Gabriele M. Rune

Hippocampal Synapses Depend on Hippocampal Estrogen Synthesis

Estrogens have been described to induce synaptogenesis in principal neurons of the hippocampus and have been shown to be synthesized and released by exactly these neurons. Here, we have focused on the significance of local estrogen synthesis on spine synapse formation and the synthesis of synaptic proteins. To this end, we reduced hippocampal estrogen synthesis in vitro with letrozole, a reversible nonsteroidal aromatase inhibitor. In hippocampal slice cultures, letrozole treatment resulted in a dose-dependent decrease of 17β-estradiol as quantified by RIA. This was accompanied by a significant decrease in the density of spine synapses and in the number of presynaptic boutons. Quantitative immunohistochemistry revealed a downregulation of spinophilin, a marker of dendritic spines, and synaptophysin, a protein of presynaptic vesicles, in response to letrozole. Surprisingly, no increase in the density of spines, boutons, and synapses and in spinophilin expression was seen after application of estradiol to the medium of cultures that had not been treated with letrozole. However, synaptophysin expression was upregulated under these conditions. Our results point to an essential role of endogenous hippocampal estrogen synthesis in the maintenance of hippocampal spine synapses.

The Potential for β-Structure in the Repeat Domain of Tau Protein Determines Aggregation, Synaptic Decay, Neuronal Loss, and Coassembly with Endogenous Tau in Inducible Mouse Models of Tauopathy

We describe two new transgenic mouse lines for studying pathological changes of Tau protein related to Alzheimers disease. They are based on the regulatable expression of the four-repeat domain of human Tau carrying the FTDP17 (frontotemporal dementia and parkinsonism linked to chromosome 17) mutation ΔK280 (TauRD/ΔK280), or the ΔK280 plus two proline mutations in the hexapeptide motifs (TauRD/ΔK280/I277P/I308P). The ΔK280 mutation accelerates aggregation (“proaggregation mutant”), whereas the proline mutations inhibit Tau aggregation in vitro and in cell models (“antiaggregation mutant”). The inducible transgene expression was driven by the forebrain-specific CaMKIIα (calcium/calmodulin-dependent protein kinase IIα) promoter. The proaggregation mutant leads to Tau aggregates and tangles as early as 2–3 months after gene expression, even at low expression (70% of endogenous mouse Tau). The antiaggregation mutant does not aggregate even after 22 months of gene expression. Both mutants show missorting of Tau in the somatodendritic compartment and hyperphosphorylation in the repeat domain [KXGS motifs, targets of the kinase MARK (microtubule affinity regulating kinase)]. This indicates that these changes are related to Tau expression rather than aggregation. The proaggregation mutant causes astrogliosis, loss of synapses and neurons from 5 months of gene expression onward, arguing that Tau toxicity is related to aggregation. Remarkably, the human proaggregation mutant TauRD coaggregates with mouse Tau, coupled with missorting and hyperphosphorylation at multiple sites. When expression of proaggregation TauRD is switched off, soluble and aggregated exogenous TauRD disappears within 1.5 months. However, tangles of mouse Tau, hyperphosphorylation, and missorting remain, suggesting an extended lifetime of aggregated wild-type Tau once a pathological conformation and aggregation is induced by a proaggregation Tau species.

Tau-Induced Defects in Synaptic Plasticity, Learning, and Memory Are Reversible in Transgenic Mice after Switching Off the Toxic Tau Mutant

This report describes the behavioral and electrophysiological analysis of regulatable transgenic mice expressing mutant repeat domains of human Tau (TauRD). Mice were generated to express TauRD in two forms, differing in their propensity for β-structure and thus in their tendency for aggregation (“pro-aggregant” or “anti-aggregant”) (Mocanu et al., 2008). Only pro-aggregant mice show pronounced changes typical for Tau pathology in Alzheimers disease (aggregation, missorting, hyperphosphorylation, synaptic and neuronal loss), indicating that the β-propensity and hence the ability to aggregate is a key factor in the disease. We now tested the mice with regard to neuromotor parameters, behavior, learning and memory, and synaptic plasticity and correlated this with histological and biochemical parameters in different stages of switching TauRD on or off. The mice are normal in neuromotor tests. However, pro-aggregant TauRD mice are strongly impaired in memory and show pronounced loss of long-term potentiation (LTP), suggesting that Tau aggregation specifically perturbs these brain functions. Remarkably, when the expression of human pro-aggregant TauRD is switched on for ∼10 months and off for ∼4 months, memory and LTP recover, whereas aggregates decrease moderately and change their composition from mixed human plus mouse Tau to mouse Tau only. Neuronal loss persists, but synapses are partially rescued. This argues that continuous presence of amyloidogenic pro-aggregant TauRD constitutes the main toxic insult for memory and LTP, rather than the aggregates as such.

Steroidogenic factor-1 expression in marmoset and rat hippocampus: co-localization with StAR and aromatase

Steroidogenic factor‐1 (SF‐1), an orphan nuclear receptor, was studied with respect to the expression of steroidogenic enzymes in the hippocampus of rat and marmoset, since SF‐1 is a regulator of steroid biosynthesis in the gonads. We used the steroidogenic acute regulatory protein (StAR) as a marker of the first step in the cascade of oestrogen synthesis and aromatase as a marker of the last. StAR transports cholesterol to the inner mitochondrial membrane where it is converted by the cytochrome P‐450 enzyme complex. This is the rate‐limiting step in steroid biosynthesis. Aromatase metabolizes testosterone to oestrogen. Using an anti‐SF‐1 antibody we show that SF‐1 is highly expressed in neuronal cells of the pyramidal layer (CA1–CA3) and in the dentate gyrus of rat and marmoset hippocampi. Binding of the antibody was seen in more than 60% of all cells in the pyramidal layer and in the fascia dentata. In situ hybridization studies revealed the same expression pattern for StAR and aromatase. StAR and aromatase‐positive cells were strictly correlated with SF‐1 as shown by computer‐assisted confocal microscopy in double labelling experiments (immunohistochemistry and in situ hybridization). This coexpression may imply SF‐1 as a possible regulator of steroidogenesis in the hippocampus. However, a few interneurones express solely SF‐1 and aromatase but are negative for StAR. Since the expression of StAR represents the first step in steroidogenesis its expression is suggestive for a de novo synthesis of steroids. A small population of interneurones must import precursors for oestrogen synthesis from other sources. Responsive cells, as evidenced by the presence of oestrogen receptor transcripts, were also found in the pyramidal layer and dentate gyrus. In conclusion, (1) SF‐1 could play a regulatory role in steroidogenesis in the hippocampus of marmoset and rat and (2) with respect to the capacity of steroidogenesis two populations of hippocampal neurones coexist.

The β-Propensity of Tau Determines Aggregation and Synaptic Loss in Inducible Mouse Models of Tauopathy

Neurofibrillary lesions are characteristic for a group of human diseases, named tauopathies, which are characterized by prominent intracellular accumulations of abnormal filaments formed by the microtubule-associated protein Tau. The tauopathies are accompanied by abnormal changes in Tau protein, including pathological conformation, somatodendritic mislocalization, hyperphosphorylation, and aggregation, whose interdependence is not well understood. To address these issues we have created transgenic mouse lines in which different variants of full-length Tau are expressed in a regulatable fashion, allowing one to switch the expression on and off at defined time points. The Tau variants differ by small mutations in the hexapeptide motifs that control the ability of Tau to adopt a β-structure conformation and hence to aggregate. The “pro-aggregation” mutant ΔK280, derived from one of the mutations observed in frontotemporal dementias, aggregates avidly in vitro, whereas the “anti-aggregation” mutant ΔK280/PP cannot aggregate because of two β-breaking prolines. In the transgenic mice, the pro-aggregation Tau induces a pathological conformation and pre-tangle aggregation, even at low expression levels, the anti-aggregation mutant does not. This illustrates that abnormal aggregation is primarily controlled by the molecular structure of Tau in vitro and in the organism. Both variants of Tau become mislocalized and hyperphosphorylated independently of aggregation, suggesting that localization and phosphorylation are mainly a consequence of increased concentration. These pathological changes are reversible when the expression of Tau is switched off. The pro-aggregation Tau causes a strong reduction in spine synapses.

Direct and indirect effects of estrogen on rat hippocampus.

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. Recent findings question this direct endocrine regulation of synaptogenesis by estradiol. We have shown, for the first time, that estrogens are synthesized de novo in rat hippocampal neurons. By using letrozole, an inhibitor of aromatase, estradiol levels in hippocampal dispersion cultures as well as in hippocampal slice cultures were significantly suppressed. Letrozole treatment resulted in a significant decrease in the density of spines and spine synapses and in the number of presynaptic boutons. Quantitative immunohistochemistry revealed a dose-dependent downregulation of spinophilin, a spine marker, and of synaptophysin, a presynaptic marker, in the hippocampus. Surprisingly, exogenous application of estradiol to the cultures had no effect. Indirect effects of estrogens, mediated via subcortical nuclei, may help to explain this phenomenon. Implantation of estrogen-filled cannulae into the median raphe, which projects to the hippocampus, resulted in a significant increase in spine density in the hippocampus after seven days of treatment. This increase was paralleled by a decrease in the density of serotonergic innervation of the strata lacunosum moleculare and radiatum of the CA1 region. Apart from direct endocrine mechanisms our findings suggest that estradiol-induced spinogenesis in the hippocampus is also mediated by indirect mechanisms and is furthermore regulated endogenously, in a paracrine manner.

Estrogen up-regulates estrogen receptor α and synaptophysin in slice cultures of rat hippocampus

Previous studies have shown that estrogen application increases the density of synaptic input and the number of spines on CA1 pyramidal neurons. Here, we have investigated whether Schaffer collaterals to CA1 pyramidal cells are involved in this estrogen-induced synaptogenesis on CA1 pyramidal neurons. To this end, we studied estrogen-induced expression of both estrogen receptor (ER) subtypes (ERalpha and ERbeta) together with the presynaptic marker synaptophysin in the rat hippocampus. In tissue sections as well as in slice cultures mRNA expression of ERalpha, ERbeta and synaptophysin was higher in CA3 than in CA1, and mRNA expression and immunoreactivity for both ER subtypes were found in both principal cells and interneurons. By using quantitative image analysis we found stronger nuclear immunoreactivity for ERalpha in CA3 than in CA1. In slice cultures, supplementation of the medium with 10(-8) M estradiol led to an increase of nuclear immunoreactivity for ERalpha, but not for ERbeta, which was accompanied by a dramatic up-regulation of synaptophysin immunoreactivity in stratum radiatum of CA1. Together these findings indicate that estrogen effects on hippocampal neurons are more pronounced in CA3 than in CA1 and that ER activation in CA3 neurons leads to an up-regulation of a presynaptic marker protein in the axons of these cells, the Schaffer collaterals. We conclude that estradiol-induced spine formation on CA1 pyramidal cells may be mediated presynaptically, very likely by activation of ERalpha in CA3 pyramidal cells, followed by an increase in Schaffer collateral synapses.

Gonadotropin-releasing hormone regulates spine density via its regulatory role in hippocampal estrogen synthesis

Spine density in the hippocampus changes during the estrus cycle and is dependent on the activity of local aromatase, the final enzyme in estrogen synthesis. In view of the abundant gonadotropin-releasing hormone receptor (GnRH-R) messenger RNA expression in the hippocampus and the direct effect of GnRH on estradiol (E2) synthesis in gonadal cells, we asked whether GnRH serves as a regulator of hippocampal E2 synthesis. In hippocampal cultures, E2 synthesis, spine synapse density, and immunoreactivity of spinophilin, a reliable spine marker, are consistently up-regulated in a dose-dependent manner at low doses of GnRH but decrease at higher doses. GnRH is ineffective in the presence of GnRH antagonists or aromatase inhibitors. Conversely, GnRH-R expression increases after inhibition of hippocampal aromatase. As we found estrus cyclicity of spine density in the hippocampus but not in the neocortex and GnRH-R expression to be fivefold higher in the hippocampus compared with the neocortex, our data strongly suggest that estrus cycle–dependent synaptogenesis in the female hippocampus results from cyclic release of GnRH.

NKCC1-Dependent GABAergic Excitation Drives Synaptic Network Maturation during Early Hippocampal Development

A high intracellular chloride concentration in immature neurons leads to a depolarizing action of GABA that is thought to shape the developing neuronal network. We show that GABA-triggered depolarization and Ca2+ transients were attenuated in mice deficient for the Na–K–2Cl cotransporter NKCC1. Correlated Ca2+ transients and giant depolarizing potentials (GDPs) were drastically reduced and the maturation of the glutamatergic and GABAergic transmission in CA1 delayed. Brain morphology, synaptic density, and expression levels of certain developmental marker genes were unchanged. The expression of lynx1, a protein known to dampen network activity, was decreased. In mice deficient for the neuronal Cl−/HCO3− exchanger AE3, GDPs were also diminished. These data show that NKCC1-mediated Cl− accumulation contributes to GABAergic excitation and network activity during early postnatal development and thus facilitates the maturation of excitatory and inhibitory synapses.

Proliferation and apoptosis of hippocampal granule cells require local oestrogen synthesis

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.