Lepu Zhou

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.

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.

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.

Aromatase Inhibition Abolishes LTP Generation in Female But Not in Male Mice

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.

Aromatase Inhibitors Induce Spine Synapse Loss in the Hippocampus of Ovariectomized Mice

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.

Cholesterol-promoted synaptogenesis requires the conversion of cholesterol to estradiol in the hippocampus

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.

Roles of 17β-Estradiol Involve Regulation of Reelin Expression and Synaptogenesis in the Dentate Gyrus

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.