Search

Previously, we found that in dissociated hippocampal cultures the proportion of large spines (head diameter ≥ 0.6 μm) was larger in cultures from female than from male animals. In order to rule out that this result is an in vitro phenomenon, we analyzed the density of large spines in fixed hippocampal vibratome sections of Thy1-GFP mice, in which GFP is expressed only in subpopulations of neurons. We compared spine numbers of the four estrus cycle stages in females with those of male mice. Remarkably, total spine numbers did not vary during the estrus cycle, while estrus cyclicity was evident regarding the number of large spines and was highest during diestrus, when estradiol levels start to rise. The average total spine number in females was identical with the spine number in male animals. The density of large spines, however, was significantly lower in male than in female animals in each stage of the estrus cycle. Interestingly, the number of spine apparatuses, a typical feature of large spines, did not differ between the sexes. Accordingly, NMDA-R1 and NMDA-R2A/B expression were lower in the hippocampus and in postsynaptic density fractions of adult male animals than in those of female animals. This difference could already be observed at birth for NMDA-R1, but not for NMDA-R2A/B expression. In dissociated embryonic hippocampal cultures, no difference was seen after 21 days in culture, while the difference was evident in postnatal cultures. Our data indicate that hippocampal neurons are differentiated in a sex-dependent manner, this differentiation being likely to develop during the perinatal period.

Brain : a journal of neurology 145(9), 3131–3146 (2022). doi:10.1093/brain/awac122, Published by Oxford Univ. Press, Oxford

Numerous studies provide increasing evidence, which supports the ideas that every cell in the brain of males may differ from those in females due to differences in sex chromosome complement as well as in response to hormonal effects. In this study, we address the question as to whether actions of neurosteroids, thus steroids, which are synthesized and function within the brain, contribute to sex-specific hippocampal synaptic plasticity. We have previously shown that predominantly in the female hippocampus, does inhibition of the conversion of testosterone to estradiol affect synaptic transmission. In this study, we show that testosterone and its metabolite dihydrotestosterone are essential for hippocampal synaptic transmission specifically in males. This also holds true for the density of mushroom spines and of spine synapses. We obtained similar sex-dependent results using primary hippocampal cultures of male and female animals. Since these cultures originated from perinatal animals, our findings argue for sex-dependent differentiation of hippocampal neurons regarding their responsiveness to sex neurosteroids up to birth, which persist during adulthood. Hence, our in vitro findings may point to a developmental effect either directly induced by sex chromosomes or indirectly by fetal testosterone secretion during the perinatal critical period, when developmental sexual priming takes place.

Simple Summary In cancer patients, adoptive T cell transfer shall increase the number of circulating cytotoxic T cells to foster anti-tumor immune responses. In solid tumors, however, lack of lymphocyte infiltration into the tumor impairs treatment efficacy due to the immune-suppressive tumor microenvironment. To make cells controllable by external forces, we loaded primary human T cells with citrate-coated superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs were tightly attached to the plasma membrane and also taken up intracellularly into vesicles. With their nanoparticle cargo, we were able to magnetically accumulate them, which is a promising finding for future magnetic delivery of immune cells after adoptive transfer. Abstract T cell infiltration into a tumor is associated with a good clinical prognosis of the patient and adoptive T cell therapy can increase anti-tumor immune responses. However, immune cells are often excluded from tumor infiltration and can lack activation due to the immune-suppressive tumor microenvironment. To make T cells controllable by external forces, we loaded primary human CD3+ T cells with citrate-coated superparamagnetic iron oxide nanoparticles (SPIONs). Since the efficacy of magnetic targeting depends on the amount of SPION loading, we investigated how experimental conditions influence nanoparticle uptake and viability of cells. We found that loading in the presence of serum improved both the colloidal stability of SPIONs and viability of T cells, whereas stimulation with CD3/CD28/CD2 and IL-2 did not influence nanoparticle uptake. Furthermore, SPION loading did not impair cytokine secretion after polyclonal stimulation. We finally achieved 1.4 pg iron loading per cell, which was both located intracellularly in vesicles and bound to the plasma membrane. Importantly, nanoparticles did not spill over to non-loaded cells. Since SPION-loading enabled efficient magnetic accumulation of T cells in vitro under dynamic conditions, we conclude that this might be a good starting point for the investigation of in vivo delivery of immune cells.

Background About 3%–5% of adults with membranous nephropathy have autoantibodies directed against thrombospondin type 1 domain–containing 7A (THSD7A), a podocyte-expressed transmembrane protein. However, the temporal and spatial expression of THSD7A and its biologic function for podocytes are unknown, information that is needed to understand the effects of THSD7A autoantibodies in this disease. Methods Using a variety of microscopic techniques, we analyzed THSD7A localization in postnatal, adult, and autoantibody-injected mice as well as in human podocytes. We also analyzed THSD7A function in human podocytes using confocal microscopy; Western blotting; and adhesion and migration assays. Results We found that THSD7A expression begins on glomerular vascularization with slit diaphragm formation in development. THSD7A localizes to the basal aspect of foot processes, closely following the meanders of the slit diaphragm in human and mice. Autoantibodies binding to THSD7A localize to the slit diaphragm. In human podocytes, THSD7A expression is accentuated at filopodia and thin arborized protrusions, an expression pattern associated with decreased membrane activity of cytoskeletal regulators. We also found that, phenotypically, THSD7A expression in human podocytes is associated not only with increases in cell size, enhanced adhesion, and reduced detachment from collagen type IV–coated plates but also, with decreased ability to migrate. Conclusions Our findings suggest that THSD7A functions as a foot process protein involved in the stabilization of the slit diaphragm of mature podocytes and that autoantibodies to THSD7A, on the basis of their localization, might structurally and functionally alter the slit diaphragm’s permeability to protein.

In general, hippocampal neurons are capable of synthesizing sex steroids de novo from cholesterol, since the brain is equipped with all the enzymes required for the synthesis of estradiol and testosterone, the end products of sex steroidogenesis. Regarding estradiol, its synthesis in hippocampal neurons is homeostatically controlled by Ca2+ transients and is regulated by GnRH. Locally synthesized estradiol and testosterone maintain synaptic transmission and synaptic connectivity. Remarkably, the neurosteroid estradiol is effective in females, but not in males, and vice versa dihydrotestosterone (DHT) is effective in males, but not in females. Experimentally induced inhibition of estradiol synthesis in females and DHT synthesis in males resp. results in synapse loss, impaired LTP, and downregulation of synaptic proteins. GnRH-induced increase in estradiol synthesis appears to provide a link between the hypothalamus and the hippocampus, which may underlie estrous cyclicity of spine density in the female hippocampus. Hippocampal neurons are sex-dependently differentiated with respect to the responsiveness of hippocampal neurons to sex neurosteroids.

It was a widely held belief that sex steroids, namely testosterone and 17β-estradiol (E2) of gonadal origin, control synaptic plasticity in the hippocampus. A new paradigm emerged when it was shown that these sex steroids are synthesized in the hippocampus. The inhibition of sex steroids in the hippocampus impairs synaptic plasticity sex-dependently in this region of the brain. In gonadectomized animals and in hippocampal cultures, inhibition of estradiol synthesis in female animals and in cultures from female animals, and inhibition of dihydrotestosterone synthesis in male animals and in cultures of male animals, cause synapse loss and impair LTP in the hippocampus, but not vice versa. Since the hippocampal cultures originated from perinatal animals, and due to the similarity of in vivo and in vitro findings, it appears that hippocampal neurons are differentiated in a sex-specific manner during the perinatal period when sexual imprinting takes place.

Locally synthesized estradiol plays an important role in synaptic plasticity in the hippocampus. We have previously shown that in hippocampal neurons, activity of the enzyme aromatase, which converts testosterone into estradiol, is reduced via Ca2+ -dependent phosphorylation. Synaptopodin is a highly estrogen responsive protein, and it has been shown that it is an important regulator of synaptic plasticity, mediated by its close association with internal calcium stores. In this study, we show that the expression of synaptopodin is stronger in the hippocampus of female animals than in that of male animals. Phosphorylation of aromatase, using letrozole, however, down-regulates synaptopodin immunohistochemistry in the hippocampus of both male and females. Similarly, in aromatase knock-out mice synaptopodin expression in the hippocampus is reduced sex independently. Using primary-dissociated hippocampal neurons, we found that evoked release of Ca2+ from internal stores down-regulates aromatase activity, which is paralleled by reduced expression of synaptopodin. Opposite effects were achieved after inhibition of the release. Calcium-dependent regulation of synaptopodin expression was abolished when the control of aromatase activity by the Ca2+ transients was disrupted. Our data suggest that the regulation of aromatase activity by Ca2+ transients in neurons contributes to synaptic plasticity in the hippocampus of male and female animals as an on-site regulatory mechanism.

Across phyla, the ribosomes—the central molecular machines for translation of genetic information—exhibit an overall preserved architecture and a conserved functional core. The natural heterogeneity of the ribosome periodically phases a debate on their functional specialization and the tissue-specific variations of the ribosomal protein (RP) pool. Using sensitive differential proteomics, we performed a thorough quantitative inventory of the protein composition of ribosomes from 3 different mouse brain tissues, i.e., hippocampus, cortex, and cerebellum, across various ages, i.e., juvenile, adult, and middle-aged mouse groups. In all 3 brain tissues, in both monosomal and polysomal ribosome fractions, we detected an invariant set of 72 of 79 core RPs, RACK1 and 2 of the 8 RP paralogs, the stoichiometry of which remained constant across different ages. The amount of a few RPs punctually varied in either one tissue or one age group, but these fluctuations were within the tight bounds of the measurement noise. Further comparison with the ribosomes from a high-metabolic-rate organ, e.g., the liver, revealed protein composition identical to that of the ribosomes from the 3 brain tissues. Together, our data show an invariant protein composition of ribosomes from 4 tissues across different ages of mice and support the idea that functional heterogeneity may arise from factors other than simply ribosomal protein stoichiometry.

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, and one-third of patients develop end-stage renal disease (ESRD). Circulating autoantibodies against the podocyte surface antigens phospholipase A2 receptor 1 (PLA2R1) and the recently identified thrombospondin type 1 domain-containing 7A (THSD7A) are assumed to cause the disease in the majority of patients. The pathogenicity of these antibodies, however, has not been directly proven. Here, we have reported the analysis and characterization of a male patient with THSD7A-associated MN who progressed to ESRD and subsequently underwent renal transplantation. MN rapidly recurred after transplantation. Enhanced staining for THSD7A was observed in the kidney allograft, and detectable anti-THSD7A antibodies were present in the serum before and after transplantation, suggesting that these antibodies induced a recurrence of MN in the renal transplant. In contrast to PLA2R1, THSD7A was expressed on both human and murine podocytes, enabling the evaluation of whether anti-THSD7A antibodies cause MN in mice. We demonstrated that human anti-THSD7A antibodies specifically bind to murine THSD7A on podocyte foot processes, induce proteinuria, and initiate a histopathological pattern that is typical of MN. Furthermore, anti-THSD7A antibodies induced marked cytoskeletal rearrangement in primary murine glomerular epithelial cells as well as in human embryonic kidney 293 cells. Our findings support a causative role of anti-THSD7A antibodies in the development of MN.

Long-lasting synaptic plasticity is often accompanied by morphological changes as well as formation and/or loss of dendritic spines. Since the spine cytoskeleton mainly consists of actin filaments, morphological changes are primarily controlled by actin binding proteins (ABPs). Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) is a neuron-specific, actin bundling protein concentrated at dendritic spines. Here, we demonstrate that ITPKA depletion in mice increases the number of hippocampal spine-synapses while reducing average spine length. By employing actin to ABP ratios similar to those occurring at post synaptic densities, in addition to cross-linking actin filaments, ITPKA strongly inhibits Arp2/3-complex induced actin filament branching by displacing the complex from F-actin. In summary, our data show that in vivo ITPKA negatively regulates formation and/or maintenance of synaptic contacts in the mammalian brain. On the molecular level this effect appears to result from the ITPKA-mediated inhibition of Arp2/3-complex F-actin branching activity.

Sexual neurosteroids (SN), namely 17β-estradiol (E2) and 5α-dehydrotestosterone (DHT), are synthesized in the hippocampus, where they induce circuit modifications by changing the number of excitatory spine synapses in a paracrine and sex-specific manner. The mechanisms of this sex-specific synapse turnover, which are likely to affect cognitive functions, are poorly understood. We found that hippocampal neurons synthesize estradiol, which maintains LTP and synapses in females but not in males. In females, inhibition of estradiol synthesis results in impairment of LTP and synapse loss. These effects were not seen in males. The essential role of local estrogen on the stability and maintenance of connectivity in the hippocampus is consistent with age-related cognitive decline in women after menopause. In male animals the regulation of synaptic stability and plasticity by locally synthesized sexual steroids remains to be clarified. This article is part of a Special Issue entitled SI: Brain and Memory.

Reelin plays an important role in cerebral cortex development and synaptogenesis. In the hippocampus, the neurosteroid estrogen affects reelin expression. In this study we tested a potential crosstalk between estradiol and reelin, thus the possibility of a reelin-induced activation of the estradiol synthesizing enzyme aromatase. As a model system, we used ovaries, which express reelin and are a major source of estradiol. We found that in wild-type mice, reelin and aromatase are expressed in granulosa cells of growing follicles. The expression of reelin varies with the estrus cycle and is highest shortly before ovulation, when estradiol serum levels are at their maximum. In ovaries of reelin-deficient reeler mice, aromatase mRNA and protein are significantly reduced, as evidenced by real-time PCR, western blot analysis, and quantitative immunohistochemistry in granulosa cells of preovulatory follicles. In line with reduced estradiol synthesis, ovarian estrus cycle length is prolonged in reeler mice. Most importantly, treating cultured granulosa cells with recombinant reelin results in significant upregulation of aromatase mRNA and protein and increased secretion of estradiol into the supernatant. Our data provide evidence of a local increase of aromatase expression by reelin. Regarding reproduction, this crosstalk may contribute to follicular stability and counteract luteinization in ovaries.

In the hippocampus, synaptic proteins, as studied so far, have been shown to be upregulated by 17β-estradiol, while inhibition of local estradiol synthesis consistently downregulates them. As an exception to this rule, we have previously shown that synaptopodin, an actin-associated postsynaptic protein, is downregulated in response to estradiol in dissociated cultured hippocampal neurons. In this study, we show, unexpectedly, that synaptopodin is downregulated in the hippocampus of aromatase knock-out mice and that inhibition of neuronal estradiol synthesis using the aromatase inhibitor letrozole also downregulates synaptopodin in these cultures. Moreover, the effects of estradiol and letrozole are additive, suggesting a subtle balance between available ligand and receptor. Using selective estrogen receptor agonists and antagonists, we consequently studied the effects of estrogen receptor subtypes on synaptopodin expression in our hippocampal cultures. We found that estradiol-induced downregulation of synaptopodin is mediated by estrogen receptor β. Estrogen receptor β in turn, is upregulated in response to intracellular estradiol ablation following inhibition of estradiol synthesis by letrozole in dissociated hippocampal cultures, as well as in the hippocampus of the aromatase knock-out mouse. Thus, it appears that both the application of estradiol, via binding to estrogen receptor β, and letrozole, via upregulation of estrogen receptor β, eventually result in a downregulation of synaptopodin. Our data show that the synaptic plasticity caused by estradiol is subject to a subtle balance of the levels of estrogen receptor subtypes regulated by the available ligands. In addition, both seem to be part of a homeostatic feedback mechanism.

Estradiol synthesis in the ovaries is regulated via feedback mechanisms mediated by gonadotrophin-releasing hormone (GnRH) and gonadotrophins, secreted by the hypothalamus and the pituitary, respectively. Estradiol synthesis also takes place in the hippocampus. In hippocampal slice cultures of female animals, GnRH regulates estradiol synthesis dose-dependently. Hence, both hippocampal and ovarian estradiol synthesis are synchronized by GnRH. Hippocampus-derived estradiol is essential to synapse stability and maintenance because it stabilizes the spine cytoskeleton of hippocampal neurons. Inhibition of hippocampal estradiol synthesis in mice, however, results in loss of spines and spine synapses in females, but not in males. Stereotaxic application of GnRH to the hippocampus of female rats confirms the regulatory role of GnRH on estradiol synthesis and synapse density in the female hippocampus in vivo. This regulatory role of GnRH necessarily results in estrus cyclicity of spine density in the hippocampus of females.

Hippocampal neurons are capable of synthesizing estradiol de novo. Estradiol synthesis can be suppressed by aromatase inhibitors and by knock-down of steroid acute regulatory protein (StAR), whereas elevated levels of substrates of steroidogenesis enhance estradiol synthesis. In rat hippocampal cultures, the expression of estrogen receptors (ERs) and synaptic proteins, as well as synapse density, correlated positively with aromatase activity, regardless of whether the cultures originated from males or females. All effects induced by the inhibition of aromatase activity were rescued by application of estradiol to the cultures. In vivo, however, systemic application of letrozole, an aromatase inhibitor, induced synapse loss in female rats, but not in males. Furthermore, in the female hippocampus, density of spines and spine synapses varied with the estrus cycle. In addressing this in vivo-in vitro discrepancy, we found that gonadotropin-releasing hormone (GnRH) regulated estradiol synthesis via an aromatase-mediated mechanism and consistently regulated spine synapse density and the expression of synaptic proteins. Along these lines, GnRH receptor density was higher in the hippocampus than in the cortex and hypothalamus, and estrus cyclicity of spinogenesis was found in the hippocampus, but not in the cortex. Since GnRH receptor expression also varies with the estrus cycle, the sexual dimorphism in estrogen-regulated spine synapse density in the hippocampus very likely results from differences in the GnRH responsiveness of the male and the female hippocampus. This article is part of a Special Issue entitled 'Neurosteroids'.

Estradiol plays essential roles in the modulation of synaptic plasticity and neuroprotection in males as well as in females, as has been shown particularly in the hippocampus. Although it has long been known that aromatase, the final enzyme in estrogen synthesis, is expressed in the hippocampus, a new paradigm emerged when it was shown that estradiol is actually synthesized de novo in this part of the brain. Increasing evidence indicates that hippocampus-derived estradiol plays a role in synaptic plasticity and neuroptrotection, rather than estradiol originating from the gonads. In recent years, a number of in vivo and in vitro studies have shown that hippocampus-derived estradiol substantially contributes to hippocampal function, in particular to structural synaptic plasticity.

Studies on the role of 17β-estradiol (E2) in the hippocampus have mainly focused on CA1 and CA3 regions, whereas in dentate gyrus (DG), its role is largely unknown. Here, we examined potential functions of E2 in DG, particularly during development. Immunohistochemistry and in situ hybridization revealed abundance of estrogen receptor (ER)α, but not ERβ, expression in DG. Similar to CA1, analysis of synapse densities revealed a reduction in spine synapse number in DG molecular layer of immature rats and adult mice after inhibition of estradiol synthesis using letrozole. Interestingly, strong expression of ERα was found in Cajal-Retzius (CR) cells, which regulate neuronal migration and synaptogenesis via the extracellular matrix protein reelin. Immunoreactivity of aromatase, the final enzyme of estradiol synthesis, was strongest in mature granule cells. In hippocampal slice cultures, exogenous application of E2 caused an increase in reelin expression in CR cells, which was abolished after blockade of ERs using ICI182,780. Vice versa, inhibition of aromatase activity by letrozole resulted in reduced reelin expression, suggesting that E2 deriving from hippocampal sources contributes to the regulation of reelin as well as to the maintenance of spine synapses in DG. E2 further regulated Notch1, a signaling protein involved in neuronal differentiation.

Estrogen-induced synaptic plasticity was frequently shown by an increase of spines at apical dendrites of CA1 pyramidal neurons after systemic application of estradiol to ovariectomized rats. Surprisingly, exogenous application of estradiol to hippocampal cultures had no effect on spines and on spine synapses, although quantitative immunohistochemistry revealed an upregulation of spinophilin and of synaptophysin, in these cultures. The role of synaptophysin as a presynaptic marker and of spinophilin as a postsynaptic marker, appears questionable from these discrepancies. In contrast, synaptopodin, a marker protein of "mature" mushroom-shaped spines, was downregulated after treatment of hippocampal cultures with estradiol. Synaptopodin is strongly associated to the spine apparatus, a spine-specific cell organelle, which is present in 80% of all mushroom-shaped spines. Consistently, we found a reduction in the number of spines, containing a spine apparatus in response to estradiol, suggesting that the presence of a spine apparatus in many but not all spines is very likely a result of their dynamic character. In summary, synaptic proteins appear to be regulated by estradiol, independent of its function on spine and spine synapse formation.

Cholesterol of glial origin promotes synaptogenesis (Mauch et al., (2001) Science 294:1354–1357). Because in the hippocampus local estradiol synthesis is essential for synaptogenesis, we addressed the question of whether cholesterol-promoted synapse formation results from the function of cholesterol as a precursor of estradiol synthesis in this brain area. To this end, we treated hippocampal cultures with cholesterol, estradiol, or with letrozole, a potent aromatase inhibitor. Cholesterol increased neuronal estradiol release into the medium, the number of spine synapses in hippocampal slice cultures, and immunoreactivity of synaptic proteins in dispersed cultures. Simultaneous application of cholesterol and letrozole or blockade of estrogen receptors by ICI 182 780 abolished cholesterol-induced synapse formation. As a further approach, we inhibited the access of cholesterol to the first enzyme of steroidogenesis by knock-down of steroidogenic acute regulatory protein, the rate-limiting step in steroidogenesis. A rescue of reduced synaptic protein expression in transfected cells was achieved by estradiol but not by cholesterol. Our data indicate that in the hippocampus cholesterol-promoted synapse formation requires the conversion of cholesterol to estradiol. © 2009 Wiley-Liss, Inc.

Zusammenfassung Unsere Untersuchungen der letzten Jahre haben gezeigt, dass nicht das Ovar die Quelle für Estrogen induzierte synaptische Plastizität im Hippokampus ist, sondern dieses aus dem Hippokampus selber stammt und haben damit einen Paradigmawechsel eingeleitet, der Estrogen als Neuromodulator unabhängig vom Geschlecht identifiziert. Hippokampale Neurone von Ratten beiderlei Geschlechts sind in der Lage, aus Cholesterol Estrogene de novo zu synthetisieren. Diese hippokampale Estrogensynthese ist sowohl für den Erhalt von Spinesynapsen in vivo als auch in vitro essenziell. Die Hemmung der Estrogensynthese zieht einen Synapsenverlust nach sich und Langzeitpotenzierung ist nicht mehr induzierbar. Die Effekte von hippokampalem Estrogen sind auto-/parakriner Natur, die über die bekannten Estrogenrezeptor-Subtypen, ERα und ERβ, vermittelt werden. Die Regulation der hippokampalen Estrogensynthese erfolgt über GnRH und erklärt die Korrelation der Spinesynapsendichte mit dem weiblichen genitalen Zyklus, die für den Hippokampus spezifisch ist.

Our knowledge on estradiol-induced modulation of synaptic function in the hippocampus is widely based on results following the application of the steroid hormone to either cell cultures, or after the treatment of gonadectomized animals, thus ignoring local neuronal estrogen synthesis. We and others, however, have shown that hippocampus-derived estradiol also controls synaptic plasticity in the hippocampus. Estradiol synthesis in the hippocampus is regulated by several mechanisms, which are reviewed in this report. The regulation of the activity of aromatase, the final enzyme of estrogen biosynthesis, by Ca(2+) transients, is of particular interest. Aromatase becomes inactivated as soon as it is phosphorylated by Ca(2+)-dependent kinases upon calcium release from internal stores. Accordingly, thapsigargin dephosphorylates aromatase and stimulates estradiol synthesis by depletion of internal Ca(2+) stores. Vice versa, letrozole, an aromatase inhibitor, phosphorylates aromatase and reduces estradiol synthesis. Treatment of the cultures with 17β-estradiol results in phosphorylation of the enzyme and increased aromatase protein expression, which suggests that estradiol synthesis in hippocampal neurons is regulated in an autocrine manner.

During the oestrus cycle, varying spine synapse density correlates positively with varying local synthesis of oestradiol in the hippocampus. In this context, the roles of the oestrogen receptor (ER) subtypes ERα and β are not fully understood. In the present study, we used neonatal hippocampal slice cultures from female rats because these cultures synthesise oestradiol and express both receptor subtypes, and inhibition of oestradiol synthesis in these cultures results in spine synapse loss. Using electron microscopy, we tested the effects on spine synapse density in response to agonists of both ERα and ERβ. Application of agonists to the cultures had no effect. After inhibition of oestradiol synthesis, however, agonists of ERα induced spine synapse formation, whereas ERβ agonists led to a reduction in spine synapse density in the CA1 region of these cultures. Consistently, up-regulation of ERβ in the hippocampus of adult female aromatase-deficient mice is paralleled by hippocampus-specific spine synapse loss in this mutant. Finally, we found an increase in spine synapses in the adult female ERβ knockout mouse, but no effect in the adult female ERα knockout mouse. Our data suggest antagonistic roles of ERβ and ERα in spine synapse formation in the female hippocampus, which may contribute to oestrus cyclicity of spine synapse density in the hippocampus.

Inhibitors of aromatase, the final enzyme of estradiol synthesis, are suspected of inducing memory deficits in women. In previous experiments, we found hippocampal spine synapse loss in female mice that had been treated with letrozole, a potent aromatase inhibitor. In this study, we therefore focused on the effects of letrozole on long-term potentiation (LTP), which is an electrophysiological parameter of memory and is known to induce spines, and on phosphorylation of cofilin, which stabilizes the spine cytoskeleton and is required for LTP in mice. In acute slices of letrozole-treated female mice with reduced estradiol serum concentrations, impairment of LTP started as early as after 6 h of treatment and progressed further, together with dephosphorylation of cofilin in the same slices. Theta-burst stimulation failed to induce LTP after 1 week of treatment. Impairment of LTP was followed by spine and spine synapse loss. The effects were confirmed in vitro by using hippocampal slice cultures of female mice. The sequence of effects in response to letrozole were similar in ovariectomized female and male mice, with, however, differences as to the degree of downregulation. Our data strongly suggest that impairment of LTP, followed by loss of mushroom spines and spine synapses in females, may have implications for memory deficits in women treated with letrozole.

Recently, inhibition of estrogen synthesis by aromatase inhibitors has become a favored therapy for breast cancer in postmenopausal women. Estrogen is, however, important for synapse formation in the hippocampus. Inhibition of aromatase induces spine synapse loss in organotypic hippocampal slice cultures. We therefore studied the effect of systemic treatment with the potent aromatase inhibitor letrozole on spine synapse formation and synaptic proteins in the hippocampi of female mice for periods of 7 d and 4 wk. In cyclic, letrozole-treated females and in ovariectomized, letrozole-treated females, the number of spine synapses was significantly reduced in the hippocampus but not in the prefrontal or cerebellar cortex. Consequently, the expression of the N-methyl-D-aspartate receptor NR1 was significantly down-regulated after treatment with letrozole. In cyclic animals the expression of the synaptic proteins synaptophysin and spinophilin was down-regulated in response to letrozole. In ovariectomized animals, however, protein expression was down-regulated after 7 d of treatment, whereas the expression was up-regulated after 4 wk of treatment. Our results indicate that systemic inhibition of aromatase in mice affects structural synaptic plasticity in the hippocampus. This may contribute to cognitive deficits in postmenopausal women treated with aromatase inhibitors.

Ovarian oestrogens have been postulated to be neuroprotective. It has also been shown that considerable amounts of oestrogens are synthesised in hippocampal neurones. In the present study, we focused on a potential role of hippocampus-derived oestradiol compared to gonad-derived oestradiol on axon outgrowth of hippocampal neurones. To address the role of hippocampus-derived oestradiol, we inhibited oestrogen synthesis by treatment of neonatal hippocampal cell cultures with letrozole, a specific aromatase inhibitor. As an alternative, we used siRNA against steroidogenic acute regulatory protein (StAR). Axon outgrowth and GAP-43 expression were significantly down-regulated in response to letrozole and in siRNA-StAR transfected cells. The effects after inhibition of oestrogen synthesis in response to letrozole and in siRNA-StAR transfected cells were reversed by oestrogen supplementation. No difference was found between ovariectomised animals, cycling animals at pro-oestrus and ovariectomised and subsequently oestradiol-treated animals. However, high pharmacological doses of oestradiol promoted axon outgrowth, which was possible to abolish by the oestrogen receptor antagonist ICI 182,780. Our results show that oestradiol-induced neurite outgrowth is very likely mediated by genomic oestrogen receptors and requires higher doses of oestradiol than physiological serum concentrations derived from the gonads.

Ovarian oestrogens have been demonstrated to influence neurogenesis in the dentate gyrus. As considerable amounts of oestrogens are synthesized in hippocampal neurones, we focused on the role of hippocampus-derived estradiol on proliferation and apoptosis of granule cells in vitro. We used hippocampal dispersion cultures, which allowed for cultivation of the cells under steroid- and serum-free conditions and monitoring of oestrogen synthesis. To address the influence of hippocampus-derived estradiol on neurogenesis, we inhibited oestrogen synthesis by treatment of hippocampal cell cultures with letrozole, a specific aromatase inhibitor. Alternatively, we used siRNA against steroidogenic acute regulatory protein (StAR). The number of proliferative cells decreased whereas the number of apoptotic cells increased dose-dependently, in response to reduced estradiol release into the medium after treatment with letrozole. This also held true for siRNA against StAR transfected cell cultures. Application of estradiol to the medium had no effect on proliferation and apoptosis whereas the anti-proliferative and pro-apoptotic effects of StAR knock-down and letrozole treatment were restored by treatment of the cultures with estradiol. Our findings suggest that neurogenesis and apoptosis in the hippocampus require a defined range of estradiol concentrations that is physiologically provided by hippocampal cells but not by gonads.

Previous studies have shown that synapses and expression of synaptic proteins in hippocampal neurons are regulated by hippocampus-derived estradiol. Here, we compared the effects of this paracrine regulation in different hippocampal regions. In tissue sections, immunohistochemistry followed by semiquantitative image analysis revealed a three-fold higher expression of steroidogenic acute regulatory protein (StAR) and aromatase in neurons of the CA3 than that of the CA1 region and in granule cells. Next, we treated hippocampal cell cultures with letrozole, an aromatase inhibitor, which resulted in a dose-dependent decrease in the release of 17beta-estradiol into the medium and in a dose-dependent downregulation of spinophilin and synaptophysin expression in dissociated hippocampal neurons. The downregulation of synaptic protein expression was restored by simultaneous application of letrozole together with estradiol. In response to a defined dose of letrozole, the downregulation of spinophilin expression was significantly stronger in CA1 neurons and in granule cells, than in cells of the CA3 region in slice cultures. With synaptophysin, downregulation was stronger in stratum lucidum of CA3 than in stratum radiatum of CA1. Both region-specific expression of steroidogenic enzymes and region-specific downregulation of synaptic proteins in response to a defined dose of letrozole may suggest different levels of estrogen concentrations within the hippocampus. Varying concentrations of estradiol in the hippocampus in turn may contribute to region-specific differentiation of hippocampal neurons.

Different effects of estrogen on synaptic plasticity have [corrected] been reported. Here, we summarise effects of low, gonad-derived serum estrogen concentrations, of intermediate concentrations, provided by hippocampal cells, and of pharmacological doses of estrogen on synapses and spines and on the expression of synaptic proteins. No effects of low concentrations were found. To study the effects of hippocampus-derived estradiol, we inhibited hippocampal estrogen synthesis by treatment of hippocampal cell cultures with letrozole, an aromatase inhibitor. Alternatively, we used siRNA against Steroidogenic acute regulatory protein (StAR). Spines, synapses, and synaptic proteins were significantly down regulated in response to letrozole and in siRNA-StAR transfected cells. Application of high pharmacological doses of estradiol promoted only synaptophysin expression, a presynaptic protein, but did not increase the number of boutons. Our results point to an essential role of endogenous hippocampal estrogen in hippocampal synaptic plasticity rather than to a direct influence of estrogens derived from peripheral sources, such as the gonads.

Introduction: Sex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17β-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue. Methods: We analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes. Results: Aromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10 �11 Mt o 10 �7 M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-β. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole. Conclusions: We demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-β .I n addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated.

Dr. Gabriele M. Rune apologizes for having listed Dr. Christian Peters as a co-author of the paper (Fester et al., 2009) ‘‘The opposing roles of estradiol on synaptic protein expression in hippocampal cultures’’ (Psychoneuroendocrinology 34 (Suppl. 1) 123—129) and having cited the results of his work without his signed authorization. Dr. Peters was not involved in the writing of the paper nor was he informed that his name was in the list of authors of the two versions of the publication. Therefore, the sentence cited below, which refers to the data contributed by Dr. Peters, needs to be deleted from the publication and Dr. Peters’ name needs to be removed from the list of authors. ‘‘In fact, according to real time RT-PCR, which was used to compare the amount of synaptopodin mRNA in estradiol-treated slice cultures to that in non-treated cultures, we found that synaptopodin mRNA expression was clearly downregulated compared to the controls’’ (pages 126—127). Psychoneuroendocrinology (2011) 36, 148