Epigenetic impact of the social and physical environment on brain and body.

Modern biomedical scientists are often trapped in silos of knowledge and practice, such as those who study brain structure, function and behavior, on the one hand, and body systems and disorders, on the other. Scientists and physicians in each of those silos have not often paid attention to the brain-body communication that leads to multi-morbidity of systemic and brain-related disorders [eg. depression with diabetes or cardiovascular disease]. Outside of biomedicine, social scientists have long recognized the impact of the social and physical environment on individuals and populations but have not usually connected these effects with changes in underlying biology. However, with the rise of epigenetics, science and the public understanding of science is leaving an era in which the DNA sequence was thought to be "destiny" and entering an era where the environment shapes the biology and behavior of individuals and groups through its interactive effects on brain and body. It does so, at least in part, by shaping epigenetically the structure and function of brain and body systems that show a considerable amount of adaptive plasticity throughout development and adult life. This results in substantial individual differences even between identical twins. These individual differences are produced epigenetically by the two-way interaction between the brain and hormones, immune system mediators and the autonomic nervous system. Disorders, then, are often multimorbid involving both brain and body, such as depression with diabetes and cardiovascular disease. It is therefore imperative to incorporate into "precision medicine" a better understanding of how these differences affect the efficacy of pharmacological, behavioral and psychosocial interventions. This article presents an overview of this new synthesis, using as an example emerging evidence about the linkages between systemic inflammation, insulin resistance and mental health and neurodegenerative diseases.

A New Chapter in Genetic Medicine: RNA Editing and its Role in Disease Pathogenesis.

The transfer of genomic information from DNA to mRNA to protein usually occurs with high fidelity, but can also be subverted by a programmed RNA sequence alteration termed 'RNA editing', involving deamination of adenosine to inosine (decoded as guanosine), or of cytosine to uracil. These sequence changes can lead to cellular heterogeneity by generating variable sets of transcripts within otherwise identical cells. Recent studies have demonstrated that editing is most prevalent in cells and tissues with high propensity for plasticity. Within those, RNA editing reproducibly targets transcripts of related function, altering the outcomes of entire pathways at once. In ongoing work, changes in patterns of editing have been correlated with neuronal disease pathogenesis, suggesting that RNA editing harbors diagnostic potential.

Epitranscriptomic profiling across cell types reveals associations between APOBEC1-mediated RNA editing, gene expression outcomes, and cellular function.

Epitranscriptomics refers to posttranscriptional alterations on an mRNA sequence that are dynamic and reproducible, and affect gene expression in a similar way to epigenetic modifications. However, the functional relevance of those modifications for the transcript, the cell, and the organism remain poorly understood. Here, we focus on RNA editing and show that Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-1 (APOBEC1), together with its cofactor RBM47, mediates robust editing in different tissues. The majority of editing events alter the sequence of the 3'UTR of targeted transcripts, and we focus on one cell type (monocytes) and on a small set of highly edited transcripts within it to show that editing alters gene expression by modulating translation (but not RNA stability or localization). We further show that specific cellular processes (phagocytosis and transendothelial migration) are enriched for transcripts that are targets of editing and that editing alters their function. Finally, we survey bone marrow progenitors and demonstrate that common monocyte progenitor cells express high levels of APOBEC1 and are susceptible to loss of the editing enzyme. Overall, APOBEC1-mediated transcriptome diversification is required for the fine-tuning of protein expression in monocytes, suggesting an epitranscriptomic mechanism for the proper maintenance of homeostasis in innate immune cells.

Loss of APOBEC1 RNA-editing function in microglia exacerbates age-related CNS pathophysiology.

Microglia (MG), a heterogeneous population of phagocytic cells, play important roles in central nervous system (CNS) homeostasis and neural plasticity. Under steady-state conditions, MG maintain homeostasis by producing antiinflammatory cytokines and neurotrophic factors, support myelin production, and remove synapses and cellular debris, as well as participating in "cross-correction," a process that supplies neurons with key factors for executing autophagy-lysosomal function. As sentinels for the immune system, MG also detect "danger" signals (pathogenic or traumatic insult), become activated, produce proinflammatory cytokines, and recruit monocytes and dendritic cells to the site of damage through a breached blood-brain barrier or via brain lymphatics. Failure to effectively resolve MG activation can be problematic and can lead to chronic inflammation, a condition proposed to underlie CNS pathophysiology in heritable brain disorders and age-related neurodegenerative and cognitive decline. Here, we show that APOBEC1-mediated RNA editing occurs within MG and is key to maintaining their resting status. Like bone marrow-derived macrophages, RNA editing in MG leads to overall changes in the abundance of edited proteins that coordinate the function of multiple cellular pathways. Conversely, mice lacking the APOBEC1 editing function in MG display evidence of dysregulation, with progressive age-related signs of neurodegeneration, characterized by clustering of activated MG, aberrant myelination, increased inflammation, and lysosomal anomalies that culminate in behavioral and motor deficiencies. Collectively, our study identifies posttranscriptional modification by RNA editing as a critical regulatory mechanism of vital cellular functions that maintain overall brain health.

Nuclear receptor REV-ERBα mediates circadian sensitivity to mortality in murine vesicular stomatitis virus-induced encephalitis.

Certain components and functions of the immune system, most notably cytokine production and immune cell migration, are under circadian regulation. Such regulation suggests that circadian rhythms may have an effect on disease onset, progression, and resolution. In the vesicular stomatitis virus (VSV)-induced encephalitis model, the replication, caudal penetration, and survivability of intranasally applied VSV depends on both innate and adaptive immune mechanisms. In the current study, we investigated the effect of circadian time of infection on the progression and outcome of VSV-induced encephalitis and demonstrated a significant decrease in the survival rate in mice infected at the start of the rest cycle, zeitgeber time 0 (ZT0). The lower survival rate in these mice was associated with higher levels of circulating chemokine (C-C motif) ligand 2 (CCL2), a greater number of peripherally derived immune cells accumulating in the olfactory bulb (OB), and increased production of proinflammatory cytokines, indicating an immune-mediated pathology. We also found that the acrophase of molecular circadian clock component REV-ERBα mRNA expression in the OB coincides with the start of the active cycle, ZT12, when VSV infection results in a more favorable outcome. This result led us to hypothesize that REV-ERBα may mediate the circadian effect on survival following VSV infection. Blocking REV-ERBα activity before VSV administration resulted in a significant increase in the expression of CCL2 and decreased survival in mice infected at the start of the active cycle. These data demonstrate that REV-ERBα-mediated inhibition of CCL2 expression during viral-induced encephalitis may have a protective effect.

Pituitary dendritic cells communicate immune pathogenic signals.

This study reveals the presence of dendritic cells (DCs) in the pituitary gland, which play a role in communicating immune activation to the hypothalamic pituitary adrenal (HPA) axis. Using enhanced yellow fluorescent protein (eyfp) expression as a reporter for CD11c, a marker of DCs, we demonstrate anatomically the presence of CD11c/eyfp+ cells throughout the pituitary. Flow cytometric analysis shows that the predominant cellular phenotype of pituitary CD11c/eyfp+ cells resembles that of non-lymphoid DCs. In vivo and in vitro immune challenge with lipopolysaccharide (LPS) stimulates these pituitary CD11c/eyfp+ DCs, but not eyfp(neg) cells, to increase levels of pro-inflammatory cytokines, IL-6, IL-1ß, and TNF-α. In vivo analysis of plasma glucocorticoid (GC) and adrenocorticotropic hormone (ACTH) levels at this early phase of the immune response to LPS suggest that pro-inflammatory cytokine production by DCs within the pituitary may activate the release of GCs from the adrenals via ACTH. Pituitary CD11c/eyfp+ cells also express annexin A1 (ANXA1), indicating a role in GC signal attenuation. In summary, our data demonstrate that a resident DC population of the pituitary gland coordinates GC release in the early phase of systemic immune activation, thereby providing an essential immune signaling sentinel for the initial shaping of the systemic immune response to LPS.

Epigenetic Modulators of Monocytic Function: Implication for Steady State and Disease in the CNS.

Epigenetic alterations are necessary for the establishment of functional and phenotypic diversity in the populations of immune cells of the monocytic lineage. The epigenetic status of individual genes at different time points defines their transcriptional responses throughout development and in response to environmental stimuli. Epigenetic states are defined at the level of DNA modifications, chromatin modifications, as well as at the level of RNA base changes through RNA editing. Drawing from lessons regarding the epigenome and epitranscriptome of cells of the monocytic lineage in the periphery, and from recently published RNAseq data deriving from brain-resident monocytes, we discuss the impact of modulation of these epigenetic states and how they affect processes important for the development of a healthy brain, as well as mechanisms of neurodegenerative disease and aging. An understanding of the varied brain responses and pathologies in light of these novel gene regulatory systems in monocytes will lead to important new insights in the understanding of the aging process and the treatment and diagnosis of neurodegenerative disease.

Adverse consequences of glucocorticoid medication: psychological, cognitive, and behavioral effects.

Glucocorticoids are the most commonly prescribed anti-inflammatory/immunosuppressant medications worldwide. This article highlights the risk of clinically significant and sometimes severe psychological, cognitive, and behavioral disturbances that may be associated with glucocorticoid use, as well as ways to prevent and treat these disturbances. An illustrative case vignette is presented describing a patient's experience of cycles of manic-like behavior and depression while on high-dosage prednisone, with long-term cognitive disorganization, vulnerability to stress, and personality changes. Severe neuropsychiatric consequences (including suicide, suicide attempt, psychosis, mania, depression, panic disorder, and delirium, confusion, or disorientation) have been reported to occur in 15.7 per 100 person-years at risk for all glucocorticoid courses, and 22.2 per 100 person-years at risk for first courses. The majority of patients experience less severe but distressing and possibly persistent changes in mood, cognition, memory, or behavior during glucocorticoid treatment or withdrawal. Although prediction of such effects is difficult, risks vary with age, gender, dosage, prior psychiatric history, and several biological markers. Key mechanisms thought to underlie these risk factors are briefly described. Recommendations are given for identifying individual risk factors and for monitoring and managing adverse neuropsychiatric effects of glucocorticoids.

7α-hydroxylation of dehydroepiandrosterone does not interfere with the activation of glucocorticoids by 11ß-hydroxysteroid dehydrogenase in E(t)C cerebellar neurons.

The neuroprotective action of dehydroepiandrosterone (DHEA) in the absence of a known specific receptor has been attributed to its metabolism by different cell types in the brain to various steroids, with a preference to its 7-hydroxylated products. The E(t)C cerebellar granule cell line converts DHEA almost exclusively to 7α-hydroxy-DHEA (7α-OH-DHEA). It has been postulated that DHEA's 7-OH and 7-oxo metabolites can decrease glucocorticoid levels by an interactive mechanism involving 11ß-hydroxysteroid dehydrogenase (11ß-HSD). In order to study the relationship of 7-hydroxylation of DHEA and glucocorticoid metabolism in intact brain cells, we examined whether E(t)C cerebellar neurons, which are avid producers of 7α-OH-DHEA, could also metabolize glucocorticoids. We report that E(t)C neuronal cells exhibit 11ß-HSD1 reductase activity, and are able to convert 11-dehydrocorticosterone into corticosterone, whereas they do not demonstrate 11ß-HSD2 dehydrogenase activity. Consequently, E(t)C cells incubated with DHEA did not yield 7-oxo- or 7ß-OH-DHEA. Our findings are supported by the reductive environment of E(t)C cells through expression of hexose-6-phosphate dehydrogenase (H6PDH), which fosters 11ß-HSD1 reductase activity. To further explore the role of 7α-OH-DHEA in E(t)C neuronal cells, we examined the effect of preventing its formation using the CYP450 inhibitor ketoconazole. Treatment of the cells with this drug decreased the yield of 7α-OH-DHEA by about 75% without the formation of alternate DHEA metabolites, and had minimal effects on glucocorticoid conversion. Likewise, elevated levels of corticosterone, the product of 11ß-HSD1, had no effect on the metabolic profile of DHEA. This study shows that in a single population of whole-cells, with a highly reductive environment, 7α-OH-DHEA is unable to block the reducing activity of 11ß-HSD1, and that 7-hydroxylation of DHEA does not interfere with the activation of glucocorticoids. Our investigation on the metabolism of DHEA in E(t)C neuronal cells suggest that other alternate mechanisms must be at play to explain the in vivo anti-glucocorticoid properties of DHEA and its 7-OH-metabolites.

Brain dendritic cells: biology and pathology.

Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. In their quiescent and mature form, the presentation of self-antigens by DC leads to tolerance; whereas, antigen presentation by mature DC, after stimulation by pathogen-associated molecular patterns, leads to the onset of antigen-specific immunity. DC have been found in many of the major organs in mammals (e.g. skin, heart, lungs, intestines and spleen); while the brain has long been considered devoid of DC in the absence of neuroinflammation. Consequently, microglia, the resident immune cell of the brain, have been charged with many functional attributes commonly ascribed to DC. Recent evidence has challenged the notion that DC are either absent or minimal players in brain immune surveillance. This review will discuss the recent literature examining DC involvement within both the young and aged steady-state brain. We will also examine DC contributions during various forms of neuroinflammation resulting from neurodegenerative autoimmune disease, injury, and CNS infections. This review also touches upon DC trafficking between the central nervous system and peripheral immune compartments during viral infections, the new molecular technologies that could be employed to enhance our current understanding of brain DC ontogeny, and some potential therapeutic uses of DC within the CNS.

Viral-induced encephalitis initiates distinct and functional CD103+ CD11b+ brain dendritic cell populations within the olfactory bulb.

Dendritic cells (DC) are antigen-presenting cells found in both lymphoid and nonlymphoid organs, including the brain (bDC) of Cd11c/eyfp transgenic C57BL/6 mice. Using an intranasal vesicular stomatitis virus infection, we demonstrated that EYFP(+) cells amass in areas associated with viral antigens, take on an activated morphology, and project their processes into infected neuronal tissue within the olfactory bulb. These bDC separated into three EYFP(+) CD45(+) CD11b(+) populations, all but one being able to functionally promote both T lymphocyte proliferation and T(H)1 cytokine production. One population was shown to emanate from the brain and a second population was peripherally derived. The third population was of indeterminate origin, being both radiosensitive and not replenished by donor bone marrow. Finally, each EYFP(+) population contained CD11b(+) CD103(+) subpopulations and could be distinguished in terms of CD115, Gr-1, and Ly-6C expression, highlighting mucosal and monocyte-derived DC lineages.

Accumulation of resident and peripheral dendritic cells in the aging CNS.

Dendritic cells (DC) are specialized antigen-presenting cells, responsible for peripheral immune responses. Recently, resident brain dendritic cells (bDC) were identified and functionally characterized in the young adult Itgax (CD11c) EYFP+ transgenic mouse brain. In the present study, we describe changes in number, phenotype, and source of bDC in the aging mouse brain. Immunohistochemistry and fluorescent activated cell sorting (FACS) analysis revealed an age-related increase in bDC with a concomitant rise in the expression of immune activation markers MHCII, CD80, and CD86. Quantification of immunolabeled bDC in the cortex, corpus callosum, and cerebellum of the aged brain revealed a 2- to 5-fold increase. In contrast, either no change or a decrease in bDC was noted in regions of adult neurogenesis. Chimeras (wild type host/EYFP+ bone marrow) suggest that the increase of EYFP+ cells in the aging brain is in part due to an accumulation of peripherally derived cells. Collectively, the numerical and phenotypic changes in bDC indicate these cells may serve as an important immune component in the functional and anatomic alterations associated with aging.

Microglia express functional 11 beta-hydroxysteroid dehydrogenase type 1.

Glucocorticoids are potent regulators of inflammation exerting permissive, stimulatory, and suppressive effects. Glucocorticoid access to intracellular receptors is regulated by the activity of two distinct enzymes known as 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) Type 1 and Type 2, which catalyze the activation or deactivation of glucocorticoids. Although expression of these enzymes in major organ systems and their roles in the metabolic effects of glucocorticoids have been described, their role in the inflammatory response has only recently started to be addressed. In this report, we have studied the expression and activity of 11 beta HSD Type 1 and Type 2 in microglia cells. Microglia, the brain's resident macrophages, initiate and orchestrate CNS inflammatory responses. Importantly, activated microglia are implicated in most neurodegenerative conditions, making them key subjects of study. We found that microglia expressed 11 beta HSD-1, but not 11 beta HSD-2, both in ex vivo FACS-sorted adult cells and in vitro primary cultures. 11 beta HSD-1 expression was increased in LPS-activated microglia. Moreover, 11 beta HSD-1 catalyzed the metabolic conversion of 11-dehydro-corticosterone into corticosterone (CORT), which potently reduced cytokine production in activated microglia. We propose that 11 beta HSD-1 may provide microglia with an intrinsic mechanism to autoregulate and inhibit proinflammatory mediator production through CORT formation.

Brain dendritic cells in ischemic stroke: time course, activation state, and origin.

The immune response to stroke is comprised of inflammatory and regulatory processes. One cell type involved in both innate and adaptive immunity is the dendritic cell (DC). A DC population residing in the healthy brain (bDC) was identified using a transgenic mouse expressing enhanced yellow fluorescent protein (EYFP) under the promoter for the DC marker, CD11c (CD11c/EYFP Tg). To determine if bDC are involved in the immune response to cerebral ischemia, transient (40 min) middle cerebral artery occlusion (MCAO) followed by 6, 24, or 72 h reperfusion was conducted in CD11c/EYFP Tg mice. Our results demonstrated that DC accumulated in the ischemic hemisphere at 24 h post-MCAO-reperfusion, particularly in the border region of the infarct where T lymphocytes accrued. To distinguish resident bDC from the infiltrating peripheral DC, radiation chimeras [1. wild type (WT) hosts restored with CD11c/EYFP Tg bone marrow (BM) or 2. CD11c/EYFP Tg hosts restored with WT BM] were generated and examined by immunocytochemistry. These data confirmed that DC populating the core of the infarct at 72 h were of peripheral origin, whereas those in the border region were comprised primarily of resident bDC. The brain resident (CD45 intermediate) cells of CD11c/EYFP Tg mice were analyzed by flow cytometry. Compared to microglia, bDC displayed increased major histocompatibility class II (MHC II) and co-stimulatory molecules following MCAO-reperfusion. High levels of MHC II and the co-stimulatory molecule CD80 on bDC at 72 h corresponded to peak lymphocyte infiltration, and suggested a functional interaction between these two immune cell populations.

Acute in vivo exposure to interferon-gamma enables resident brain dendritic cells to become effective antigen presenting cells.

Dendritic cells (DC) are the professional antigen presenting cells (APC) that bridge the innate and adaptive immune system. Previously, in a CD11c/EYFP transgenic mouse developed to study DC functions, we anatomically mapped and phenotypically characterized a discrete population of EYFP(+) cells within the microglia that we termed brain dendritic cells (bDC). In this study, we advanced our knowledge of the function of these cells in the CD11c/EYFP transgenic mouse and its chimeras, using acute stimuli of stereotaxically inoculated IFNgamma or IL-4 into the CNS. The administration of IFNgamma increased the number of EYFP(+)bDC but did not recruit peripheral DC into the CNS. IFNgamma, but not IL-4, upregulated the expression levels of major histocompatibility class II (MHC-II). In addition, IFNgamma-activated EYFP(+)bDC induced antigen-specific naïve CD4 T cells to proliferate and secrete Th1/Th17 cytokines. Activated bDC were also able to stimulate naïve CD8 T cells. Collectively, these data reveal the Th1 cytokine IFNgamma, but not the Th2 cytokine IL4, induces bDC to up-regulate MHC-II and become competent APC.

CD11c/EYFP transgene illuminates a discrete network of dendritic cells within the embryonic, neonatal, adult, and injured mouse brain.

The CD11c enhanced yellow fluorescent protein (EYFP) transgenic mouse was constructed to identify dendritic cells in the periphery (Lindquist et al. [2004] Nat. Immunol. 5:1243-1250). In this study, we used this mouse to characterize dendritic cells within the CNS. Our anatomic results showed discrete populations of EYFP(+) brain dendritic cells (EYFP(+) bDC) that colocalized with a small fraction of microglia immunoreactive for Mac-1, Iba-1, CD45, and F4/80 but not for NeuN, Dcx, NG2 proteoglycan, or GFAP. EYFP(+) bDC, isolated by fluorescent activated cell sorting (FACS), expressed mRNA for the Itgax (CD11c) gene, whereas FACS anlaysis of EYFP(+) bDC cultures revealed the presence of CD11c protein. Light microscopy studies revealed that EYFP(+) bDC were present in the embryonic CNS when the blood-brain barrier is formed and postnatally when brain cells are amenable to culturing. In adult male mice, EYFP(+) bDC distribution was prominent within regions of the CNS that 1) are subject to structural plasticity and neurogenesis, 2) receive sensory and humoral input from the external environment, and 3) lack a blood-brain barrier. Ultrastructural analysis of EYFP(+) bDC in adult neurogenic niches showed their proximity to developing neurons and a morphology characteristic of immune/microglia cells. Kainic acid-induced seizures revealed that EYFP(+) bDC responded to damage of the hippocampus and displayed morphologies similar to those described for seizure-activated EGFP(+) microglia in the hippocampus of cfms (CSF-1R) EGFP mice. Collectively, these findings suggest a new member of the dendritic cell family residing among the heterogeneous microglia population.

Brain microglia express steroid-converting enzymes in the mouse.

In the CNS, steroid hormones play a major role in the maintenance of brain homeostasis and it's response to injury. Since activated microglia are the pivotal immune cell involved in neurodegeneration, we investigated the possibility that microglia provide a discrete source for the metabolism of active steroid hormones. Using RT-PCR, our results showed that mouse microglia expressed mRNA for 17beta-hydroxysteroid dehydrogenase type 1 and steroid 5alpha-reductase type 1, which are involved in the metabolism of androgens and estrogens. Microglia also expressed the peripheral benzodiazepine receptor and steroid acute regulatory protein; however, the enzymes required for de novo formation of progesterone and DHEA from cholesterol were not expressed. To test the function of these enzymes, primary microglia cultures were incubated with steroid precursors, DHEA and AD. Microglia preferentially produced delta-5 androgens (Adiol) from DHEA and 5alpha-reduced androgens from AD. Adiol behaved as an effective estrogen receptor agonist in neuronal cells. Activation of microglia with pro-inflammatory factors, LPS and INFgamma did not affect the enzymatic properties of these proteins. However, PBR ligands reduced TNFalpha production signifying an immunomodulatory role for PBR. Collectively, our results suggest that microglia utilize steroid-converting enzymes and related proteins to influence inflammation and neurodegeneration within microenvironments of the brain.

Steroid hormone receptor expression and function in microglia.

Steroid hormones such as glucocorticoids and estrogens are well-known regulators of peripheral immune responses and also show anti-inflammatory properties in the brain. However, the expression of steroid hormone receptors in microglia, the pivotal immune cell that coordinates the brain inflammatory response, is still controversial. Here we use real time RT-PCR to show that microglia, isolated from adult fms-EGFP mice by FACS, express glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and estrogen receptor alpha (ERalpha). GR was the most abundant steroid hormone receptor transcript in microglia. The presence of GR and ERalpha immunoreactivity was further confirmed in vivo at the ultrastructural level. To understand the role of steroid hormone receptors during the inflammation process, we evaluated the expression of steroid hormone receptors after inflammatory challenge and found a significant down-regulation of GR, MR, and ERalpha in microglia. Finally, we tested the immunomodulatory properties of estrogens and glucocorticoids. Estradiol benzoate did not have any significant impact on the inflammatory profile of ex vivo sorted microglia, either in resting conditions or after challenge. Furthermore, corticosterone was a more consistent anti-inflammatory agent than 17beta-estradiol in vitro. Our results support the hypothesis that adult microglia are a direct target of steroid hormones and that glucocorticoids, through the predominant expression of GR and MR, are the primary steroid hormone regulators of microglial inflammatory activity. The down-regulation of steroid hormone receptors after LPS challenge may serve as a prerequisite to suppressing the anti-inflammatory actions of endogenous steroid hormones on the immune system, and contribute to a sustained activation of microglia.

Transcriptional activity of estrogen receptors ERalpha and ERbeta in the EtC.1 cerebellar granule cell line.

Estrogen receptors alpha and beta (ERalpha and ERbeta) are expressed in the cerebellum throughout development and in the adult suggesting an important role of 17-beta-estradiol (E2) in this brain structure. In the present study, we have characterized the functionality of estrogen receptors (ERs) expressed in the immature cerebellar granule cell line E(t)C.1 by transfecting such cells with a luciferase reporter gene (ERE-Luc) coupled to an estrogen response element promoter. The induction of luciferase activity in E(t)C.1 cells by E2 and ER-subtype selective agonists was compared in normal cells and in cells overexpressing human ERalpha or ERbeta (hERalpha or hERbeta). E2-mediated transcription of the reporter gene was blocked by the ER antagonist ICI 182,780 (ICI), demonstrating the presence of functional native ERs. The selective agonist for ERalpha (PPT) showed a reduced response in luciferase induction compared to E2. Moreover, the ERbeta agonist (DPN) was unable to induce luciferase activity. E2-induced ERE-Luc transcription was not increased by overexpression of hERalpha. In contrast, hERbeta overexpression reduced the efficacy of E2 and abolished ERalpha-selective agonist activity. The ERbeta-specific agonist did not induce gene reporter activity unless hERbeta was overexpressed in the cells, suggesting that the endogenous ERbeta in E(t)C.1 cells is transcriptionally inactive. ICI inhibition of E2 responses was not affected by overexpression of the human ERs. The data suggest that ERalpha plays a predominant role in E2-mediated transcription in E(t)C.1 cells. Our data are discussed in view of other reports alluding to the complexity and cell-type specificity of E2-mediated transcription.

Characterization of a cerebellar granule progenitor cell line, EtC.1, and its responsiveness to 17-beta-estradiol.

Mouse cerebellar development occurs at late embryonic stages and through the first few weeks of postnatal life. Hormones such as 17-beta-estradiol (E2) have been implicated in cerebellar development, through the expression of E2 receptors (ER). However, the role of E2 in the development and function of cerebellar neurons has yet to be fully elucidated. To gain insight into E2's actions on the developing cerebellum, we characterized a cloned neuronal cell line, E(t)C.1, derived from late embryonic cerebellum for its neural properties and responsiveness to E2. Our results revealed that E(t)C.1 cells express markers characteristic of neural progenitor cells such as Nestin, Musashi, and Doublecortin (DCX), and of the granule cell lineage such as Math1 and Zipro1. The ER alpha and beta (ERalpha and ERbeta) were also identified in this cell line. Functionality of ERs was verified using an Estrogen Response Element (ERE)-Luciferase reporter plasmid. E2 modulated ERalpha, FMRP, and IL-6, which were expressed in these cells. However, E2 did not induce changes in neural proteins nor induce maturation of E(t)C.1 cells. CREB and ERK(1/2) protein kinases were not modulated by E2 either. Interestingly, E(t)C.1 expressed active p450 Aromatase (P450arom), which was confirmed by the aromatization of androstenedione (AD) to E2 and other estrogen metabolites. Collectively, our results show that the E(t)C.1 cell line may serve as a model to study early development of cerebellar progenitor granule cells, and their responsiveness to E2.