Phospholipase Cascades in OX1 Orexin Receptor Signaling

Orexins (orexin-A and -B) are neuropeptides with multiple physiological functions, among which regulation of wakefulness and appetite are the best known. Neurons producing orexins are localized in the lateral hypothalamus, from where they send projections to many parts of the brain. Orexins exert their functions by activating two G-protein-coupled receptors, OX1 and OX2. Orexin receptor expression was first reported in the brain, but they are also expressed outside of the central nervous system (CNS). Upon activation, OX1 and OX2 receptors can couple to heterotrimeric G-proteins of different families, including Gq, Gs and Gi/o. Orexin receptor activation can evoke increases in intracellular Ca2+ concentrations via multiple mechanisms, including activation of phospholipase C and increased Ca2+ influx, and it also regulates adenylyl cyclase activity, both positively and negatively. Multiple kinases have also been reported in their signaling cascades, including protein kinases C and D, extracellular signal-regulated kinase, p38, Src, and phosphatidylinositol-3-kinase. Previous studies have suggested interactions between the orexin and endocannabinoid systems. The endocannabinoid system consists of endocannabinoids, which are neuromodulatory lipids produced on demand by neurons, their effectors (CB1 and CB2 cannabinoid receptors, as well as other receptors and channels), and finally the enzymes that degrade them. The cannabinoid and orexin systems have several overlapping functions, such as regulation of pain transmission, appetite, learning, and reward. Evidence for the existence of interactions between these systems has been gained from physiological, but also molecular studies. Even heteromerization of CB1 and OX1 receptors has been reported. In this thesis, the signaling of the OX1 receptor was further investigated, with special emphasis on lipid mediators. Recombinant cells (mainly CHO cells) were employed as model systems. We were able to directly demonstrate the activation of phospholipase D upon OX1 stimulation, and consequently add PLD to the signaling cascades mediating orexin responses. Phospholipase D activation was mediated by a novel protein kinase C isoform, most likely protein kinase Cδ. OX1 receptor activation also leads to the release of two other messengers: arachidonic acid and the endocannabinoid 2-arachidonoylglycerol. In this thesis, the release of these messengers and the pathways leading to their production, upon orexin receptor activation, were investigated in detail for the first time. Powerful arachidonic acid release by cytosolic phospholipase A2 (cPLA2) was observed in recombinant CHO cells. In contrast, 2-arachidonoylglycerol was released by a cascade involving activation of phospholipase C and diacyglycerol lipase; this was observed in CHO, neuro-2a, and HEK293 cells. By utilizing CHO cells in an artificial cell−cell communication assay, we saw that the released 2-arachidonoyl¬glycerol can act as a paracrine messenger, activating neighboring cells expressing CB1 cannabinoid receptors. 2-Arachidonoylglycerol similarly also acted as an autocrine messenger, and co-signaling of OX1 and CB1 receptors upon orexin stimulation of the receptor-co-expressing cells via the "2-arachidonoylglycerol loop" led to the potentiation of ERK activation. This implies that the significance of the previously reported OX1−CB1 interaction is more likely to originate from functional than physical interaction of the receptors. However, the idea of heteromerization between OX1 and CB1 receptors is interesting, and in the final study of the present series, we utilized the bioluminescence resonance energy transfer (BRET) method to investigate constitutive homo- and heteromerization between OX1, OX2, and CB1 receptors. According to our results, all receptor combinations readily form heteromeric complexes when expressed in CHO cells.Oreksiinit (oreksiini-A ja -B) ovat neuropeptidejä, jotka säätelevät monenlaisia keskeisiä fysiologisia toimintoja, joista eniten tutkittuja ovat valveilla olon ja ruokahalun säätely. Oreksiineja tuotetaan lateraalisen hypotalamuksen neuroneissa, jotka lähettävät haarakkeita useisiin aivojen osiin. Oreksiinien vaikutukset välittyvät G-proteiinikytkentäisten OX1- ja OX2-reseptoreiden kautta. Vaikka oreksiinireseptorit löydettiin alun perin aivoista, on niitä huomattu ilmentyvän myös keskushermoston ulkopuolisissa kudoksissa. Aktivoituneet oreksiinireseptorit voivat kytkeytyä useisiin eri G-proteiineihin, esimerkiksi Gq-, Gs- ja Gi/o-perheiden jäseniin. Solunsisäinen kalsiumpitoisuus nousee oreksiinireseptorien aktivoituessa. Tämä tapahtuu usealla eri mekanismilla kuten fosfolipaasi C:n aktivoitumisen ja kalsiumin sisäänvirtauksen lisääntymisen kautta. Oreksiinireseptorien aktivaation on myös näytetty johtavan muun useiden eri kinaasien aktivaatioon, mukaanlukien proteiinikinaasi C ja D, p38, Src ja fosfoinositidi-3-kinaasi, sekä adenylaattisyklaasin positiiviseen tai negatiiviseen säätelyyn. Aiemmissa tutkimuksissa on saatu viitteitä oreksiini- ja endokannabinoidijärjestelmien välisestä vuorovaikutuksesta. Endokannabinoidijärjestelmään kuuluvat endokannabinoidit, jotka ovat hermosolun toimintaa muokkaavia lipidejä, joita tuotetaan tarpeen mukaan, endo¬kanna-binoidien efektorit (G-proteiinikytkentäiset CB1- ja CB2-kannabinoidireseptorit sekä muut reseptorit ja ionikanavat, joiden toimintaa endokannabinoidit säätelevät), sekä entsyymit, jotka hajottavat endokannabinoideja. Oreksiini- ja endokannabinoidijärjestelmillä on useita päällekkäisiä toimintoja. Ne molemmat ovat mukana muun muassa ruokahalun, kivuntunteen ja oppimisen säätelyssä sekä palkitsemisjärjestelmissä. Sekä fysiologiset että molekulaariset tutkimukset tukevat käsitystä näiden järjestelmien vuorovaikutuksesta, ja jopa OX1- ja CB1-reseptorien heteromerisaatiosta on näyttöä. Tässä väitöskirjassa on tutkittu OX1-oreksiinireseptorien viestintää ja erityisesti lipidi-viestimolekyylien osaa siinä. Tutkimuksissa on käytetty rekombinanttisoluja, erityisesti CHO (lyhenne sanoista Chinese hamster ovary eli kiinanhamsterin munasarja) -soluja. Onnistuimme osoittamaan, että fosfolipaasi D aktivoituu OX1-oreksiinireseptoria stimuloitaessa; täten fosfolipaasi D voitiin lisätä oreksiinireseptorien säätelemiin viestireitteihin. Proteiinikinaasi Cδ näyttäisi aktivoivan fosfolipaasi D:n. OX1-oreksiinireseptorin aktivoituminen johtaa myös kahteen muuhun tärkeän lipidiviestiin, arakidonihappon ja endokannabinoidi 2-arakidonyyliglyseroliin. Tässä väitöskirjassa näiden tärkeiden viestimolekyylien vapautumiseen johtavia reittejä oreksiinireseptorien aktivoituessa tutkittiin yksityiskohtaisesti ensimmäistä kertaa. CHO-soluissa sytosolinen/Ca2+-riippuvainen fosfolipaasi A2 aktivoituu, mikä johtaa voimakkaaseen arakidonihapon vapautukseen. 2-arakidonyyliglyserolin vapautus taas tapahtuu fosfolipaasi C:n ja diasyyligyserolilipaasin aktivoitumisen kautta; tämä havaittiin CHO, neuro-2a- ja HEK293-soluissa. Kehitimme CHO-soluille solujenvälistä viestintää mittaavan koejärjestelyn, jossa vapautuneen 2-arakidonyyliglyseroli pystyi parakriinisesti aktivoimaan läheisiä soluja, kun nämä ilmensivät CB1-reseptoreita. Kun samat solut ilmensivät sekä OX1- että CB1-reseptoreita, oreksiini,reseptoristimulaation vapauttama 2-arakidonyyliglyseroli toimi myös autokriinisenä viestimolekyylinä. Tämä autokriininen viesti vahvisti ERK-viestintää näissä soluissa, ja niinpä aiemmin raportoitu ERK-viestinnän vahvistuminen molempia reseptoreita ilmentävissä soluissa onkin todennäköisemmin tämän toiminnallisen vuorovaikutuksen kuin suoran fysikaalisen vuorovaikutuksen (kuten reseptoriheteromerisaation) tulosta. Koska OX1- ja CB1-reseptorien heteromerisaatio on kuitenkin mielenkiintoinen ilmiö, tutkimme viimeisessä osatyössä tätä BRET (bioluminesenssin resonanssienergian siirtyminen) -menetelmän avulla. Kaikkien kahdenvälisten kombinaatioiden OX1-, OX2- ja CB1-reseptorien välillä nähtiin muodostavan konstitutiivisia homo- ja heteromerisiä komplekseja CHO-soluissa

Focus Localis ry kunnallistutkimuksen suunnannäyttäjänä 50 vuotta

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Six Strategies for Strong cultures How upper echelon leaders enable diffusion of innovation in health care

Master in Business Administration (MBA) - Nord universitet 201

Protein Kinase A-Mediated Effects of Protein Kinase C Partial Agonist 5-(Hydroxymethyl)Isophthalate 1a3 in Colorectal Cancer Cells

Colorectal cancer is the third most commonly occurring cancer in men and the second in women. The global burden of colorectal cancer is projected to increase to over 2 million new cases with over 1 million deaths within the next 10 years and there is a great need for new compounds with novel mechanisms of action. Our group has developed PKC modulating isophthalic acid derivatives that induce cytotoxicity towards human cervical and prostate cancer cell lines. In this study, we investigated the effects of 5-(hydroxymethyl)isophthalate 1a3 (HMI-1a3) on colorectal cancer cell lines (Caco2, Colo205 and HT29). HMI-1a3 inhibited cell proliferation, decreased cell viability and induced an apoptotic response in all studied cell lines. These effects, however, were independent of PKC. Using serine/threonine kinome profiling and pharmacological kinase inhibitors we identified activation of the cAMP/PKA pathway as a new mechanism-of-action for HMI-1a3-induced anti-cancer activity in colorectal cancer cell lines. Our current results strengthen the hypothesis for HMI-1a3 as a potential anti-cancer agent against various malignancies. Significance Statement Colorectal cancer (CRC) is a common solid organ malignancy. Here, we demonstrate that the protein kinase C (PKC) C1 domain-targeted isophthalatic acid derivative HMI-1a3 has anti-cancer activity on CRC cell lines independently of PKC. We identified protein kinase A (PKA) activation as a mechanism of HMI-1a3 induced anti-cancer effects. Our results reveal a new anti-cancer mechanism of action for the partial PKC agonist HMI-1a3 and thus provide new insights for the development of PKC and PKA modulators for cancer therapy.Peer reviewe

New tricks of prolyl oligopeptidase inhibitors - A common drug therapy for several neurodegenerative diseases

Changes in prolyl oligopeptidase (PREP) expression levels, protein distribution, and activity correlate with aging and are reported in many neurodegenerative conditions. Together with decreased neuropeptide levels observed in aging and neurodegeneration, and PREP's ability to cleave only small peptides, PREP was identified as a druggable target. Known PREP non-enzymatic functions were disregarded or attributed to PREP enzymatic activity, and several potent small molecule PREP inhibitors were developed during early stages of PREP research. These showed a lot of potential but with variable results in experimental memory models, however, the initial excitement was short-lived and all of the clinical trials were discontinued in either Phase I or II clinical trials for unknown reasons. Recently, PREP's ability to form protein-protein interactions, alter cell proliferation and autophagy has gained more attention than earlier recognized catalytical activity. Of new findings, particularly the aggregation of alpha-synuclein (aSyn) that is seen in the presence of PREP is especially interesting because PREP inhibitors are capable of altering aSyn-PREP interaction in a manner that reduces the aSyn dimerization process. Therefore, it is possible that PREP inhibitors that are altering interactions could have different characteristics than those aimed for strong inhibition of catalytic activity. Moreover, PREP co-localization with aSyn, tau, and amyloid-beta hints to PREP's possible role not only in the synucleinopathies but in other neurodegenerative diseases as well. This commentary will focus on less well-acknowledged non-enzymatic functions of PREP that may provide a better approach for the development of PREP inhibitors for the treatment of neurodegenerative disorders.Peer reviewe

Ajassa ja paikallisesti merkittävää tutkimusjulkaisemista: – Kauan eläköön Focus Localis!

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Redefining Local Self-Government: Finnish Municipalities Seeking Their Essence

acceptedVersionPeer reviewe

Ajassa ja paikallisesti merkittävää tutkimusjulkaisemista : Kauan eläköön Focus Localis!

publishedVersionNon peer reviewe

Prolyl oligopeptidase inhibition activates autophagy via protein phosphatase 2A

Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurode-generative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclinl-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 knock-out 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.Peer reviewe