Endogén glutamát jelentősége neuroendokrin rendszerek szabályozásában = Role of endogenous glutamate in the regulation of neurosecretory systems

A kutatási támogatás segítségével a kutatócsoport tanulmányozta és feltérképezte az átvivőanyagként glutamátot használó (vezikuláris glutamát transzportereket tartalmazó) idegsejtek pontos anatómiai megoszlását rágcsálók hipotalamuszában. Leírta ezen glutamáterg idegsejtek részvételét az emberi hipotalamusz, és ezen belül, az emberi szaporodást irányító GnRH idegsejtek beidegzésében. Igazolta glutamáterg idegi fenotípus jegyeinek meglétét a szuprakiazmatikus mag egy elszórt neuron populációjában, továbbá korábban peptidergként megismert olyan neuronrendszerekben, melyek az agyalapi mirigy mellső és hátsó lebenyének működését szabályozzák. Tanulmányozta és endokrin fenotípus szerint azonosította a mellső hipofízis hámsejtjeiben is a glutamáterg neuronokra jellemző VGLUT1 és VGLUT2 enzim izoformákat és vizsgálta azok termelődésének szabályozását endokrin állatmodelleken. Elektronmikroszkópos vizsgálatokkal megállapította, hogy míg a neuroendokrin rendszerekben a VGLUT2 marker enzim mikrovezikulákhoz asszociált, addig az agyalapi mirigy mellső lebenyének hámsejtjeiben szekretoros granulumokban fordul elő. In situ hibridizáció használatával részletesen feltérképezte az egér hipotalamuszában az endokannabinoid érzékeny glutamáterg és GABAerg idegsejtek megoszlását. A projekthez kapcsolódó egyéb tanulmányokban új eredményeket szolgáltatott a reprodukció és az ösztrogén szignalizáció hipotalamikus és agykérgi mechanizmusainak jobb megértéséhez is. | Using this grant support, the research group described the topographic distribution of neurons that use glutamate (and contain one of the two major isoforms of vesicular glutamate transporter enzymes, VGLUT1 and VGLUT2) as synaptic transmitter in the rodent hypothalamus. They described the contribution of glutamatergic neurons to the innervation of the human hypothalamus and specifically, its GnRH neurons. They provided evidence for the occurrence of scattered glutamatergic neurons in the suprachiasmatic nucleus and in parvi- and magnocellular neurons known to regulate the anterior and posterior pituitary lobes solely via peptidergic mechanisms. They characterized epithelial cells in the anterior pituitary that express the VGLUT1 and VGLUT2 enzyme isoforms and studied the regulation of these enzymes under different endocrine challenges. They used electron microscopy and established that the glutamatergic marker enzyme VGLUT2 is associated with small-clear synaptic vesicles in neuroendocrine neuronal cells of the hypothalamus and with dense-core vesicles in glutamatergic endothelial cells of the adenohypophysis. They provided a detalied in situ hybridization map on the distribution of endocannabinoid-sensitive (CB1 mRNA expressing) hypothalamic neurons that exhibit glutamatergic and GABAergic phenotypes. In other studies linked to the project they provided new data about the regulation of reproduction and about estrogen signaling in the hypothalamus and the cerebral cortex

Endokannabinoid szignalizáció szerepe a reprodukció hypothalamikus szintű szabályozásában = Endocannabinoid signaling in hypothalamic regulation of reproduction

A szaporodás idegrendszeri szabályozásában kulcs szerepet tölt be a gonadotropin-releasing hormone (GnRH) neuronrendszer. A rendszer működését perifériás hormonhatások és más agyi neuronhálózatok szabályozzák. Multidiszciplináris megközelítés alkalmazásával tanulmányoztuk a GnRH neuronrendszer kapcsolatait és szignál transzdukciós mechanizmusait, különös tekintettel a retrográd endokannabinoid szignalizáció szabályozó szerepére. Kísérleti eredményeinkről 24 tudományos közleményben adtunk számot, 96 összesített impakt értékkel. Feltártuk a hypothalamus kannabinoid receptor 1 (CB1) hírvivő RNS-t termelő idegsejtjeinek lokalizációját, valamint azok glutamáterg és GABA-erg fenotípusait. Igazoltuk, hogy a GnRH sejteken végződő GABA tartalmú idegvégződések CB1-t tartalmaznak, valamint bebizonyítottuk, hogy a GnRH idegsejtekből felszabaduló endokannabinoidok befolyásolják a GABA közvetítette információ átadást a GnRH neuronok felé. Feltártuk a ghrelin és endokannabinoid szignalizációs útvonalak kapcsolt jellegét a hypothalamusban. Igazoltuk a humán GnRH idegsejtek glutamát- és GABA-erg beidegzését. A GnRH neuronrendszer kisspeptinerg afferensei vonatkozásában új regulációs adatokat szolgáltattunk. Vizsgáltuk az ösztrogén szignalizáció szerepét a GnRH idegsejtek működésében, valamint az agykéregben. A GnRH idegsejtek működésének elmélyültebb tanulmányozására matematikai modellt alkottunk. Összegezve, eredményeink a reprodukció szabályozásának új mechanizmusait tárták fel. | Gonadotropin-releasing hormone (GnRH)-synthesizing neurons play a pivotal role in the central regulation of reproduction. Their operation depends on signaling by peripheral hormones and interactions with other neuronal circuits. By means of a multidisciplinary approach, the networking and signal transduction mechanisms of GnRH neurons were studied, with special reference to a putative retrograde endogenous cannabinoid signaling mechanism. The research results were published in 24 original papers representing a cumulative impact value of 96. Specifically, we mapped the hypothalamic distribution of cannabinoid receptor 1 (CB1) mRNA-expressing neurons and their GABA- and glutamatergic phenotypes, proved the presence of CB1 in neuronal afferents of GnRH neurons and characterized the impact of endocannabinoids liberated from GnRH neurons on the GABA-ergic signal transduction to GnRH cells. We provided evidence for the coupled nature of the ghrelin and the endocannabinoid signaling mechanisms. New GABA- and glutamatergic afferents of human GnRH neurons were also identified. In addition, novel regulatory mechanisms executed by kisspeptinergic circuits upon GnRH cells were revealed. We elucidated further characteristics of estradiol feedback effects to GnRH and cortical neurons. We established a mathematical model for the better understanding of GnRH cell performance. Collectively, our results shed light on novel mechanisms regulating reproduction at the hypothalamic level

Novel cyclic C5-curcuminoids penetrating the blood-brain barrier: design, synthesis and antiproliferative activity against astrocytoma and neuroblastoma cells

Novel series of cyclic C5-curcuminoids 17a-j and 19-22 were prepared as cytotoxic agents and evaluated against human neuroblastoma (SH-SY5Y) or human grade IV astrocytoma (CCF-STTG1) cell lines in low (∼0.1 nM - 10 nM) concentrations. Among the tested 21 derivatives, 16 displayed potent antiproliferative activity with IC50 values in the low nanomolar to picomolar range (IC50 = 7.483-0.139 nM). Highly active compounds like N-monocarboxylic derivative 19b with IC50 = 0.139 nM value against neuroblastoma and N-alkyl substituted 11 with IC50 = 0.257 nM against astrocytoma proved some degree of selectivity toward non-cancerous astrocytes and kidney cells. This potent anticancer activity did not show a strong correlation with experimental logPTLC values, but the most potent antiproliferative molecules 11-13 and 19-22 are belonging to discrete subgroups of the cyclic C5-curcuminoids. Compounds 12, 17c and 19b were subjected to blood-brain barrier (BBB) penetration studies, too. The BBB was revealed to be permeable for all of them but, as the apparent permeability coefficient (Papp) values mirrored, in different ratios. Lower toxicity of 12, 17c and 19b was observed toward primary rat brain endothelial cells of the BBB model, which means they remained undamaged under 10 µM concentrations. Penetration depends, at least in part, on albumin binding of 12, 17c and 19b and the presence of monocarboxylic acid transporters in the case of 19b. Permeation through the BBB and albumin binding, we described here, is the first example of cyclic C5-curcuminoids as to our knowledge

Ghrelin Modulates the fMRI BOLD Response of Homeostatic and Hedonic Brain Centers Regulating Energy Balance in the Rat

The orexigenic gut-brain peptide, ghrelin and its G-protein coupled receptor, the growth hormone secretagogue receptor 1a (GHS-R1A) are pivotal regulators of hypothalamic feeding centers and reward processing neuronal circuits of the brain. These systems operate in a cooperative manner and receive a wide array of neuronal hormone/transmitter messages and metabolic signals. Functional magnetic resonance imaging was employed in the current study to map BOLD responses to ghrelin in different brain regions with special reference on homeostatic and hedonic regulatory centers of energy balance. Experimental groups involved male, ovariectomized female and ovariectomized estradiol-replaced rats. Putative modulation of ghrelin signaling by endocannabinoids was also studied. Ghrelin-evoked effects were calculated as mean of the BOLD responses 30 minutes after administration. In the male rat, ghrelin evoked a slowly decreasing BOLD response in all studied regions of interest (ROI) within the limbic system. This effect was antagonized by pretreatment with GHS-R1A antagonist JMV2959. The comparison of ghrelin effects in the presence or absence of JMV2959 in individual ROIs revealed significant changes in the prefrontal cortex, nucleus accumbens of the telencephalon, and also within hypothalamic centers like the lateral hypothalamus, ventromedial nucleus, paraventricular nucleus and suprachiasmatic nucleus. In the female rat, the ghrelin effects were almost identical to those observed in males. Ovariectomy and chronic estradiol replacement had no effect on the BOLD response. Inhibition of the endocannabinoid signaling by rimonabant significantly attenuated the response of the nucleus accumbens and septum. In summary, ghrelin can modulate hypothalamic and mesolimbic structures controlling energy balance in both sexes. The endocannabinoid signaling system contributes to the manifestation of ghrelin’s BOLD effect in a region specific manner. In females, the estradiol milieu does not influence the BOLD response to ghrelin

Expression of p16INK4a, p53 and Rb proteins are independent from the presence of human papillomavirus genes in oral squamous cell carcinoma

BACKGROUND:The overexpression of p16(INK4A) and suppression of p53 and Rb proteins are key features of oncogenic transformation by human papillomaviruses (HPV) in anogenital cancers. HPV genomes are often detected in cancers of the oral cavity, but it is unclear whether HPV has a specific oncogenic role there.OBJECTIVES:The objectives of the study were to investigate the expression of p53, Rb, and p16(INK4A) proteins and identify HPV infection and viral integration into the host genome.METHODS:Seventy-nine cases of oral squamous cell carcinoma (OSCC) were studied by immunohistochemistry. Polymerase chain reaction (PCR) was performed to identify HPV DNA from the samples. The results were correlated with clinical data.RESULTS:Thirty-three cases were HPV positive for high-risk HPV (HR-HPV) types, of which 27 harbored HPV16. In 25 of 27 HPV16-positive tumors, the HPV16 genome was fully integrated into the host genome, as evidenced by the lack of PCR-amplifiable E2 gene sequences. Forty-five patients were p53 overexpressing, 20 with HR-HPV-positive and 25 with HR-HPV-negative tumors. p16(INK4A) protein was overexpressed in 4 of 31 HR-HPV-positive and 9of 45 HR-HPV-negative cases. Twenty-six of 32 HR-HPV-positive and 37 of 44 HR-HPV-negative samples exhibited pRb nuclear staining. These differences between HR-HPV-positive and -negative tumors were not statistically significant. No correlation was found between these biological factors and tumor location, stage, differentiation grade, or alcohol or tobacco abuse.CONCLUSIONS:A tumor immunophenotype, similar to HPV-related anogenital cancers, is not present in OSCC and highly oncogenic HPV types are therefore unlikely to be specific or independent risk factors for oral cancer