Chitinase 3-like proteins as diagnostic and prognostic biomarkers of multiple sclerosis.

BACKGROUND: Despite sensitivity of MRI to diagnose multiple sclerosis (MS), prognostic biomarkers are still needed for optimized treatment. OBJECTIVE: The objective of this paper is to identify cerebrospinal fluid (CSF) diagnostic biomarkers of MS using quantitative proteomics and to analyze their expression at different disease stages. METHODS: We conducted differential analysis of the CSF proteome from control and relapsing-remitting MS (RRMS) patients followed by verification by ELISA of candidate biomarkers in CSF and serum in control, clinically isolated syndrome (CIS), RRMS and progressive MS (PMS) patients. RESULTS: Twenty-two of the 527 quantified proteins exhibited different abundances in control and RRMS CSF. These include chitinase 3-like protein 1 (CHI3L1) and 2 (CHI3L2), which showed a strong expression in brain of MS patients, especially in astrocytes and microglial cells from white matter plaques. CSF and serum CHI3L1 levels increased with the disease stage and CIS patients with high CSF (>189 ng/ml) and serum (>33 ng/ml) CHI3L1 converted more rapidly to RRMS (log rank test, p < 0.05 and p < 0.001, respectively). In contrast, CSF CHI3L2 levels were lower in PMS than in RRMS patients. Accordingly, CSF CHI3L1/CHI3L2 ratio accurately discriminated PMS from RRMS. CONCLUSIONS: CSF CHI3L1 and CHI3L2 and serum CHI3L1 might help to define MS disease stage and have a prognostic value in CIS.

The nucleus accumbens 5-HTR4-CART pathway ties anorexia to hyperactivity.

In mental diseases, the brain does not systematically adjust motor activity to feeding. Probably, the most outlined example is the association between hyperactivity and anorexia in Anorexia nervosa. The neural underpinnings of this 'paradox', however, are poorly elucidated. Although anorexia and hyperactivity prevail over self-preservation, both symptoms rarely exist independently, suggesting commonalities in neural pathways, most likely in the reward system. We previously discovered an addictive molecular facet of anorexia, involving production, in the nucleus accumbens (NAc), of the same transcripts stimulated in response to cocaine and amphetamine (CART) upon stimulation of the 5-HT(4) receptors (5-HTR(4)) or MDMA (ecstasy). Here, we tested whether this pathway predisposes not only to anorexia but also to hyperactivity. Following food restriction, mice are expected to overeat. However, selecting hyperactive and addiction-related animal models, we observed that mice lacking 5-HTR(1B) self-imposed food restriction after deprivation and still displayed anorexia and hyperactivity after ecstasy. Decryption of the mechanisms showed a gain-of-function of 5-HTR(4) in the absence of 5-HTR(1B), associated with CART surplus in the NAc and not in other brain areas. NAc-5-HTR(4) overexpression upregulated NAc-CART, provoked anorexia and hyperactivity. NAc-5-HTR(4) knockdown or blockade reduced ecstasy-induced hyperactivity. Finally, NAc-CART knockdown suppressed hyperactivity upon stimulation of the NAc-5-HTR(4). Additionally, inactivating NAc-5-HTR(4) suppressed ecstasy's preference, strengthening the rewarding facet of anorexia. In conclusion, the NAc-5-HTR(4)/CART pathway establishes a 'tight-junction' between anorexia and hyperactivity, suggesting the existence of a primary functional unit susceptible to limit overeating associated with resting following homeostasis rules.

Metabotropic glutamate receptors: from the workbench to the bedside.

Metabotropic glutamate (mGlu) receptors were discovered in the mid 1980s and originally described as glutamate receptors coupled to polyphosphoinositide hydrolysis. Almost 6500 articles have been published since then, and subtype-selective 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, chronic pain, and gastroesophageal reflux disorder. Prof. Erminio Costa was linked to the early times of the mGlu receptor history, when a few research groups challenged the general belief that glutamate could only activate ionotropic receptors and all metabolic responses to glutamate were secondary to calcium entry. This review moves from those nostalgic times to the most recent advances in the physiology and pharmacology of mGlu receptors, and highlights the role of individual mGlu receptor subtypes in the pathophysiology of human disorders. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Physical interaction between the serotonin transporter and neuronal nitric oxide synthase underlies reciprocal modulation of their activity.

The spatiotemporal regulation of neurotransmitter transporters involves proteins that interact with their intracellular domains. Using a proteomic approach, we identified several proteins that interact with the C terminus of the serotonin transporter (SERT). These included neuronal nitric oxide synthase (nNOS), a PSD-95/Disc large/ZO-1 (PDZ) domain-containing protein recruited by the atypical PDZ binding motif of SERT. Coexpression of nNOS with SERT in HEK293 cells decreased SERT cell surface localization and 5-hydroxytryptamine (5-HT) uptake. These effects were absent in cells transfected with SERT mutated in its PDZ motif to prevent physical association with nNOS, and 5-HT uptake was unaffected by activation or inhibition of nNOS enzymatic activity. 5-HT uptake into brain synaptosomes was increased in both nNOS-deficient and wild-type mice i.v. injected with a membrane-permeant peptidyl mimetic of SERT C terminus, which disrupted interaction between SERT and nNOS, suggesting that nNOS reduces SERT activity in vivo. Furthermore, treating cultured mesencephalic neurons with the mimetic peptide similarly increased 5-HT uptake. Reciprocally, indicating that 5-HT uptake stimulates nNOS activity, NO production was enhanced on exposure of cells cotransfected with nNOS and SERT to 5-HT. This effect was abolished by 5-HT uptake inhibitors and absent in cells expressing SERT mutated in its PDZ motif. In conclusion, physical association between nNOS and SERT provides a molecular substrate for their reciprocal functional modulation. In addition to showing that nNOS controls cell surface localization of SERT, these findings provide evidence for regulation of cellular signaling (NO production) by a substrate-carrying transporter.

[The Lemierre syndrome: a complicated oropharyngeal infection]. / Het Syndroom van Lemierre: een gecompliceerde orofaryngeale infectie.

The Lemierre syndrome or 'necrobacillosis' is a post angina sepsis caused by an acute oropharyngeal infection with a secondary thrombophlebitis of the internal jugular vein. There are often septic emboli in the lungs, although intestinal organs can also be affected. This syndrome is caused by the strictly anaerobic gram-negative pathogen Fusobacterium necrophorum, sometimes in combination with other pathogens. The patient typically presents with high fever, pain in the neck, malaise and dyspnoea one week after the start of an angina. Plain chest radiograph shows bilateral nodular infiltrates, ultrasound reveals a thrombophlebitis of the internal jugular vein. CT scan can be useful to confirm the diagnosis and possible complications. In the beginning there is often a transient hyperbilirubinemia with toxic inflammatory blood results. Under the correct antibiotic regime complete recovery can be obtained.

Modulation of memory processes and cellular excitability in the dentate gyrus of freely moving rats by a 5-HT4 receptors partial agonist, and an antagonist.

Firstly, olfactory association learning was used to determine the modulating effect of 5-HT4 receptor involvement in learning and long-term memory. Secondly, the effects of systemic injections of a 5-HT4 partial agonist and an antagonist on long-term potentiation (LTP) and depotentiation in the dentate gyrus (DG) were tested in freely moving rats. The modulating role of the 5-HT4 receptors was studied by using a potent, 5-HT4 partial agonist RS 67333 [1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-n-butyl-4-piperidinyl)-1-propanone] and a selective 5-HT4 receptor antagonist RS 67532 [1-(4-amino-5-chloro-2-(3,5-dimethoxybenzyloxyphenyl)-5-(1-piperidinyl)-1-propanone]. Agonist or antagonist systemic chronic injections prior to five training sessions yielded a facilitatory effect on procedural memory during the first session only with the antagonist. Systemic injection of the antagonist only before the first training session improved procedural memory during the first session and associative memory during the second session. Similar injection with the 5-HT4 partial agonist had an opposite effect. The systemic injection of the 5-HT4 partial agonist prior to the induction of LTP in the dentate gyrus by high-frequency stimulation was followed by a population spike increase, while the systemic injection of the antagonist accelerated the depotentiation 48 h later. The behavioural and physiological results pointed out the involvement of 5-HT4 receptors in processing related to the long-term hippocampal-dependent memory system, and suggest that specific 5-HT4 agonists could be used to treat amnesic patients with a dysfunction in this particular system.

GPCR-interacting proteins (GIPs): nature and functions.

The simplistic idea that seven transmembrane receptors are single monomeric proteins that interact with heterotrimeric G-proteins after agonist binding is definitively out of date. Indeed, GPCRs (G-protein-coupled receptors) are part of multiprotein networks organized around scaffolding proteins. These GIPs (GPCR-interacting proteins) are either transmembrane or cytosolic proteins. Proteomic approaches can be used to get global pictures of these 'receptosomes'. This approach allowed us to identify direct but also indirect binding partners of serotonin receptors. GIPs are involved in a wide range of functions including control of the targeting, trafficking and signalling of GPCRs. One of them, Shank, which is a secondary and tertiary partner of metabotropic and ionotropic glutamate receptors, respectively, can induce the formation of a whole functional glutamate 'receptosome' and the structure to which it is associated, the dendritic spine.

5-HT4 receptors.

Serotonin 4 receptors (5-HT(4)Rs) were discovered 15 years ago. They are coded by a very complex gene (700Kb, 38 exons) which generates eight carboxy-terminal variants (a, b, c, d, e, f, g, n). Their sequences differ after position L(358). Another variant is characterized by a 14 residue insertion within the extracellular loop 2. Highly selective potent 5-HT(4) receptor antagonists and partial agonists which cross the blood-brain barrier have been synthesized, but a specific full agonist for brain studies is still missing. Based on physiological and behavioral experiments, 5-HT(4)Rs may be targets to treat cognitive deficits, abdominal pain and feeding disorders. One 5-HT(4)R-directed drug (SL65.0155) is already in phase II to treat patients suffering from memory deficits or dementia.

Neuroprotective activity of antazoline against neuronal damage induced by limbic status epilepticus.

Imidazoline drugs exert neuroprotective effects in cerebral ischaemia models. They also have effects against mouse cerebellar and striatal neuronal death induced by N-methyl-D-aspartate (NMDA) through the blockade of NMDA currents. Here, we investigated the effects of antazoline on NMDA toxicity and current in rat hippocampal neuronal cultures, and on an in vivo model of status epilepticus. In hippocampal cultures, antazoline (30 microM) decreased NMDA-mediated neurotoxicity and also blocked the NMDA current with voltage-dependent and fast-reversible action (inhibition by 85+/-3% at -60 mV). Status epilepticus was induced by injecting pilocarpine (200 nmol) directly into the right pyriform cortex of male adult rats. The rats then received immediately three consecutive i.p. injections at 30-min intervals of either PBS (control group) or antazoline at 10 mg/kg (low-dose group) or at 45 mg/kg (high-dose group). During the 6-h recording, status epilepticus lasted more than 200 min in all groups. In the high-dose group only, seizures completely ceased 1 h after the third injection of antazoline, then started again 1 h later. Rats were killed 1 week later, and Cresyl Violet-stained sections of their brain were analysed for damage quantification. On the ipsilateral side to the pilocarpine injection, pyriform cortex and hippocampal CA1 and CA3 areas were significantly protected in both antazoline-treated groups, whilst prepyriform and entorhinal cortices were only in the high-dose group. On the contralateral side to the pilocarpine injection, only the hippocampal CA3 area was significantly protected in the low-dose group, but all investigated structures were in the high-dose group. In conclusion, antazoline is a potent neuroprotective drug in different models of neuronal primary culture, as previously shown in striatal and cerebellar granule neurons [Neuropharmacology 39 (2000) 2244], and here in hippocampal neurons. Antazoline is also neuroprotective in vivo in the intra-pyriform pilocarpine-induced status epilepticus model.

Impaired somatodendritic responses to pituitary adenylate cyclase-activating polypeptide (PACAP) of supraoptic neurones in PACAP type I -receptor deficient mice.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor (PAC1 receptor) in regulating hypothalamic supraoptic neurones was investigated using PAC1 receptor-deficient male mice (PAC1-/-). The effects of PACAP on [Ca2+]i were investigated in freshly dissociated supraoptic neurones and on the somatodendritic release of vasopressin and oxytocin, examined on intact supraoptic nuclei. In supraoptic neurones from wild-type mice (PAC1+/+), 100 nm PACAP induced an increase in [Ca2+]i and release of vasopressin and oxytocin, whereas in heterozygous (PAC1+/-) and null-mutant mice (PAC1-/-), PACAP was much less effective. PACAP had no effect on these two parameters when applied to isolated neurohypophysial nerve terminals of PAC1+/+ and PAC1-/- mice, and rats. In conclusion, the PAC1 receptor is solely responsible for the PACAP-induced [Ca2+]i signalling and secretion of vasopressin and oxytocin in the somatodendritic region of supraoptic neurones.

PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7.

Both postsynaptic density and presynaptic active zone are structural matrix containing scaffolding proteins that are involved in the organization of the synapse. Little is known about the functional role of these proteins in the signaling of presynaptic receptors. Here we show that the interaction of the presynaptic metabotropic glutamate (mGlu) receptor subtype, mGlu7a, with the postsynaptic density-95 disc-large zona occludens 1 (PDZ) domain-containing protein, PICK1, is required for specific inhibition of P/Q-type Ca(2+) channels, in cultured cerebellar granule neurons. Furthermore, we show that activation of the presynaptic mGlu7a receptor inhibits synaptic transmission and this effect also requires the presence of PICK1. These results indicate that the scaffolding protein, PICK1, plays an essential role in the control of synaptic transmission by the mGlu7a receptor complex.

Group 2 metabotropic glutamate receptors induced long term depression in mouse striatal slices.

We studied the roles of mGlu2/3 receptors (mGlu2/3) in glutamatergic transmission at corticostriatal synapses in mice brain slices. Perfusion of the selective mGlu2/3 agonists LY354740 and L-CCG1 caused the long term depression (LTD) of evoked synaptic responses. Photonic and electronic microscopy showed mGlu2/3 on axonal fibers and glial processes but not on striatal dendrites. mGlu2/3-LTD was independent of synaptic activity and insensitive to specific antagonists of dopamine D1, D2, GABA(B), N-methyl-D-aspartate or adenosine A1 receptors. Manipulation of the cAMP/protein kinase A cascade had no effect on the mGlu2/3-LTD. In contrast, MEK1-2 inhibitors reduced both mGlu2/3 initial depression and LTD suggesting the involvement of the mitogen activated kinase pathway in mGlu2/3-LTD.

Molecular events involved in neuronal death induced in the mouse hippocampus by in-vivo injection of kainic acid.

Apoptosis results from the activation of a programmed cellular cascade involving several mechanisms. In the present study, we have investigated the implication of three molecules of this cascade, p53, Bax and caspase-3, in neuronal death induced by kainic acid (KA) administration in mouse hippocampus. Using immunocytochemistry, western blot and quantification of enzyme activity, we observed in p53+/+ and p53-/- animals that KA induced neuronal death by both p53-dependent and independent pathways. Moreover, apoptosis (labeled by TUNEL) and the increase of bax and caspase-3 protein expression after the neurotoxic insult appeared to clearly depend on p53 expression.

The C terminus of the metabotropic glutamate receptor subtypes 2 and 7 specifies the receptor signaling pathways.

There is accumulating evidence that the specificity of the transduction cascades activated by G protein-coupled receptors cannot solely depend on the nature of the coupled G protein. To identify additional structural determinants, we studied two metabotropic glutamate (mGlu) receptors, the mGlu2 and mGlu7 receptors, that are both coupled to G(o) proteins but are known to affect different effectors in neurons. Thus, the mGlu2 receptor selectively blocks N- and L-type Ca(2+) channels via a protein kinase C-independent pathway, whereas the mGlu7 receptor selectively blocks P/Q-type Ca(2+) channels via a protein kinase C-dependent pathway, and both effects are pertussis toxin-sensitive. We examined the role of the C-terminal domain of these receptors in this coupling. Chimeras were constructed by exchanging the C terminus of these receptors and transfected into neurons. Different chimeric receptors bearing the C terminus of mGlu7 receptor blocked selectively P/Q-type Ca(2+) channels, whereas chimeras bearing the C terminus of mGlu2 receptor selectively blocked N- and L-type Ca(2+) channels. These results show that the C terminus of mGlu2 and mGlu7 receptors is a key structural determinant that allows these receptors to select a specific signaling pathway in neurons.

Distribution of metabotropic glutamate receptor DmGlu-A in Drosophila melanogaster central nervous system.

L-glutamate is the excitatory neurotransmitter at neuromuscular junctions in insects. It may also be involved in neurotransmission within the central nervous system (CNS), but its function therein remains elusive. The roles of glutamatergic synapses in the Drosophila melanogaster CNS were investigated, with focus on the study of DmGluRA, a G-protein-coupled glutamate receptor. In a first attempt to determine the function of this receptor, we describe its distribution in the larval and adult Drosophila CNS, using a polyclonal antibody raised against the C-terminal sequence of the protein. DmGluRA is expressed in a reproducible pattern both in the larva and in the adult. In particular, DmGluRA can be found in the antennal lobes, the optic lobes, the central complex, and the median bundle in the adult CNS. However, DmGluRA-containing neurons represented only a small fraction of all CNS neurons. DmGluRA immunoreactivity was not detected at the larval neuromuscular junction nor in the body wall muscles. The correlations between DmGluRA distribution and previously described glutamate-like immunoreactivity patterns, as well as the implications of these observations concerning the possible functions of DmGluRA in the Drosophila CNS, are discussed.

Agonist-independent activation of metabotropic glutamate receptors by the intracellular protein Homer.

G-protein-coupled receptors (GPCRs) transduce signals from extracellular transmitters to the inside of the cell by activating G proteins. Mutation and overexpression of these receptors have revealed that they can reach their active state even in the absence of agonist, as a result of a natural shift in the equilibrium between their inactive and active conformations. Such agonist-independent (constitutive) activity has been observed for the glutamate GPCRs (the metabotropic glutamate receptors mGluR1a and mGluR5) when they are overexpressed in heterologous cells. Here we show that in neurons, the constitutive activity of these receptors is controlled by Homer proteins, which bind directly to the receptors' carboxy-terminal intracellular domains. Disruption of this interaction by mutagenesis or antisense strategies, or expression of endogenous Homer1a (H1a), induces constitutive activity in mGluR1a or mGluR5. Our results show that these glutamate GPCRs can be directly activated by intracellular proteins as well as by agonists.

BAY36-7620: a potent non-competitive mGlu1 receptor antagonist with inverse agonist activity.

L-Glutamate (Glu) activates at least eight different G protein-coupled receptors known as metabotropic glutamate (mGlu) receptors, which mostly act as regulators of synaptic transmission. These receptors consist of two domains: an extracellular domain in which agonists bind and a transmembrane heptahelix region involved in G protein activation. Although new mGlu receptor agonists and antagonists have been described, few are selective for a single mGlu subtype. Here, we have examined the effects of a novel compound, BAY36-7620 [(3aS,6aS)- 6a-Naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on], on mGlu receptors (mGlu1-8), transiently expressed in human embryonic kidney 293 cells. BAY36-7620 is a potent (IC(50) = 0.16 microM) and selective antagonist at mGlu1 receptors and inhibits >60% of mGlu1a receptor constitutive activity (IC(50) = 0.38 microM). BAY36-7620 is therefore the first described mGlu1 receptor inverse agonist. To address the mechanism of action of BAY36-7620, Glu dose-response curves were performed in the presence of increasing concentrations of BAY36-7620. The results show that BAY36-7620 largely decreases the maximal effect of Glu. Moreover, BAY36-7620 did not displace the [(3)H]quisqualate binding from the Glu-binding pocket, further indicating that BAY36-7620 is a noncompetitive mGlu1 antagonist. Studies of chimeric receptors containing regions of mGlu1 and regions of DmGluA, mGlu2, or mGlu5, revealed that the transmembrane region of mGlu1 is necessary for activity of BAY36-7620. Transmembrane helices 4 to 7 are shown to play a critical role in the selectivity of BAY36-7620. This specific site of action of BAY36-7620 differs from that of competitive antagonists and indicates that the transmembrane region plays a pivotal role in the agonist-independent activity of this receptor. BAY36-7620 will be useful to further delineate the functional importance of the mGlu1 receptor, including its putative agonist-independent activity.

Alternative splicing of the imprinted candidate tumor suppressor gene ZAC regulates its antiproliferative and DNA binding activities.

ZAC encodes a zinc finger protein with antiproliferative activity, is maternally imprinted and is a candidate for the tumor suppressor gene on 6q24. ZAC expression is frequently lost in breast and ovary tumor-derived cell lines and down-regulated in breast primary tumors. In this report, we describe ZACDelta2, an alternatively spliced variant of ZAC lacking the sequence encoding the two N-terminal zinc fingers. Messenger RNAs encoding ZAC or ZACDelta2 were equally abundant and both proteins were nuclear. ZACDelta2 displayed an improved transactivation activity and an enhanced affinity for a ZAC binding site, suggesting that the two N-terminal zinc fingers negatively regulated ZAC binding to its target DNA sequences. Both proteins were equally efficient in preventing colony formation, indicating similar overall antiproliferative activities. However, these activities resulted from a differential regulation of apoptosis vs cell cycle progression since ZACDelta2 was more efficient at induction of cell cycle arrest than ZAC, whereas it was the reverse for apoptosis induction. Hence, these data further underline that ZAC gene is critically controlled, both at the transcriptional level through imprinting and at the functional level through alternative splicing.

Characterization of the methylation-sensitive promoter of the imprinted ZAC gene supports its role in transient neonatal diabetes mellitus.

ZAC is a recently isolated zinc finger protein that induces apoptosis and cell cycle arrest. The corresponding gene is imprinted maternally through an unknown mechanism and maps to 6q24-q25, within the minimal interval harboring the gene responsible for transient neonatal diabetes mellitus (TNDM) and a tumor suppressor gene involved in breast cancer. Because of its functional properties, imprinting status, and expression pattern in mammary cell lines and tumors, ZAC is the best candidate so far for both disease conditions. In the present work, we delineated ZAC genomic organization and mapped its transcriptional start site. It is noteworthy that the ZAC promoter localized to the CpG island harboring the methylation imprint associated with TNDM and methylation of this promoter silenced its activity. These data indicate that the methylation mark may have a direct effect on the silencing of the ZAC imprinted allele. Our findings further strengthen the hypothesis that ZAC is the gene responsible for TNDM and suggest a novel mechanism for ZAC inactivation in breast tumors.

Constitutively active mutants of 5-HT4 receptors are they in unique active states?

Somatic mutations leading to constitutively active G-protein coupled receptors (GPCRs) are responsible for certain human diseases. A consistent structural description of the molecular change underlying the conversion of GPCRs from an inactive R state to an active R* state is lacking. Here, we show that a series of constitutively active 5-HT4 receptors (mutated or truncated in the C-terminal and the third intracellular loop) were characterized by an increase in their denaturation rate at 55 degrees C. The thermal denaturation kinetics were monophasic, suggesting that we were measuring mainly the denaturation rate of R*. Analysis of these kinetics revealed that constitutively active C-terminal domain mutants, were due to a change in the J constant governing the R/R* equilibrium. However, the constitutive activity of the receptor mutated within the third intracellular loop was the result of both a change in the allosteric J constant and a change in the R* conformation.