BACKGROUND AND PURPOSEOrexin receptors potently signal to lipid messenger systems, and our previous studies have suggested that PLD would be one of these. We thus wanted to verify this by direct measurements and clarify the molecular mechanism of the coupling. EXPERIMENTAL APPROACHOrexin receptor-mediated PLD activation was investigated in CHO cells stably expressing human OX1 orexin receptors using KEY RESULTSOrexin stimulation strongly increased PLD activity -even more so than the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate), a highly potent activator of PLD. Both orexin and TPA responses were mediated by PLD1. Orexin-A and -B showed approximately 10-fold difference in potency, and the concentration-response curves were biphasic. Using pharmacological inhibitors and activators, both orexin and TPA were shown to signal to PLD1 via the novel PKC isoform, PKCd. In contrast, pharmacological or molecular biological inhibitors of Rho family proteins RhoA/B/C, cdc42 and Rac did not inhibit the orexin (or the TPA) response, nor did the molecular biological inhibitors of PKD. In addition, neither cAMP elevation, Gai/o nor Gbg seemed to play an important role in the orexin response. CONCLUSIONS AND IMPLICATIONSStimulation of OX1 receptors potently activates PLD (probably PLD1) in CHO cells and this is mediated by PKCd but not other PKC isoforms, PKDs or Rho family G-proteins. At present, the physiological significance of orexin-induced PLD activation is unknown, but this is not the first time we have identified PKCd in orexin signalling, and thus some specific signalling cascade may exist between orexin receptors and PKCd. Abbreviations bARK1, b-adrenoceptor kinase 1; c-and nPKC, conventional and novel PKC, respectively; GF109203X (bisindolylmaleimide I, Gö6850), 2-(1-[3-dimethylaminopropyl]-1H-indol-3-yl)-3-(1H-indol-3-yl)-maleimide; GGTI-2133, N-([4-(imidazol-4-yl)methylamino]-2- [1-naphthyl]benzoyl)leucine trifluoroacetate salt; Gö6976, 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo(2,3-a)pyrrolo(3,4-c)carbazole-12-propanenitrile; HBM, HEPES-buffered medium; KAC1-1, a peptide cPKC activator; KIC1-1, a peptide cPKC inhibitor; KAD1-1, a peptide PKCd activator; KAE1-1, a peptide PKCe activator, KIE1-1, a peptide PKCe inhibitor; MAFP, methyl arachidonyl fluorophosphonate; PA, phosphatidic acid; pEC50, -logEC50; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP5K, type I

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Increased levels of glutamate causing excitotoxic damage accompany neurological disorders such as ischemia/stroke, epilepsy and some neurodegenerative diseases. Cyclin-dependent kinase-5 (Cdk5) is important for synaptic plasticity and is deregulated in neurodegenerative diseases. However, the mechanisms by which kainic acid (KA)-induced excitotoxic damage involves Cdk5 in neuronal injury are not fully understood. In this work, we have thus studied involvement of Cdk5 in the KA-mediated degeneration of glutamatergic synapses in the rat hippocampus. KA induced degeneration of mossy fiber synapses and decreased glutamate receptor (GluR)6/7 and post-synaptic density protein 95 (PSD95) levels in rat hippocampus in vivo after intraventricular injection of KA. KA also increased the cleavage of Cdk5 regulatory protein p35, and Cdk5 phosphorylation in the hippocampus at 12 h after treatment. Studies with hippocampal neurons in vitro showed a rapid decline in GluR6/7 and PSD95 levels after KA treatment with the breakdown of p35 protein and phosphorylation of Cdk5. These changes depended on an increase in calcium as shown by the chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N ',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and glycol-bis (2-aminoethylether)-N,N,N ',N '-tetra-acetic acid. Inhibition of Cdk5 using roscovitine or employing dominant-negative Cdk5 and Cdk5 silencing RNA constructs counteracted the decreases in GluR6/7 and PSD95 levels induced by KA in hippocampal neurons. The dominant-negative Cdk5 was also able to decrease neuronal degeneration induced by KA in cultured neurons. The results show that Cdk5 is essentially involved in the KA-mediated alterations in synaptic proteins and in cell degeneration in hippocampal neurons after an excitotoxic injury. Inhibition of pathways activated by Cdk5 may be beneficial for treatment of synaptic degeneration and excitotoxicity observed in various brain diseases.

It has been proposed that OX 1 orexin receptors and CB 1 cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX 1 receptors and potentiate OX 1 receptor signaling to extracellular signal-regulated kinase (ERK). We have recently shown that OX 1 receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX 1 -CB 1 receptor interaction in signaling, for instance, in retrograde synaptic transmission. In the current study, we set out to investigate this possibility utilizing recombinant Chinese hamster ovary K1 cells. 2-AG released from OX 1 receptor-expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB 1 receptor-expressing cells. When OX 1 and CB 1 receptors were expressed in the same cells, OX 1 stimulation-induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX 1 response as such was fully abolished by specific inhibition of CB 1 receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX 1 -CB 1 heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX 1 -CB 1 synergism and thus suggest a functional rather than a molecular interaction of OX 1 and CB 1 receptors.

BACKGROUND AND PURPOSEOne of the major responses upon orexin receptor activation is Ca 2+ influx, and this influx seems to amplify the other responses mediated by orexin receptors. However, the reduction in Ca 2+, often used to assess the importance of Ca 2+ influx, might affect other properties, like ligand−receptor interactions, as suggested for some GPCR systems. Hence, we investigated the role of the ligand−receptor interaction and Ca 2+ signal cascades in the apparent Ca 2+ requirement of orexin-A signalling. EXPERIMENTAL APPROACHReceptor binding was assessed in CHO cells expressing human OX1 receptors with [ 125 I]-orexin-A by conventional ligand binding as well as scintillation proximity assays. PLC activity was determined by chromatography. KEY RESULTSBoth orexin receptor binding and PLC activation were strongly dependent on the extracellular Ca 2+ concentration. The relationship between Ca 2+ concentration and receptor binding was the same as that for PLC activation. However, when Ca 2+ entry was reduced by depolarizing the cells or by inhibiting the receptor-operated Ca 2+ channels, orexin-A-stimulated PLC activity was much more strongly inhibited than orexin-A binding. CONCLUSIONS AND IMPLICATIONS Ca2+ plays a dual role in orexin signalling by being a prerequisite for both ligand−receptor interaction and amplifying orexin signals via Ca 2+ influx. Some previous results obtained utilizing Ca 2+ chelators have to be re-evaluated based on the results of the current study. From a drug discovery perspective, further experiments need to identify the target for Ca 2+ in orexin-A−OX1 receptor interaction and its mechanism of action. Abbreviations extracellular Ca 2+; [Ca 2+

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a b s t r a c tWe wanted to analyze the basis for the distinction between OX 1 and OX 2 orexin receptors by the known agonists, orexin-A, orexin-B and Ala 11 , D-Leu 15 -orexin-B, of which the latter two show some selectivity for OX 2 . For this, chimaeric OX 1 /OX 2 and OX 2 /OX 1 orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca 2+ elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor's ligand profile.

Lynch syndrome (LS) is the most common hereditary cancer syndrome. Early diagnosis improves prognosis and reduces healthcare costs, through existing cancer surveillance methods. The problem is finding and diagnosing the cancer predisposing genetic condition. The current workup involves a complex array of tests that combines family cancer history and clinical phenotypes with tumor characteristics and sequencing data, followed by a challenging task to interpret the found variant(s). Based on the knowledge that an inherited mismatch repair (MMR) deficiency is a hallmark of LS, we have developed and validated a functional MMR test, DiagMMR, that detects inherited MMR deficiency directly from healthy tissue without need of tumor and variant information.¬ The validation included 119 skin biopsies collected from clinically pathogenic MMR variant carriers (MSH2, MSH6) and controls, and was followed by a small clinical pilot study. The repair reaction was performed on proteins extracted from primary fibroblasts and the interpretation was based on the MMR capability of the sample in relation to cut-off, which distinguishes MMR proficient (non-LS) from MMR deficient (LS) function. The results were compared to the reference standard (germline NGS). The test was shown to have exceptional specificity (100%) with high sensitivity (89%) and accuracy (97%). The ability to efficiently distinguish LS carriers from controls was further shown with a high AUROC value (0.97). This test offers an excellent tool for detecting inherited MMR deficiency linked to MSH2 or MSH6 and can be used alone or with conventional tests to recognize genetically predisposed individuals.