Estradiol protects against β-amyloid (25–35)-induced toxicity in SK-N-SH human neuroblastoma cells

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Neuroscience Letters 218 (1996) 165-168

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Estradiol protects against/ -amyloid (25-35)-induced toxicity in SK-N-SH human neuroblastoma cells P a t t i e S. G r e e n , K e l l y E. G r i d l e y , J a m e s W . S i m p k i n s * Department of Pharmacodynamics and the Center for Neurobiology of Aging, University of Florida, Gainesville, FL 32610, USA Received 5 August 1996; revised version received 3 October 1996; accepted 3 October 1996

Abstract

Estrogen-replacement therapy has been associated with a reduced incidence of Alzheimer's disease (AD) and improved cognition in several small open clinical trials. We assessed the possibility that estrogens may reduce toxicity of ~-amyloid (A/3)by testing the effects of/~-estradiol on the toxic.ityof the neurotoxic fragment of ~-amyloid (AB 25-35) in SK-N-SH neuroblastoma cells. A/3 25-35 caused a dose-dependent death in SK-N-SH cells with a LDs0of 28.9 #M. In cultures simultaneously exposed to 20 #M A/3 and 17 ~-estradiol (2 nM), A/~-induced toxicity was reduced by 83 and 51% in two separate studies. Further studies show that 0.2 nM 17 ~-estradiol was as effective as the 2 nM concentration. 17 ~-Estradiol (2 riM) conferred neuroprotection equivalent to that of 17 ~-estradiol. These data support the hypothesis that estrogens reduce/3-amyloid toxicity and this may help explain the beneficial effects of estrogens in AD.

Keywords: Estradiol; Estrogens;/3-Amyloid; SK-N-SH cells; Alzheimer's disease

/3-Amyloid (A/3) is a hydrophobic, non-glycosylated peptide of 39-43 amino acids that is thought to be generated from amyloid precursor protein (APP) by its processing through a lysoson~tal pathway, as opposed to the nonamyloidogenic secretory pathway (see [16] for review). The production of these insoluble fragments results in their aggregation which forms the core substance in senile plaques, a hallmark of post-mortem diagnosis of Alzheimer's disease (AD). The neurotoxicity of A/~ 1-40 and 1-42 has been demonstrated through studies of primary neuronal cultures and PC-12 cells, where the addition of the pre-aggregated forms of these peptides results in a profound disruption of cellular morphology and cell death [27,28]. These effects of A/~ appear to require the formation of aggregates [5,20] and physical contact with the plasma membrane [7]. Additionally, A/~ toxicity has been shown in transgenic mice that over express APP [6]. The toxic portion of the A/3 molecule appears to be the 25-35 fragment [14]. This A/3 25-35 fragment causes cell death in primary neuronal cultures with a dose-response and time-course similar to * Corresponding author. Box 100487, JHMHC, University of Florida, Gainesville, F1 32610, USA. Tel.: +1 352 3928509; fax: +1 352 3929364.

that observed with A/3 1-40 [7] and has the added feature of rapid aggregation in aqueous solutions [7,14]. Paganini-Hill and Henderson showed that post-menopausal estrogen-replacement therapy (ERT) is associated with a 40% reduction in the incidence of Alzheimer's disease in a retrospective evaluation of a large Southern California population [9,19]. Their data further indicate that the effects of estrogens are dose and duration dependent. Estrogens could influence A/3 toxicity by altering APP processing, preventing the formation of A/~ aggregates, or interfering with the effects of A/3 fragment on neurons once fragments aggregate. Using a high estrogen-receptor expressing human breast carcinoma cell line, Jaffe et al. [11] demonstrated that exposure to 2 x 10 -9 M 17 ~-estradiol increased by 1.75-fold the amount of secreted, soluble form of APP, suggesting that estrogens may stimulate the non-amyloidogenic processing of APP. Alternatively, estrogens could prevent A/3 toxicity by slowing the aggregation of A/3 or by directly protecting neurons. The present study was undertaken to determine if in vitro exposure to estrogens affects A/~ neurotoxicity. SK-N-SH cells were obtained from American Type Tissue Collection (Rockville, MD, USA). Cell cultures were grown to confluency in RPMI-1640 media supplemented

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with 10% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100/zg/ml streptomycin (all reagents from Sigma Chemical Corporation, St. Louis, MO, USA) in monolayers in plastic Coming 150 cm 2 flasks (Fisher Scientific Inc., Orlando, FL, USA) at 37°C and under 5% CO2 and 95% air. Media was changed three times weekly. Cells were observed with a phase contrast microscope (Nikon Diaphot-300). SK-N-SH cells were backcultured every 5-7 days to maintain the cell line and the ceils used in the following experiments were in passes four to seven. The growth media was initially decanted and the cells were rinsed with 0.02% EDTA which was discarded. Cells were suspended after incubation with 0.02% EDTA for 30 min at 37°C. Cells were then counted on a Neubauer hemacytometer (Fisher Scientific) and resuspended in appropriate media. Cells were plated at a density of 1.0 x 106 cells/ml in 24-well plates (Fisher Scientific Inc.). Experiments were initiated by the backculturing of SKN-SH cells with 0.02% EDTA as described above. Cells were suspended and then centrifuged at 1000 rev./min for 5 min. The cell pellet was resuspended in the appropriate treatment media at a concentration of 1.0 × l 0 6 cells/ml. In all studies, cells were cultured in RPMI-1640 with 10% FBS alone or supplemented with the addition/3-amyloid peptide, (A/3 25-35; Bachem, Torrance, CA, USA), 17/3estradiol (17 ~-E2; Pharmos Inc., Alachua, FL, USA) or 17 ot-estradiol (17 c~-E2; Sigma Chemical Corp.). Both estrogens were initially dissolved at 1 mg/ml in absolute ethanol and diluted in RPMI-1640 to a final concentration of 0.2 or 2 nM. Lyophilized A~ 25-35 (1 mg) was initially dissolved in 200/zl ddH20 then 800/xl phosphate-buffered saline (PBS) was added. A~ 25-35 was then diluted in RPMI-1640 to a final concentration of 10, 20, 40 or 80 #M. Cell viability was assessed at 96 h of treatment using the trypan blue dye exclusion method [4]. At the appropriate A m

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time, cell suspensions were made by decanting media, rinsing each well with 0.02% EDTA, then incubating cells with 0.2 ml 0.02% EDTA for 30 min at 37°C. Cells were suspended by repeated pipetting. Aliquots (100 #1) from each cell suspension were incubated with 100/~1 of 0.4% trypan blue stain (Sigma Chemical Corp.) for 5 min at room temperature. All suspensions were counted on a Neubauer hemacytometer within 10 min of addition of trypan blue. Two independent counts of live and dead cells were made for each aliquot. All statistical evaluations used analysis of variance followed by Scheffe's test. P = 0.05 was considered significant. Each group consisted of 4- or 5-wells per group. Four days of exposure of SK-N-SH cells to A~ 25-35 resulted in a dose-dependent loss of trypan blue excluding cells with a LDs0 of 28.9/~M (Fig. 1). A~ reduced cell number by 36, 65, 70 and 83% at the 10, 20, 40 and 80 #M doses, respectively. This neurotoxic fragment of A/3 caused shrinking of soma, fragmentation of neurites and the accumulation of cellular debris in the culture wells. Addition of 17/~-E2 (2 nM) reduced A/3 (20/zM) toxicity by 83 and 51% in two separate studies that used different A/3 lots and SK-N-SH cultures (Fig. 2). The estrogen-treated cultures showed evidence of more neuronal-type cell morphology and intact neurites. In a third and final study we assessed the dose-dependence of the effects of 17/3-E2 on the neurotoxicity of A~ and assessed a single dose of 17 c~-E2. Treatment with 20 /xM A/3 25-35 caused a 43% death of SK-N-SH cells,

P.S. Green et aL / Neuroscience Letters 218 (1996) 165-168

while treatment with 17/3-E2 (0.2 and 2 nM) or 17 o~-E2 (2 nM) alone caused no significant effect on cell number (data not shown). Both doses of 17 /3-E2 and the single dose of 17 or-E2 tested eliminated most of the cell loss due to Al3 25-35 treatment (Fig. 3). The present study demonstrates for the first time that physiologically relevant concentrations of estradiol can attenuate the toxicity associated with cellular exposure to a neurotoxic fragment of A/3. Recently published results by Behl et al.[2] and Goodman et al. [8] have shown that 17 /3-E2 at #M concentrations demonstrates beneficial effects in primary rat cultures in response to A/3 toxicity. These observations may help explain the observed association of ERT with reduced incidence of AD [9,19]. The neuroprotective concentrations of 17/3-E2 used in the present study are in the range of circulating levels in premenopausal women (0.2 n M = 54 pg/ml) and in women on postmenopausal ERT (2 nM = 544 pg/ml). Estrogens are neuroprotective in a variety of in vitro models. We have shown estrogens to be effective neuroprotectants in SK-N-SH cultures against serum-deprivation [3,23] and hypoglycemia [23]; further we [23] and Singer et al. [24] observed a protection of rat primary cortical neurons from N-methyl-D-aspartate (NMDA)induced and glutamate-induced toxicity, respectively. The present observation regarding the efficacy of estradiol at physiologically relevant concentrations against A#induced toxicity suggests that this ovarian steroid may play a fundamental role in the protection of neurons from a variety of insults. The SK-N-SH cell line is an appropriate in vitro model for the assessment of the neurotoxic effects of A/3. This 1.2 A

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cell line is derived from a human female subject, shows a neuronal phenotype and is estrogen responsive [3,22,23]. Additionally, the dose-dependence of the A/3 toxicity in SK-N-SH cells is very similar to that reported for primary cortical neurons and PC 12 cells in vitro [27,28] and the ease with which SK-N-SH cells can be cultured makes them very useful in the assessment of neurotoxicity and neuroprotection, Estrogens could prevent Al3-induced toxicity through a variety of mechanisms. A/3 has been shown to increase membrane lipid peroxidation [1,10] and estrogens have been shown in several systems to reduce peroxidation [18,26]. Additionally, the protective effects of estrogen against NMDA-induced toxicity [22,24] may be important as A/3 has been shown to increase the neurotoxicity of NMDA [12,14] and estrogens could exert the observed effects by reducing excitatory amino acid effects on cells. Finally, estrogens have been shown to induce the expression of neurotrophins and their receptors [17,25], an effect that could provide protection from A/3 toxicity

]15j. While the precise mechanism of the estrogen protection from A/3 insult is not known, the present results suggest that interaction with the estrogen receptor may not be required. 17 or-E2 is only a weak estrogen-receptor agonist [13], but it appears to be as effective as the potent estrogen, 17/3-E2, against A~ toxicity (present report) and serumdeprivation toxicity [21]. Tamoxifen antagonized only a small part of the estrogen effect [21]. Recently, Behl et al. [2] have shown that high concentrations of 17 /3-E2 reduced A/3 toxicity in a cell line lacking the estrogen receptor, adding support to a non-genomic mechanism of the neuroprotective effects of estrogens. Collectively, these results suggest that estrogens may normally serve to protect neurons from the toxicity of A/3 and that ERT may be a useful therapy for preventing the toxic effects of A/3 in AD. The authors thank Victoria Redd for typing and editorial review of this manuscript and Dr. Christopher de Fiebre for a program to calculate LDs0. Supported by NIH grants AG 10485, T32-NS07333 and a grant from Apollo Genetics, Inc. (Cambridge, MA, USA).

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Treatment Fig. 3. Effects of 17/3-estradiol (0.2 or 2 nM) and 17 a-estradiol (2 nM) on the toxicity induced by A/3 25-35 (20/xM; lot QM501). Cell were plated at 106 cells/ml and were exposed to vehicle, 17/3-estradiol, 17 otestradiol and A/3 25-35, alone or in combination. Four days later, the number of trypan blue-excluding cells was determined. Depicted are mean + SEM for 5-wells/group. *P < 0.05 versus control group. Separate groups (n = 5-wells/group) were exposed to 17 t3-estradiol (0.2 or 2 nM) or 17 a-estradiol (2 nM) without the addition of A/3. The steroid addition had no effect on cell number (data not shown).

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