Repeated Estradiol Treatment Attenuates Chronic Cerebral Hypoperfusion-Induced Neurodegeneration in Rat Hippocampus.

Although a substantial number of pre-clinical and experimental studies have investigated effects of 17ß-estradiol, its precise molecular mechanism of action in the early state of chronic cerebral hypoperfusion remains controversial. The present study attempted to verify whether post-ischemic estradiol treatment (33.3 µg/kg for seven consecutive days) affects previously reported number of hippocampal apoptotic cells and amount of DNA fragmentation characteristic for apoptosis as well as the expression of key elements within synaptosomal Akt and Erk signal transduction pathways (NF-κB, Bax, Bcl-2, cytochrome C, caspase 3, and PARP). Additionally, alterations of aforementioned molecules linked to protection in various neurodegenerative disorders were monitored in the cytosolic, mitochondrial, and nuclear fractions associating investigated kinases and NF-κB with gene expression of their downstream effectors-Bcl-2, Bax, and caspase 3. The results revealed that an initial increase in the number of apoptotic cells and amount of DNA fragmentation induced by chronic cerebral hypoperfusion was significantly reduced by 17ß-estradiol. In synaptic regions, an altered profile with respect to the protein expression of Bcl-2 and phosphorylated Akt was detected, although the level of other examined proteins was not modified. In other investigated sub-cellular fractions, 17ß-estradiol elicited phosphorylation and translocation of Akt and Erk along with modulation of the expression of their subsequent effectors. Our findings support the concept that repeated post-ischemic 17ß-estradiol treatment attenuates neurodegeneration induced by chronic cerebral hypoperfusion in hippocampus through the activation of investigated kinases and regulation of their downstream molecules in sub-cellular manner indicating a time window and regime of its administration as a valid therapeutic intervention.

Regional and sex-related differences in modulating effects of female sex steroids on ecto-5'-nucleotidase expression in the rat cerebral cortex and hippocampus.

Ecto-5'-nucleotidase (eN), a membrane rate-limiting enzyme of the purine catabolic pathway, catalyzes the conversion of AMP to adenosine involved in the regulation of many brain physiological and pathological processes. Since gender fundamentally determines hormonal milieu in the body and brain, it is reasonable to assume that sex differences in the activity of various signaling systems, including adenosine, may be generated by gonadal steroids. Thus, we examined expression of eN as a component of adenosine signaling system in the basal state in cerebral cortex and hippocampus of male and female rats at gene, protein and functional level, as well as in the state of gonadal hormone deprivation, induced by ovariectomy (OVX), whereas impact of steroid hormones was explored after repeated administration of 17α-estradiol, 17ß-estradiol and progesterone for seven consecutive days. Results showed regional and sex-related differences in basal eN activity level, with the highest AMP hydrolysis observed in the hippocampus of male rats. Furthermore, ovarian steroids do not contribute to basal gene eN expression or the activity in cortical and hippocampal region of female rats. However, protein eN expression was increased in OVX rats in both investigated region. Investigated exogenous steroids had no influence on eN expression in male brain, while in OVX females alterations in eN activity were induced. The observed effects in female rats were different between examined regions e.g. in cortex, applied treatments predominantly decreased whereas in hippocampus increased eN activity. Based on the presented results, eN exerts regional and sex-related response in basal state as well as after treatment with female gonadal hormones, however the exact mechanisms of sex steroids actions on eN remain unclear and should be fully explored.

Protective effect of Hsp70i against chronic social isolation stress in the rat hippocampus.

Stress-related glucocorticoids and glutamate release has been implicated in depression. Glutamate neurotoxicity is mediated, in part, by the production of nitric oxide via nitric oxide synthase (NOS) isoforms and mitochondrial damage. We previously reported that chronic social isolation stress triggers proapoptotic signaling in the rat prefrontal cortex, but not in the hippocampus. Given that the hippocampus is highly sensitive to stress, we examined signaling cascades underlying the hippocampal cellular protection through the NOS pathway, antioxidant capacity and heat shock protein (Hsp) expression. We investigated neuronal (nNOS) and inducible (iNOS) protein levels, subcellular protein distributions of nuclear factor-κB (NF-κB), CuZnSOD and MnSOD activity, reduced glutathione (GSH), stress-inducible Hsp70 (Hsp70i) protein expression and serum corticosterone (CORT) levels of rats exposed to 21 days of chronic social isolation, an animal model of depression, alone or in combination with 2 h of acute immobilization or cold stress (combined stress). Both acute stressors elevated CORT, with lesser magnitude increase in chronically isolated rats exposed to novel acute stress as compared to acute stressors alone, indicating compromised HPA axis activity. Acute cold decreased nuclear CuZnSOD activity and stimulated NF-κB nuclear translocation. Chronic social isolation resulted in no activation of NF-κB, but led to decreased GSH, iNOS and increased nNOS and Hsp70i levels, alterations that remained following combined stressors. Decreased mitochondrial MnSOD activity after combined stressors suggests compromised detoxifying capacity. These data indicate that Hsp70i upregulation may provide hippocampal cellular protection against chronic social isolation stress mediated by downregulation of iNOS protein expression through suppression of NF-κB activation.

Different susceptibility of prefrontal cortex and hippocampus to oxidative stress following chronic social isolation stress.

Chronic oxidative stress plays an important role in depression. The aim of present study was to examine the stress-induced changes in serum corticosterone (CORT) levels, cytosolic protein carbonyl groups, malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO) and total superoxide dismutase (SOD) activity in the prefrontal cortex versus hippocampus of male Wistar rats exposed to acute (2 h of immobilization or cold), chronic (21d of social isolation) stress, and their combination (chronic + acute stress). The subcellular distribution of nuclear factor-κB (NF-κB) and cytosolic cyclooxygenase 2 (COX-2) protein expressions were also examined. Depressive- and anxiety-like behaviors were assessed via the forced swim, sucrose preference, and marble burying tests in chronically isolated rats. Although both acute stressors resulted in elevated CORT, increased MDA in the prefrontal cortex and NF-κB activation accompanied by increased NO in the hippocampus were detected only following acute cold stress. Chronic isolation resulted in no change in CORT levels, but disabled appropriate response to novel acute stress and led to depressive- and anxiety-like behaviors. Increased oxidative/nitrosative stress markers, likely by NF-κB nuclear translocation and concomitant COX-2 upregulation, associated with decreased SOD activity and GSH levels, suggested the existence of oxidative stress in the prefrontal cortex. In contrast, hippocampus was less susceptible to oxidative damage showing only increase in protein carbonyl groups and depleted GSH. Taken together, the prefrontal cortex seems to be more sensitive to oxidative stress than the hippocampus following chronic isolation stress, which may be relevant for further research related to stress-induced depressive-like behavior.

Chronic isolation stress compromises JNK/c-Jun signaling in rat brain.

The c-Jun NH2-terminal kinases (JNKs) are important stress-responsive kinases. They regulate cellular activities by sequential phosphorylation and activation through a mitogen-activated protein kinase cascade, whereas JNKs activation is altered in response to various stressors. In the present study, we used immunoblotting to assess the effect of 21 day of social isolation as the chronic stressor, either sole and in combination with 2 h of acute immobilization or cold (4°C) stress on circulating corticosterone level and phosphorylation status of p46 (phospho-p46/total p46) and p54 (phospho-p54/total p54) JNK isoforms in the cytosolic and nuclear fraction of the prefrontal cortex and hippocampus of male Wistar rats. Also, the phosphorylation status of JNK nuclear down-stream target c-Jun (p-c-Jun/c-Jun) on Ser63 was examined. Both acute stressors with elevated CORT levels led to increased phosphorylation status of cytosolic p54 JNK isoforms but not p46 JNK isoforms only in the hippocampus and no change in phosphorylation status of c-jun in both brain regions. Chronic isolation with unaltered CORT level and reduced responsiveness to novel acute stressors, led to unchanged or reduced phosphorylation status of p46 and p54 JNK isoforms in both fractions and both brain regions, whereas the decrease of c-Jun phosphorylation status was found only in the prefrontal cortex. Our results suggest that compromised JNKs activation following chronic isolation may lead to interruption of JNK signaling, which could be related with neuropsychiatric disorders such as depression or long-lasting neuronal remodeling.

Stress-induced alternations in CuZnSOD and MnSOD activity in cellular compartments of rat liver.

Exposure to different stressors initiates generation of reactive oxygen species (ROS), which create harmful environment for cellular macromolecules. Superoxide dismutases (SODs) represent the first line of antioxidant defense. Hence, any alternation in their function might be potentially damaging. To better define the role of SODs, we investigated the CuZnSOD activity in cytosolic and the nuclear fraction as well as mitochondrial MnSOD activity in the liver of Wistar male rats after exposure to 2 h of acute immobilization (IM) or cold (4°C) stress, 21 days of chronic social isolation (IS) or their combination (chronic stress followed by acute stress). Serum corticosterone (CORT) was monitored as an indicator of the stress response. Acute IM stress, with elevated CORT level, led to increased hepatic CuZnSOD activity in the nuclear fraction. Chronic isolation stress, where CORT was close to control value, did not change the CuZnSOD activity either in nuclei or in cytosolic fraction, while combined stress IS+Cold led to increased cytosolic CuZnSOD activity. MnSOD activity in mitochondrial fraction was decreased in all treated groups. Data have shown that different stressors have diverse effect on hepatic CuZnSOD and MnSOD activity as well as on serum CORT level. Increased nuclear CuZnSOD activity after acute stress represents physiological response since the named activity protects cells against oxidative stress, while chronic IS stress compromises CuZnSOD function, suggesting an inefficient defense against ROS. Observed decrease of MnSOD activities indicate inadequate elimination of ROS after acute or chronic stress, which is characteristic of the oxidative stress.

The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins.

A primary pathologic component of Alzheimer's disease (AD) is the formation of neurofibrillary tangles composed of hyperphosphorylated tau (p-tau). Expediting the removal of these p-tau species may be a relevant therapeutic strategy. Here we report that inhibition of Hsp90 led to decreases in p-tau levels independent of heat shock factor 1 (HSF1) activation. A critical mediator of this mechanism was carboxy terminus of Hsp70-interacting protein (CHIP), a tau ubiquitin ligase. Cochaperones were also involved in Hsp90-mediated removal of p-tau, while those of the mature Hsp90 refolding complex prevented this effect. This is the first demonstration to our knowledge that blockade of the refolding pathway promotes p-tau turnover through degradation. We also show that peripheral administration of a novel Hsp90 inhibitor promoted selective decreases in p-tau species in a mouse model of tauopathy, further suggesting a central role for the Hsp90 complex in the pathogenesis of tauopathies. When taken in the context of known high-affinity Hsp90 complexes in affected regions of the AD brain, these data implicate a central role for Hsp90 in the development of AD and other tauopathies and may provide a rationale for the development of novel Hsp90-based therapeutic strategies.

Proteomic profiling of KATP channel-deficient hypertensive heart maps risk for maladaptive cardiomyopathic outcome.

KCNJ11 null mutants, lacking Kir6.2 ATP-sensitive K(+) (K(ATP)) channels, exhibit a marked susceptibility towards hypertension (HTN)-induced heart failure. To gain insight into the molecular alterations induced by knockout of this metabolic sensor under hemodynamic stress, wild-type (WT) and Kir6.2 knockout (Kir6.2-KO) cardiac proteomes were profiled by comparative 2-DE and Orbitrap MS. Despite equivalent systemic HTN produced by chronic hyperaldosteronism, 114 unique proteins were altered in Kir6.2-KO compared to WT hearts. Bioinformatic analysis linked the primary biological function of the K(ATP) channel-dependent protein cohort to energetic metabolism (64% of proteins), followed by signaling infrastructure (36%) including oxidoreductases, stress-related chaperones, processes supporting protein degradation, transcription and translation, and cytostructure. Mapped protein-protein relationships authenticated the primary impact on metabolic pathways, delineating the K(ATP) channel-dependent subproteome within a nonstochastic network. Iterative systems interrogation of the proteomic web prioritized heart-specific adverse effects, i.e., "Cardiac Damage", "Cardiac Enlargement", and "Cardiac Fibrosis", exposing a predisposition for the development of cardiomyopathic traits in the hypertensive Kir6.2-KO. Validating this maladaptive forecast, phenotyping documented an aggravated myocardial contractile performance, a massive interstitial fibrosis and an exaggerated left ventricular size, all prognostic indices of poor outcome. Thus, Kir6.2 ablation engenders unfavorable proteomic remodeling in hypertensive hearts, providing a composite molecular substrate for pathologic stress-associated cardiovascular disease.

ATP-sensitive K+ channel knockout induces cardiac proteome remodeling predictive of heart disease susceptibility.

Forecasting disease susceptibility requires detection of maladaptive signatures prior to onset of overt symptoms. A case-in-point are cardiac ATP-sensitive K+ (K(ATP)) channelopathies, for which the substrate underlying disease vulnerability remains to be identified. Resolving molecular pathobiology, even for single genetic defects, mandates a systems platform to reliably diagnose disease predisposition. High-throughput proteomic analysis was here integrated with network biology to decode consequences of Kir6.2 K(ATP) channel pore deletion. Differential two-dimensional gel electrophoresis reproducibly resolved >800 protein species from hearts of asymptomatic wild-type and Kir6.2-knockout counterparts. K(ATP) channel ablation remodeled the cardiac proteome, significantly altering 71 protein spots, from which 102 unique identities were assigned following hybrid linear ion trap quadrupole-Orbitrap tandem mass spectrometry. Ontological annotation stratified the K(ATP) channel-dependent protein cohort into a predominant bioenergetic module (63 resolved identities), with additional focused sets representing signaling molecules (6), oxidoreductases (8), chaperones (6), and proteins involved in catabolism (6), cytostructure (8), and transcription and translation (5). Protein interaction mapping, in conjunction with expression level changes, localized a K(ATP) channel-associated subproteome within a nonstochastic scale-free network. Global assessment of the K(ATP) channel deficient environment verified the primary impact on metabolic pathways and revealed overrepresentation of markers associated with cardiovascular disease. Experimental imposition of graded stress precipitated exaggerated structural and functional myocardial defects in the Kir6.2-knockout, decreasing survivorship and validating the forecast of disease susceptibility. Proteomic cartography thus provides an integral view of molecular remodeling in the heart induced by K(ATP) channel deletion, establishing a systems approach that predicts outcome at a presymptomatic stage.

The effect of acute or/and chronic stress on the MnSOD protein expression in rat prefrontal cortex and hippocampus.

Manganese superoxide dismutase (MnSOD) is the major antioxidant in mitochondria that protect brain from neuroendocrine stress. Although MnSOD is localized in the mitochondria, the immediate subcellular distribution of MnSOD protein level in the prefrontal cortex and hippocampus of Wistar male rats exposed to acute stressors immobilization or cold, chronic stress isolation or their combinations (acute/chronic) have not been studied. Western immunoblotting revealed that acute immobilization stress resulted in an increase in mitochondrial MnSOD protein level, whereas chronic isolation compromises MnSOD protein level. Chronically stressed animals exposed to novel acute stressors showed a significant decrease in mitochondrial MnSOD protein level and reciprocal increase in this protein in the cytosolic fraction. At the same time, a significant increase in serum corticosterone level was observed after acute stressors, whereas chronic isolation led to negligable changes and caused a reduced responsiveness to a novel acute stressors. Presence of cytochrome c in mitochondrial and cytosolic fraction of both brain structures was also confirmed. Results suggest that chronic stress isolation results in mitochondrial dysfunction and MnSOD release into the cytosol.

Repeated low-dose 17ß-estradiol treatment prevents activation of apoptotic signaling both in the synaptosomal and cellular fraction in rat prefrontal cortex following cerebral ischemia.

Disturbance in blood circulation is associated with numerous pathological conditions characterized by cognitive decline and neurodegeneration. Activation of pro-apoptotic signaling previously detected in the synaptosomal fraction may underlie neurodegeneration in the prefrontal cortex of rats submitted to permanent bilateral common carotid arteries occlusion (two-vessel occlusion, 2VO). 17ß-Estradiol (E) exerts potent neuroprotective effects in the brain affecting, among other, ischemia-induced pathological changes. As most significant changes in rats submitted to 2VO were observed on 7th day following the insult, of interest was to examine whether 7 day treatment with low dose of E (33.3 µg/kg/day) prevents formerly reported neurodegeneration and may represent additional therapy during the early post-ischemic period. Role of E treatment on apoptotic pathway was monitored on Bcl-2 family members, cytochrome c, caspase 3 and PARP protein level in the synaptosomal (P2) fraction of the prefrontal cortex. Furthermore, changes of these proteins were examined in the cytosolic, mitochondrial and nuclear fraction, with the emphasis on potential involvement of extracellular signal-regulated kinases (ERK) and protein kinase B (Akt) activation and their role in nuclear translocation of transcriptional nuclear factor kappa B (NF-kB) associated with alteration of Bax and Bcl-2 gene expression. The extent of cellular damage was determined using DNA fragmentation and Fluoro-Jade B staining. The absence of activation of apoptotic cascade both in the P2 and cell accompanied with decreased DNA fragmentation and number of degenerating neurons clearly indicates that E treatment ensures the efficient protection against ischemic insult. Moreover, E-mediated modulation of pro-apoptotic signaling in the cortical cellular fractions involves cooperative activation of ERK and Akt, which may be implicated in the observed prevention of neurodegenerative changes.

Chronic administration of fluoxetine or clozapine induces oxidative stress in rat liver: a histopathological study.

Chronic exposure to stress contributes to the etiology of mood disorders, and the liver as a target organ of antidepressant and antipsychotic drug metabolism is vulnerable to drug-induced toxicity. We investigated the effects of chronic administration of fluoxetine (15mg/kg/day) or clozapine (20mg/kg/day) on liver injury via the measurement of liver enzymes, oxidative stress and histopathology in rats exposed to chronic social isolation (21days), an animal model of depression, and controls. The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the liver content of carbonyl groups, malonyldialdehyde (MDA), reduced glutathione (GSH), cytosolic glutathione S-transferase (GST) and nitric oxide (NO) metabolites were determined. We also characterized nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2) and CuZn-superoxide dismutase (CuZnSOD) protein expression as well as histopathological changes. Increased serum ALT activity in chronically-isolated and control animals treated with both drugs was found while increased AST activity was observed only in fluoxetine-treated rats (chronically-isolated and controls). Increased carbonyl content, MDA, GST activity and decreased GSH levels in drug-treated controls/chronically-isolated animals suggest a link between drugs and hepatic oxidative stress. Increased NO levels associated with NF-κB activation and the concomitant increased COX-2 expression together with compromised CuZnSOD expression in clozapine-treated chronically-isolated rats likely reinforce oxidative stress, observed by increased lipid peroxidation and GSH depletion. In contrast, fluoxetine reduced NO levels in chronically-isolated rats. Isolation induced oxidative stress but histological changes were similar to those observed in vehicle-treated controls. Chronic administration of fluoxetine in both chronically-isolated and control animals resulted in more or less normal hepatic architecture, while clozapine in both groups resulted in liver injury. These data suggest that clozapine appears to have a higher potential to induce liver toxicity than fluoxetine.

Chronic social isolation induces NF-κB activation and upregulation of iNOS protein expression in rat prefrontal cortex.

Exposure of an organism to stress, results in oxidative stress and increased nitric oxide (NO) production in the brain. The role of the processes caused by chronic stress in the prefrontal cortex has not been fully investigated. Considering that chronic stress increases NO production by the enzyme nitric oxide synthase (NOS), we examined the cytosolic neuronal (nNOS) or inducible (iNOS) protein levels in the prefrontal cortex of rats exposed to 21d of chronic social isolation stress, an animal model of depression, alone or in combination with 2h of acute immobilization or cold (4°C) stress (combined stress). Antioxidative status via cytosolic CuZnSOD and mitochondrial MnSOD activity, cytosolic redox status via reduced glutathione (GSH) concentration were determined. Furthermore, cytosolic inducible heat shock protein 70 (Hsp70i), cytosolic/nuclear distributions of NF-κB and serum corticosterone (CORT) were also investigated to elucidate the possible mechanism involved in the cellular NOS pathway. Our results showed that both acute stressors led to increases of CORT and nNOS protein while iNOS protein expression was unaffected. In contrast to the acute stress, chronic social isolation compromised hypothalamic-pituitary-adrenal axis functioning such that the normal stress response was impaired following subsequent acute stressors. Downregulated redox GSH status as well as decreased activity of CuZnSOD and MnSOD suggests the existence of oxidative stress which remained as such following combined stressors. Changes in redox-status associated with decreased Hsp70i protein expression enabled NF-κB translocation into the nucleus, causing increased cytosolic nNOS and iNOS protein expression. Results suggest that NOS signaling pathway plays a differential role between acute and chronic stress whereby state of oxidative/nitrosative stress after chronic social isolation is caused, at least in part, by NF-κB activation and increased iNOS protein expression.

Human hydroxysteroid sulfotransferase SULT2B1 pharmacogenomics: gene sequence variation and functional genomics.

The human hydroxysteroid sulfotransferase (SULT) 2B1 gene is a member of the cytosolic SULT gene superfamily. The two SULT2B1 isoforms, SULT2B1a and SULT2B1b, are encoded by a single gene as a result of alternative transcription initiation and alternative splicing. SULT2B1b catalyzes the sulfonation of 3beta-hydroxysteroid hormones and cholesterol, whereas SULT2B1a preferentially catalyzes pregnenolone sulfonation. We used a genotype-to-phenotype approach to identify and characterize common sequence variation in SULT2B1. Specifically, we resequenced all exons, splice junctions, and approximately 2.5 kb of the 5'-flanking regions (FRs) for each isoform using 60 DNA samples each from African-American and Caucasian-American subjects. We observed 100 polymorphisms, including four nonsynonymous coding single nucleotide polymorphisms and one 6-base pair deletion-all within the "shared" region of the open reading frame. Functional genomic studies of the wild type (WT) and five variant allozymes for each isoform performed with a mammalian expression system showed that variant allozyme activities ranged from 64 to 88% of WT for SULT2B1a and from 76 to 98% for SULT2B1b. Relative levels of immunoreactive protein were similar to those for enzyme activity. Luciferase reporter gene constructs for 2.5 kb of the SULT2B1b 5'-FR displayed a cell line-dependent pattern of variation in activity. Finally, deletion of the proline-rich SULT2B1 carboxyl terminus resulted in intracellular protein aggregate formation and accelerated degradation of the truncated protein. These studies resulted in the identification of common SULT2B1 gene sequence variation, as well as insight into the effects of that variation on the function of this important steroid-metabolizing enzyme.