Nonclassical Ligand-Independent Regulation of Go Protein by an Orphan Class C G-Protein-Coupled Receptor.

The orphan G-protein-coupled receptor (GPCR) GPR158 is expressed in the brain, where it is involved in the osteocalcin effect on cognitive processes, and at the periphery, where it may contribute to glaucoma and cancers. GPR158 forms a complex with RGS7-ß5, leading to the regulation of neighboring GPCR-induced Go protein activity. GPR158 also interacts with αo, although no canonical Go coupling has been reported. GPR158 displays three VCPWE motifs in its C-terminal domain that are putatively involved in G-protein regulation. Here, we addressed the scaffolding function of GPR158 and its VCPWE motifs on Go. We observed that GPR158 interacted with and stabilized the amount of RGS7-ß5 through a 50-residue region downstream of its transmembrane domain and upstream of the VCPWE motifs. We show that two VCPWE motifs are involved in αo binding. Using a Gαo-ßγ bioluminescence resonance energy transfer (BRET) sensor, we found that GPR158 decreases the BRET signal as observed upon G-protein activation; however, no constitutive activity of GPR158 could be detected through the measurement of various G-protein-mediated downstream responses. We propose that the effect of GPR158 on Go is unlikely due to a canonical activation of Go, but rather to the trapping of Gαo by the VCPWE motifs, possibly leading to its dissociation from ßγ Such action of GPR158 is expected to prolong the ßγ activity, as also observed with some activators of G-protein signaling. Taken together, our data revealed a complex functional scaffolding or signaling role for GPR158 controlling Go through an original mechanism.

Inhibition of Serine Palmitoyltransferase by a Small Organic Molecule Promotes Neuronal Survival after Astrocyte Amyloid Beta 1-42 Injury.

Alzheimer's disease (AD) is a slow-progressing disease of the brain characterized by symptoms such as impairment of memory and other cognitive functions. AD is associated with an inflammatory process that involves astrocytes and microglial cells, among other components. Astrocytes are the most abundant type of glial cells in the central nervous system (CNS). They are involved in inducing neuroinflammation. The present study uses astrocyte-neuron cocultures to investigate how ARN14494, a serine palmitoyltransferase (SPT) inhibitor, affects the CNS in terms of anti-inflammation and neuroprotection. SPT is the first rate-limiting enzyme in the de novo ceramide synthesis pathway. Consistent evidence suggests that ceramide is increased in AD brain patients. After ß-amyloid 1-42 injury in an in vitro model of AD, ARN14494 inhibits SPT activity and the synthesis of long-chain ceramides and dihydroceramides that are involved in AD progression. In mouse primary cortical astrocytes, ARN14494 prevents the synthesis of proinflammatory cytokines TNFα and IL1ß, growth factor TGFß1, and oxidative stress-related enzymes iNOS and COX2. ARN14494 also exerts neuroprotective properties in primary cortical neurons. ARN14494 decreases neuronal death and caspase-3 activation in neurons, when the neuroinflammation is attenuated in astrocytes. These findings suggest that ARN14494 protects neurons from ß-amyloid 1-42 induced neurotoxicity through a variety of mechanisms, including antioxidation, antiapoptosis, and anti-inflammation. SPT inhibition could therefore be a safe therapeutic strategy for ameliorating the pathology of Alzheimer's disease.

A Triazolotriazine-Based Dual GSK-3ß/CK-1δ Ligand as a Potential Neuroprotective Agent Presenting Two Different Mechanisms of Enzymatic Inhibition.

Glycogen synthase kinase 3ß (GSK-3ß) and casein kinase 1δ (CK-1δ) are emerging targets for the treatment of neuroinflammatory disorders, including Parkinson's disease. An inhibitor able to target these two kinases was developed by docking-based design. Compound 12, 3-(7-amino-5-(cyclohexylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)-2-cyanoacrylamide, showed combined inhibitory activity against GSK-3ß and CK-1δ [IC50 (GSK-3ß)=0.17 µm; IC50 (CK-1δ)=0.68 µm]. In particular, classical ATP competition was observed against CK-1δ, and a co-crystal of compound 12 inside GSK-3ß confirmed a covalent interaction between the cyanoacrylamide warhead and Cys199, which could help in the development of more potent covalent inhibitors of GSK-3ß. Preliminary studies on in vitro models of Parkinson's disease revealed that compound 12 is not cytotoxic and shows neuroprotective activity. These results encourage further investigations to validate GSK-3ß/CK-1δ inhibition as a possible new strategy to treat neuroinflammatory/degenerative diseases.

Differential activation of the calcium/protein kinase C and the canonical beta-catenin pathway by Wnt1 and Wnt7a produces opposite effects on cell proliferation in PC12 cells.

We examined the effect of Wnt1 and Wnt7a on cell proliferation using undifferentiated PC12 cells, which originate from the neural crest and are widely employed as a neuronal cell model. Heterologous expression of Wnt1 enhanced [3H]thymidine incorporation and expression of cyclin D1 and cylin E in PC12 cells. Opposite effects were observed in PC12 cells expressing Wnt7a. Searching for the mechanisms underlying the opposite effects of Wnt1 and Wnt7a on PC12 cell proliferation, we examined the activation of the canonical beta-catenin/T-cell-lymphoid enhancer-binding protein transcription factor pathway and the 'calcium pathway' by co-transfecting the cells with a reporter gene controlled by either T-cell-lymphoid enhancer-binding protein transcription factor or the calcium-activated transcription factor, NFAT. Wnt1 and Wnt7a activated both pathways, but to a different extent. While Wnt1 preferentially activated the calcium pathway, Wnt7a mainly activated the canonical pathway. Pharmacological inhibition of protein kinase C, which is a component of the calcium pathway, abrogated the increase in cell proliferation induced by Wnt1 without affecting the antiproliferative action of Wnt7a. The action of Wnt7a was instead occluded by lithium ions, which mimic the activation of the canonical pathway, and was largely reduced by Dickkopf-1, which acts as an inhibitor of the canonical pathway. In addition, expression of a constitutively active mutant of beta-catenin potently activated the canonical Wnt pathway and reduced [3H]thymidine incorporation. These data challenge the view that the canonical Wnt pathway invariably supports cell growth and suggest that, at least in PC12 cells, cell proliferation is regulated by the balance between the calcium/protein kinase C pathway and the canonical pathway.

Metabotropic glutamate receptors: beyond the regulation of synaptic transmission.

Metabotropic glutamate (mGlu) receptors are G-protein coupled receptors activated by glutamate, the major excitatory neurotransmitter of the CNS. A growing body of evidence suggests that the function of mGlu receptors is not restricted to the regulation of synaptic transmission. mGlu receptors are expressed in a variety of peripheral cells, including inter alia hepatocytes, pancreatic cells, osteoblasts and immune cells. Within the immunological synapses, mGlu receptors expressed by T cells might contribute to the vast array of signals generated by the antigen-presenting cells. mGlu receptors are also found in embryonic and neural stem cells. This suggests their involvement in the pathophysiology of brain tumors, which likely originates from cancer stem cells similar to neural stem cells. Ligands of mGlu3 and mGlu4 receptors are potential candidates for the experimental treatment of malignant gliomas and medulloblastomas, respectively.

The 1,2,4-Triazolo[4,3-a]pyrazin-3-one as a Versatile Scaffold for the Design of Potent Adenosine Human Receptor Antagonists. Structural Investigations to Target the A2A Receptor Subtype.

In this work, we describe the identification of the 1,2,4-triazolo[4,3-a]pyrazin-3-one as a new versatile scaffold for the development of adenosine human (h) receptor antagonists. The new chemotype ensued from a molecular simplification approach applied to our previously reported 1,2,4-triazolo[4,3-a]quinoxalin-1-one series. Hence, a set of novel 8-amino-2-aryl-1,2,4-triazolopyrazin-3-one derivatives, featured by different substituents on the 2-phenyl ring (R) and at position 6 (R6), was synthesized with the main purpose of targeting the hA2A adenosine receptor (AR). Several compounds possessed nanomolar affinity for the hA2A AR (Ki = 2.9-10 nM) and some, very interestingly, also showed high selectivity for the target. One selected potent hA2A AR antagonist (12, R = H, R6 = 4-methoxyphenyl) demonstrated some ability to counteract MPP+-induced neurotoxicity in cultured human neuroblastoma SH-SY5Y cells, a widely used in vitro Parkinson's disease model. Docking studies at hAR structures were performed to rationalize the observed affinity data.

Endogenous activation of mGlu5 metabotropic glutamate receptors supports self-renewal of cultured mouse embryonic stem cells.

Cultured mouse embryonic stem (ES) cells maintained under undifferentiated conditions (i.e. grown in medium containing 15% FCS and leukemia inhibitory factor--LIF) expressed mGlu5 metabotropic glutamate receptors. Activation of these receptors with quisqualate increased [Ca2+]i but only when cultures were deprived of extracellular glutamate, indicating that the receptor was saturated by the endogenous glutamate. Pharmacological blockade of mGlu5 receptors with 2-methyl-6-(phenylethynyl)pyridine (MPEP) or antisense-induced knock-down of mGlu5 receptors decreased the expression of the two main transcription factors that sustain ES cell self-renewal, i.e. Oct-4 and Nanog, as assessed by real-time PCR and immunoblotting. Exposure of ES cell cultures to MPEP also reduced alkaline phosphatase activity, a marker of undifferentiated ES cells. These data support a critical role for mGlu receptors in early development showing that mGlu5 receptors are expressed by ES cells and their activation sustains ES cell self-renewal in culture.

Endogenously activated mGlu5 metabotropic glutamate receptors sustain the increase in c-Myc expression induced by leukaemia inhibitory factor in cultured mouse embryonic stem cells.

We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-3-K, both effects resulting from the activation of PKC.