Jyrki P. Kukkonen

NAIP interacts with hippocalcin and protects neurons against calcium-induced cell death through caspase-3-dependent and -independent pathways

Inhibitor‐of‐apoptosis proteins (IAPs), including neuronal apoptosis inhibitory protein (NAIP), inhibit cell death. Other IAPs inhibit key caspase proteases which effect cell death, but the mechanism by which NAIP acts is unknown. Here we report that NAIP, through its third baculovirus inhibitory repeat domain (BIR3), binds the neuron‐restricted calcium‐binding protein, hippocalcin, in an interaction promoted by calcium. In neuronal cell lines NSC‐34 and Neuro‐2a, over‐expression of the BIR domains of NAIP (NAIP‐BIR1–3) counteracted the calcium‐induced cell death induced by ionomycin and thapsigargin. This protective capacity was significantly enhanced when NAIP‐BIR1–3 was co‐expressed with hippocalcin. Over‐expression of the BIR3 domain or hippocalcin alone did not substantially enhance cell survival, but co‐expression greatly increased their protective effects. These data suggest synergy between NAIP and hippocalcin in facilitating neuronal survival against calcium‐induced death stimuli mediated through the BIR3 domain. Analysis of caspase activity after thapsigargin treatment revealed that caspase‐3 is activated in NSC‐34, but not Neuro‐2a, cells. Thus NAIP, in conjunction with hippocalcin, can protect neurons against calcium‐induced cell death in caspase‐3‐activated and non‐activated pathways.

OX1 orexin receptors couple to adenylyl cyclase regulation via multiple mechanisms

In this study, the mechanism of OX1 orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC50 ≈ 1nm) inhibition and a strong (4-fold), low potency (EC50 = ≈300 nm) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of Gi/o proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC50 = ≈500 nm). cAMP elevation was not caused by Ca2+ elevation or by Gβγ. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCδ, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of Gs activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC50 of orexin-A = ≈10 nm and EC50 of orexin-B = ≈100 nm), and the response was fully dependent on PKCδ. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Gαs antibodies. We conclude that OX1 receptors stimulate adenylyl cyclase via a low potency Gs coupling and a high potency phospholipase C → PKC coupling. The former or some exogenous Gs activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX1 receptors couple to Gs almost as efficiently as the orexin-A-activated receptors, in contrast to Ca2+ elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.

Physiology of the orexinergic/hypocretinergic system: a revisit in 2012

The neuropeptides orexins and their G protein-coupled receptors, OX(1) and OX(2), were discovered in 1998, and since then, their role has been investigated in many functions mediated by the central nervous system, including sleep and wakefulness, appetite/metabolism, stress response, reward/addiction, and analgesia. Orexins also have peripheral actions of less clear physiological significance still. Cellular responses to the orexin receptor activity are highly diverse. The receptors couple to at least three families of heterotrimeric G proteins and other proteins that ultimately regulate entities such as phospholipases and kinases, which impact on neuronal excitation, synaptic plasticity, and cell death. This article is a 10-year update of my previous review on the physiology of the orexinergic/hypocretinergic system. I seek to provide a comprehensive update of orexin physiology that spans from the molecular players in orexin receptor signaling to the systemic responses yet emphasizing the cellular physiological aspects of this system.

Orexin/hypocretin receptor signalling cascades

Orexin (hypocretin) peptides and their two known G‐protein‐coupled receptors play essential roles in sleep–wake control and powerfully influence other systems regulating appetite/metabolism, stress and reward. Consequently, drugs that influence signalling by these receptors may provide novel therapeutic opportunities for treating sleep disorders, obesity and addiction. It is therefore critical to understand how these receptors operate, the nature of the signalling cascades they engage and their physiological targets. In this review, we evaluate what is currently known about orexin receptor signalling cascades, while a sister review (Leonard & Kukkonen, this issue) focuses on tissue‐specific responses. The evidence suggests that orexin receptor signalling is multifaceted and is substantially more diverse than originally thought. Indeed, orexin receptors are able to couple to members of at least three G‐protein families and possibly other proteins, through which they regulate non‐selective cation channels, phospholipases, adenylyl cyclase, and protein and lipid kinases. In the central nervous system, orexin receptors produce neuroexcitation by postsynaptic depolarization via activation of non‐selective cation channels, inhibition of K+ channels and activation of Na+/Ca2+ exchange, but they also can stimulate the release of neurotransmitters by presynaptic actions and modulate synaptic plasticity. Ca2+ signalling is also prominently influenced by these receptors, both via the classical phospholipase C−Ca2+ release pathway and via Ca2+ influx, mediated by several pathways. Upon longer‐lasting stimulation, plastic effects are observed in some cell types, while others, especially cancer cells, are stimulated to die. Thus, orexin receptor signals appear highly tunable, depending on the milieu in which they are operating.

*Orexin signaling in recombinant neuron-like cells

To assess the role of orexin receptor signaling in neuron‐like cells, Neuro‐2a murine neuroblastoma and PC12 human pheochromocytoma cells were stably transfected with human OX1 or OX2 receptors. Activation of both receptors strongly elevated cellular inositol phosphates and Ca2+. A difference in the potency between orexin‐A and ‐B was seen for OX1, but not OX2 receptors. Dependence of the orexin‐mediated Ca2+ response on extracellular Ca2+ and the observed Ba2+ influx indicate that in addition to phospholipase C, orexin receptors also may couple to similar non‐voltage‐gated Ca2+ channels in neuronal cells as previously characterized in non‐neuronal cells.

G-protein-coupled OX1 Orexin/hcrtr-1 Hypocretin Receptors Induce Caspase-dependent and -independent Cell Death through p38 Mitogen-/Stress-activated Protein Kinase

We have investigated the signaling of OX1 receptors to cell death using Chinese hamster ovary cells as a model system. OX1 receptor stimulation with orexin-A caused a delayed cell death independently of cytosolic Ca2+ elevation. The classical mitogen-activated protein kinase (MAPK) pathways, ERK and p38, were strongly activated by orexin-A. p38 was essential for induction of cell death, whereas the ERK pathway appeared protective. A pathway often implicated in the p38-mediated cell death, activation of p53, did not mediate the cell death, as there was no stabilization of p53 or increase in p53-dependent transcriptional activity, and dominant-negative p53 constructs did not inhibit cell demise. Under basal conditions, orexin-A-induced cell death was associated with compact chromatin condensation and it required de novo gene transcription and protein synthesis, the classical hallmarks of programmed (apoptotic) cell death. However, though the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (Z-VAD-fmk) fully inhibited the caspase activity, it did not rescue the cells from orexin-A-induced death. In the presence of Z-VAD-fmk, orexin-A-induced cell death was still dependent on p38 and de novo protein synthesis, but it no longer required gene transcription. Thus, caspase inhibition causes activation of alternative, gene transcription-independent death pathway. In summary, the present study points out mechanisms for orexin receptor-mediated cell death and adds to our general understanding of the role of G-protein-coupled receptor signaling in cell death by suggesting a pathway from G-protein-coupled receptors to cell death via p38 mitogen-/stress-activated protein kinase independent of p53 and caspase activation.

Orexin receptors couple to Ca2+ channels different from store-operated Ca2+ channels.

We have investigated Ca2+ release and receptor- and store-operated Ca2+ influxes in Chinese hamster ovary-K1 cells expressing human OX1 orexin receptor. Receptor-operated Ca2+ influx-response to 3 nM orexin-A was not affected by Gd3+ or 2-APB (2-aminoethoxydiphenyl borate), but was inhibited by Ni2+. Store-operated Ca2+ influx was blocked by Ni2+, Gd3+ and 2-APB, whereas the thapsigargin-induced release was unaffected. 2-APB did not block inositol-1,4,5- trisphosphate-dependent Ca2+ release in these cells. Thus, low concentrations of orexin-A cause activation of two Ca2+ influxes in the cells: primarily, a receptor-operated Ca2+ influx, and secondarily, a store-depletion activated Ca2+ influx, which is subsequent to receptor-activated Ca2+ influx and the therewith-caused IP3 production. The results show that these two rely on different molecular entities.

Orexin/hypocretin receptor signalling: a functional perspective

Multiple homeostatic systems are regulated by orexin (hypocretin) peptides and their two known GPCRs. Activation of orexin receptors promotes waking and is essential for expression of normal sleep and waking behaviour, with the sleep disorder narcolepsy resulting from the absence of orexin signalling. Orexin receptors also influence systems regulating appetite/metabolism, stress and reward, and are found in several peripheral tissues. Nevertheless, much remains unknown about the signalling pathways and targets engaged by native receptors. In this review, we integrate knowledge about the orexin receptor signalling capabilities obtained from studies in expression systems and various native cell types (as presented in Kukkonen and Leonard, this issue of British Journal of Pharmacology) with knowledge of orexin signalling in different tissues. The tissues reviewed include the CNS, the gastrointestinal tract, the pituitary gland, pancreas, adrenal gland, adipose tissue and the male reproductive system. We also summarize the findings in different native and recombinant cell lines, especially focusing on the different cascades in CHO cells, which is the most investigated cell line. This reveals that while a substantial gap exists between what is known about orexin receptor signalling and effectors in recombinant systems and native systems, mounting evidence suggests that orexin receptor signalling is more diverse than originally thought. Moreover, rather than being restricted to orexin receptor ‘overexpressing’ cells, this signalling diversity may be utilized by native receptors in a site‐specific manner.

Hippocalcin protects against caspase-12-induced and age-dependent neuronal degeneration

Hippocalcin is a neuronal calcium binding protein, but its physiological function in brain is unknown. We show here that hippocampal neurons from hippocalcin-deficient mice are more vulnerable to degeneration, particularly using thapsigargin, elevating intracellular calcium. Caspase-12 was activated in neurons lacking hippocalcin, while calpain was unchanged. Neuronal viability was accompanied by endoplasmic reticulum (ER) stress and a change in the relative induction of the ER chaperone, BiP/GRP78. Neuronal apoptosis inhibitor protein (NAIP), known to interact with hippocalcin, was not altered, but hippocampal neurons from gene-deleted mice were more sensitive to excitotoxicity caused by kainic acid. In addition, an age-dependent increase in neurodegeneration occurred in the gene-deleted mice, showing that hippocalcin contributes to neuronal viability during aging.

Regulation of OX1 orexin/hypocretin receptor‐coupling to phospholipase C by Ca2+ influx

Orexin (OX) receptors induce Ca2+ elevations via both receptor‐operated Ca2+ channels (ROCs) and the “conventional” phospholipase C (PLC)–Ca2+ release–store‐operated Ca2+ channel (SOC) pathways. In this study we assessed the ability of these different Ca2+ influx pathways to amplify OX1 receptor signalling to PLC in response to stimulation with the physiological ligand orexin‐A.