Krisztina Kovacs

Regulation of stress-induced transcriptional changes in the hypothalamic neurosecretory neurons.

Transcriptional changes in corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) gene expression were studied byin situ hybridization histochemistry using cRNA probes directed against intronic sequences. Acute ether stress resulted in a rapid induction of CRF and a delayed activation of vasopressin heteronuclear (hn)RNA in the parvocellular neurosecretory neurons within the paraventricular nucleus (PVN) of the hypothalamus. To explore possible molecular mechanisms regulating stress-related neuropeptide expression in vivo, the time-courses of stress-induced activation of different transcription factor classes were compared to that of changes in neuropeptide transcription. The peak of CRF transcription was parallel to that of cAMP response-element binding protein (CREB) phosphorylation but preceded the induction of c-fos and NGFI-B mRNAs and Fos protein. In contrast, AVP expression occurred in step with immediate-early gene (IEG) responses, suggesting involvement of different mechanisms underlying stress-induced neuropeptide responses. The interference of glucocorticoid hormones with stress-induced neuropeptide and transcription-factor responses has also been revealed in rats acutely or chronically pretreated with glucocorticoids. Acute dexamethasone injection did not prevent neuropeptide and transcription factor responses to ether inhalation, whereas chronic corticosterone administration completely blocked IEG and neuropeptide induction in the stress-related neurosecretory neurons.

Prevention of doxorubicin-induced acute cardiotoxicity by an experimental antioxidant compound.

Doxorubicin is a widely used anticancer agent, but its application is restricted by its cardiotoxic side effects. The current theory of its cardiotoxicity is based on free radical formation. The compound H-2545, having a 3-carboxamido-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole moiety, was reported to exhibit antioxidant properties and accumulate in cell membranes, scavenging free radicals at the site of formation. Therefore, we hypothesized that H-2545 could reduce the doxorubicin-induced acute deterioration of cardiac function. Langendorff-perfused rat hearts were treated with doxorubicin and/or H-2545, its metabolite H-2954, or dihydrolipoamide. High-energy phosphate levels, contractile function, lipid peroxidation, protein oxidation, and Akt phosphorylation were investigated. We also determined whether the antioxidants influenced doxorubicin toxicity on malignant cells. During perfusion with doxorubicin, the energetic and functional parameters of the myocardium were improved by adding H-2545. H-2545 significantly diminished doxorubicin-induced lipid and protein damage. On H-2545 treatment, the doxorubicin-triggered Akt phosphorylation was markedly reduced, whereas dihydrolipoamide had such an effect only at higher concentrations. H-2545 did not alter the anticancer effect of doxorubicin on malignant cell lines. We propose that the coadministration of the antioxidant H-2545 attenuates doxorubicin-induced acute cardiotoxicity without interfering with its anticancer effects. Prevention of the acute adverse effects of doxorubicin on myocardium may hinder the later development of cardiomyopathy.

Adolescents’ awareness of HPV infections and attitudes towards HPV vaccination 3 years following the introduction of the HPV vaccine in Hungary

Hungary takes the fourth place regarding the incidence and the fifth regarding the mortality of cervical cancer among the member countries of the European Union, with 500 deaths due to this preventable illness and nearly 1200 new cases diagnosed every year. Although the vaccines have been available for 3 years, the estimated rate of the female population vaccinated against HPV is approximately 10% in the 12-26-year-age cohort. The aim of this study was to determine factors and motivations affecting the uptake of HPV vaccination among Hungarian adolescents. Examining the effects of some possible sociodemographic predictors (age and gender) and the exposure to health information on HPV vaccine acceptability were also focused on, as well as assessing the most trusted sources of information about sexually transmitted diseases (STDs). A nationwide anonymous questionnaire survey with a sample of 1769 students attending public primary or secondary schools was organised by the authors in 16 Hungarian cities and towns. Data were analysed using the Statistical Package for the Social Sciences (SPSS). Adolescents awareness of HPV was relatively low. Only 35% of the participants reported they had heard about HPV prior to the survey. Almost 70% of the potentially affected study population had not heard about the vaccine previously. Every fourth student did not believe that vaccination against HPV can prevent cervical cancer. If the vaccination was available free of charge, almost 80% of respondents would request it, but in case they had to pay for it, this number would significantly decrease. Significantly better knowledge and also more positive attitudes towards HPV vaccination was found in relation to the number of information sources. The majority of respondents (62-83%) were open for further information about STDs. The main trusted mediators were school-health services (61.3%), education on health at school (49.2%), health professionals (42.2%) and electronic media (24.6%). Since Hungarian adolescent students expect guidance about STDs principally from school health education, an urgent need for well-designed, HPV-focused educational programmes emerges. Launching such programmes would be especially important for the adolescent population to increase their awareness of the risks associated with HPV infection thus reducing the high incidence of cervical cancer in Hungary in the future.

Akt activation induced by an antioxidant compound during ischemia-reperfusion

Molecular mechanisms of cardioprotection afforded by modified mexiletine compounds were investigated during ischemia-reperfusion (IR) in Langendorff perfused hearts. Rat hearts were subjected to a global 25 min ischemia followed by reperfusion, either untreated or treated with mexiletine, or three substituted mexiletine derivates (5 muM). A modified mexiletine derivative (H-2693) promoted best the recovery of myocardial energy metabolism (assessed by (31)P NMR spectroscopy) compared to untreated and mexiletine-treated hearts. H-2693 also preserved cardiac contractile function and attenuated the IR-induced lipid peroxidation (TBARS formation) and protein oxidation (carbonyl content). Western blot revealed that H-2693 propagated the phosphorylation of Akt (activation) and its downstream substrate glycogen synthase kinase-3beta (GSK-3beta, inactivation) compared to untreated IR. Parallel treatment with the phosphatidylinositol-3-kinase (upstream activator of Akt) inhibitor wortmannin (100 nM) abolished the beneficial effects of H-2693 on energetics and function, and reduced Akt and GSK-3beta phosphorylation. As a result of the antiapoptotic impacts of Akt activation, H-2693 decreased caspase-3 activity, which was neutralized by wortmannin. Here we first demonstrated that a free radical-entrapping compound could activate the prosurvival Akt pathway beyond its proven ability to scavenge reactive oxygen species. In conclusion, the favorable influence of H-2693 on signaling events during IR may have considerably contributed to its cardioprotective effect.

Regulation of MKP-1 expression and MAPK activation by PARP-1 in oxidative stress: A new mechanism for the cytoplasmic effect of PARP-1 activation

Previously, it was suggested that the release of nuclearly formed ADP-ribose polymers or ADP-ribosylated proteins could be responsible for the cytosolic and mitochondrial effects of poly(ADP-ribose) polymerase (PARP)-1 activation in oxidative stress. In this report, we provide a novel alternative mechanism. We found that reactive oxygen species-activated PARP-1 regulated the activation of JNK and p38 mitogen-activated protein kinases (MAPKs) because inhibition of PARP-1 by pharmacons, small interfering RNA silencing of PARP-1 expression, or the transdominant expression of enzymatically inactive PARP-1 resulted in the inactivation of these MAPKs. This regulation was achieved by increased expression and enlarged cytoplasmic localization of MAPK phosphatase-1 (MKP-1) upon PARP-1 inhibition in oxidative stress because changes in MKP-1 expression were reflected in the phosphorylation states of JNK and p38. Furthermore, we found that in MKP-1-silenced cells, PARP inhibition was unable to exert its protective effect, indicating the pivotal roles of JNK and p38 in mediating the oxidative-stress-induced cell death as well as that of increased MKP-1 expression in mediating the protective effect of PARP inhibition. We suggest that regulation of a protein that can directly influence cytoplasmic signaling cascades at the expression level represents a novel mechanism for the cytoplasmic action of PARP-1 inhibition.

Protective effects of the neuropeptide PACAP in diabetic retinopathy

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly potent neurotrophic and neuroprotective effects. PACAP and its receptors occur in the retina and PACAP has been applied in animal models of metabolic retinal disorders to reduce structural and functional damage. Furthermore, PACAP has been implicated as a potential anti-diabetic peptide. Our aim has been to investigate, by using a complex morphological, immunochemical and molecular biological approach, whether PACAP attenuates diabetic retinopathy. Diabetes was induced in rats with a single streptozotocin injection. PACAP was injected intravitreally into one eye (100 pmol) three times during the last week of a 3-week survival period. Retinas were processed for the following procedures: routine histology, immunohistochemistry (single and double labeling, whole-mount), quantitative reverse transcription with the polymerase chain reaction and Western blotting. Cone photoreceptors and dopaminergic amacrine and ganglion cells degenerated in diabetic retinas and glial fibrillary acidic protein were upregulated in Müller glial cells. The number of cones, the length of their outer segments and the cell number in the ganglion cell layer were decreased. PACAP ameliorated these structural changes. Moreover, PACAP increased the levels of PAC1-receptor and tyrosine-hydroxylase as detected by molecular biological methods. Thus, PACAP has significant protective effects in the diabetic retina. PACAP treatment attenuates neuronal cell loss in diabetic retinopathy, the protective effects of PACAP probably being mediated through the activation of PAC1-receptor. These results suggest that PACAP has a therapeutic potential in diabetic retinopathy.

Mice deficient in pituitary adenylate cyclase activating polypeptide (PACAP) show increased susceptibility to in vivo renal ischemia/reperfusion injury

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with well-known cytoprotective effects. We have reported earlier that PACAP decreases mortality and the degree of tubular atrophy in a rat model of renal ischemia/reperfusion injury. Recently, we have shown that kidney cultures isolated from PACAP deficient mice show increased susceptibility to renal oxidative stress. Based on these previous studies, we raised the question whether PACAP deficient mice display increased sensitivity to in vivo kidney ischemia/reperfusion. PACAP⁻/⁻ mice underwent 45 or 60 min of renal ischemia followed by 2 weeks reperfusion. Kidneys were processed for histological analysis. Sections stained with PAS-haematoxylin were graded for the following parameters: degree of tubular dilation, Bowmanns capsule dilation, lymphocyte and macrophage infiltration, thyroidization and the disappearance of the PAS-positive glycocalyx from under the brush border. In other sets of experiments, tissue cytokine expression and the level of the endogenous antioxidant superoxide dismutase (SOD) were also determined after 60 min ischemia/reperfusion. Our results show that while intact kidneys were not different between wild-type and PACAP deficient mice, marked differences were observed in the histological structures in groups that underwent ischemia/reperfusion. PACAP deficient mice had a worse histological outcome, with significantly higher histological scores for all tested parameters. Cytokine expression was also markedly different between wild-type and PACAP deficient mice. In addition, the level of SOD was significantly lower in PACAP⁻/⁻ animals after ischemia/reperfusion. In conclusion, the lack of endogenous PACAP leads to higher susceptibility to in vivo renal ischemia/reperfusion, suggesting that PACAP has an endogenous renoprotective effect.

PACAP promotes neuron survival in early experimental diabetic retinopathy.

Metabolic changes induced by diabetes lead to a multifactorial progressive disease of the retina with an extremely complex pathogenesis. One of the mechanisms of retinal cell death in diabetes is via apoptosis. Our previous results show that pituitary adenylate cyclase activating polypeptide (PACAP) attenuates the morphological and neurochemical changes in a rat model of diabetic retinopathy. The aim of this study was to investigate the mechanisms of this protective effect. Retinas of streptozotocin-induced diabetic rats were analyzed using apoptosis detection combined with immunolabeling. Western blot was used to measure levels of pro- and anti-apoptotic pathways. Intraocular PACAP injection markedly attenuated diabetic retinal injury: increased levels of the anti-apoptotic p-Akt, p-ERK1, p-ERK2, PKC, Bcl-2, while decreased levels of the pro-apoptotic p-p38MAPK and activated caspases (8, 3, 12) were detected. The number of apoptotic cells increased in all nuclear layers of diabetic retinas, but significantly decreased after PACAP treatment. Our results clearly demonstrate that the protective effects of PACAP are mediated, at least partly, by attenuating apoptosis, including also that of the dopaminergic amacrine cells. Inhibition of apoptosis is one of the PACAP-induced pathways with therapeutic potential in early experimental diabetic retinopathy.

Ontogeny of calbindin immunoreactivity in the human hippocampal formation with a special emphasis on granule cells of the dentate gyrus

Calbindin (CB) is a calcium‐binding protein that is present in principal cells as well as in interneurons of the hippocampal formation of various species including humans. Studies with transgenic mice revealed that CB is essential for long‐term potentiation and synaptic plasticity which are the cellular basis of learning and memory. In a previous study we have shown that CB expression in granule cells of the dentate gyrus correlates with the functional maturation of the hippocampal formation in the rat.

Protection Against Chronic Hypoperfusion-Induced Retinal Neurodegeneration by PARP Inhibition via Activation of PI-3-kinase Akt Pathway and Suppression of JNK and p38 MAP Kinases

Poly(ADP-ribose) polymerase (PARP) activation is considered as a major regulator of cell death in various pathophysiological conditions, however, no direct information is available about its role in chronic hypoperfusion-induced neuronal death. Here, we provide evidence for the protective effect of PARP inhibition on degenerative retinal damage induced by bilateral common carotid artery occlusion (BCCAO), an adequate chronic hypoperfusion murine model. We found that BCCAO in adult male Wistar rats led to severe degeneration of all retinal layers that was attenuated by a carboxaminobenzimidazol-derivative PARP inhibitor (HO3089) administered unilaterally into the vitreous body immediately following carotid occlusion and then 4 times in a 2-week-period. Normal morphological structure of the retina was preserved and the thickness of the retinal layers was increased in HO3089-treated eyes compared to the BCCAO eyes. For Western blot studies, HO3089 was administered immediately after BCCAO and retinas were removed 4xa0h later. According to Western blot analysis utilizing phosphorylation-specific primary antibodies, besides activating poly-ADP-ribose (PAR) synthesis, BCCAO induced phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). HO3089 inhibited PAR synthesis, and decreased the phosphorylation of these proapoptotic MAPKs. In addition, HO3089 treatment induced phosphorylation, that is activation, of the protective Akt/glycogen synthase kinase (GSK)-3beta and extracellular signal-regulated kinase (ERK1/2) signaling pathways. These data indicate that PARP activation has a major role in mediating chronic hypoperfusion-induced neuronal death, and inhibition of the enzyme prevents the pathological changes both in the morphology and the kinase signaling cascades involved. These results identify PARP inhibition as a possible molecular target in the clinical management of chronic hypoperfusion-induced neurodegenerative diseases including ocular ischemic syndrome.