EGCG functions through estrogen receptor-mediated activation of ADAM10 in the promotion of non-amyloidogenic processing of APP

Estrogen depletion following menopause has been correlated with an increased risk of developing Alzheimer’s disease (AD). We previously explored the beneficial effect of (−)-epigallocatechin-3-gallate (EGCG) on AD mice and found increased non-amyloidogenic processing of amyloid precursor protein (APP) through the α-secretase a disintegrin and metallopeptidase domain 10 (ADAM10). Our results in this study suggest that EGCG-mediated enhancement of non-amyloidogenic processing of APP is mediated by the maturation of ADAM10 via an estrogen receptor-α (ERα)/phosphoinositide 3-kinase/Ak-transforming dependent mechanism, independent of furin-mediated ADAM10 activation. These data support prior assertions that central selective ER modulation could be a therapeutic target for AD and support the use of EGCG as a well-tolerated alternative to estrogen therapy in the prophylaxis and treatment of this disease.     

Brain Endogenous Estrogen Levels Determine Responses to Estrogen Replacement Therapy via Regulation of BACE1 and NEP in Female Alzheimer’s Transgenic Mice

Estrogens have been found to improve memory and reduce risk of dementia, although conflicting results such as failure of estrogen replacement therapy for treatment of Alzheimer's disease (AD) also has been reported. Only recently, our published human brain studies showed a depletion of brain estrogen in women with AD, while other studies have demonstrated cognitive impairment believed to be caused by inhibition of endogenous estrogen synthesis in females. To investigate whether the shortage of brain estrogen alters the sensitivity of response to estrogen replacement therapy, we have used genetic and surgical animal models to examine the response of estrogen treatment in AD neuropathology. Our studies have shown that early treatment with 17β-estradiol (E2) or genistein could reduce brain amyloid levels by increasing Aβ clearance in both APP23 mice with genetic deficiency of aromatase (APP/Ar^+/?), in which the brains contain nondetectable levels of estrogen, and in APP23 mice with an ovariectomy (APP/OVX), in which the brains still contain certain levels of estrogen. However, only APP/Ar^+/? mice showed a great reduction in brain amyloid plaque formation after E2 or genistein treatment along with downregulation of β-secretase (BACE1) mRNA and protein expression. Our results suggest that early and long-term usage of E2 and/or genistein may prevent AD pathologies in a dependent manner on endogenous brain estrogen levels in aged females.     

Estrogen Reduces 11β‐Hydroxysteroid Dehydrogenase Type 1 in Liver and Visceral,but Not Subcutaneous,Adipose Tissue in Rats

Following menopause, body fat is redistributed from peripheral to central depots. This may be linked to the age related decrease in estrogen levels. We hypothesized that estrogen supplementation could counteract this fat redistribution through tissue‐specific modulation of glucocorticoid exposure. We measured fat depot masses and the expression and activity of the glucocorticoid‐activating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) in fat and liver of ovariectomized female rats treated with or without 17β‐estradiol. 11βHSD1 converts inert cortisone, or 11‐dehydrocorticosterone in rats into active cortisol and corticosterone. Estradiol‐treated rats gained less weight and had significantly lower visceral adipose tissue weight than nontreated rats (P < 0.01); subcutaneous adipose weight was unaltered. In addition, 11βHSD1 activity/expression was downregulated in liver and visceral, but not subcutaneous, fat of estradiol‐treated rats (P < 0.001 for both). This downregulation altered the balance of 11βHSD1 expression and activity between adipose tissue depots, with higher levels in subcutaneous than visceral adipose tissue of estradiol‐treated animals (P < 0.05 for both), opposite the pattern in ovariectomized rats not treated with estradiol (P < 0.001 for mRNA expression). Thus, estrogen modulates fat distribution, at least in part, through effects on tissue‐specific glucocorticoid metabolism, suggesting that estrogen replacement therapy could influence obesity related morbidity in postmenopausal women.     

Usefulness of experimental models to understand the vascular effects of estrogens

Hormonal replacement therapy does not prevent cardiovascular events in postmenopausal women. In contrast, the incidence of cardiovascular diseases is higher in men than in premenopausal women but increases in postmenopausal women, and all animal studies demonstrate a prevention of fatty streak deposit by estradiol. Although estradiol improves the lipoprotein profile, this effect can account for only a minor part of the protective effect. Endothelium appears to be an important target for estradiol, because this hormone potentiates endothelial nitric oxide (NO) production, thus promoting the beneficial effects of NO, such as vasorelaxation and inhibition of platelet aggregation. Estradiol accelerates endothelial regrowth, thus favoring vascular healing, and prevents apoptosis of endothelial cells. Estradiol prevents fatty streak deposit through a mechanism which is clearly independent of NO. The immuno-inflammatory system appears to play a key role in the development of fatty streak deposit as well as in atherosclerotic plaque rupture. Mice deficient either in monocyte-macrophages or in lymphocytes are partially protected against fatty streak deposit. Interestingly, the atheroprotective effect of estradiol is absent in mice deficient in T and B lymphocytes. Most of these effects of estradiol are mediated by estrogen receptor alpha, and are independent of estrogen receptor beta. Thus, the inflammatory-immune system appears to be also a major target of estrogens. However, the effects of estrogens on the immuno-inflammatory system appear ambiguous, as in some models, estradiol rather promotes inflammation (by increasing interferon gamma which could elicit plaque destabilization). A better understanding of the mechanisms of estrogens on the normal and atheromatous arteries is required and should help to optimize the prevention of cardiovascular disease after menopause.     

Regulation of nitric oxide-dependent vasodilation in coronary arteries of estrogen receptor-alpha-deficient mice

Estrogen has been shown to increase endothelium-dependent vasodilation and expression of endothelial nitric oxide (NO) synthase (eNOS); however, the role of estrogen receptors in mediating estrogen effects on endothelial function remains to be elucidated. The purpose of this study was to test the hypothesis that estrogen modulates NO-dependent vasodilation of coronary arteries through its action on estrogen receptor-alpha (ER-alpha) to increase protein levels of eNOS and Cu/Zn superoxide dismutase (SOD-1). Vasodilation to acetylcholine (ACh) and sodium nitroprusside was assessed in isolated coronary arteries from intact and ovariectomized female wild-type (WT) and ER-alpha knockout (ERalphaKO) mice. Protein levels for eNOS and SOD-1 were also evaluated. Vasodilation to ACh was not significantly altered in ERalphaKO mice compared with WT mice. Ovariectomy reduced responsiveness to ACh in ERalphaKO mice but not WT mice. Responses to sodium nitroprusside were not altered by disruption of ER-alpha or by ovariectomy. Supplementation with estrogen restored ACh-induced vasodilation in ovariectomized ERalphaKO mice. eNOS protein was reduced in ERalphaKO mice compared with WT mice. Ovariectomy caused a further reduction in eNOS protein in ERalphaKO mice, but this reduction was reversed by estrogen treatment. SOD-1 protein levels were increased by disruption of ER-alpha. Ovariectomy reduced SOD-1 protein in ERalphaKO mice, but this reduction was partially reversed by estrogen replacement. These results suggest that estrogen modulation of eNOS protein content is mediated in part through ER-alpha. NO-dependent responses are preserved in ERalphaKO mice, possibly through increased SOD-1 expression and enhanced bioavailability of NO.     

Effects of diet-induced hypercholesterolemia on amyloid accumulation in ovariectomized mice

A central hypothesis in the study of Alzheimer’s disease (AD) is the accumulation and aggregation of β-amyloid peptide (Aβ). Recent epidemiological studies suggest that patients with elevated cholesterol and decreased estrogen levels are more susceptible to AD through Aβ accumulation. To test the above hypothesis, we used ovariectomized with diet-induced hypercholesterolemia (OVX) and hypercholesterolemia (HCL) diet alone mouse models. HPLC analysis reveals the presence of beta amyloid in the OVX and HCL mice brain. Congo red staining analysis revealed the extent of amyloid deposition in OVX and hypercholesterolemia mice brain. Overall, Aβ levels were higher in OVX mice than in HCL. Secondly, estrogen receptors α (ERα) were assessed by immunohistochemistry and this suggested that there was a decreased expression of ER α in OVX animals when compared to hypercholesterolemic animals. Aβ was quantified by Western blot and ELISA analysis. Overall, Aβ levels were higher in OVX mice than in HCL mice. Our experimental results suggested that OVX animals were more susceptible to AD with significant increase in Aβ peptide.     

The Pathogenic Implication of Abnormal Interaction Between Apolipoprotein E Isoforms, Amyloid-beta Peptides, and Sulfatides in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common cause of dementia in the aging population. Prior work has shown that the ε4 allele of apolipoprotein E (apoE4) is a major risk factor for “sporadic” AD, which accounts for >99% of AD cases without a defined underlying mechanism. Recently, we have demonstrated that sulfatides are substantially and specifically depleted at the very early stage of AD. To identify the mechanism(s) of sulfatide loss concurrent with AD onset, we have found that: (1) sulfatides are specifically associated with apoE-associated particles in cerebrospinal fluid (CSF); (2) apoE modulates cellular sulfatide levels; and (3) the modulation of sulfatide content is apoE isoform dependent. These findings not only lead to identification of the potential mechanisms underlying sulfatide depletion at the earliest stages of AD but also serve as mechanistic links to explain the genetic association of apoE4 with AD. Moreover, our recent studies further demonstrated that (1) apoE mediates sulfatide depletion in amyloid-β precursor protein transgenic mice; (2) sulfatides enhance amyloid β (Aβ) peptides binding to apoE-associated particles; (3) Aβ42 content notably correlates with sulfatide content in CSF; (4) sulfatides markedly enhance the uptake of Aβ peptides; and (5) abnormal sulfatide-facilitated Aβ uptake results in the accumulation of Aβ in lysosomes. Collectively, our studies clearly provide a link between apoE, Aβ, and sulfatides in AD and establish a foundation for the development of effective therapeutic interventions for AD.     

Design, synthesis, and evaluation of estradiol-linked genotoxicants as anti-cancer agents

A series of bifunctional compounds was prepared consisting of 17beta estradiol linked to a DNA damaging N,N-bis-(2-chloroethyl)aniline. The objective of our studies was to determine the characteristics of the linker that permitted both reaction with DNA and binding of the resultant covalent adducts to the estrogen receptor. Linker characteristics were pivotal determinants underlying the ability of the compounds to kill selectively breast cancer cells that express the estrogen receptor.     

Non-steroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease: old and new mechanisms of action

Alzheimer's disease (AD) is characterized by cerebral deposits of beta-amyloid (A beta) peptides and neurofibrillary tangles (NFT) which are surrounded by inflammatory cells. Epidemiological studies have shown that prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD and delays the onset of the disease. It has been postulated that some NSAIDs target pathological hallmarks of AD by interacting with several pathways, including the inhibition of cyclooxygenases (COX) and activation of the peroxisome proliferator-activated receptor gamma. A variety of experimental studies indicate that a subset of NSAIDs such as ibuprofen, flurbiprofen, indomethacin and sulindac also possess A beta-lowering properties in both AD transgenic mice and cell cultures of peripheral, glial and neuronal origin. While COX inhibition occurs at low concentrations in vitro (nM-low microm range), the A beta-lowering activity is observed at high concentrations (< or = 50 microm). Nonetheless, studies with flurbiprofen or ibuprofen in AD transgenic mice show that the effects on A beta levels or deposition are attained at plasma levels similar to those achieved in humans at therapeutic dosage. Still, it remains to be assessed whether adequate concentrations are reached in the brain. This is a crucial aspect that will allow defining the dose-window and the length of treatment in future clinical trials. Here, we will discuss how the combination of anti-amyloidogenic and anti-inflammatory activities of certain NSAIDs may produce a profile potentially relevant to their clinical use as disease-modifying agents for the treatment of AD.     

Modulation of aberrant CDK5 signaling rescues impaired neurogenesis in models of Alzheimer's disease

Recent studies show that in Alzheimer''s disease (AD), alterations in neurogenesis contribute to the neurodegenerative process. Neurodegeneration in AD has been associated with aberrant signaling through the cyclin-dependent kinase-5 (CDK5) pathway via its activators p35/p25; however, the role of CDK5 in the mechanisms of defective adult neurogenesis in AD is unknown. First, to study AD-like abnormal activation of CDK5 signaling in an in vitro model of neurogenesis, neuronal progenitor cells (NPCs) were infected with a viral vector expressing p35, and exposed to amyloid-β protein (Aβ₁₋₄₂). These conditions resulted in impaired maturation and neurite outgrowth in vitro, and these effects were reversed by pharmacological or genetic inhibition of CDK5. Similarly, neurogenesis was impaired in a transgenic mouse model of AD that expresses high levels of amyloid precursor protein (APP), and this effect was reversed in transgenic mice crossed with a CDK5 heterozygous-deficient mouse line. A similar rescue effect was observed in APP transgenic mice treated with Roscovitine, a pharmacological inhibitor of CDK5. Taken together, these data suggest that the CDK5 signaling pathway has a critical role in maintaining the integrity of NPCs and neuronal maturation in the adult hippocampus. Moreover, potential therapeutic approaches could focus on modulating the aberrant activity of CDK5 to target the neurogenic and neurodegenerative alterations in AD.     

Regulation of Alzheimer beta-amyloid precursor trafficking and metabolism

Alzheimer's disease (AD) is characterized by the intracranial accumulation of the 4 kDa amyloid-beta peptide (Abeta), following proteolysis of a approximately 700-amino acid, integral membrane precursor, the Alzheimer amyloid precursor protein (APP). The best evidence causally linking APP to AD has been provided by the discovery of mutations within the APP coding sequence that segregate with disease phenotypes in autosomal dominant forms of familial AD (FAD). Though FAD is rare ( < 10% of all AD), the hallmark features (amyloid plaques, neurofibrillary tangles, synaptic and neuronal loss, neurotransmitter deficits and dementia) are indistinguishable when FAD is compared with typical, common, 'non-familial', or sporadic, AD (SAD). Studies of some clinically relevant mutant APP molecules from FAD families have yielded evidence that APP mutations can lead to the enhanced generation or aggregability of Abeta, consistent with a pathogenic role in AD. Other genetic loci for FAD have been discovered which are distinct from the immediate regulatory and coding regions of the APP gene, indicating that defects in molecules other than APP can also specify cerebral amyloidogenesis and FAD. To date, all APP and non-APP FAD mutations can be demonstrated to have the common feature of promoting amyloidogenesis of Abeta. Epidemiological studies indicate that postmenopausal women on estrogen replacement therapy (ERT) have their relative risk of developing SAD diminished by about one third as compared with age-matched women not receiving ERT [M.X. Tang, D. Jacobs, Y. Stern, K. Marder, P. Schofield, B. Gurland, H. Andrews, R. Mayeux, Effect of estrogen during menopause on risk and age at onset of Alzheimer's disease, Lancet 348 (2000) 429432]. Because of the key role of cerebral Abeta accumulation in initiating AD pathology, it is most attractive that estradiol might modulate SAD risk or age-at-onset by inhibiting Abeta accumulation. A possible mechanistic basis for such a scenario is reviewed here.     

Increased memory load-related frontal activation after estradiol treatment in postmenopausal women

Prior research shows that menopause is associated with changes in cognition in some older women. However, how estrogen loss and subsequent estrogen treatment affects cognition and particularly the underlying brain processes responsible for any cognitive changes is less well understood. We examined the ability of estradiol to modulate the manipulation of information in working memory and related brain activation in postmenopausal women. Twenty healthy postmenopausal women (mean age (SD) = 59.13 (5.5)) were randomly assigned to three months of 1 mg oral 17-β estradiol or placebo. At baseline and three months later each woman completed a visual verbal N-back sequential letter test of working memory during functional magnetic resonance imaging (fMRI). The fMRI data showed that women who were treated with estradiol for three months had increased frontal activation during the more difficult working memory load conditions compared to women treated with placebo. Performance on the verbal working memory task showed no difference between estradiol and placebo treated subjects. These data are consistent with prior work showing increases in frontal activation on memory tasks after estrogen treatment. However, this is the first study to show that estrogen-induced increases in brain activity were tied to cognitive load during a verbal working memory task. These data suggest that estradiol treatment effects on cognition may be in part produced through modulation of frontal lobe functioning under difficult task conditions.     

Effects of human apolipoprotein E isoforms on the amyloid beta-protein concentration and lipid composition in brain low-density membrane domains

Apolipoprotein E4 (apoE4) encoded by epsilon 4 allele is a strong genetic risk factor for Alzheimer's disease (AD). ApoE4 carriers have accelerated amyloid beta-protein (A beta) deposition in their brains, which may account for their unusual susceptibility to AD. We hypothesized that the accelerated A beta deposition in the brain of apoE4 carriers is mediated through cholesterol-enriched low-density membrane (LDM) domains. Thus, the concentrations of A beta and various lipids in LDM domains were quantified in the brains of homozygous apoE3 and apoE4 knock-in (KI) mice, and in the brains of those mice bred with beta-amyloid precursor protein (APP) transgenic mice (Tg2576). The A beta 40 and A beta 42 concentrations and the A beta 42 proportions in LDM domains did not differ between apoE3 and apoE4 KI mice up to 18 months of age. The A beta 40 concentration in the LDM domains was slightly, but significantly higher in apoE3/APP mice than in apoE4/APP mice. The lipid composition of LDM domains was modulated in an apoE isoform-specific manner, but its significance for A beta deposition remains unknown. These data show that the apoE isoform-specific effects on the A beta concentration in LDM domains do not occur in KI mouse models.     

Executive function is less sensitive to estradiol than spatial memory: performance on an analog of the card sorting test in ovariectomized aged rhesus monkeys

Functions supported by the frontal lobes are particularly sensitive to the detrimental effects of aging. Recent studies on postmenopausal women find that estrogen replacement therapy benefits performance on tasks dependent on the frontal lobes. To determine whether estrogen has a similar influence in a rhesus monkey model of menopause, we tested five aged, long-term ovariectomized rhesus monkeys in a modified version of the Wisconsin Card Sort test which had been adapted to the nonhuman primate. In this test, monkeys had to select 3-D objects based either on color (blue, red, yellow) or shape (block, tube, cup) and had to be able to switch their response as a function of reinforcement contingencies. The monkeys were treated with placebo and ethinyl estradiol (EE2, 450 ng/kg/day) in alternation with each successive test. Contrary to our hypothesis, estradiol treatment did not affect performance. Because previous studies in the same monkeys [Neurobiol. Aging 23 (2002) 589] had shown that EE2 improves performance on a spatial memory task dependent on the hippocampus, but not on another task dependent upon the frontal lobes (the delayed response), we conclude that executive processes may be less sensitive to the effects of estradiol than hippocampal-dependent tasks.     

Estrogenic action of estriol fatty acid esters

Recent studies suggest that, estriol, like estradiol, is biosynthetically esterified with fatty acids. We have synthesized the stearate estriol, at C-16 alpha, C-17 beta and the diester, C-16 alpha,17 beta and tested these D-ring esters for their estrogenic action both in vivo and in vitro, comparing them to estradiol, estriol and estradiol-17-stearate. None of the estriol esters bind to the estrogen receptor. They are only weakly estrogenic in a microtiter plate estrogen bioassay: stimulation of alkaline phosphatase in the Ishikawa endometrial cells. However, both estriol monoesters are extremely potent estrogens when injected subcutaneously (in aqueous alcohol) into ovariectomized mice. Compared to the free steroids, they produced a dramatically increased uterine weight with a greatly prolonged duration of stimulation. The 16 alpha,17 beta-diester also induced a protracted uterotrophic response, but the stimulation of uterine weight was comparatively low. Since the esters of estradiol and estriol do not bind to the estrogen receptor, their estrogenic signal must be generated through the action of esterase enzymes. These naturally occurring esters have the potential of being extremely useful pharmacological agents for long-lived estrogenic stimulation.