Opioid peptidek sejtproliferációt gátló hatásmechanizmusának további vizsgálata uterusban = Further studies on the mechanism of the cell proliferation-inhibitory action of opioid peptides in the uterus

Az opioid peptidek sejtproliferációt gátló hatását vizsgáltuk tenyésztett patkány és humán uterus sejtekben. Méréseinkhez sejtszámlálást, DNS meghatározást, MTT sejtproliferációs assay-t és immunoblot módszereket alkalmaztunk. Adataink alapján az opioid peptidek patkány uterus sejtek osztódását gátló hatásában két eltérő fázis különíthető el az egyedfejlődés alatt, melyeket egy érzéketlen periódus választ el egymástól. Az opioid peptid és progeszteron receptor rendszerek között kétirányú kapcsolat működik felnőtt patkány uterus sejtek, valamint humán myometrium és endometrium sejtek szaporodásának gátló jellegű szabályozásában. Fiatal, éretlen patkányok uterus sejtjeiben e molekuláris kapcsolat még nem mutatható ki. A naloxon- és az RU486-hatás időfüggésének eltérései alapján e kétirányú kapcsolat eltérő támadáspontokon keresztül valósulhat meg. Az opioid peptidek sejtosztódást gátló mechanizmusa és az ópiát-progeszteron kölcsönhatás működőképes humán leiomyoma sejtekben is. Azonban [D-Met2-Pro5]-enkefalinamid (ENK) a leiomyoma sejtek nem-stimulált szaporodását is gátolta, és az ENK és DAMGO mellett itt [Met5]-enkefalin is hatásosnak bizonyult a 7 napos patkány uterusban leírtakhoz hasonlóan. A myometrium sejtekben találtakkal ellentétben, RU486 önmagában nem serkentette a leiomyoma sejtek a szaporodását, azonban az ENK gátló hatását kivédte. Az eltérések a sejtosztódás szabályozásának részleges dedifferentációját mutathatják, ami a pathomechanizmusban is szerepet játszhat. | The inhibitory action of opioid peptides on the proliferation of cultured rat and human uterine cells was investigated. Cell counting, DNA determination, MTT cell proliferation assay and immunoblot technique were used to obtain data. Our data demonstrate two different phases of the inhibitory action of opioid peptides on rat uterine cell proliferation during ontogeny with an insensitive interval in between. A bidirectional interaction exists between the opioid peptide and progesterone signaling systems in the inhibitory regulation of cell proliferation in adult rat uterus and in human myometrium and endometrium. Uteri of immature rats lack this molecular connection. The difference between the time dependence of naloxon and RU486 actions suggests that, this bidirectional action is realized by targeting different elements in the mechanism of action. The opioid peptides' growth inhibitory system and the opiate-progesterone interaction are functional in human uterine leiomyoma cells. However, [D-Met2-Pro5]-enkephalinamide (ENK) inhibited the non-stimulated proliferation of leiomyoma cells and, besides ENK and DAMGO, [Met5]-enkephalin was effective as well, similar to that of found in 7-day-old rats. In contrast to myometrial cells, RU486 alone did not stimulate the proliferation of leiomyoma cells however; it antagonized the inhibitory effect of ENK. These alterations suggest a partial dedifferentiation of cell-division regulation and, might play a role in the pathomechanism

Membran ösztrogén receptorok sejtszintű hatásainak vizsgálata human uterus hormonális szabályozásában és daganatos elváltozások patomechanizmusában = Cellular function of membrane estrogen receptors in hormonal control of human uterus and in pathomechanism of tumor formation

Patkány uterusban E2 kezelés hatására az Akt foszforilációja a Ser473 és a FOXO1 Ser 256 csoportokon fokozódik, amit ICI 182, 780 és Wortmannin gátol. Az Akt expresszió nem változott, a pAkt myomában jelentősen fokozódott. A pAkt a proliferációs fázisban volt a legmagasabb. Myomákban a ciklus alatti Akt foszforiláciő fokozódásával párhuzamosan az ER foszforilációja, a cyclin D1 és Bcl-2 proteinek expressziója is fokozódott. Myomában az inaktív pPTEN-Ser380 fokozódott a pAkt változásokkal megegyező módon. A myometriumokban pPTEN szintekben ciklus fázisoktól függő és pAkt-tól független változásokat találtunk. A PTEN ínaktivációja és Akt aktivációja myomában a menstruációs ciklus alatt párhuzamosan változott. A pFOXO1 (Ser256) protein szintje magasabb volt myomában és változott a menstruációs ciklus alatt ill. menopauzában. Myomában a pFOXO-Ser256 elsősorban a magban volt kimutatható, feltehetően a nucleocytoplasmatikus transport zavara miatt, aminek az oka valószínűleg a párhuzamosan meghatározott 14-3-3 protein expresszió csökkenése.Myomában a megváltozott PTEN/ER/PI3K/Akt/FOXO1 jelátvitel a sejtekben sejttúlélést biztosító (prosurvival) programok előtérbe kerülését serkenti. Az ERalpha expresszió postmenopauzás endometriumban irregulárisan változott. Az ER(Ser167) és Akt foszforilációja magasabb vér E2 szintnél (>50pmol/L) fokozódott. A pAkt pozitívan korrelált se E2 koncentrációval (r: 0.811, p50pmol/L). The pAkt (Ser473) was abundant in endometrium of women with higher (>50 pmol/L) E2 concentration. The pAkt significantly correlated with Se E2 (r: 0.811, p<0.001) and pERalpha (r: 0.879, p< 0.001)

Regulation of mouse microglia activation and effector functions by bone marrow-derived mesenchymal stem cells

Mesenchymal stem or stromal cells (MSCs) are rare multipotent cells with potent regenerative and immunomodulatory properties. Microglial cells are specialized tissue macrophages of the central nervous system (CNS) that continuously survey their environment with highly motile extensions. Recently several studies have shown that MSCs are capable of reprogramming microglia into an “M2-like” phenotype characterized by increased phagocytic activity and upregulated expression of anti- inflammatory mediators in vitro. However, the precise polarization states of microglia in the presence of MSCs under physiological or under inflammatory conditions remain largely unknown. In this study, we found that MSCs induce a mixed microglia phenotype defined as Arg-1-high, CD86-high, CD206-high, IL-10-high, PGE2-high, MCP-1/CCL2-high, IL-1β- moderate, NALP-3-low, and TNF-α-low cells. These MSC-elicited microglial cells have high phagocytic activity and antigen-presenting ability. Lipopolysaccharide (LPS) is able to shape this microglia phenotype quantitatively, but not qualitatively in the presence of MSCs. This unique polarization state resembles a novel regulatory microglia phenotype, which might contribute to the resolution of inflammation and to tissue repair in the CNS

Pozícionális gének aktivitásának szerepe az idegsejt-fenotípus meghatározásában = Role of positional genes in the determination of the neuronal phenotype

A jellegzetes mintázatban aktiválódó pozícionális gének agyfejlődésben játszott szerepét vizsgálva az alábbi eredményeket nyertük: 1. A korai NE-4C embrionális idegi őssejtek indukálatlan állapotban regionálisan nem elkötelezettek; a neuron-képzés időszakában a regionális gének széles skálája aktiválódik; a sejtekből GABAerg, glutamaterg és szerotonin termelő neuronok is fejlődnek. 2. Az NE-4C sejtvonal transzfekciójával 11 idegi őssejtklónt alapítottunk; ezek a fejlődés minden szakaszában expresszálták az emx2 (antero-dorzális telencephalon) regionális gént; az NE-4Cemx2+ sejtek adhéziós sajátságai megváltoztak és katekolamin termelő neuronokat is képeztek. 3. Idegi őssejtvonalakat izoláltunk embrionális és kifejlett egéragyból új, szintetikus adhezív peptidkonjugátumok alkalmazásával; 4. A különböző agyi régiókból származó idegi őssejtvonalak és a belőlük in vitro fejlődő idegszöveti sejttípusok sok eredetre jellemző sajátságot megőriztek, de a hagyományos regionális gén-mintázat ezt az „emlékezetet” nem tükrözte; 5. Az egyes őssejt-klónok retinoid-érzékenysége és inherens retinsav metabolizmusa eltérő. Igazoltuk, hogy felnőtt agy neurogén régiói magas retinsav-tartalommal bírnak. 6. A kifejlett agyi parenchyma nem nyújt befogadó környeztet az őssejtek számára; a sérült előagyban az ős/progenitor sejtek szaporodnak, de nem differenciálódnak; túlnyomásos O2-kezelés hatására sporadikus idegsejt irányú fejlődés indítható. | Studies on developmental roles of „positional genes” in the formation of regional brain features led to the following results: 1. Early embryonic neural stem cells (NE-4C) are regionally not determined; in neuron-fromation phase, however, many regional genes got activated; GABAergic and glutamatergic neurons developed from the clon. 2. Inserting the Emx2 (antero-dorsal telencephalic) positional gene into NE-4C cells, 11 sub-clones were established, all expressing Emx2 throughout the entire differentiation period. The NE-4Cemx2+ cells displayed altered adhesive characteristics, and could generate catecholamine producing neurons. 3. By using novel synthetic adhesive peptide-conjugates, neural stem/progenitor clones had been established from different ages and regions of the mouse brain. 4. Neural stem/progenitor clones preserved several features characteristic to their origin, while the expression profile of traditional „regional genes” failed to reflect the regional „memory”. 5. The stem/progenitor clones displayed important differences in retinoid sentitivity and metabolism. In the neurogenic zones of the adult brain, enhanced retinoic acid contaent was demonstrated. 6. The adult brain parenhcyma is not permissive for the implanted stem/progenitor cells, regardless of their origin. In cortical lesion sites, the stem/progenitor cells proliferate, but do not differentiate. Hyperbaric O2-treatment was shown to allow sporadic neuronal differentiation

Versatility of microglial bioenergetic machinery under starving conditions

Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear. In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary - and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies. All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation. It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism

Retinoid Machinery in Distinct Neural Stem Cell Populations with Different Retinoid Responsiveness

Retinoic acid (RA) is present at sites of neurogenesis in both the embryonic and adult brain. While it is widely accepted that RA signaling is involved in the regulation of neural stem cell differentiation, little is known about vitamin A utilization and biosynthesis of active retinoids in the neurogenic niches, or about the details of retinoid metabolism in neural stem cells and differentiating progenies. Here we provide data on retinoid responsiveness and RA production of distinct neural stem cell/neural progenitor populations. In addition, we demonstrate differentiation-related changes in the expression of genes encoding proteins of the retinoid machinery, including components responsible for uptake (Stra6) and storage (Lrat) of vitamin A, transport of retinoids (Rbp4, CrbpI, CrabpI-II), synthesis (Rdh10, Raldh1-4), degradation of RA (Cyp26a1-c1) and RA signaling (Raralpha,beta,gamma, Rxralpha,beta,gamma). We show that both early embryonic neuroectodermal (NE-4C) stem cells and late embryonic or adult derived radial glia like progenitors (RGl cells) are capable to produce bioactive retinoids but respond differently to retinoid signals. However, while neuronal differentiation of RGl cells can not be induced by RA, neuron formation by NE-4C cells is initiated by both RA and RA-precursors (retinol or retinyl acetate). The data indicate that endogenous RA production, at least in some neural stem cell populations, may result in autocrine regulation of neuronal differentiation

Co-transmission of acetylcholine and GABA regulates hippocampal states

The basal forebrain cholinergic system is widely assumed to control cortical functions via non-synaptic transmission of a single neurotransmitter. Yet, we find that mouse hippocampal cholinergic terminals invariably establish GABAergic synapses, and their cholinergic vesicles dock at those synapses only. We demonstrate that these synapses do not co-release but co-transmit GABA and acetylcholine via different vesicles, whose release is triggered by distinct calcium channels. This co-transmission evokes composite postsynaptic potentials, which are mutually cross-regulated by presynaptic autoreceptors. Although postsynaptic cholinergic receptor distribution cannot be investigated, their response latencies suggest a focal, intra- and/or peri-synaptic localisation, while GABAA receptors are detected intra-synaptically. The GABAergic component alone effectively suppresses hippocampal sharp wave-ripples and epileptiform activity. Therefore, the differentially regulated GABAergic and cholinergic co-transmission suggests a hitherto unrecognised level of control over cortical states. This novel model of hippocampal cholinergic neurotransmission may lead to alternative pharmacotherapies after cholinergic deinnervation seen in neurodegenerative disorders

Atomic Force Microscopy of height fluctuations of fibroblast cells

We investigated the nanometer scale height fluctuations of 3T3 fibroblast cells with the atomic force microscope (AFM) under physiological conditions. Correlation between these fluctuations and lateral cellular motility can be observed. Fluctuations measured on leading edges appear to be predominantly related to actin polymerization-depolymerization processes. We found fast (5 Hz) pulsatory behavior with 1--2 nm amplitude on a cell with low motility showing emphasized structure of stress fibres. Myosin driven contractions of stress fibres are thought to induce this pulsation.Comment: 6 pages, 5 figures, 1 tabl

Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke

Microglia are the main immune cells of the brain and contribute to common brain diseases. However, it is unclear how microglia influence neuronal activity and survival in the injured brain in vivo. Here we develop a precisely controlled model of brain injury induced by cerebral ischaemia combined with fast in vivo two-photon calcium imaging and selective microglial manipulation. We show that selective elimination of microglia leads to a striking, 60% increase in infarct size, which is reversed by microglial repopulation. Microglia-mediated protection includes reduction of excitotoxic injury, since an absence of microglia leads to dysregulated neuronal calcium responses, calcium overload and increased neuronal death. Furthermore, the incidence of spreading depolarization (SD) is markedly reduced in the absence of microglia. Thus, microglia are involved in changes in neuronal network activity and SD after brain injury in vivo that could have important implications for common brain diseases

Microglial control of neuronal development via somatic purinergic junctions

Microglia, the resident immune cells of the brain, play important roles during development. Although bi-directional communication between microglia and neuronal progenitors or immature neurons has been demonstrated, the main sites of interaction and the underlying mechanisms remain elusive. By using advanced methods, here we provide evidence that microglial processes form specialized contacts with the cell bodies of developing neurons throughout embryonic, early postnatal, and adult neurogenesis. These early developmental contacts are highly reminiscent of somatic purinergic junctions that are instrumental for microglia-neuron communication in the adult brain. The formation and maintenance of these junctions is regulated by functional microglial P2Y12 receptors, and deletion of P2Y12Rs disturbs proliferation of neuronal precursors and leads to aberrant cortical cytoarchitecture during development and in adulthood. We propose that early developmental formation of somatic purinergic junctions represents an important interface for microglia to monitor the status of immature neurons and control neurodevelopment