Identification of Prominin-2 as a new player of cardiomyocyte senescence in the aging heart.

The aging heart is characterized by a number of structural changes leading to ventricular stiffness, impaired resistance to stress and increased risk of developing heart failure (HF). Genetic or pharmacological removal of senescent cells has recently demonstrated the possibility to relieve some cardiac aging features such as hypertrophy and fibrosis. However, the contribution of the different cell types in cardiac aging remains fragmentary due to a lack of cell-specific markers. Cardiomyocytes undergo post-mitotic senescence in response to telomere damage, characterized by persistent DNA damage response and expression of the classical senescence markers p21 and p16, which are shared by many other cell types. In the present study, we used transcriptomic approaches to discover new markers specific for cardiomyocyte senescence. We identified Prominin2 (Prom2), encoding a transmembrane glycoprotein, as the most upregulated gene in cardiomyocytes of aged mice compared to young mice. We showed that Prom2 was upregulated by a p53-dependent pathway in stress-induced premature senescence. Prom2 expression correlated with cardiomyocyte hypertrophy in the hearts of aged mice and was increased in atrial samples of patients with HF with preserved ejection fraction. Consistently, Prom2 overexpression was sufficient to drive senescence, hypertrophy and resistance to cytotoxic stress while Prom2 shRNA silencing inhibited these features in doxorubicin-treated cardiac cells. In conclusion, we identified Prom2 as a new player of cardiac aging, linking cardiomyocyte hypertrophy to senescence. These results could provide a better understanding and targeting of cell-type specific senescence in age-associated cardiac diseases.

Towards a Large-Scale Assessment of the Relationship between Biological and Chronological Aging: The INSPIRE Mouse Cohort.

Aging is the major risk factor for the development of chronic diseases. After decades of research focused on extending lifespan, current efforts seek primarily to promote healthy aging. Recent advances suggest that biological processes linked to aging are more reliable than chronological age to account for an individual's functional status, i.e. frail or robust. It is becoming increasingly apparent that biological aging may be detectable as a progressive loss of resilience much earlier than the appearance of clinical signs of frailty. In this context, the INSPIRE program was built to identify the mechanisms of accelerated aging and the early biological signs predicting frailty and pathological aging. To address this issue, we designed a cohort of outbred Swiss mice (1576 male and female mice) in which we will continuously monitor spontaneous and voluntary physical activity from 6 to 24 months of age under either normal or high fat/high sucrose diet. At different age points (6, 12, 18, 24 months), multiorgan functional phenotyping will be carried out to identify early signs of organ dysfunction and generate a large biological fluids/feces/organs biobank (100,000 samples). A comprehensive correlation between functional and biological phenotypes will be assessed to determine: 1) the early signs of biological aging and their relationship with chronological age; 2) the role of dietary and exercise interventions on accelerating or decelerating the rate of biological aging; and 3) novel targets for the promotion of healthy aging. All the functional and omics data, as well as the biobank generated in the framework of the INSPIRE cohort will be available to the aging scientific community. The present article describes the scientific background and the strategies employed for the design of the INSPIRE Mouse cohort.

Prucalopride and donepezil act synergistically to reverse scopolamine-induced memory deficit in C57Bl/6j mice.

It is known that 5-HT(4) receptor agonists increase sAPPalpha levels in the cortex and hippocampus of mice as well as in a model of Alzheimer's disease (AD). As sAPPalpha is thought to have pro-mnesic properties, we assessed whether its increase induces cognitive improvement in a spatial memory task and whether it reverses a scopolamine-induced memory deficit. Mice treated or not treated with scopolamine were trained in the Morris water maze for 3 days. Before the probe test, they received an injection of either a 5-HT(4) receptor agonist (prucalopride or RS 67333), or an acetylcholinesterase inhibitor (donepezil), or both drugs. As expected, scopolamine decreased performance, an effect that was not reversed by the drugs tested when injected alone. However, prucalopride (5 mg kg(-1), s.c.) acted synergistically with donepezil (0.75 mg kg(-1), s.c.) to counteract completely scopolamine-induced amnesia. Western blot analysis of tissue homogenates in the cortex and hippocampus shows that sAPPalpha levels did not differ between saline- and scopolamine-treated mice. Furthermore, a region-dependent drug action was observed since the scopolamine-treated mice display a tendency to increase sAPPalpha levels in the hippocampus after donepezil or in the cortex after prucalopride. Our results suggest that a combined treatment with a 5-HT(4) receptor agonist with an acetylcholinesterase inhibitor has beneficial effects on memory in mice. Moreover, it seems to enhance sAPPalpha levels in two brain regions highly affected in AD. Thus, a drug polytherapy could be interesting not only to enhance cognitive performance and decrease drawbacks but also to get the best action in each brain region.

5-HT4 receptor agonists increase sAPPalpha levels in the cortex and hippocampus of male C57BL/6j mice.

BACKGROUND AND PURPOSE: A strategy to treat Alzheimer's disease (AD) is to increase the soluble form of amyloid precursor protein (sAPPalpha), a promnesic protein, in the brain. Because strong evidence supports beneficial effects of 5-hydroxytryptamine 5-HT(4) receptor agonists in memory and learning, we investigated the role of 5-HT(4) receptors on APP processing in 8 weeks-old male C57BL/6j mice. EXPERIMENTAL APPROACH: Mice were given, subcutaneously, prucalopride or ML 10302 (s.c.), two highly selective 5-HT(4) receptor agonists and, up to 240 min later, the hippocampus and cortex were analysed by Western blot for sAPPalpha determination. KEY RESULTS: Prucalopride (5 or 10 mg kg(-1)) significantly increased sAPPalpha levels in the hippocampus and cortex, but did not modify the expression level of APP mRNA as detected by quantitative RT-PCR. A selective 5-HT(4) receptor antagonist, GR125487 (1 mg kg(-1), s.c.) inhibited prucalopride induced- increase in sAPPalpha levels. In addition, levels of sAPPalpha were increased by ML10302 only at 20 mg kg(-1) and was limited to the cortex. Also, prucalopride increased sAPPalpha levels in the cortex of a transgenic mouse model of AD, expressing the London mutation of APP. Furthermore, the combined injection of a selective acetylcholinesterase inhibitor, donepezil and prucalopride induced a synergic increase in sAPPalpha levels in the cortex and hippocampus. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that the 5-HT(4) receptor plays a key role in the non-amyloidogenic pathway of APP metabolism in vivo and give support to the beneficial use of 5-HT(4) agonists for AD treatment.

Corticotropin-releasing hormone-mediated neuroprotection against oxidative stress is associated with the increased release of non-amyloidogenic amyloid beta precursor protein and with the suppression of nuclear factor-kappaB.

The neuropeptide CRH is the central regulator of the hypothalamic-pituitary-adrenal (HPA) stress response system and is implicated in various stress-related conditions. In the neurodegenerative disorder Alzheimer's disease (AD), levels of CRH are decreased. AD pathology is characterized by the deposition of the nonsoluble amyloid beta protein (A beta), oxidative stress, and neuronal cell death. Employing primary neurons and clonal cells, we demonstrate that CRH has a neuroprotective activity in CRH-receptor type 1 (CRH-R1)-expressing neurons against oxidative cell death. The protective effect of CRH was blocked by selective and nonselective CRH-R1 antagonists and by protein kinase A inhibitors. Overexpression of CRH-R1 in clonal hippocampal cells lacking endogenous CRH-receptors established neuroprotection by CRH. The activation of CRH-R1 and neuroprotection are accompanied by an increased release of non-amyloidogenic soluble A beta precursor protein. At the molecular level CRH caused the suppression of the DNA-binding activity and transcriptional activity of the transcription factor NF-kappaB. Suppression of NF-kappaB by overexpression of a super-repressor mutant form of IkappaB-alpha, a specific inhibitor of NF-kappaB, led to protection of the cells against oxidative stress. These data demonstrate a novel cytoprotective effect of CRH that is mediated by CRH-R1 and downstream by suppression of NF-kappaB and indicate CRH as an endogenous protective neuropeptide against oxidative cell death in addition to its function in the HPA-system. Moreover, the protective function of CRH proposes a molecular link between oxidative stress-related degenerative events and the CRH-R1 system.

Assignment of the beta-thyroid hormone receptor to 3,5,3'-triiodothyronine-dependent inhibition of transcription from the thyrotropin-releasing hormone promoter in chick hypothalamic neurons.

Thyroid hormone, T3, is essential to the normal development and metabolism of vertebrates. Fine tuning of circulating levels of T3 is critical and involves feedback inhibition of the TRH and TSH genes by T3 at the hypothalamic and hypophyseal levels. However, the molecular basis of T3 inhibition of TRH gene expression in the hypothalamus is not known. The actions of T3 on target gene expression are mediated through nuclear receptor proteins, TR alpha and TR beta. To examine their effects on T3-dependent transcription from the rat TRH promoter, we used a gene transfer technique to express TR alpha and TR beta in cultured embryonic chick hypothalamic cells. Transcription from the TRH promoter construct transfected into these cultures was depressed in the presence of 10(-9) M T3. Cotransfecting TR alpha or TR beta activated transcription from the TRH promoter. However, only TR beta-dependent TRH transcription was differentially modulated by T3. Physiological concentrations of T3 decreased TR beta-dependent TRH transcription 4-fold. Thus, when T3 levels increase, TR beta mediates inhibition of TRH expression, a key step in down-regulating the hypophyseal-thyroid axis. This study demonstrates for the first time a T3-dependent differential regulation of the TRH promoter by TR beta and not TR alpha. Thus, the negative regulation of the TRH promoter in transiently transfected primary embryonic chick hypothalamic neurons provides a useful system for studying the molecular actions of thyroid hormone receptors.

Glucocorticoids enhance oxidative stress-induced cell death in hippocampal neurons in vitro.

In patients with Alzheimer's disease, hippocampal cells are among the first neuronal cells of the brain to degenerate. Both rat primary hippocampal neurons and cells of the clonal mouse hippocampal cell line HT22 express endogenous functional glucocorticoid receptors (GRs), as shown by transient transfection of cells with a luciferase reporter plasmid containing GR-responsive elements. The influence of activated GRs on oxidative stress-induced neuronal cell death in vitro was investigated employing these hippocampal model systems. Two oxidative stressors were investigated, the free radical-inducing Alzheimer's disease-associated amyloid beta-protein, which is toxic to hippocampal neurons, and the excitatory amino acid glutamate, which induces oxidative cell death in HT22 cells via an increase in intracellular peroxides. Cellular viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test and trypan exclusion staining, followed by microscopical cell counting. Glucocorticoids strongly increased the vulnerability of the hippocampal cells to amyloid beta-protein and glutamate. This increase could be blocked by the specific GR antagonist RU486. Our data suggest that changes in hippocampal GR homeostasis and regulation may render hippocampal neurons more vulnerable to oxidative stress-induced neuronal degeneration.

Inhibition of Zac1, a new gene differentially expressed in the anterior pituitary, increases cell proliferation.

Zac1 is a new zinc finger protein that concomitantly controls apoptosis and cell cycle arrest through separate pathways. The mouse Zac1 gene is mainly expressed in the pituitary gland and in different brain areas. In this study regional and cellular expression of Zac1 in the pituitary gland was determined by in situ hybridization. Zac1 messenger RNA was abundantly expressed in the anterior pituitary lobe compared with that in the intermediate and posterior lobes. Zac1 transcripts were found in all hormone-secreting cell types, with the highest levels in GH- and PRL-producing cells. To investigate the impact of Zac1 in pituitary cell proliferation, we ablated the endogenous Zac1 gene by antisense treatment in two murine cell types, AtT-20 and TtT/GF, that are representative of granular and agranular cell lineages, respectively. The decline in Zac1 protein levels under antisense treatment was accompanied by increased DNA synthesis in clonal corticotroph and folliculo-stellate cells, as demonstrated by enhanced [3H]thymidine incorporation (36% and 50%, respectively). Antisense oligonucleotides against Zac1 controlled cell proliferation in a dose-dependent way, and mutagenized antisense oligonucleotides were inert. Conclusively, our data provide the first evidence of a role for Zac1 in pituitary growth control.

New arylpiperazine derivatives as antagonists of the human cloned 5-HT(4) receptor isoforms.

New derivatives of arylpiperazine 9 were designed from ML 10302, a potent 5-HT(4) receptor agonist in the gastrointestinal system. Compounds were synthesized by condensation of a number of available arylpiperazines or heteroarylpiperazines with 2-bromoethyl 4-amino-5-chloro-2-methoxybenzoate. They were evaluated in binding assays on the recently cloned human 5-HT(4(e)) isoform stably expressed in C6 glial cells with [(3)H]GR 113808 as the radioligand. The affinity values (K(i)) depended upon the substituent on the aromatic ring. A chlorine atom produced a marked drop in activity (K(i) > 100 nM), while a m-methoxy group gave a compound with nanomolar affinity (K(i) = 3 nM). The most potent compounds were the heterocyclic derivatives with pyrimidine, pyrazine, pyridazine, or pyridine moieties (compounds 9r, 9t, 9u, 9x, respectively). K(i) values for 9a and 9r were determined for the 5-HT(4(a)), 5-HT(4(b)), 5-HT(4(c)), and 5-HT(4(d)) receptor isoforms transiently expressed in COS cells. The results indicated that the compounds were not selective. They produced an inhibition of the 5-HT-stimulated cyclic AMP synthesis in the C6 glial cells stably expressing the 5-HT(4(e)) receptor and shifted the 5-HT concentration-effect curve on adenylyl cyclase activity with pK(D) values of 7.44 and 8.47, respectively. In isolated human atrial myocytes, 9r antagonized the stimulatory effect of 5-HT on the L-type calcium current (I(Ca)) with a K(D) value of 0.7 nM.

Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytes.

The effects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (I(Ca)) and intracellular cyclic AMP concentration ([cAMP]i) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the different cardiac PDE subtypes was confirmed by RT-PCR. IBMX (100 microM), a broad-spectrum PDE inhibitor, increased basal I(Ca) by 120% and [cAMP]i by 70%, similarly to a saturating concentration of the beta-adrenoceptor agonist isoprenaline (1 microM). However, MIMX (1 microM), a PDE1 inhibitor, EHNA (10 microM), a PDE2 inhibitor, cilostamide (0.1 microM), a PDE3 inhibitor, or Ro20-1724 (0.1 microM), a PDE4 inhibitor, had no effect on basal I(Ca) and little stimulatory effects on [cAMP]i (20-30%). Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any effect on I(Ca) or [cAMP]i. While all combinations tested significantly increased [cAMP]i (40-50%), only cilostamide (0.1 microM)+ Ro20-1724 (0.1 microM) produced a significant stimulation of I(Ca) (50%). Addition of EHNA (10 microM) to this mix increased I(Ca) to 110% and [cAMP]i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 microM) or isoprenaline (1 microM). When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I(Ca) by (approximately 40% and had negligible effect on [cAMP]i, each selective PDE inhibitor induced a clear stimulation of [cAMP]i and an additional increase in I(Ca). Maximal effects on I(Ca) were approximately 8% for MIMX (3 microM), approximately 20% for EHNA (1-3 microM), approximately 30% for cilostamide (0.3-1 microM) and approximately 50% for Ro20-1724 (0.1 microM). Our results demonstrate that PDE1-4 subtypes regulate I(Ca) in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal I(Ca), all four PDE subtypes determine the response of I(Ca) to a stimulus activating cyclic AMP production, with the rank order of potency PDE4>PDE3>PDE2>PDE1.

Pharmacological characterization of the human 5-HT(4(d)) receptor splice variant stably expressed in Chinese hamster ovary cells.

The recently identified C-terminal splice variant of the human 5-HT(4) receptor, the h5-HT(4(d)) receptor, was stably expressed in a CHO cell line at 493+/-25 fmol mg(-1) protein. We analysed its pharmacological properties by measuring binding affinities and 5-HT(4) ligand-induced cyclic AMP production. The pharmacological binding profile determined in competition studies with the specific antagonist [(3)H]-GR113808 revealed a rank order of affinity of 5-HT(4) ligands for the h5-HT(4(d)) receptor that was consistent with those previously reported for other 5-HT(4) receptor isoforms. In adenylyl cyclase functional assays, the h5-HT(4(d)) receptor displayed equipotent coupling for all 5-HT(4) agonists tested (EC(50) in the range of 1 - 6 nM). EC(50) values were lower than those previously obtained with the 5-HT(4(e)) receptor stably expressed in CHO cells indicating that the 5-HT(4(d)) receptor was more efficiently coupled to its effector than the 5-HT(4(e)) receptor isoform. Moreover, in terms of agonist efficacy (E(max)), the benzamide derivative, renzapride displayed full agonist properties at the h5-HT(4(d)) receptor (same E(max) as 5-HT) whereas it was previously shown to be a partial agonist at the h5-HT(4(e)) receptor. A constitutive activity of the h5-HT(4(d)) receptor was observed in CHO cells in the absence of any 5-HT(4) ligand. Surprisingly, two 5-HT(4) ligands, SB204070 and RS39604 which are described as highly potent antagonists in various biological models, revealed partial agonist properties at the h5-HT(4(d)) receptor. We conclude that C-terminal tails of 5-HT(4) receptor isoforms may directly influence their functional properties.

Exploration of the ligand binding site of the human 5-HT(4) receptor by site-directed mutagenesis and molecular modeling.

Among the five human 5-HT(4) (h5-HT(4)) receptor isoforms, the h5-HT(4(a)) receptor was studied with a particular emphasis on the molecular interactions involved in ligand binding. For this purpose, we used site-directed mutagenesis of the transmembrane domain. Twelve mutants were constructed with a special focus on the residue P4.53 of helix IV which substitutes in h5-HT(4) receptors the highly conserved S residue among the rhodopsin family receptors. The mutated receptors were transiently expressed in COS-7 cells. Ligand binding or competition studies with two h5-HT(4) receptor agonists, serotonin and ML10302 and two h5-HT(4) receptor antagonists, [(3)H]-GR113808 and ML10375 were performed on wild type and mutant receptors. Functional activity of the receptors was evaluated by measuring the ability of serotonin to stimulate adenylyl cyclase. Ligand binding experiments revealed that [(3)H]-GR113808 did not bind to mutants P4.53A, S5.43A, F6.51A, Y7.43A and to double mutant F6.52V/N6.55L. On the other hand mutations D3.32N, S5.43A and Y7.43A appeared to promote a dramatic decrease of h5-HT(4(a)) receptor functional activity. From these studies, S5.43 and Y7.43 clearly emerged as common anchoring sites to antagonist [(3)H]-GR113808 and to serotonin. According to these results, we propose ligand-receptor complex models with serotonin and [(3)H]-GR113808. For serotonin, three interaction points were selected including ionic interaction with D3.32, a stabilizing interaction of this ion pair by Y7.43 and a hydrogen bond with S5.43. [(3)H]-GR113808 was also docked, based on the same type of interactions with S5.43 and D3.32: the proposed model suggested a possible role of P4.53 in helix IV structure allowing the involvement of a close hydrophobic residue, W4.50, in a hydrophobic pocket for hydrophobic interactions with the indole ring of [(3)H]-GR113808.

Isolation of the serotoninergic 5-HT4(e) receptor from human heart and comparative analysis of its pharmacological profile in C6-glial and CHO cell lines.

RT - PCR technique was used to clone the human 5-HT(4(e)) receptor (h5-HT(4(e))) from heart atrium. We showed that this h5-HT(4(e)) receptor splice variant is restricted to brain and heart atrium. Recombinant h5-HT(4(e)) receptor was stably expressed in CHO and C6-glial cell lines at 347 and 88 fmol mg(-1) protein, respectively. Expression of h5-HT(4(e)) receptors at the cell membrane was confirmed by immunoblotting. The receptor binding profile, determined by competition with [(3)H]-GR113808 of a number of 5-HT(4) ligands, was consistent with that previously reported for other 5-HT(4) receptor isoforms. Surprisingly, we found that the rank order of potencies (EC(50)) of 5-HT(4) agonists obtained from adenylyl cyclase functional assays was inversely correlated to their rank order of affinities (K(i)) obtained from binding assays. Furthermore, EC(50) values for 5-HT, renzapride and cisapride were 2 fold lower in C6-glial cells than in CHO cells. ML10302 and renzapride behaved like partial agonists on the h5-HT(4(e)) receptor. These results are in agreement with the reported low efficacy of the these two compounds on L-type Ca(2+) currents and myocyte contractility in human atrium. A constitutive activity of the h5-HT(4(e)) receptor was observed in CHO cells in the absence of any 5-HT(4) ligand and two 5-HT(4) antagonists, GR113808 and ML10375, behaved as inverse agonists. These data show that the h5-HT(4(e)) receptor has a pharmacological profile which is close to the native h5-HT(4) receptor in human atrium with a functional potency which is dependent on the cellular context in which the receptor is expressed.

Quantitative mRNA analysis of five C-terminal splice variants of the human 5-HT4 receptor in the central nervous system by TaqMan real time RT-PCR.

5-HT4 receptors mediate several physiological effects of 5-HT, particularly in the central nervous system (CNS), heart and gut. Recently, several C-terminal splice variants of the human 5-HT4 (h5-HT4) receptor have been described, namely h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g). Previous tissue distribution data suggest some degree of specificity in the mRNA expression patterns of the different h5-HT4 receptor splice variants. However, comparison of the mRNA expression profiles of these splice variants is difficult due to the non-quantitative methods used, and in addition, there is very limited data on the expression of each splice variant in human CNS subregions. In the present study we used a single technique, TaqMan real time quantitative RT-PCR, to investigate the mRNA distribution of 5-HT4 receptor C-terminal splice variants in multiple human CNS and peripheral tissues. Using a primer/probe set that amplified all 5-HT4 splice variants (5-HT4pan), the highest CNS expression of 5-HT4 receptor mRNA was observed in basal ganglia, amygdala and hippocampus, consistent with previous studies. h5-HT4(a), h5-HT4(b), h5-HT4(c) and h5-HT4(g) were predominantly expressed in various CNS tissues, compared to most peripheral tissues, but there were differences in expression levels and distribution patterns of each variant. The distribution profile and expression levels observed for the 5-HT4(b) splice variant were virtually identical to that obtained with the 5-HT4pan primer/probe set, whilst the other splice variants were expressed at much lower levels and with different expression patterns obtained with both 5-HT4(b) and 5-HT4pan primer/probe sets. Highest levels of 5-HT4(g) were observed in the hypothalamus and cortex, whilst the 5-HT4(a) variant was highest in the amygdala. 5-HT4(c) expression was highest in the pituitary gland whilst 5-HT4(d) mRNA was only detected in the small intestine at very low levels and not in the CNS. In conclusion, we have shown quantitative differences in the mRNA distribution profiles of the 5-HT4 receptor C-terminal splice variants in human CNS subregions as well as peripheral tissues. In addition, our data suggests that the h5-HT4(b) variant is the most predominant form of the 5-HT4 receptor in humans.

Estrogens with an intact phenolic group prevent death of neuronal cells following glutathione depletion.

Oxidative stress-induced neurodegeneration has been implicated in a variety of neuropsychiatric disorders including Alzheimer's disease (AD). Therefore, neuroprotection is of central interest in basic and preclinical neuroscience. Recently, we reported that the AD-associated amyliod beta protein can induce neuronal cell death via the generation of free radicals, oxidative stress and lipid peroxidation. The depletion of the intracellular pool of glutathione (GSH), an important intracellular oxidant, can also induce oxidative events. Various lipophilic antioxidants, including the female sex hormone estrogen, can protect neurons against oxidative cell death. Here, we report that estrogens prevent oxidative cell death induced by GSH depletion in murine clonal hippocampal HT22 cells and in cells of the sympathetic precursor-like cell line PC12. Estrogens act as free radical scavengers and inhibit the intracellular accumulation of peroxides caused by GSH depletion and, ultimately, prevent neuronal cell death. This protective activity is independent of the presence or activation of estrogen receptors but is dependent on the presence of an intact hydroxyl group in the steroid ring A of the estrogen molecule. The modification or the absence of this group led to a loss of the neuroprotective activity. These data further support the important role of antioxidants in neuroprotection and may help in the design of novel antioxidant drugs.

Melatonin prevents oxidative stress-induced cell death in hippocampal cells.

The pineal hormone melatonin has been suggested to be a very effective antioxidant in the brain. Oxidative stress may play a role in certain neuropathological conditions and the hippocampus is an early target of neurodegeneration. Using cells of the clonal hippocampal cell line HT22 and organotypic hippocampal rat brain slice cultures, we investigated a possible protective role of melatonin against oxidative stress-induced hippocampal cell death. Glutamate alone induced oxidative apoptotic cell death in HT22 cells as detected with Hoechst staining and DNA fragmentation analysis. Preincubation with 1 mM melatonin protected HT22 cells against glutamate-induced cell death and protected organotypic hippocampal slices against H2O2-induced cell death. These findings suggest that this neurohormone may be useful in the prevention of neurodegenerative diseases.

Bcl-2 prevents hippocampal cell death induced by the neuroleptic drug haloperidol.

The proto-oncogene bcl-2 can rescue cells from apoptosis and necrosis and its mechanism of action may involve antioxidant activity. The possible involvement of free radicals necrotic cell death induced by the neuroleptic drug haloperidol has led us to investigate the potential protective effect of Bcl-2 against haloperidol toxicity. We found that clonal mouse hippocampal cells stably transfected with the antioxidant Bcl-2 are protected against haloperidol toxicity. This data strongly supports the causative involvement of free radicals in haloperidol toxicity.

Precocious auto-induction of thyroid hormone receptors in embryonic chick hypothalamic neurons.

Qualitative immunocytochemical and quantitative autoradiographic approaches were used to examine the presence of thyroid hormone receptor (TR) proteins in embryonic chick hypothalamic cultures and the effect of T3 on their expression. We used antibodies raised against rat TRs having first verified that they recognised nuclear T3 binding proteins in extracts from chick brains. TRs were expressed in embryonic hypothalamic neurons from 6 day-old chick embryos maintained 4 days in vitro, and their expression was up-regulated by T3. This demonstration of TRs in embryonic hypothalamic neurons suggests that a physiological mechanism for early regulation of the hypothalamic neuropeptide, thyrotropin releasing hormone, is installed prior to the onset of thyroid function.

Transcription factor NF-kappaB: friend or foe of neurons?

It is now well established that a number of neurological disorders such as Parkinson's disease and Alzheimer's disease are characterized by the formation of reactive oxygen species (ROS). When produced in excess, ROS can induce oxidative damage to cellular macromolecules and ultimately cell death. Cells 'employ a number of defense systems against oxidative damage, including antioxidants and antioxidant enzymes. In an effort to develop a better understanding of the molecular mechanisms of the cellular response to a challenge by ROS as observed under certain neuropathological conditions, recent studies have focused on the regulation of gene expression by neurotoxins, including oxidative stressors. One transcription factor that can be activated by oxidative stress is the nuclear transcription factor-kappa B (NF-kappaB). Initially, this factor has been shown to play a major role in the activation of defensive genes during immune and inflammatory responses. But as evidence accumulates suggesting a close association of NF-kappaB activation also with the neuropathology occurring in neurodegenerative processes and neuronal cell death, the search is on to define potential roles for this transcription factor that are specific for neurons. The present article summarizes some of the recent studies that are trying to clarify NF-kappaB's role during neuronal degeneration.

Insulin-like growth factor-1-mediated neuroprotection against oxidative stress is associated with activation of nuclear factor kappaB.

The role of insulin-like growth factor 1 (IGF-1) for the treatment of neurodegenerative disorders, such as Alzheimer's disease, has recently gained attention. The present study demonstrates that IGF-1 promotes the survival of rat primary cerebellar neurons and of immortalized hypothalamic rat GT1-7 cells after challenge with oxidative stress induced by hydrogen peroxide (H2O2). Neuroprotective concentrations of IGF-1 specifically induce the transcriptional activity and the DNA binding activity of nuclear factor kappaB (NF-kappaB), a transcription factor that has been suggested to play a neuroprotective role. This induction is associated with increased nuclear translocation of the p65 subunit of NF-kappaB and with degradation of the NF-kappaB inhibitory protein IkappaBalpha. IGF-1-mediated protection of GT1-7 cells against oxidative challenges was mimicked by overexpression of the NF-kappaB subunit c-Rel. Partial inhibition of NF-kappaB baseline activity by overexpression of a dominant-negative IkappaBalpha mutant enhanced the toxicity of H2O2 in GT1-7 cells. The pathway by which IGF-1 promotes neuronal survival and activation of NF-kappaB involves the phosphoinositol (PI) 3-kinase, because both effects of IGF-1 are blocked by LY294002 and wortmannin, two specific PI 3-kinase inhibitors. Taken together, our results provide evidence for a novel molecular link between IGF-1-mediated neuroprotection and induction of NF-kappaB that is dependent on the PI 3-kinase pathway.