Małgorzata Kajta

Antidepressant drugs inhibit glucocorticoid receptor-mediated gene transcription – a possible mechanism

Antidepressant drugs are known to inhibit some changes evoked by glucocorticoids, as well as a hyperactivity of hypothalamic‐pituitary‐adrenal (HPA) axis, often observed in depression. The aim of present study was to investigate effects of various antidepressant drugs on the glucocorticoid‐mediated gene transcription in fibroblast cells, stably transfected with an MMTV promoter (LMCAT cells). The present study have shown that antidepressants (imipramine, amitriptyline, desipramine, fluoxetine, tianeptine, mianserin and moclobemide), but not cocaine, inhibit the corticosterone‐induced gene transcription in a concentration‐ and a time‐dependent manner. Drugs which are known to augment clinical effects of medication in depressed patients (lithium chloride, amantadine, memantine), do not affect the inhibitory effects of imipramine on the glucocorticoid receptor (GR)‐mediated gene transcription. Inhibitors of phospholipase C (PLC), protein kinase C (PKC), Ca2+/calmodulin‐dependent protein kinase (CaMK) and antagonists of the L‐type Ca2+ channel also inhibit the corticosterone‐induced gene transcription. Inhibitors of protein kinase A (PKA) and protein kinase G (PKG) are without effect on the GR‐induced gene transcription. Phorbol ester (an activator of PKC) attenuates the inhibitory effect of imipramine on the GR‐induced gene transcription. Imipramine decreases binding of corticosterone‐receptor complex to DNA. It is concluded that antidepressant drugs inhibit the corticosterone‐induced gene transcription, and that the inhibitory effect of imipramine depends partly on the PLC/PKC pathway.

Genistein inhibits glutamate-induced apoptotic processes in primary neuronal cell cultures: An involvement of aryl hydrocarbon receptor and estrogen receptor/glycogen synthase kinase-3β intracellular signaling pathway

Phytoestrogens prevent neuronal damage, however, mechanism of their neuroprotective action has not been fully elucidated. This study aimed to evaluate the effects of genistein on glutamate-induced apoptosis in mouse primary neuronal cell cultures. Glutamate (1 mM) enhanced caspase-3 activity and lactate dehydrogenase (LDH) release in the hippocampal, neocortical and cerebellar neurons in time-dependent manner, and these data were confirmed at the cellular level with Hoechst 33342 and calcein AM staining. Genistein (10-10,000 nM) significantly inhibited glutamate-induced apoptosis, and the effect of this isoflavone was most prominent in the hippocampal cells. Next, we studied an involvement of estrogen and aryl hydrocarbon receptors in anti-apoptotic effects of genistein. A high-affinity estrogen receptor antagonist, ICI 182, 780 (1 microM), reversed, whereas less specific antagonist/partial agonist, tamoxifen (1 microM), either intensified or partially inhibited genistein effects. Aryl hydrocarbon receptor antagonist, alpha-naphthoflavone (1 microM), exhibited a biphasic action: it enhanced genistein action toward a short-term exposure (3 h) to glutamate, but antagonized genistein action toward prolonged exposure (24 h) to that insult. SB 216763 (1 microM), which preferentially inhibits glycogen synthase kinase-3beta (GSK-3beta), potentiated genistein effects. These data point to strong effects of genistein at low micromolar concentrations in various brain tissues against glutamate-evoked apoptosis. Moreover, this study provided evidence for involvement of aryl hydrocarbon receptor and estrogen receptor/GSK-3beta intracellular signaling pathway in anti-apoptotic action of genistein.

Valproate inhibits the conversion of testosterone to estradiol and acts as an apoptotic agent in growing porcine ovarian follicular cells.

Summary:  Purpose: Long‐term valproate (VPA) treatment has been associated with hyperandrogenism and polycystic ovaries in women with epilepsy. The exact mechanisms of action of the drug on sex steroid hormone function are still unsettled. The aim of the present study was to investigate the action of VPA on basal and gonadotropin‐stimulated steroid secretion in porcine ovarian follicular cells and to measure the conversion of testosterone to estradiol. Second, the action of VPA on proliferation and apoptosis of follicular cells was investigated.

ARYL HYDROCARBON RECEPTOR-MEDIATED APOPTOSIS OF NEURONAL CELLS: A POSSIBLE INTERACTION WITH ESTROGEN RECEPTOR SIGNALING

Activation of aryl hydrocarbon receptors (AhRs) induces neuronal damage, but the mechanism by which this occurs is largely unknown. This study evaluated the effects of an AhR agonist, beta-naphthoflavone, on apoptotic pathways in mouse primary neuronal cell cultures. beta-Naphthoflavone (0.1-100 micronhanced caspase-3 activity and lactate dehydrogenase (LDH) release in neocortical and hippocampal cells. These data were supported at the cellular level with Hoechst 33342 and calcein AM staining. alpha-Naphthoflavone inhibited the action of beta-naphthoflavone, thus confirming specific activation of AhRs. A high-affinity estrogen receptor (ER) antagonist, ICI 182,780, and a selective estrogen receptor modulator (SERM), tamoxifen, enhanced beta-naphthoflavone-mediated apoptosis. Another SERM, raloxifene, and an ERalpha antagonist, methyl-piperidino-pyrazole, did not affect beta-naphthoflavone-induced caspase-3 activity. However, they inhibited beta-naphthoflavone-induced LDH release at a late hour of treatment, thus suggesting delayed control of AhR-mediated neuronal cell death. The apoptotic effects of beta-naphthoflavone were accompanied by increased levels of AhRs, and these receptors colocalized with ERbeta as demonstrated by confocal microscopy. These data strongly support apoptotic effects of AhR activation in neocortical and hippocampal tissues. Moreover, this study provides evidence for direct interaction of the AhR-mediated apoptotic pathway with estrogen receptor signaling, which provides insight into new strategies to treat or prevent AhR-mediated neurotoxicity.

Neuroprotective effects of neuropeptide Y-Y2 and Y5 receptor agonists in vitro and in vivo

It is generally assumed that neurodegeneration is connected with glutamatergic hyperactivity, and that neuropeptide Y (NPY) inhibits glutamate release. Some earlier studies indicated that NPY may have neuroprotective effect; however, the results obtained so far are still divergent, and the role of different Y receptors remains unclear. Therefore in the presented study we investigated the neuroprotective potential of NPY and its Y2, Y5 or Y1 receptor (R) ligands against the kainate (KA)-induced excitotoxicity in neuronal cultures in vitro, as well as in vivo after intrahippocampal KA injection and also in an ischemic middle cerebral artery occlusion model after intraventricular injection of Y2R agonist. NPY compounds were applicated 30 min, 1, 3 or 6 h after the start of the exposure to KA, or 30 min after the onset of ischemia. Our results indicate the neuroprotective activity of NPY and its Y2R and Y5R ligands against the kainate-induced excitotoxicity in primary cortical and hippocampal cultures. Importantly, NPY was effective when given as late as 6 h, while Y2R or Y5R agonists 3 h, after starting the exposure to KA. In in vitro studies those protective effects were inhibited by the respective receptor antagonists. Neuroprotection was also observed in vivo after intrahippocampal injection of Y2R and Y5R agonists 30 min or 1 h after KA. No protection was found either in vitro or in vivo after the Y1R agonist. The Y2R agonist also showed neuroprotective activity in the ischemic model. The obtained results indicate that neuropeptide Y produces neuroprotective effect via Y2 and Y5 receptors, and that the compounds may be effective after delayed application.

Cellular strategies of estrogen-mediated neuroprotection during brain development

The role of estrogen during brain development is well documented. Estrogen influences cell survival and differentiation and also controls the formation and maintenance of neural networks. Knowledge of trophic estrogen action in the central nervous system (CNS) was the basis for the establishment of research programs directed toward a potential function of estrogen as a neuroprotective factor in the adult brain. Considerable evidence has accumulated over the years supporting this hypothesis. Experimental and epidemiologic studies as well as clinical trials have demonstrated that estrogen is beneficial for the course of neurodegenerative disorders such as Parkinson and Alzheimer diseases but may also protect neurons from postischemic neuronal degeneration. In this article, we aim to unravel potential physiologic responses and cell survival strategies that allow a more detailed understanding of estrogen-mediated neuroprotection in the brain. In particular, we focus on the participation of estrogen in the regulation of apoptotic processes. Furthermore, we present data on reciprocal estrogen-growth factor interactions. Both of these mechanisms were found to operate during brain development and to conciliate estrogen effects on neurons. This makes them likely candidates for taking part in conveying estrogen-dependent neuroprotection in the adult CNS.

The key involvement of estrogen receptor β and G-protein-coupled receptor 30 in the neuroprotective action of daidzein.

Phytoestrogens have received considerable attention because they provide an array of beneficial effects, such as neuroprotection. To better understand the molecular and functional link between phytoestrogens and classical as well as membrane estrogen receptors (ERs), we investigated the effect of daidzein on the glutamate-mediated apoptotic pathway. Our study demonstrated that daidzein (0.1-10μM) inhibited the pro-apoptotic and neurotoxic effects caused by glutamate treatment. Hippocampal, neocortical and cerebellar tissues responded to the inhibitory action of daidzein on glutamate-activated caspase-3 and lactate dehydrogenase (LDH) release in a similar manner. Biochemical data were supported at the cellular level by Hoechst 33342 and calcein AM staining. The sensitivity of neuronal cells to daidzein-mediated protection was most prominent in hippocampal cultures at an early stage of development 7th day in vitro. A selective estrogen receptor β (ERβ) antagonist, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5,-a]pyrimidin-3-yl]phenol (PHTPP), and a selective G-protein-coupled receptor 30 (GPR30) antagonist, 3aS(∗),4R(∗),9bR(∗))-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G15), reversed the daidzein-mediated inhibition of glutamate-induced loss of membrane mitochondrial potential, caspase-3 activity, and LDH release. A selective ERα antagonist, methyl-piperidino-pyrazole (MPP), did not influence any anti-apoptotic effect of daidzein. However, a high-affinity estrogen receptor antagonist, 7α,17β-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI) 182,780, and a selective GPR30 agonist, (±)-1-[(3aR(∗),4S(∗),9bS(∗))-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone (G1), intensified the protective action of daidzein against glutamate-induced loss of membrane mitochondrial potential and LDH release. In siRNA ERβ- and siRNA GPR30-transfected cells, daidzein did not inhibit the glutamate-induced effects. Twenty-four hour exposure to glutamate did not affect the cellular distribution of ERβ and GPR30, but caused greater than 100% increase in the levels of the receptors. Co-treatment with daidzein decreased the level of ERβ without significant changing of the GPR30 protein level. Here, we elucidated neuroprotective effects of daidzein at low micromolar concentrations and demonstrated that the phytoestrogens may exert their effects through novel extranuclear GPR30 and the classical transcriptionally acting ERβ. These studies uncover key roles of the ERβ and GPR30 intracellular signaling pathways in mediating the anti-apoptotic action of daidzein and may provide insight into new strategies to treat or prevent neural degeneration.

Impact of endocrine-disrupting chemicals on neural development and the onset of neurological disorders

Even though high doses of organic pollutants are toxic, relatively low concentrations have been reported to cause long-term alterations in functioning of individual organisms, populations and even next generations. Among these pollutants are dioxins, polychlorinated biphenyls, pesticides, brominated flame retardants, plasticizers (bisphenol A, nonylphenol, and phthalates) as well as personal care products and drugs. In addition to toxic effects, they are able to interfere with hormone receptors, hormone synthesis or hormone conversion. Because these chemicals alter hormone-dependent processes and disrupt functioning of the endocrine glands, they have been classified as endocrine-disrupting chemicals (EDCs). Because certain EDCs are able to alter neural transmission and the formation of neural networks, the term neural-disrupting chemicals has been introduced, thus implicating EDCs in the etiology of neurological disorders. Recently, public concern has been focused on the effects of EDCs on brain function, concomitantly with an increase in neuropsychiatric disorders, including autism, attention deficit and hyperactivity disorder as well as learning disabilities and aggressiveness. Several lines of evidence suggest that exposure to EDCs is associated with depression and could result in neural degeneration. EDCs act via several classes of receptors with the best documented mechanisms being reported for nuclear steroid and xenobiotic receptors. Low doses of EDCs have been postulated to cause incomplete methylation of specific gene regions in the young brain and to impair neural development and brain functions across generations. Efforts are needed to develop systematic epidemiological studies and to investigate the mechanisms of action of EDCs in order to fully understand their effects on wildlife and humans.

The mechanism of 1,2,3,4‐tetrahydroisoquinolines neuroprotection: the importance of free radicals scavenging properties and inhibition of glutamate‐induced excitotoxicity

1‐Methyl‐1,2,3,4‐tetrahydroisoquinoline (1MeTIQ), unlike several other tetrahydroisoquinolines, displays neuroprotective properties. To elucidate this action we compared the effects of 1MeTIQ with 1,2,3,4‐tetrahydroisoquinoline (TIQ), a compound sharing many activities with 1MeTIQ (among them reducing free radicals formed during dopamine catabolism), but offering no clear neuroprotection. We found that the compounds similarly inhibit free‐radical generation in an abiotic system, as well as indices of neurotoxicity (caspase‐3 activity and lactate dehydrogenase release) induced by glutamate in mouse embryonic primary cell cultures (a preparation resistant to NMDA toxicity). However, in granular cell cultures obtained from 7‐day‐old rats, 1MeTIQ prevented the glutamate‐induced cell death and 45Ca2+ influx, whereas TIQ did not. This suggested a specific action of 1MeTIQ on NMDA receptors, which was confirmed by the inhibition of [3H]MK‐801 binding by 1MeTIQ. Finally, we demonstrated in an in vivo microdialysis experiment that 1MeTIQ prevents kainate‐induced release of excitatory amino acids from the rat frontal cortex. Our results indicate that 1MeTIQ, in contrast to TIQ, offers a unique and complex mechanism of neuroprotection in which antagonism to the glutamatergic system may play a very important role. The results suggest the potential of 1MeTIQ as a therapeutic agent in various neurodegenarative illnesses of the central nervous system.

Neuroprotection by co-treatment and post-treating with calcitriol following the ischemic and excitotoxic insult in vivo and in vitro.

Several in vivo and in vitro studies have demonstrated the neuroprotective potential of pretreatment with 1alpha,25-dihydroxyvitamin D3 (calcitriol). The aim of the present study was to determine the effectiveness of calcitriol administered in vivo after a brain ischemic episode in the rat model of perinatal asphyxia, or when co-applied with or without delay during 24-h exposure of mouse hippocampal, neocortical and cerebellar neuronal cultures to glutamate on their 7th and 12th day in vitro (7 DIV and 12 DIV, respectively). Calcitriol was also administered after acute exposure of rat cerebellar neurons to glutamate. In 7-day-old rat pups subjected to hypoxia-ischemia, acute application of calcitriol in a single dose of 2 microg/kg, 30 min after termination of the insult, or subchronic, 7-day post-treatment with calcitriol, effectively reduced brain damage. The level of neuroprotection exceeded that achieved by hypoxic preconditioning used as the reference neuroprotective method. The results of in vitro experiments revealed the ability of calcitriol to reduce excitotoxicity in a manner dependent on the origin of the neuronal cells, their stage of maturation in culture and the duration of exposure to the excitotoxic insult before calcitriol application. Calcitriol was neuroprotective when it was administered together with glutamate or even after a delay of up to 6h during 24-h excitotoxic challenge of hippocampal and neocortical, but not cerebellar neuronal cultures. Application of calcitriol to cultured cerebellar granule neurons after acute exposure to glutamate was ineffective. In 12 DIV hippocampal cell cultures, 50 nM calcitriol inhibited glutamate-induced caspase-3 activity, while only 100 nM concentrations were effective in 7 DIV cultures. We ascribe the protective effects of calcitriol to the rapid modulation of mechanisms that are instrumental in the direct anti-apoptotic, neuroprotective action of this compound.