We showed previously that OX 1 orexin receptor stimulation produced a strong 3 H overflow response from [ 3 H]arachidonic acid (AA)-labeled cells. Here we addressed this issue with a novel set of tools and methods, to distinguish the enzyme pathways responsible for this response. CHO-K1 cells heterologously expressing human OX 1 receptors were used as a model system. By using selective pharmacological inhibitors, we showed that, in orexin-A-stimulated cells, the AA-derived radioactivity was released as two distinct components, i.e., free AA and the endocannabinoid 2-arachidonoyl glycerol (2-AG). Two orexin-activated enzymatic cascades are responsible for this response: cytosolic phospholipase A 2 (cPLA 2 ) and diacylglycerol lipase; the former cascade is responsible for part of the AA release, whereas the latter is responsible for all of the 2-AG release and part of the AA release. Essentially only diacylglycerol released by phospholipase C but not by phospholipase D was implicated as a substrate for 2-AG production, although both phospholipases were strongly activated. The 2-AG released acted as a potent paracrine messenger through cannabinoid CB 1 receptors in an artificial cell-cell communication assay that was developed. The cPLA 2 cascade, in contrast, was involved in the activation of orexin receptor-operated Ca 2ϩ influx. 2-AG was also released upon OX 1 receptor stimulation in recombinant HEK-293 and neuro-2a cells. The results directly show, for the first time, that orexin receptors are able to generate potent endocannabinoid signals in addition to arachidonic acid signals, which may explain the proposed orexincannabinoid interactions (e.g., in neurons).

Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurodegenerative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclin1-mediated macroautophagy (autophagy), and that PREP inhibition by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson's disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, we wanted to further characterize how PREP regulates autophagy. We measured the levels of various kinases and proteins regulating beclin1-autophagy in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, and verified the results in vivo by using PREP knockout and wild-type mouse tissue where PREP was restored or overexpressed, respectively. We found that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), and activator (protein phosphatase 2 phosphatase activator, PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of deathassociated protein kinase 1 (DAPK1), beclin1 phosphorylation and induced autophagy while PREP overexpression reduced this. Lowered activity of PP2A is connected to several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy but development of such compounds has been a challenge. The concept of PREP inhibition has been proved safe, and therefore, our study supports the further development of PREP inhibitors as PP2A activators.

Huey and Slatkin's (Q Rev Biol 51:363-384, 1976) cost-benefit model of lizard thermoregulation predicts variation in thermoregulatory strategies (from active thermoregulation to thermoconformity) with respect to the costs and benefits of the thermoregulatory behaviour and the thermal quality of the environment. Although this framework has been widely employed in correlative field studies, experimental tests aiming to evaluate the model are scarce. We conducted laboratory experiments to see whether the common lizard Zootoca vivipara, an active and effective thermoregulator in the field, can alter its thermoregulatory behaviour in response to differences in perceived predation risk and food supply in a constant thermal environment. Predation risk and food supply were represented by chemical cues of a sympatric snake predator and the lizards' food in the laboratory, respectively. We also compared males and postpartum females, which have different preferred or "target" body temperatures. Both sexes thermoregulated actively in all treatments. We detected sex-specific differences in the way lizards adjusted their accuracy of thermoregulation to the treatments: males were less accurate in the predation treatment, while no such effects were detected in females. Neither sex reacted to the food treatment. With regard to the two main types of thermoregulatory behaviour (activity and microhabitat selection), the treatments had no significant effects. However, postpartum females were more active than males in all treatments. Our results further stress that increasing physiological performance by active thermoregulation has high priority in lizard behaviour, but also shows that lizards can indeed shift their accuracy of thermoregulation in response to costs with possible immediate negative fitness effects (i.e. predation-caused mortality).

Alzheimer's disease (AD), the most common cause of dementia, is an irreversible and progressive neurodegenerative disorder. It affects predominantly brain areas that are critical for memory and learning and is characterized by two main pathological hallmarks: extracellular amyloid plaques and intracellular neurofibrillary tangles. Protein kinase C (PKC) has been classified as one of the cognitive kinases controlling memory and learning. By regulating several signalling pathways involved in amyloid and tau pathologies, it also plays an inhibitory role in AD pathophysiology. Among downstream targets of PKC are the embryonic lethal abnormal vision (ELAV)-like RNA-binding proteins that modulate the stability and the translation of specific target mRNAs involved in synaptic remodelling linked to cognitive processes. This MiniReview summarizes the current evidence on the role of PKC and ELAV-like proteins in learning and memory, highlighting how their derangement can contribute to AD pathophysiology. This last aspect emphasizes the potential of pharmacological activation of PKC as a promising therapeutic strategy for the treatment of AD. Alzheimer's DiseaseAlzheimer's disease (AD) is a progressive neurodegenerative disorder, which leads to severe impairment of memory and cognitive functions, alterations in behaviour, incapacity for independent living and, finally, to death. It is the most common cause of dementia [1], which affects more than 35 million people worldwide [2]. The prevalence of both AD and dementia escalates along with increasing age. As the life span in developing countries rises, the number of people with dementia is expected to almost double every 20 years and reach 115 million in 2050 [2]. In the World Alzheimer Report 2013, it was estimated that in 2013, the global costs of dementia exceed US$600 billion, which is approximately 1% of global gross domestic product [3].The current pharmacological therapy for AD includes cholinesterase inhibitors (donepezil, rivastigmine and galantamine) as well as memantine, which is a low-affinity voltage-dependent uncompetitive antagonist of glutamatergic N-methyl-Daspartate (NMDA) receptors. Additionally, the neuropsychiatric symptoms are commonly treated with antidepressants and/or risperidone or other atypical antipsychotics. All of the available treatments are symptomatic and cannot cure or significantly inhibit the progression of the disease. Therefore, new disease-modifying treatments are urgently needed.The main neuropathological change characteristic to AD is the loss of cholinergic neurons in the nucleus basalis magnocellularis and their synapses, particularly in cerebral cortex, hippocampus and other subcortical structures that contribute to memory formation [4,5]. The underlying neuropathogenesis is not known, but the loss of neurons results from the accumulation of two distinctive pathological features: predominantly extracellular b amyloid (Ab) deposits (plaques) and intracellular neurofibrillary tangles consisting of hyperphosphorylated tau p...

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