Intercelluláris funkciós molekulák működése agyi endotélsejtekben = The function of junctional molecules in cerebral endothelial cells

Kutatásaink során az interendotheliális kapcsolatok szerepét vizsgáltuk elsősorban patológiás körülmények között. Megállapítottuk, hogy agyi endotélsejtekben az oxidatív stressz genotoxikus hatással rendelkezik és a MAPKinázok aktiválódásához illetve az occludin expressziójának jelentös csökkenéséhez vezet. Az occludin lebontásában az itch nevű ubiqiutin ligáz illetve a proteaszómák játszhatnak fontos szerepet. Kimutattuk, hogy a vérzéses shock az irreverzíbilis stádiumban a vér-agy gát áteresztőképességének fokozódásához vezet, amely hátterében az occludin downregulációja lehet. További kísérleti eredményeink arra engednek következtetni, hogy a tirozin foszforilációnak fontos szerepe lehet a junkcionális fehérjék működésének szabályozásában. Ezen túlmenően a ZO2 a sejtmagba vándorolva és a SAF-B nevű fehérjéhez kapcsolódva jeltovábbító molekulaként képes működni. Kimutattuk hogy a beta-catenin src által mediált tirozin foszforlációja jelentős szerepet játszhat a hiperozmotikus mannitol által indukált vér-agy gát permeabilitás fokozódás mechanizmusában. | Our research was focused on the study of the role of interendothelial junctions under pathological conditions. We have shown that oxidative stress has a genotoxic effect in cerebral endothelial cells, can lead to the activation of MAPKinases and to the downregulation of the tight junction molecule occludin. In the downregulation of occluding the ubiquitin ligase itch and the proteasomes may play a crucial role. We have demonstrated that hemorrhagic shock can lead an increase in the permeability of the blood-brain barrier, which could be mediated by the downregulation of occludin. Our experiments have demonstrated that tyrosine phosphorylation could play an important role in the regulation of junctional proteins. Furthermore we have shown that ZO2 may enter the nucleus and by binding to the SAF-B protein may play an active role in signal transduction. We have shown that src mediated tyrosine phosphorylation of beta-catenin could be an important step in the hyperosmotic mannitol induced opening of the blood-brain barrier

A nemi hormonok és neuroszteroidok neuroprotektív szerepe: in vivo és in vitro vizsgálatok = Neuroprotective effects of gonadal hormones and neurosteroids: in vivo and in vitro studies

A jelen pályázat keretében elvégzett vizsgálatok célja azon sejt és molekuláris szintű mechanizmusok tanulmányozása volt, melyek az öregedésből ill. az agyi sérülésekből adódó morfológiai és funkcionális károsodások enyhítését lehetővé teszik. Különböző állatmodelleken a női nemi hormon (ösztrogén) ill. az egyik neuroszteroid, dehydroepiandoszteron (DHEA) hatását tanulmányoztuk az idegi sérüléseket követő reakciókra. Legfontosabb megállapításaink: A patkány szaglógumó kísérletes deafferentációját követően kialakuló reaktív asztrogliózist az ösztrogén és a DHEA egyaránt csökkenti. Aromatáz gátló fadrozol segítségével igazoltuk, hogy a protektív hatás kialakulásáért nem maga a neuroszteroid, hanem az abból lokálisan szintetizálódó 17β-ösztadiol a felelős. Patkány agykéregben hideg lézióval előidézett sérülés mértékét dehydroepiandoszteron szulfát (DHEAS) kezelésével csökkenteni lehet. Figyelemreméltó, hogy ilyen körülmények között a neuroszteroid protektív hatása kifejezettebb, ha azt a sérülést követően alkalmazzuk. BrdU-val történő jelöléssel igazoltuk, hogy a patkány szaglógumójában az ösztrogén régió-specifikus módon hat az agyterületen folyó neurogenezisre, az újonnan integrálódó interneuronok túlélésére. | In the present project we studied the process of neurodegeneration occurring during normal ageing or induced by injury. By using different animal models we have studied the effects of estrogen and the neurosteroid dehydroepiandrosterone (DHEA) on the neuronal and glial reactions following neuronal injury. Our aim was to identify those cellular and molecular mechanisms, which were responsible for the neuroprotective effects of these steroids. In deafferentated olfactory bulb of adult rats we have demonstrated that DHEA attenuates the glial reaction to denervation and may regulate glial plasticity in the olfactory glomeruli. With the use of aromatase inhibitor fadrozole we have provided evidences that these effects are likely to be mediated by the local conversion of DHEA to 17 β-estradiol. In a focal cortical cold lesion model we have shown that the size of injury can be decreased by dehydroepiandrosterone sulphate (DHEAS). Our observations clearly show that DHEAS is neuroprotective both in pre- and post-traumatic administration and these results suggest that it may be of substantial therapeutic benefit for the treatment of traumatic injury. By using the mitotic marker BrdU we have found that cells destined to the glomerular and granule cell layers react in the same way to chronic estrogen treatment, and the effect of 17 β-estradiol on the neurogenesis and neuron survival is region specific within the adult olfactory bulb

Neurodegeneratív betegségek kialakulása során létrejövő vér-agy gát változások vizsgálata in vivo és in vitro kísérletes modelleken = Impairment of the blood-brain barrier associated with neurodegenerative diseases: study on in vivo and in vitro models

Kutatásunk során igazoltuk primer agyi endotélsejt és asztroglia ko-kultúrán alapuló vér-agy gát modellünkön, hogy a prion fehérjének és az amyloid -beta peptideknek fibrillumokat képző szakaszai közvetlen endotélsejt károsító hatást fejtenek ki. A peptidek hatására megváltozott a sejtmorfológia, citoplazmatikus vakuolizáció jött létre. Az amyloid peptid kezelés az agyi endotélsejtek egy részének pusztulását eredményezte, ebben nekrotikus és apoptotikus folyamatok is részt vettek. Ezzel párhuzamosan az endotélsejtek barrier funkciója romlott, amely mögött az endotélsejteket összekötő szoros zárókapcsolatokra (TJ) kifejtett károsító hatás állt. A HIV-1 vírus Tat fehérjéje hasonlóképpen gyengítette az agyi endotélsejtek TJ struktúráit: a TJ fehérjék mennyiségét csökkentette, eloszlását megváltoztatta. A Tat vírusfehérjének ez a hatása szerepet játszhat a neuroAIDS során létrejövő vér-agy gát károsodásban, és az ezzel szorosan összefüggő dementia kialakulásában. Kimutattuk, hogy amyloid peptidek csökkentik a vér-agy gát fontos efflux pumpáinak, a P-glikoproteinnek és az MRP-1-nek aktivitását is. Eredményeink alapján a vér-agy gát barrier és homeosztatikus működése is zavart szenvedhet neurodegeneratív betegségekben. Pentozán enyhítette az amyloid peptid kezelések okozta elváltozásokat. Az endotélsejtekre kifejtett protektív hatás a gyógyszer új klinikai alkalmazását jelentheti, és hozzájárulhat a neurodegeneratív kórfolyamatokban kialakuló vér-agy gát károsodás kivédéséhez. | During the investigations we could demonstrate on our in vitro blood-brain barrier (BBB) model, based on primary brain endothelial cell and astroglia co-culture, that fibrillogenic fragments of prion protein and amyloid -beta peptides exerted direct toxicity on endothelial cells. Peptide treatments resulted in changes of cell morphology, cytoplasmic vacuolizations. Treatments with amyloid peptides led to endothelial cell death, partly necrotic, partly apoptotic. At the same time paracellular barrier integrity of endothelial cells was deteriorated due to damaged interendothelial tight junctions (TJ). HIV-1 Tat protein weakened the brain endothelial TJ in the same way: the expression and intracellular localization of TJ proteins was disturbed. This effect of Tat viral protein can play a role in BBB dysfunction and dementia in neuroAIDS. Amyloid peptides decreased the activity of two important efflux pumps of the BBB, P-glycoprotein and MRP-1 in brain endothelial cells. Our results indicate that in neurodegenerative diseases both barrier integrity and homeostatic functions of the BBB can be damaged. Pentosan polysulphate, attenuated the brain endothelial dysfunctions caused by amyloid peptide treatments. A new clinical application of the drug may be developed based on this protective effect. Pentosan can be a potential drug candidate for the treatment of BBB dysfunctions in neurodegenerative diseases

Aromatase and estrogen receptor beta expression in the rat olfactory bulb: neuroestrogen action in the first relay station of the olfactory pathway?

The expression pattern of aromatase (ARO), the enzyme converting androgens to estrogens, was analyzed in the olfactory bulb of adult male rats and was compared with the distribution of estrogen receptor beta (ER beta), the main estrogen receptor isoform expressed in this brain region. A strong ARO immunolabeling obtained with a specificity tested antibody was observed in juxtaglomerular neurons of the glomerular layer and a weaker immunoreaction was detected in the mitral cell layer of the main olfactory bulb, while the granule cell layer of the main olfactory bulb as well as all layers in the accessory olfactory bulb showed faint immunolabeling. Fluorescence double labeling experiments revealed that ARO detected in juxtaglomerular neurons of the main olfactory bulb colocalized with tyrosine hydroxylase (TH) and glutamic acid decarboxylase 67 (GAD67), while no colocalization between ARO and the calcium binding proteins calretinin (CR) and calbindin (CB) was observed. Furthermore, the TH immunoreactive neurons expressed metabotropic glutamate receptor 1 (mGluR1) too. ER beta immunoreactivity, in contrast to ARO, was detected in all layers of both the main and accessory olfactory bulb. In the glomerular layer of the main olfactory bulb it was expressed in TH and GAD67 containing juxtaglomerular neurons, and it colocalized with CR, CB and even with glial fibrillary acidic protein too. Our morphological findings suggest that ARO expression is a novel feature of dopaminergic/GABAergic juxtaglomerular neurons in the adult rat main olfactory bulb, and raise the possibility that ARO activity may change in function of olfactory input via mGluR1. In situ estrogen production in the olfactory bulb in turn may modulate interglomerular circuits through ER beta

Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

Estrogens regulate a variety of neuroendocrine, reproductive and also non-reproductive brain functions. Estradiol biosynthesis in the central nervous system (CNS) is catalyzed by the enzyme aromatase, which is expressed in several brain regions by neurons, astrocytes and microglia. In this study, we performed a complex fluorescent immunocytochemical analysis which revealed that aromatase is colocalized with the nuclear stain in glial fibrillary acidic protein (GFAP) positive astrocytes in cell cultures. Confocal immunofluorescent Z-stack scanning analysis confirmed the colocalization of aromatase with the nuclear DAPI signal. Nuclear aromatase was also detectable in the S100 beta positive astrocyte subpopulation. When the nuclear aromatase signal was present, estrogen receptor alpha was also abundant in the nucleus. Immunostaining of frozen brain tissue sections showed that the nuclear colocalization of the enzyme in GFAP-positive astrocytes is also detectable in the adult rat brain. CD11b/c labelled microglial cells express aromatase, but the immunopositive signal was distributed only in the cytoplasm both in the ramified and amoeboid microglial forms. Immunostaining of rat ovarian tissue sections and human granulosa cells revealed that aromatase was present only in the cytoplasm. This novel observation suggests a new unique mechanism in astrocytes that may regulate certain CNS functions via estradiol production

Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

Estrogens regulate a variety of neuroendocrine, reproductive and also non-reproductive brain functions. Estradiol biosynthesis in the central nervous system (CNS) is catalyzed by the enzyme aromatase, which is expressed in several brain regions by neurons, astrocytes and microglia. In this study, we performed a complex fluorescent immunocytochemical analysis which revealed that aromatase is colocalized with the nuclear stain in glial fibrillary acidic protein (GFAP) positive astrocytes in cell cultures. Confocal immunofluorescent Z-stack scanning analysis confirmed the colocalization of aromatase with the nuclear DAPI signal. Nuclear aromatase was also detectable in the S100 beta positive astrocyte subpopulation. When the nuclear aromatase signal was present, estrogen receptor alpha was also abundant in the nucleus. Immunostaining of frozen brain tissue sections showed that the nuclear colocalization of the enzyme in GFAP-positive astrocytes is also detectable in the adult rat brain. CD11b/c labelled microglial cells express aromatase, but the immunopositive signal was distributed only in the cytoplasm both in the ramified and amoeboid microglial forms. Immunostaining of rat ovarian tissue sections and human granulosa cells revealed that aromatase was present only in the cytoplasm. This novel observation suggests a new unique mechanism in astrocytes that may regulate certain CNS functions via estradiol production

Inhibition of NHE-1 Increases Smoke-Induced Proliferative Activity of Barrett’s Esophageal Cell Line

Several clinical studies indicate that smoking predisposes its consumers to esophageal inflammatory and malignant diseases, but the cellular mechanism is not clear. Ion transporters protect esophageal epithelial cells by maintaining intracellular pH at normal levels. In this study, we hypothesized that smoking affects the function of ion transporters, thus playing a role in the development of smoking-induced esophageal diseases. Esophageal cell lines were treated with cigarettesmoke extract (CSE), and the viability and proliferation of the cells, as well as the activity, mRNA and protein expression of the Na(+)/H(+) exchanger-1 (NHE-1), were studied. NHE-1 expression was also investigated in human samples. For chronic treatment, guinea pigs were exposed to tobacco smoke, and NHE-1 activity was measured. Silencing of NHE-1 was performed by using specific siRNA. CSE treatment increased the activity and protein expression of NHE-1 in the metaplastic cells and decreased the rate of proliferation in a NHE-1-dependent manner. In contrast, CSE increased the proliferation of dysplastic cells independently of NHE-1. In the normal cells, the expression and activity of NHE-1 decreased due to in vitro and in vivo smoke exposure. Smoking enhances the function of NHE-1 in Barrett’s esophagus, and this is presumably a compensatory mechanism against this toxic agent

Cerebrovascular Pathology in Hypertriglyceridemic APOB-100 Transgenic Mice

Hypertriglyceridemia is not only a serious risk factor in the development of cardiovascular diseases, but it is linked to neurodegeneration, too. Previously, we generated transgenic mice overexpressing the human APOB-100 protein, a mouse model of human atherosclerosis. In this model we observed high plasma levels of triglycerides, oxidative stress, tau hyperphosphorylation, synaptic dysfunction, cognitive impairment, increased neural apoptosis and neurodegeneration. Neurovascular dysfunction is recognized as a key factor in the development of neurodegenerative diseases, but the cellular and molecular events linking cerebrovascular pathology and neurodegeneration are not fully understood. Our aim was to study cerebrovascular changes in APOB-100 transgenic mice. We described the kinetics of the development of chronic hypertriglyceridemia in the transgenic animals. Increased blood-brain barrier permeability was found in the hippocampus of APOB-100 transgenic mice which was accompanied by structural changes. Using transmission electron microscopy, we detected changes in the brain capillary endothelial tight junction structure and edematous swelling of astrocyte endfeet. In brain microvessels isolated from APOB-100 transgenic animals increased Lox-1, Aqp4, and decreased Meox-2, Mfsd2a, Abcb1a, Lrp2, Glut-1, Nos2, Nos3, Vim, and in transgenic brains reduced Cdh2 and Gfap-σ gene expressions were measured using quantitative real-time PCR. We confirmed the decreased P-glycoprotein (ABCB1) and vimentin expression related to the neurovascular unit by immunostaining in transgenic brain sections using confocal microscopy. We conclude that in chronic hypertriglyceridemic APOB-100 transgenic mice both functional and morphological cerebrovascular pathology can be observed, and this animal model could be a useful tool to study the link between cerebrovascular pathology and neurodegeneration

Abstracts from the 20th International Symposium on Signal Transduction at the Blood-Brain Barriers

https://deepblue.lib.umich.edu/bitstream/2027.42/138963/1/12987_2017_Article_71.pd