Neuroprotective effects of estrogen against beta-amyloid toxicity are mediated by estrogen receptors in cultured neuronal cells Hee Kim a , Oh Young Bang b , Min Whan Jung c , Sang Duk Ha a , Hyun Seok Hong a , Kyoon Huh b , Seung Up Kim a , Inhee Mook-Jung a, * a Brain Disease Research Center, Ajou University School of Medicine, Suwon 442-721, South Korea c Department of Neurology, Ajou University School of Medicine, Suwon 442-721, South Korea b Institute for Medical Sciences, Ajou University School of Medicine, Suwon 442-721, South Korea Received 15 October 2000; received in revised form 12 February 2001; accepted 18 February 2001 Abstract Although estrogen is known to exert bene®cial effects on Alzheimer's disease, its underlying cellular mechanisms have not been clear. In this study we investigated whether or not neuroprotective effects of estrogen are mediated by estrogen receptors (ERs). Treatment of estrogen (1.8 nM) reduced beta-amyloid (Ab)-induced death of ER-expressing W4 cells. This effect of estrogen was blocked by a speci®c ER blocker ICI 182,780. When estrogen was treated to HT22 cells, which lack functional ERs, Ab-induced cell death was not affected. Transfection of HT22 cells with human ERa, but not ERb, restored protective action of estrogen against Ab. Hoechst staining revealed that estrogen protected ERa-expres- sing cells by blocking Ab-induced apoptosis. These results indicate that estrogen blocks Ab-induced cell death via ERa- dependent pathways. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Estrogen; Estrogen receptor; Beta amyloid; Alzheimer's disease; Cell death; Apoptosis Recently much attention has been given to estrogen for its bene®cial effects on Alzheimer's disease (AD) [17]. Several clinical studies have shown that post-menopausal women who are under estrogen replacement therapy are less likely to develop AD compared to those who are not, and that estrogen use is associated with a delayed onset of AD [8,20]. The underlying mechanisms of the estrogen effects on AD are currently unclear, but estrogen has many actions in the brain that are potentially bene®cial. Estrogen is known to boost the production of acetylcholine [3], decrease production of beta-amyloid (Ab) [19], increase a-secretase derived amyloid precursor protein (sAPPa) production [7], improve blood ¯ow through the brain [6] and maintain the integrity of the hippocampus [18]. In addition, estrogen blocks Ab-induced neuronal cell death in vitro [1] and increases expression of an anti-apoptotic protein Bcl-xL in cultured hippocampal neurons [14]. In general, estrogen is known to exert its effects through both estrogen receptor (ER)-dependent and -independent pathways [5,10] and there exist two different receptor subtypes, a and b [9]. Currently little is known about the role of ERs in mediating the bene®cial effects of estrogen on AD. In this study we investigated whether or not neuropro- tective effects of estrogen against Ab toxicity are dependent upon ERs and, if they are, which receptor subtype plays the major role in this process. Cultured W4 and HT22 cells were used in this study. W4 cells, which express ERs, are APP-overexpressing B103 cells [15]. In contrast, HT22 cells do not express functional ERs [5]. For selective expression of ERa and ERb, HT22 cells were transfected with human ERa cDNA with subcloning into pBabe vector containing a puromycin resis- tant gene for selection (5 mg/ml of puromycin, Sigma, St. Louis, MO) or human ERb cDNA with 25 mg/ml of G418 selection. All transfections were performed using lipofecta- mine (Gibco-BRL, Grand Island, NY). Before factor treat- ment, to make a steroid-free condition, cells were cultured in phenol red-free Dulbecco's modi®ed Eagle's medium (DMEM) (Gibco-BRL, Grand Island, NY) containing 10% charcoal stripped fetal bovine serum (FBS) (Hyclone, Irvine, CA) for 48 h. Various combinations of the following factors were treated for 18 h: 25 mMAb 25±35 (US Peptides, Fullerton, CA); 1.8 nM 17-b-estradiol (Sigma, St. Louis, MO) and 100 nM ICI 182,780 (Tocris, United Kingdom). Neuroscience Letters 302 (2001) 58±62 www.elsevier.com/locate/neulet 0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(01)01659-7 * Corresponding author. Tel.: 182-31-219-4554; fax: 182-31- 216-6381. E-mail address: inhee@madang.ajou.ac.kr (I. Mook-Jung).