding pocket of mGlu4. Accordingly, cinnabarinic acid did not activate truncated mGlu4 receptors lacking the N-terminal Venus-flytrap domain, as opposed to the mGlu4 receptor enhancer, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC). Finally, we could detect endogenous cinnabarinic acid in brain tissue and peripheral organs by high-performance liquid chromatography-tandem mass spectrometry analysis. Levels increased substantially during inflammation induced by lipopolysaccharide. We conclude that cinnabarinic acid is a novel endogenous orthosteric agonist of mGlu4 receptors endowed with neuroprotective activity.

Switch in the expression of mGlu1 and mGlu5 metabotropic glutamate receptors in the cerebellum of mice developing experimental autoimmune encephalomyelitis and in autoptic cerebellar samples from patients with multiple sclerosis

Recent evidence suggests that changes in the expression of membrane receptors/ion channels in cerebellar Purkinje cells contribute to the onset of cerebellar motor symptoms in patients with multiple sclerosis (MS). We examined the expression of group-I metabotropic glutamate receptors (mGlu1 and mGlu5 receptors) in the cerebellum of mice developing experimental autoimmune encephalomyelitis (EAE) and in autoptic cerebellar samples of MS patients. EAE was induced in mice by immunization with the 35-55 fragment of MOG (myelin oligodendrocyte glycoprotein). EAE mice showed a progressive loss of mGlu1a receptors in the cerebellum, associated with an increased expression of mGlu5 receptors. These changes were restricted to Purkinje cells and their dendritic arborization, as shown by immunohistochemistry. A reduced expression of mGlu1a receptors in cerebellar Purkinje cells was also found in 7 of 9 MS patients. In addition, a light/moderate to very strong mGlu5 receptor immunoreactivity was detected in Purkinje cells of 8 MS patients, but was always absent in non-MS control patients. In EAE mice, an acute treatment with the mGlu1 receptor enhancer, 9H-xanthene-9-carboxylic acid (4-trifluoromethyl-oxazol-2-yl)-amide (RO0711401), significantly improved motor coordination, whereas treatment with the mGlu5 receptor antagonists, 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and 6-methyl-2-(phenylazo)-3-pyridinol (SIB-1757), had no effect. We conclude that mGlu1 receptor enhancers improve motor symptoms associated with EAE and might be helpful as symptomatic drugs in patients with MS.

Xanthurenic Acid Activates mGlu2/3 Metabotropic Glutamate Receptors and is a Potential Trait Marker for Schizophrenia

The kynurenine pathway of tryptophan metabolism has been implicated in the pathophysiology of psychiatric disorders, including schizophrenia. We report here that the kynurenine metabolite, xanturenic acid (XA), interacts with, and activates mGlu2 and mGlu3 metabotropic glutamate receptors in heterologous expression systems. However, the molecular nature of this interaction is unknown, and our data cannot exclude that XA acts primarily on other targets, such as the vesicular glutamate transporter, in the CNS. Systemic administration of XA in mice produced antipsychotic-like effects in the MK-801-induced model of hyperactivity. This effect required the presence of mGlu2 receptors and was abrogated by the preferential mGlu2/3 receptor antagonist, LY341495. Because the mGlu2 receptor is a potential drug target in the treatment of schizophrenia, we decided to measure serum levels of XA and other kynurenine metabolites in patients affected by schizophrenia. Serum XA levels were largely reduced in a large cohort of patients affected by schizophrenia, and, in patients with first-episode schizophrenia, levels remained low after 12 months of antipsychotic medication. As opposed to other kynurenine metabolites, XA levels were also significantly reduced in first-degree relatives of patients affected by schizophrenia. We suggest that lowered serum XA levels might represent a novel trait marker for schizophrenia.

N-Acetyl-cysteine causes analgesia by reinforcing the endogenous activation of type-2 metabotropic glutamate receptors

BackgroundPharmacological activation of type-2 metabotropic glutamate receptors (mGlu2 receptors) causes analgesia in experimental models of inflammatory and neuropathic pain. Presynaptic mGlu2 receptors are activated by the glutamate released from astrocytes by means of the cystine/glutamate antiporter (System xc- or Sxc-). We examined the analgesic activity of the Sxc- activator, N-acetyl-cysteine (NAC), in mice developing inflammatory or neuropathic pain.ResultsA single injection of NAC (100 mg/kg, i.p.) reduced nocifensive behavior in the second phase of the formalin test. NAC-induced analgesia was abrogated by the Sxc- inhibitor, sulphasalazine (8 mg/kg, i.p.) or by the mGlu2/3 receptor antagonist, LY341495 (1 mg/kg, i.p.). NAC still caused analgesia in mGlu3−/− mice, but was inactive in mGlu2−/− mice. In wild-type mice, NAC retained the analgesic activity in the formalin test when injected daily for 7 days, indicating the lack of tolerance. Both single and repeated injections of NAC also caused analgesia in the complete Freund’s adjuvant (CFA) model of chronic inflammatory pain, and, again, analgesia was abolished by LY341495. Data obtained in mice developing neuropathic pain in response to chronic constriction injury (CCI) of the sciatic nerve were divergent. In this model, a single injection of NAC caused analgesia that was reversed by LY341495, whereas repeated injections of NAC were ineffective. Thus, tolerance to NAC-induced analgesia developed in the CCI model, but not in models of inflammatory pain. The CFA and CCI models differed with respect to the expression levels of xCT (the catalytic subunit of Sxc-) and activator of G-protein signaling type-3 (AGS3) in the dorsal portion of the lumbar spinal cord. CFA-treated mice showed no change in either protein, whereas CCI mice showed an ipislateral reduction in xCT levels and a bilateral increase in AGS3 levels in the spinal cord.ConclusionsThese data demonstrate that pharmacological activation of Sxc- causes analgesia by reinforcing the endogenous activation of mGlu2 receptors. NAC has an excellent profile of safety and tolerability when clinically used as a mucolytic agent or in the management of acetaminophen overdose. Thus, our data encourage the use of NAC for the experimental treatment of inflammatory pain in humans.

Targeting metabotropic glutamate receptors in neuroimmune communication

L-Glutamate (L-Glu) is the principal excitatory neurotransmitter in the Central Nervous System (CNS), where it regulates cellular and synaptic activity, neuronal plasticity, cell survival and other relevant functions. Glutamatergic neurotransmission is complex and involves both ionotropic (ligand-gated ion channels; iGluRs) and metabotropic receptors (G-protein coupled receptors). Recent evidence suggests that glutamatergic receptors are also expressed by immune cells, regulating the degree of cell activation. In this review we primarily focus on mGluRs and their role in the crosstalk between the central nervous and immune systems during neuroinflammation.

Cinnabarinic acid and xanthurenic acid: Two kynurenine metabolites that interact with metabotropic glutamate receptors.

ABSTRACT Cinnabarinic and xanthurenic acids are kynurenine metabolites generated by oxidative dimerization of 3‐hydroxyanthranilic acid and transamination of 3‐hydroxykynurenine, respectively. Recent evidence suggests that both compounds can affect brain function and neurotransmission and interact with metabotropic glutamate (mGlu) receptors. Cinnabarinic acid behaves as an orthosteric agonist of mGlu4 receptors, whereas some of the in vitro and in vivo effects produced by xanthurenic acid appear to be mediated by the activation of mGlu2 and mGlu3 receptors. Cinnabarinic acid could play an important role in mechanisms of neuroinflammation acting as a linking bridge between the immune system and the CNS. Xanthurenic acid has potential implications in the pathophysiology of schizophrenia and is a promising candidate as a peripheral biomarker of the disorder. The action of cinnabarinic acid and xanthurenic acid may extend beyond the regulation of mGlu receptors and may involve several diverse molecular targets, such as the aryl hydrocarbon receptor for cinnabarinic acid and vesicular glutamate transporters for xanthurenic acid. The growing interest on these two metabolites of the kynurenine pathway may unravel new aspects in the complex interaction between tryptophan metabolism and brain function, and lead to the discovery of new potential targets for the treatment of neurological and psychiatric disorders. This article is part of the Special Issue entitled ‘The Kynurenine Pathway in Health and Disease’. HIGHLIGHTSCinnabarinic and xanthurenic acids (CA, XA) are neuroactive kynurenines metabolites.CA acts as an orthosteric agonist of mGlu4 receptors.Some in vitro and in vivo effects of XA are mediated by mGlu2/3 receptors.Other targets of CA and XA include the Ah receptor and VGLUT, respectively.CA and XA might have a role in the pathophysiology of CNS disorders.

Fathoming the kynurenine pathway in migraine: why understanding the enzymatic cascades is still critically important

Kynurenine pathway, the quantitatively main branch of tryptophan metabolism, has been long been considered a source of nicotinamide adenine dinucleotide, although several of its products, the so-called kynurenines, are endowed with the capacity to activate glutamate receptors, thus potentially influencing a large group of functions in the central nervous system (CNS). Migraine, a largely unknown pathology, is strictly related to the glutamate system in the CNS pathologic terms. Despite the large number of studies conducted on migraine etio-pathology, the kynurenine pathway has been only recently linked to this disease. Nonetheless, some evidence suggests an intriguing role for some kynurenines, and an exploratory study on the serum kynurenine level might be helpful to better understand possible alterations of the kynurenine pathway in patients suffering from migraine.

Targeting metabotropic glutamate receptors for the treatment of neuroinflammation

HighlightsmGlu receptors are found in lymphocytes and microglia, and modulate neuroinflammation.Some kynurenine metabolites target mGlu receptors inside and outside the CNS.mGlu4 receptor PAMs restrain neuroinflammation by supporting immune tolerance.mGlu2, mGlu3, mGlu4 and mGlu5 receptors differentially regulate microglial function.This should be taken into account in the development of mGlu receptor ligands. &NA; A large body of evidence suggests that neuroinflammation lies at the core of nearly all CNS disorders, including psychiatric disorders. Invading and local immune cells orchestrate the series of events that lead to either tissue repair or damage in response to neuroinflammation. Both lymphocytes and microglia express metabotropic glutamate (mGlu) receptors, which respond to glutamate or other endogenous activators (e.g. some kynurenine metabolites of tryptophan metabolism) influencing immune phenotype and the balance between pro‐inflammatory and anti‐inflammatory cytokines. Here, we offer an up‐to‐date on the role of individual mGlu receptor subtypes in the regulation of innate and adaptive immune response, highlighting the relevance of this information in the development of subtype‐selective mGlu receptor ligands for treatment of CNS disorders.

Vasorelaxing Action of the Kynurenine Metabolite, Xanthurenic Acid: The Missing Link in Endotoxin-Induced Hypotension?

The kynurenine pathway of tryptophan metabolism is activated by pro-inflammatory cytokines. L-kynurenine, an upstream metabolite of the pathway, acts as a putative endothelium-derived relaxing factor, and has been hypothesized to play a causative role in the pathophysiology of inflammation-induced hypotension. Here, we show that xanthurenic acid (XA), the transamination product of 3-hydroxykynurenine, is more efficacious than L-kynurenine in causing relaxation of a resistance artery, but fails to relax pre-contracted aortic rings. In the mesenteric artery, XA enhanced activating phosphorylation of endothelial nitric oxide synthase (NOS), and the relaxing action of XA was abrogated by pharmacological inhibition of NOS and endothelial-derived hyperpolarizing factor. Systemic injection of XA reduced blood pressure in mice, and serum levels of XA increased by several fold in response to a pulse with the endotoxin, lipopolysaccharide (LPS). LPS-induced hypotension in mice was prevented by pre-treatment with the kynurenine monooxygenase (KMO) inhibitor, Ro-618048, which lowered serum levels of XA but enhanced serum levels of L-kynurenine. UPF 648, another KMO inhibitor, could also abrogate LPS-induced hypotension. Our data identify XA as a novel vasoactive compound and suggest that formation of XA is a key event in the pathophysiology of inflammation-induced hypotension.