Miklós Sárvári

Estrogens regulate neuroinflammatory genes via estrogen receptors α and β in the frontal cortex of middle-aged female rats

BackgroundEstrogens exert anti-inflammatory and neuroprotective effects in the brain mainly via estrogen receptors α (ERα) and β (ERβ). These receptors are members of the nuclear receptor superfamily of ligand-dependent transcription factors. This study was aimed at the elucidation of the effects of ERα and ERβ agonists on the expression of neuroinflammatory genes in the frontal cortex of aging female rats.MethodsTo identify estrogen-responsive immunity/inflammation genes, we treated middle-aged, ovariectomized rats with 17β-estradiol (E2), ERα agonist 16α-lactone-estradiol (16α-LE2) and ERβ agonist diarylpropionitrile (DPN), or vehicle by Alzet minipump delivery for 29 days. Then we compared the transcriptomes of the frontal cortex of estrogen-deprived versus ER agonist-treated animals using Affymetrix Rat230 2.0 expression arrays and TaqMan-based quantitative real-time PCR. Microarray and PCR data were evaluated by using Bioconductor packages and the RealTime StatMiner software, respectively.ResultsMicroarray analysis revealed the transcriptional regulation of 21 immunity/inflammation genes by 16α-LE2. The subsequent comparative real-time PCR study analyzed the isotype specific effects of ER agonists on neuroinflammatory genes of primarily glial origin. E2 regulated the expression of sixteen genes, including down-regulation of complement C3 and C4b, Ccl2, Tgfb1, macrophage expressed gene Mpeg1, RT1-Aw2, Cx3cr1, Fcgr2b, Cd11b, Tlr4 and Tlr9, and up-regulation of defensin Np4 and RatNP-3b, IgG-2a, Il6 and ER gene Esr1. Similar to E2, both 16α-LE2 and DPN evoked up-regulation of defensins, IgG-2a and Il6, and down-regulation of C3 and its receptor Cd11b, Ccl2, RT1-Aw2 and Fcgr2b.ConclusionsThese findings provide evidence that E2, 16α-LE2 and DPN modulate the expression of neuroinflammatory genes in the frontal cortex of middle-aged female rats via both ERα and ERβ. We propose that ERβ is a promising target to suppress regulatory functions of glial cells in the E2-deprived female brain and in various neuroinflammatory diseases.

Estradiol replacement alters expression of genes related to neurotransmission and immune surveillance in the frontal cortex of middle-aged, ovariectomized rats.

Estradiol (E2) modulates a wide range of functions of the frontal cerebral cortex. From the onset of menopause, declining levels of E2 can cause cognitive disturbances and changes in behavior that can be counterbalanced by hormone replacement. To study the effect of E2 replacement on the cortical transcriptome in a rodent model with low serum E2 level, we treated middle-aged, ovariectomized rats with E2 or vehicle using osmotic minipumps for 4 wk. Six animals for each group were selected, and samples of their frontal cortex were subjected to expression profiling using oligonucleotide microarrays. The explored E2-regulated genes were related to neurotransmission (Adora2a, Cartpt, Drd1a, Drd2, Gjb2, Nts, and Tac1), immunity (C3, C4b, Cd74, Fcgr2b, Mpeg1, and RT1-Aw2), signal transduction (Igf2, Igfbp2, Igfbp6, Rgs9, and Sncg), transport (Abca1, Hba-a2, Slc13a3, and Slc22a8), extracellular matrix (Col1a2, Col3a1, Fmod, and Lum), and transcription (Irf7 and Nupr1). Seventy-four percent of the transcriptional changes identified by microarray were confirmed by quantitative real-time PCR. The genes identified by expression profiling indicated that chronic E2 replacement significantly altered the transcriptome of the frontal cortex. The genomic effects of E2 influenced dopaminergic and peptidergic neurotransmission, immune surveillance, adenosine and insulin-like growth factor signaling and transport processes, among other functions. Identification of these novel E2-regulated mechanisms highlights the wide range of genomic responses of the aging female frontal cerebral cortex subjected to hormone replacement. Some of the genomic effects identified in this study may underlie the beneficial effects of E2 on cognition, behavior, and neuroprotection.

Ghrelin Decreases Firing Activity of Gonadotropin- Releasing Hormone (GnRH) Neurons in an Estrous Cycle and Endocannabinoid Signaling Dependent Manner

The orexigenic peptide, ghrelin is known to influence function of GnRH neurons, however, the direct effects of the hormone upon these neurons have not been explored, yet. The present study was undertaken to reveal expression of growth hormone secretagogue receptor (GHS-R) in GnRH neurons and elucidate the mechanisms of ghrelin actions upon them. Ca2+-imaging revealed a ghrelin-triggered increase of the Ca2+-content in GT1-7 neurons kept in a steroid-free medium, which was abolished by GHS-R-antagonist JMV2959 (10µM) suggesting direct action of ghrelin. Estradiol (1nM) eliminated the ghrelin-evoked rise of Ca2+-content, indicating the estradiol dependency of the process. Expression of GHS-R mRNA was then confirmed in GnRH-GFP neurons of transgenic mice by single cell RT-PCR. Firing rate and burst frequency of GnRH-GFP neurons were lower in metestrous than proestrous mice. Ghrelin (40nM-4μM) administration resulted in a decreased firing rate and burst frequency of GnRH neurons in metestrous, but not in proestrous mice. Ghrelin also decreased the firing rate of GnRH neurons in males. The ghrelin-evoked alterations of the firing parameters were prevented by JMV2959, supporting the receptor-specific actions of ghrelin on GnRH neurons. In metestrous mice, ghrelin decreased the frequency of GABAergic mPSCs in GnRH neurons. Effects of ghrelin were abolished by the cannabinoid receptor type-1 (CB1) antagonist AM251 (1µM) and the intracellularly applied DAG-lipase inhibitor THL (10µM), indicating the involvement of retrograde endocannabinoid signaling. These findings demonstrate that ghrelin exerts direct regulatory effects on GnRH neurons via GHS-R, and modulates the firing of GnRH neurons in an ovarian-cycle and endocannabinoid dependent manner.

Ovariectomy and Subsequent Treatment with Estrogen Receptor Agonists Tune the Innate Immune System of the Hippocampus in Middle-Aged Female Rats

The innate immune system including microglia has a major contribution to maintenance of the physiological functions of the hippocampus by permanent monitoring of the neural milieu and elimination of tissue-damaging threats. The hippocampus is vulnerable to age-related changes ranging from gene expression to network connectivity. The risk of hippocampal deterioration increases with the decline of gonadal hormone supply. To explore the impact of hormone milieu on the function of the innate immune system in middle-aged female rats, we compared mRNA expression in the hippocampus after gonadal hormone withdrawal, with or without subsequent estrogen replacement using estradiol and isotype-selective estrogen receptor (ER) agonists. Targeted profiling assessed the status of the innate immune system (macrophage-associated receptors, complement, inhibitory neuronal ligands), local estradiol synthesis (P450 aromatase) and estrogen reception (ER). Results established upregulation of macrophage-associated (Cd45, Iba1, Cd68, Cd11b, Cd18, Fcgr1a, Fcgr2b) and complement (C3, factor B, properdin) genes in response to ovariectomy. Ovariectomy upregulated Cd22 and downregulated semaphorin3A (Sema3a) expression, indicating altered neuronal regulation of microglia. Ovariectomy also led to downregulation of aromatase and upregulation of ERα gene. Of note, analogous changes were observed in the hippocampus of postmenopausal women. In ovariectomized rats, estradiol replacement attenuated Iba1, Cd11b, Fcgr1a, C3, increased mannose receptor Mrc1, Cd163 and reversed Sema3a expression. In contrast, reduced expression of aromatase was not reversed by estradiol. While the effects of ERα agonist closely resembled those of estradiol, ERβ agonist was also capable of attenuating the expression of several macrophage-associated and complement genes. These data together indicate that the innate immune system of the aging hippocampus is highly responsive to the gonadal hormone milieu. In ovariectomized female rats, estradiol replacement exerts potent immunomodulatory effects including attenuation of microglia sensitization, initiation of M2-like activation and modulation of complement expression by targeting hippocampal neurons and glial cells through ERα and ERβ.

Expression of hemolytically active human complement component C1r proenzyme in insect cells using a baculovirus vector

The gene of human C1r has been expressed in a baculovirus-insect-cell system via the pAc373 transplacement vector. The full-length cDNA copy was inserted into the pAc373 vector downstream from the strong polyhedrin promoter of the baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells were cotransfected with the resultant plasmid, pAcC1r, and the wild-type AcNPV DNA. Recombinant viruses, which drove the expression of C1r protein, were selected by plaque morphology and ELISA. Insect cells infected with the recombinant virus produced and secreted human C1r protein, at a level of 1-2 mg/l of medium. The expressed C1r was isolated from the medium by chromatofocusing. On reducing gels only a single Coomassie-staining band was observed, and this band migrated at 80-83 kD characteristic of the unactivated C1r proenzyme. Its identification as C1r was immunologically confirmed on Western blots. C1 reconstituted from purified C1r expressed in insect cells together with human C1q and C1s proved biologically active in a hemolytic assay. Thus, the baculovirus-insect-cell system is capable of expressing and secreting a sophisticated, multifunctional human complement subcomponent in its biologically activatable form.

Estradiol and isotype-selective estrogen receptor agonists modulate the mesocortical dopaminergic system in gonadectomized female rats

The mesocortical dopaminergic pathway projecting from the ventral tegmental area (VTA) to the prefrontal cortex (PFC) contributes to the processing of reward signals. This pathway is regulated by gonadal steroids including estradiol. To address the putative role of estradiol and isotype-selective estrogen receptor (ER) agonists in the regulation of the rodent mesocortical system, we combined fMRI, HPLC-MS and qRT-PCR techniques. In fMRI experiments adult, chronically ovariectomized rats, treated with either vehicle, estradiol, ERα agonist 16α-lactone-estradiol (LE2) or ERβ agonist diarylpropionitrile (DPN), received a single dose of d-amphetamine-sulphate (10mg/kg, i.p.) and BOLD responses were monitored in the VTA and the PFC. Ovariectomized rats showed no significant response to amphetamine. In contrast, the VTA of ER agonist-substituted ovariectomized rats showed robust amphetamine-evoked BOLD increases. The PFC of estradiol-replaced animals was also responsive to amphetamine. Mass spectroscopic analysis of dopamine and its metabolites revealed a two-fold increase in both dopamine and 3,4-dihydroxyphenylacetic acid content of the PFC in estradiol-replaced animals compared to ovariectomized controls. qRT-PCR studies revealed upregulation of dopamine transporter and dopamine receptor in the VTA and PFC, respectively, of ER agonist-treated ovariectomized animals. Collectively, the results indicate that E2 and isotype-selective ER agonists can powerfully modulate the responsiveness of the mesocortical dopaminergic system, increase the expression of key genes related to dopaminergic neurotransmission and augment the dopamine content of the PFC. In a broader sense, the findings support the concept that the manifestation of reward signals in the PFC is dependent on the actual estrogen milieu of the brain.

Hippocampal Gene Expression Is Highly Responsive to Estradiol Replacement in Middle-Aged Female Rats.

In the hippocampus, estrogens are powerful modulators of neurotransmission, synaptic plasticity and neurogenesis. In women, menopause is associated with increased risk of memory disturbances, which can be attenuated by timely estrogen therapy. In animal models of menopause, 17β-estradiol (E2) replacement improves hippocampus-dependent spatial memory. Here, we explored the effect of E2 replacement on hippocampal gene expression in a rat menopause model. Middle-aged ovariectomized female rats were treated continuously for 29 days with E2, and then, the hippocampal transcriptome was investigated with Affymetrix expression arrays. Microarray data were analyzed by Bioconductor packages and web-based softwares, and verified with quantitative PCR. At standard fold change selection criterion, 156 genes responded to E2. All alterations but 4 were transcriptional activation. Robust activation (fold change > 10) occurred in the case of transthyretin, klotho, claudin 2, prolactin receptor, ectodin, coagulation factor V, Igf2, Igfbp2, and sodium/sulfate symporter. Classification of the 156 genes revealed major groups, including signaling (35 genes), metabolism (31 genes), extracellular matrix (17 genes), and transcription (16 genes). We selected 33 genes for further studies, and all changes were confirmed by real-time PCR. The results suggest that E2 promotes retinoid, growth factor, homeoprotein, neurohormone, and neurotransmitter signaling, changes metabolism, extracellular matrix composition, and transcription, and induces protective mechanisms via genomic effects. We propose that these mechanisms contribute to effects of E2 on neurogenesis, neural plasticity, and memory functions. Our findings provide further support for the rationale to develop safe estrogen receptor ligands for the maintenance of cognitive performance in postmenopausal women.

Gene expression profiling identifies key estradiol targets in the frontal cortex of the rat

Estradiol modulates a wide range of neural functions in the frontal cerebral cortex where subsets of neurons express estrogen receptor-alpha and -beta. Through these receptors, estradiol contributes to the maintenance of normal operation of the frontal cortex. During the decline of gonadal hormones, the frequency of neurological and psychiatric disorders increases. To shed light on the etiology of disorders related to declining levels of estrogens, we studied the genomic responses to estradiol. Ovariectomized rats were treated with a sc injection of estradiol. Twenty-four hours later, samples from the frontal cortices were dissected, and their mRNA content was analyzed. One hundred thirty-six estradiol-regulated transcripts were identified on Rat 230 2.0 Expression Array. Of the 136 estrogen-regulated genes, 26 and 36 genes encoded proteins involved in the regulation of transcription and signal transduction, respectively. Thirteen genes were related to the calcium signaling pathway. They comprised five genes coding for neurotransmitter receptors. Transcription of three neuropeptides, including cocaine- and amphetamine-regulated transcript, were up-regulated. Fifty-two genes were selected for validation, and 12 transcriptional changes were confirmed. These results provided evidence that estradiol evokes broad transcriptional response in the cortex. Modulation of key components of the calcium signaling pathway, dopaminergic, serotonergic, and glutamatergic neurotransmission, may explain the influence of estrogens on cognitive function and behavior. Up-regulation of cocaine- and amphetamine-regulated transcript contributes to the neuroprotective effects of estradiol. Identification of estradiol-regulated genes in the frontal cortex helps to understand the pathomechanism of neurological and psychiatric disorders associated with altered levels of estrogens.

Ecdysteroids increase the yield of recombinant protein produced in baculovirus insect cell expression system

Spodoptera frugiperda cells are the hosts of wild type and recombinant virus in the baculovirus insect cell expression system. The expression of the foreign gene could be enhanced by the addition of ecdysteroids and increased amount of recombinant protein was secreted into the medium. The time and concentration dependence of this effect was followed in the case of 20-hydroxyecdysone-, makisterone and ecdysone. 20-hydroxyecdysone proved to be the most efficient, producing a three fold increase in the level of recombinant protein secreted into the medium, as it was measured by ELISA. This effect was also confirmed by tracing the L-(35S)methionine incorporation into the gene product. Makisterone was also effective in stimulation, while ecdysone proved to be ineffective.

FUNCTIONAL EFFECTS OF DOMAIN DELETIONS IN A MULTIDOMAIN SERINE PROTEASE, C1R

The C1r subcomponent of the first component of complement is a complex, multidomain glycoprotein containing five regulatory or binding modules in addition to the serine protease domain. To reveal the functional role of the N-terminal regulatory domains, two deletion mutants of C1r were constructed. One mutant comprises the N-terminal half of domain I joined to the second half of the highly homologous domain III, resulting in one chimeric domain in the N-terminal region, instead of domains I-III. In the second mutant most of the N-terminal portion of domain I was deleted. Both deletion mutants were expressed in the baculovirus-insect cell expression system with yields typical of wild type C1r. Both mutants maintained the ability of the wild type C1r to dimerize. The folding and secretion of the recombinant proteins was not affected by these deletions, and C1-inhibitor binding was not impaired. The stability of the zymogen was significantly decreased however, indicating that the N-terminal region of the C1r molecule contains essential elements involved in the control of activation of the serine protease module. Tetramer formation with C1s in the presence of Ca2+ was abolished by both deletions. We suggest that the first domain of C1r is essential for tetramer formation, since the deletion of domain I from C1r impairs this interaction.