Osteopontin-stimulated apoptosis in cardiac myocytes involves oxidative stress and mitochondrial death pathway: role of a pro-apoptotic protein BIK.

Increased osteopontin (OPN) expression in the heart, specifically in myocytes, associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we provided evidence that OPN interacts with CD44 receptor, and induces myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. Here we tested the hypothesis that OPN induces oxidative stress in myocytes and the heart via the involvement of mitochondria and NADPH oxidase-4 (NOX-4). Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) increased oxidative stress as analyzed by protein carbonylation, and intracellular reactive oxygen species (ROS) levels as analyzed by ROS detection kit and dichlorohydrofluorescein diacetate staining. Pretreatment with NAC (antioxidant), apocynin (NOX inhibitor), MnTBAP (superoxide dismutase mimetic), and mitochondrial KATP channel blockers (glibenclamide and 5-hydroxydecanoate) decreased OPN-stimulated ROS production, cytosolic cytochrome c levels, and apoptosis. OPN increased NOX-4 expression, while decreasing SOD-2 expression. OPN decreased mitochondrial membrane potential as measured by JC-1 staining, and induced mitochondrial abnormalities including swelling and reorganization of cristae as observed using transmission electron microscopy. OPN increased expression of BIK, a pro-apoptotic protein involved in reorganization of mitochondrial cristae. Expression of dominant-negative BIK decreased OPN-stimulated apoptosis. In vivo, OPN expression in cardiac myocyte-specific manner associated with increased protein carbonylation, and expression of NOX-4 and BIK. Thus, OPN induces oxidative stress via the involvement of mitochondria and NOX-4. It may affect mitochondrial morphology and integrity, at least in part, via the involvement of BIK.

Osteopontin stimulates apoptosis in adult cardiac myocytes via the involvement of CD44 receptors, mitochondrial death pathway, and endoplasmic reticulum stress.

Increased osteopontin (OPN) expression associates with increased myocyte apoptosis and myocardial dysfunction. The objective of this study was to identify the receptor for OPN and get insight into the mechanism by which OPN induces cardiac myocyte apoptosis. Adult rat ventricular myocytes (ARVMs) and transgenic mice expressing OPN in a myocyte-specific manner were used for in vitro and in vivo studies. Treatment with purified OPN (20 nM) protein or adenoviral-mediated OPN expression induced apoptosis in ARVMs. OPN co-immunoprecipitated with CD44 receptors, not with ß1 or ß3 integrins. Proximity ligation assay confirmed interaction of OPN with CD44 receptors. Neutralizing anti-CD44 antibodies inhibited OPN-stimulated apoptosis. OPN activated JNKs and increased expression of Bax and levels of cytosolic cytochrome c, suggesting involvement of mitochondrial death pathway. OPN increased endoplasmic reticulum (ER) stress, as evidenced by increased expression of Gadd153 and activation of caspase-12. Inhibition of JNKs using SP600125 or ER stress using salubrinal or caspase-12 inhibitor significantly reduced OPN-stimulated apoptosis. Expression of OPN in adult mouse heart in myocyte-specific manner associated with decreased left ventricular function and increased myocyte apoptosis. In the heart, OPN expression increased JNKs and caspase-12 activities, and expression of Bax and Gadd153. Thus, OPN, acting via CD44 receptors, induces apoptosis in myocytes via the involvement of mitochondrial death pathway and ER stress.

Lack of ataxia telangiectasia mutated kinase induces structural and functional changes in the heart: role in ß-adrenergic receptor-stimulated apoptosis.

Ataxia telangiectasia mutated kinase (ATM) is involved in cell cycle checkpoints, DNA repair and apoptosis. ß-Adrenergic receptor (ß-AR) stimulation induces cardiac myocyte apoptosis. Here we analysed basal myocardial structure and function in ATM knockout (KO) mice and tested the hypothesis that ATM modulates ß-AR-stimulated myocyte apoptosis. Left ventricular (LV) structure and function, myocyte apoptosis, fibrosis and expression of fibrosis-, hypertrophy- and apoptosis-related proteins were examined in wild-type (WT) and KO mice with or without l-isoprenaline treatment for 24 h. Body and heart weights were lower in KO mice. M-Mode echocardiography showed reduced septal wall thicknesses and LV diameters in KO mice. Doppler echocardiography showed an increased ratio of early peak velocity (E wave) to that of the late LV filling (A wave) in KO mice. Basal fibrosis and myocyte cross-sectional area were greater in KO hearts. Expression of fibrosis-related genes (connective tissue growth factor and plasminogen activator inhibitor-1) and hypertrophy-related gene (atrial natriuretic peptide) was higher in KO hearts. ß-Adrenergic receptor stimulation increased myocyte apoptosis to a similar extent in both groups. Activation of c-Jun N-terminal kinases and expression and phosphorylation of p53 in response to ß-AR stimulation were only observed in the WT group. Akt phosphorylation was lower in KO sham-treated animals and remained lower following ß-AR stimulation in the KO group. ß-Adrenergic receptor stimulation activated glycogen synthase kinase-3ß to a similar extent in both groups. Thus, lack of ATM induces structural and functional changes in the heart, with enhanced myocardial fibrosis and myocyte hypertrophy. ß-Adrenergic receptor-stimulated apoptosis in WT hearts is associated with a p53- and JNKs-dependent mechanism, while decreased Akt activity may play a role in increased myocyte apoptosis in the absence of ATM.

Dexamethasone-induced up-regulation of the human norepinephrine transporter involves the glucocorticoid receptor and increased binding of C/EBP-ß to the proximal promoter of norepinephrine transporter.

Previously, we have found glucocorticoids up-regulate norepinephrine (NE) transporter (NET) expression in vitro. However, the underlying transcriptional mechanism is poorly understood. In this study, the role of glucocorticoids on the transcriptional regulation of NET was investigated. Exposure of neuroblastoma SK-N-BE(2)M17 cells to dexamethasone (Dex) significantly increased NET mRNA and protein levels in a time- and dose-dependent manner. This effect was attenuated by glucocorticoid receptor (GR) antagonist mifepristone, suggesting that up-regulation of NET by Dex was mediated by the GR. In reporter gene assays, exposure of cells to Dex resulted in dose-dependent increases of luciferase activity that were also prevented by mifepristone. Serial deletions of the NET promoter delineated Dex-responsiveness to a -301 to -148 bp region containing a CCAAT/enhancer binding protein-ß (C/EBP-ß) response element. Co-immunoprecipitation experiments demonstrated that Dex treatment caused the interaction of the GR with C/EBP-ß. Chromatin immunoprecipitation (ChIP) assay revealed that Dex exposure resulted in binding of both GR and C/EBP-ß to the NET promoter. Further experiments showed that mutation of the C/EBP-ß response element abrogated C/EBP-ß- and GR-mediated transactivation of NET. These findings demonstrate that Dex-induced increase in NET expression is mediated by the GR via a non-conventional transcriptional mechanism involving interaction of C/EBP-ß with a C/EBP-ß response element.

Neuron-restrictive silencer factor-mediated hyperpolarization-activated cyclic nucleotide gated channelopathy in experimental temporal lobe epilepsy.

OBJECTIVE: Enduring, abnormal expression and function of the ion channel hyperpolarization-activated cyclic adenosine monophosphate gated channel type 1 (HCN1) occurs in temporal lobe epilepsy (TLE). We examined the underlying mechanisms, and investigated whether interfering with these mechanisms could modify disease course. METHODS: Experimental TLE was provoked by kainic acid-induced status epilepticus (SE). HCN1 channel repression was examined at mRNA, protein, and functional levels. Chromatin immunoprecipitation was employed to identify the transcriptional mechanism of repressed HCN1 expression, and the basis for their endurance. Physical interaction of the repressor, NRSF, was abolished using decoy oligodeoxynucleotides (ODNs). Video/electroencephalographic recordings were performed to assess the onset and initial pattern of spontaneous seizures. RESULTS: Levels of NRSF and its physical binding to the Hcn1 gene were augmented after SE, resulting in repression of HCN1 expression and HCN1-mediated currents (I(h) ), and reduced I(h) -dependent resonance in hippocampal CA1 pyramidal cell dendrites. Chromatin changes typical of enduring, epigenetic gene repression were apparent at the Hcn1 gene within a week after SE. Administration of decoy ODNs comprising the NRSF DNA-binding sequence (neuron restrictive silencer element [NRSE]), in vitro and in vivo, reduced NRSF binding to Hcn1, prevented its repression, and restored I(h) function. In vivo, decoy NRSE ODN treatment restored theta rhythm and altered the initial pattern of spontaneous seizures. INTERPRETATION: Acquired HCN1 channelopathy derives from NRSF-mediated transcriptional repression that endures via chromatin modification and may provide insight into the mechanisms of a number of channelopathies that coexist with, and may contribute to, the conversion of a normal brain into an epileptic one.

Epileptogenesis provoked by prolonged experimental febrile seizures: mechanisms and biomarkers.

Whether long febrile seizures (FSs) can cause epilepsy in the absence of genetic or acquired predisposing factors is unclear. Having established causality between long FSs and limbic epilepsy in an animal model, we studied here if the duration of the inciting FSs influenced the probability of developing subsequent epilepsy and the severity of the spontaneous seizures. We evaluated if interictal epileptifom activity and/or elevation of hippocampal T2 signal on magnetic resonance image (MRI) provided predictive biomarkers for epileptogenesis, and if the inflammatory mediator interleukin-1beta (IL-1beta), an intrinsic element of FS generation, contributed also to subsequent epileptogenesis. We found that febrile status epilepticus, lasting an average of 64 min, increased the severity and duration of subsequent spontaneous seizures compared with FSs averaging 24 min. Interictal activity in rats sustaining febrile status epilepticus was also significantly longer and more robust, and correlated with the presence of hippocampal T2 changes in individual rats. Neither T2 changes nor interictal activity predicted epileptogenesis. Hippocampal levels of IL-1beta were significantly higher for >24 h after prolonged FSs. Chronically, IL-1beta levels were elevated only in rats developing spontaneous limbic seizures after febrile status epilepticus, consistent with a role for this inflammatory mediator in epileptogenesis. Establishing seizure duration as an important determinant in epileptogenesis and defining the predictive roles of interictal activity, MRI, and inflammatory processes are of paramount importance to the clinical understanding of the outcome of FSs, the most common neurological insult in infants and children.

Trafficking and surface expression of hyperpolarization-activated cyclic nucleotide-gated channels in hippocampal neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated current I(h) and thus play important roles in the regulation of brain excitability. The subcellular distribution pattern of the HCN channels influences the effects that they exert on the properties and activity of neurons. However, little is known about the mechanisms that control HCN channel trafficking to subcellular compartments or that regulate their surface expression. Here we studied the dynamics of HCN channel trafficking in hippocampal neurons using dissociated cultures coupled with time lapse imaging of fluorophore-fused HCN channels. HCN1-green fluorescence protein (HCN1-GFP) channels resided in vesicle-like organelles that moved in distinct patterns along neuronal dendrites, and these properties were isoform-specific. HCN1 trafficking required intact actin and tubulin and was rapidly inhibited by activation of either NMDA or AMPA-type ionotropic glutamate receptors in a calcium-dependent manner. Glutamate-induced inhibition of the movement of HCN1-GFP-expressing puncta was associated with increased surface expression of both native and transfected HCN1 channels, and this surface expression was accompanied by augmented I(h). Taken together, the results reveal the highly dynamic nature of HCN1 channel trafficking in hippocampal neurons and provide a novel potential mechanism for rapid regulation of I(h), and hence of neuronal properties, via alterations of HCN1 trafficking and surface expression.

Corticosterone up-regulates expression and function of norepinephrine transporter in SK-N-BE(2)C cells.

Glucocorticoids affect cellular and molecular events in brains by modulating the expression of many genes during stress. In the present study, we examined the regulatory effect of corticosterone on the expression and function of the norepinephrine transporter (NET) in vitro. The results show that exposure of SK-N-BE(2)C cells to corticosterone for 14 days significantly increased mRNA (up to 43%) and protein (up to 71%) levels of NET in the concentration-dependent manner. Longer exposure (21 days) resulted in greater increases in the levels of mRNAs (up to about 160%) and proteins (up to about 250%) of the NET. The up-regulatory effect of corticosterone on NET expression lasted a persistent period after cessation of exposure. Associated with the corticosterone-induced enhancement in NET expression, there was a parallel increase in the uptake of [(3)H]norepinephrine by SK-N-BE(2)C cells. Increased NET expression and function were abolished after exposure of cells to corticosterone in combination with mifepristone or spironolactone, two specific antagonists of corticosteroid receptors. This is consistent with the hypothesis that corticosterone-induced NET up-regulation is mediated by corticosteroid receptors. Nevertheless, there was no synergistic effect for a combination of both corticosteroid receptor antagonists. A similar up-regulation of NET protein levels was also observed after exposing PC12 cells to corticosterone. The present findings demonstrate that corticosterone up-regulates the expression and function of NET in vitro, indicating the action of corticosterone on the noradrenergic phenotype may play an important role in the correlation between stress and the development of depression.

Activity-dependent heteromerization of the hyperpolarization-activated, cyclic-nucleotide gated (HCN) channels: role of N-linked glycosylation.

Formation of heteromeric complexes of ion channels via co-assembly of different subunit isoforms provides an important mechanism for enhanced channel diversity. We have previously demonstrated co-association of the hyperpolarization activated cyclic-nucleotide gated (HCN1/HCN2) channel isoforms that was regulated by network (seizure) activity in developing hippocampus. However, the mechanisms that underlie this augmented expression of heteromeric complexes have remained unknown. Glycosylation of the HCN channels has been implicated in the stabilization and membrane expression of heteromeric HCN1/HCN2 constructs in heterologous systems. Therefore, we used in vivo and in vitro systems to test the hypothesis that activity modifies HCN1/HCN2 heteromerization in neurons by modulating the glycosylation state of the channel molecules. Seizure-like activity (SA) increased HCN1/HCN2 heteromerization in hippocampus in vivo as well as in hippocampal organotypic slice cultures. This activity increased the abundance of glycosylated HCN1 but not HCN2-channel molecules. In addition, glycosylated HCN1 channels were preferentially co-immunoprecipitated with the HCN2 isoforms. Provoking SA in vitro in the presence of the N-linked glycosylation blocker tunicamycin abrogated the activity-dependent increase of HCN1/HCN2 heteromerization. Thus, hippocampal HCN1 molecules have a significantly higher probability of being glycosylated after SA, and this might promote a stable heteromerization with HCN2.

Mechanisms of seizure-induced 'transcriptional channelopathy' of hyperpolarization-activated cyclic nucleotide gated (HCN) channels.

Epilepsy may result from abnormal function of ion channels, such as those caused by genetic mutations. Recently, pathological alterations of the expression or localization of normal channels have been implicated in epilepsy generation, and termed 'acquired channelopathies'. Altered expression levels of the HCN channels - that conduct the hyperpolarization-activated current, I(h) - have been demonstrated in hippocampus of patients with severe temporal lobe epilepsy as well as in animal models of temporal lobe and absence epilepsies. Here we probe the mechanisms for the altered expression of HCN channels which is provoked by seizures. In organotypic hippocampal slice cultures, seizure-like events selectively reduced HCN type 1 channel expression and increased HCN2 mRNA levels, as occurs in vivo. The mechanisms for HCN1 reduction involved Ca(2+)-permeable AMPA receptor-mediated Ca(2+) influx, and subsequent activation of Ca(2+)/calmodulin-dependent protein kinase II. In contrast, upregulation of HCN2 expression was independent of these processes. The data demonstrate an orchestrated program for seizure-evoked transcriptional channelopathy involving the HCN channels that may contribute to certain epilepsies.

Fever, febrile seizures and epilepsy.

Seizures induced by fever (febrile seizures) are the most common type of pathological brain activity in infants and children. These febrile seizures and their potential contribution to the mechanisms of limbic (temporal lobe) epilepsy have been a topic of major clinical and scientific interest. Key questions include the mechanisms by which fever generates seizures, the effects of long febrile seizures on neuronal function and the potential contribution of these seizures to epilepsy. This review builds on recent advances derived from animal models and summarizes our current knowledge of the mechanisms underlying febrile seizures and of changes in neuronal gene expression and function that facilitate the enduring effects of prolonged febrile seizures on neuronal and network excitability. The review also discusses the relevance of these findings to the general mechanisms of epileptogenesis during development and points out gaps in our knowledge, including the relationship of animal models to human febrile seizures and epilepsy.

[Deletion mutation of beta-amyloid precursor protein decreases the secretion of beta-amyloid in SH-SY5Y neuroblastoma cells].

OBJECTIVE: To explore the effect of A beta secretion lacking exons 2 and 3 of APP695 (delta APP695) on SH-SY5Y neuroblastoma cells. METHODS: APP695 cDNA and delta APP695 cDNA eukaryotic expression vectors were constructed by gene recombination. Immunoprecipitation Western blotting was used to analyse the A beta secretion and MTT assay to observe the cell viability's change after being stably transfected with the plasmids by lipofectamine method. RESULTS: Stable transfectants were selected in 0.6 g.L-1 G418. delta APP695 cDNA construct transfection decreased significantly the production of beta amyloid in the SH-SY5Y compared with APP695 cDNA construct. It did not affect the cell viability by MTT method. CONCLUSION: This result suggests that the alternative splicing of APP exons 2 and 3 modulates the secretion of A beta by influencing the processing of APP.