Mechanisms of Estrogenic Protection against gp120-Induced Neurotoxicity

Experimental Neurology 168, 385–391 (2001) doi:10.1006/exnr.2000.7619, available online at http://www.idealibrary.com on

Mechanisms of Estrogenic Protection against gp120-Induced Neurotoxicity Sarah A. Howard, Sheila M. Brooke, and Robert M. Sapolsky Department of Biological Sciences, Stanford University, Stanford, California 94305 Received June 27, 2000; accepted November 27, 2000

gp120, an HIV coat glycoprotein that may play a role in AIDS-related dementia complex (ADC), induces neuronal toxicity characterized by NMDA receptor activation, accumulation of intracellular calcium, and downstream degenerative events including generation of reactive oxygen species and lipid peroxidation. We have previously demonstrated estrogenic protection against gp120 neurotoxicity in primary hippocampal cultures. We here characterize the mechanism of protection by blocking the classical cytosolic estrogen receptors and by measuring oxidative end points including accumulation of extracellular superoxide and lipid peroxidation. Despite blocking ER␣ and ER␤ with 1 ␮M tamoxifen, we do not see a decrease in the protection afforded by 100 nM 17 ␤-estradiol against 200 pM gp120. Additionally, 17␣-estradiol, which does not activate estrogen receptors, protects to the same extent as 17␤-estradiol. 17␤-Estradiol does, however, decrease gp120-induced lipid peroxidation and accumulation of superoxide. Together the data suggest an antioxidant mechanism of estrogen protection that is independent of receptor binding. ©

2001 Academic Press

Key Words: gp120; ROS; estradiol; tamoxifen; superoxide; lipid peroxidation; estrogen; hippocampus.

INTRODUCTION

Approximately 20% of HIV patients develop AIDSrelated dementia complex (ADC), consisting of neurologic and neuropsychologic impairments. The HIV glycoprotein gp120 has been implicated in neurotoxicity in vivo, in vitro, and in transgenic models. It may play a role in ADC through directly toxic actions on neurons, perhaps via chemokine receptors, or indirectly by inducing macrophages or microglia to secrete neurotoxins. gp120’s toxicity is characterized by release of cytokines, NMDA-receptor activation, calcium mobilization, increased expression of nitric oxide synthase, decreased expression of nerve growth factor, generation of reactive oxygen species (ROS), and decreased glucose utilization (reviewed in (30)).

Estrogens have been cited to promote neuronal survival and health in a number of scenarios. They protect against excitatory amino acids (47), ␤-amyloid (20), and glucose and serum deprivation (4, 13, 19). They enhance the health of neurons by promoting regulation of calcium homeostasis (34, 38), by decreasing the production of oxygen radicals (3, 27, 36), by increasing release of neurotrophins, and by promoting dendritic growth (33). In clinical studies, estrogen treatment has decreased the severity of Alzheimer’s disease in postmenopausal women (22, 43). We have previously demonstrated 17␤-estradiol attenuation of gp120-induced neurotoxicity and of gp120-induced calcium mobilization in hippocampal cultures (6). We now look to the mechanism of protection. Estrogens can operate either genomically by binding to receptors and translocating to the DNA to modify transcription or through nongenomic/non-receptor-mediated mechanisms, which include anti-oxidation (reviewed in (32)). Receptor independent mechanisms typically occur more rapidly than receptor mediated ones, and are characterized by incremental increases in effect at concentrations above the K d for the estrogen receptor. We here explore whether estrogen’s protection against gp120 neurotoxicity is receptor dependent by blocking the estrogen receptors with tamoxifen, an antagonist with partial agonist properties (2, 44). Additionally, we test whether 17␣-estradiol, which shares 17␤-estradiol’s antioxidant properties (36) but lacks the ability to activate the estrogen receptors (reviewed in (18)), is capable of protecting against gp120 neurotoxicity. Finally, we question if estrogenic protection against gp120 neurotoxicity could depend on estrogenic antioxidant properties by examining to what extent estrogen inhibits gp120-induced lipid peroxidation and accumulation of superoxide. MATERIALS AND METHODS

Preparation of Cultures Hippocampi were removed from 18-day-old fetal Sprague–Dawley rats. Cells were dissociated with pa-

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0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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pain, filtered through an 80-␮m cell strainer and resuspended in a modified MEM media (UCSF Tissue Culture Facility, San Francisco, CA) containing 30 mM glucose and 10% horse serum (Hyclone, Logan, UT) (6). Cells were plated at a density of 20,000/cm 2 on 12-well plates for the ROS studies, 24-well plates for the lipid peroxidation studies, and on 96-well plates for the toxicity studies. Plates used were dictated by the demands of each assay; lipid peroxidation and superoxide studies require a large quantity of cells while the toxicity studies require reading 96 well plates on a fluorescence plate reader. Cells were maintained in this media for 10 –12 days before use at which time approximately 20 –30% of cells were neuronal, as assessed by cell counting. Solutions Recombinant gp120 (HIVSF2gp120 Austral, Novato, CA and HIV-1 IIIB from Bartels, Inc., Issaquah, WA) was dissolved in a phosphate-buffered saline solution containing 1 mM EDTA and 1 mM EGTA. Aliquots of stock solution were kept at ⫺80°C and diluted in MEM Eagle’s media (UCSF Tissue Culture facility, San Francisco, CA) to 200 pM just prior to use. Previous studies of ours and of other labs have shown that this gp120 from a T-cell tropic strain of HIV induces toxicity in neuronal cultures (6, 8, 23, 24, 35). In order to study the previously observed estrogenic effect on its toxicity, we have chosen to continue to work with this gp120. Additionally, heat denaturing the gp120 eliminated its toxicity (6). 17␤-Estradiol, 17␣-estradiol, and tamoxifen (Sigma, St. Louis, MO) were stored at 1 and 10 mM in ethanol. In control cultures, equivalent volumes of respective vehicles were used. In all experiments, 17␤-estradiol is used at 100 nM, which has been shown to attenuate gp120 neurotoxicity (6). Tamoxifen in 10-fold excess of estradiol has been shown to block its receptor-mediated effects (19) and was used here at 1 and 3 ␮M, 10- and 30-fold excess over the estradiol concentration used. Toxicity Studies Cells were incubated in MEM containing gp120 alone; gp120 ⫹ 17␤-estradiol, 17␣-estradiol, tamoxifen; gp120 ⫹ 17␤-estradiol ⫹ tamoxifen; or control vehicle for 72 h. Amount of toxicity was determined by an immunocytochemistry protocol previously described (7). Briefly, cells were incubated with a primary antibody against neuronal MAP-2 (Sigma) diluted 1:1000 in phosphate-buffered saline (PBS) plus 5% milk followed by incubation with a rat adsorbed biotinylated secondary IgG antibody (Vector, Burlingame, CA). Manufacturer’s protocols for preparing and incubating with ABC reagent were followed (Vector). Finally ABTS (2,2⬘-azino-bis(3ethylbenzthiazoline-6-sulfonic acid), Vector) was added and produced a color change

in proportion to the amount of MAP-2. Absorbance at 405 nm was measured on a plate reader. Wells treated with cold media to selectively kill neurons served as blanks and were subtracted from absorbance readings. Data were presented as a percentage of control for each plate. Lipid Peroxidation Studies The amount of lipid peroxidation was determined by measuring the loss of fluorescence due to peroxidation of the naturally fluorescent fatty acid cis-parinaric acid (21, 23, 26, 29). Hippocampal cultures were treated for 24 h, a time at which little toxicity is seen and at which damage from dangerous radicals might be expected, with control or experimental solutions (gp120, estradiol, gp120 ⫹ estradiol, or control). After pretreatment, cis-parinaric acid (Molecular Probes, Eugene, OR) was added to the wells to a final concentration of 10 ␮M and incubated an additional 3 h. Media was aspirated and cells were suspended in phosphate-buffered saline solution for analysis. Fluorescence was measured on a Perkin-Elmer LS50B spectrometer with excitation 312 nm and emission 414 nm. Blanks received no cis-parinaric acid and were subtracted from fluorescence readings. Data were expressed as a percentage of control for each experiment. Because cis-parinaric acid is light sensitive, all manipulations were performed in the dark. Superoxide Studies Extracellular superoxide levels were determined by the amount of reduction of cell impermeable acetylated cytochrome c. Cultures were exposed for 24 h to media ⫾ estradiol. For superoxide determination, medium was removed and cells washed once with KRPH buffer at 37°C. KRPH buffer containing 60 ␮M partially acetylated cytochrome c (Sigma) and one of the treatments (gp120, estradiol, gp120 ⫹ estradiol, or control) was added. After 30 min, the KRPH solution was removed and diluted to 1.5 times the initial concentration in distilled, deionized water. The amount of reduction of acetylated cytochrome c was measured on a spectrophotometer as the difference between the second derivatives of the absorption at 248 and 256 nm (1, 28). This amount was standardized to the total amount of cytochrome c present by dividing it by the corresponding value after complete reduction with sodium hydrosulfite (Sigma). Values were standardized for protein levels with a Pierce assay (Pierce, Rockford, IL), and data were expressed as a percentage of control for each experiment. Data Analysis and Statistics For all toxicity studies, only plates showing at least a 10% decrease in toxicity with gp120 were used. In the

ESTROGENIC PROTECTION AGAINST gp120 NEUROTOXICITY

toxicity studies, each plate’s data were expressed as a percentage of control for that plate. The average of all control wells on each plate was set to zero toxicity, and the data were scaled appropriately. Data from all plates over multiple experiments were then averaged and the standard error calculated. For the lipid peroxidation and superoxide studies, all of the control wells for a single experiment, including several plates, were averaged and set to zero. As in the toxicity studies, data were scaled appropriately and standard errors figured after the accumulation of data from multiple experiments. For all studies, one-way analysis of variance (ANOVA) was carried out followed by Student– Newman–Keuls post hoc tests. Error bars indicate standard error, and significance was set at the 0.05 level. RESULTS

In concurrence with our previous results, 200 pM gp120 was significantly toxic compared to control in hippocampal cultures (Fig. 1). As our prior data indicated (6), simultaneous treatment with 100 nM 17␤estradiol completely attenuated the gp120-induced toxicity. Addition of the estrogen receptor antagonist tamoxifen (1 or 3 ␮M) did not block the estradiol protective effect (Figs. 1a and 1b). 17␣-Estradiol (100 nM), like 17␤-estradiol, completely attenuated the gp120 toxicity (Fig. 1c). A lower concentration (10 nM) of 17␣-estradiol with gp120 did not significantly change levels of toxicity from control or from gp120 alone (data not shown). We also examined gp120’s effects on ROS. gp120 caused a significant increase in extracellular superoxide accumulation relative to control cultures (Fig. 2). In contrast, 17␤-estradiol alone caused a significant decline. Treatment with gp120 and 17␤-estradiol together did not change accumulation from control levels. Downstream of an increase in ROS, gp120 induced a significant increase in lipid peroxidation above control cultures (Fig. 3). In contrast, 17␤-estradiol alone had no significant effect on lipid peroxidation. 17␤-Estradiol significantly attenuated gp120-induced lipid peroxidation. DISCUSSION

The well-established neurotoxicity of gp120 suggests that it might contribute to ADC. As reviewed in the introduction, gp120 induces an NMDA receptor-dependent cascade involving calcium mobilization and downstream degenerative events. We have previously demonstrated that gp120 increases superoxide and hydrogen peroxide levels, as well as lipid peroxidation in primary hippocampal cultures (23). Additionally, gp120 has been found to increase hydrogen peroxide

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levels and to decrease the ratio of glutathione to glutathione disulfide in CD4⫹ Jurkat cells (45). Downstream of an increase in ROS, gp120 increases lipid peroxidation in human neuroblastoma CHP100 cells (31) and in human monocytic U93 cells (15). Generation of ROS can occur through a number of pathways including conversion of xanthine dehydrogenase to xanthine oxidase, activation of nitric oxide synthase, liberation of arachidonic acid by phospholipases, and disruption of oxidative phosphorylation. One mechanism by which gp120 in particular may induce accumulation of ROS is through increased activity of iNOS and nitric oxide (14). There is evidence that the oxidative responses to gp120 are required for toxicity, in that the antioxidants ascorbic acid and glutathione block gp120 toxicity in Th1 cells (40), and superoxide dismutase decreases toxicity in primary cortical cultures (10). Additionally, blocking lipid peroxidation prevents gp120 toxicity in neuroblastoma cells (8). As reported previously, estradiol attenuates gp120 toxicity; this appears to be steroid specific in that protection was not seen with progesterone, or a variety of glucocorticoids (brooke endocrine). This protection may occur through a receptor-dependent and/or independent mechanism. There is evidence for beneficial estrogen effects through either mechanism. Estrogen protection against glutamate toxicity in primary cortical cultures is thought to require receptor binding (47) and evidence is accumulating to explain the mechanism of this receptor mediated estrogenic effect. Downstream of receptor binding, estradiol modulates bcl-2 levels after ischemia in the cerebral cortex (12, 46). Receptor binding also leads to activation of the AP-1 responsive element (48, 50), increased NMDAR1 protein production in the hippocampus (16), increased CREB expression (39), and decreased ␤-amyloid generation (54). The classical cytosolic receptor, however, is not required for protection against hemoglobin or kainate toxicity in cortical cultures (42). Receptor binding is also not required for the estrogenic effects on dendritic growth in cultured neocortical neurons (5). These receptor-independent estrogen effects may reflect the antioxidant properties of the hormone. In concurrence, estradiol decreased lipid peroxidation induced by iron sulfate and A␤ both in cells and in isolated membranes (17). Additionally, estrogenic molecules including 17␤estradiol, 17␣-estradiol, and estriol reduced accumulation of free radicals in the absence of living cells or cell extracts. Other steroids including testosterone, progesterone, and cortisol did not display antioxidant properties (36). This leads to the well established hypothesis that receptor independent effects are due to the specific structure of estrogens. A comparison of various phenolic molecules, including 17␤-estradiol, showed similar protection against glutamate or hydrogen peroxide

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FIG. 2. 17␤-Estradiol (100 nM) attenuated 200 pM gp120-induced increase in superoxide. gp120 significantly increases superoxide accumulation. Addition of estradiol with gp120 prevents an increase above control levels. * P ⬍ 0.05 significant difference from control. n, number of wells analyzed.

in HT22 cells, different degrees of binding ER and induction of ERE controlled genes, but similar prevention of membrane and LDL oxidation (37). Because phenolic compounds with as few as one ring have comparable antioxidant properties with estradiol, estradiol’s antioxidant properties have been attributed to its single phenolic ring (37). In agreement, those steroids, including testosterone, progesterone, and the glucocorticoids, which lack a phenolic ring also do not have antioxidant properties. Receptor-independent effects may also be due to the four-ring steroidal structure and specific stereochemistry of estrogens which allow 17␤estradiol and tamoxifen, but to a much less extent 17␣-estradiol, to decrease membrane fluidity (11, 53). In addition to the cases in which there is evidence for either antioxidant- or receptor-mediated effects, it is not clear which mechanism is involved in estrogenic protection against NMDA toxicity (42), glucose deprivation and FeSO 4 (17), and, until now, gp120-induced neurotoxicity. In our study, blocking the estrogen receptor with tamoxifen caused no change in estrogen’s effects on gp120 toxicity. We have used concentrations of this estrogen receptor antagonist at a 10-fold molar excess over estradiol. This excess of tamoxifen has previously been shown to reduce estrogenic effects (19). Additionally, tamoxifen binds ER␣ and ER␤ with similar affin-

FIG. 1. Estrogenic protection against gp120 neurotoxicity in hippocampal cultures is not receptor mediated. (a, b, c) Exposure to gp120 for 72 h induced significant toxicity. Addition of 17␤-estradiol (␤E) completely attenuated the toxicity. Tamoxifen (3 ␮M (a), 1 ␮M (b)) did not block 17␤-estradiol protection against gp120-induced toxicity. (c) 17 ␣-Estradiol (␣E) alone was protective against gp120

toxicity. Conditions: 200 pM gp120, 100 nM 17␤-estradiol; all other concentrations as shown. gp, gp120; ␣E, 17␣-estradiol; ␤E, 17␤estradiol; and T, tamoxifen. Numbers refer to number of wells analyzed. (#) and (##) refer to significant difference from control with P ⬍ 0.05 and P ⬍ 0.01, respectively. (*) and (**) refer to significant differences from gp120 with P ⬍ 0.05 and P ⬍ 0.01, respectively.

ESTROGENIC PROTECTION AGAINST gp120 NEUROTOXICITY

FIG. 3. 17␤-Estradiol (100 nM) attenuated 200 pM gp120-induced increase in lipid peroxidation in hippocampal cultures. Gp120 induced a significant increase in lipid peroxidation. Addition of estradiol with gp120 decreased lipid peroxidation significantly below that of gp120 alone. ## P ⬍ 0.01 significant increase from control. * P ⬍ 0.05 significant difference from gp120. n, number of wells analyzed.

ity (25). This leads to the interpretation that the protective estradiol effect against gp120 toxicity is not mediated through either receptor. There is a trend toward the higher concentration (3 ␮M) of tamoxifen enhancing protection when combined with estradiol. The level of toxicity, however, is not significantly different than control or than gp120 ⫹ 17␤-estradiol. This trend may be due to tamoxifen’s minor agonist (2, 41, 44, 49) or antioxidant properties (51–53). Finally, 17␣-estradiol, which has a steroidal ring structure and antioxidant properties (36) but does not activate estrogen receptors (reviewed in (18)), was also protective. This further supports our interpretation that the protective estrogenic effect is not receptor mediated. A receptor independent mechanism is typically supported by evidence demonstrating an increase in effect even at concentrations above the receptor K d. We previously carried out a dose–response study and observed (6) that a minimum of 1 nM estradiol was needed for protection against gp120, which would be considerably above the K d for ER␣ (0.1 nM) and somewhat higher than that of ER␤ (0.4 nM). Under the paradigm used in our previous study (6), 1 nM estradiol had completely eliminated gp120-induced damage, making it impossible to detect further protection. If estradiol is not protecting via its receptor, it may instead depend on its antioxidant and steroidal properties to decrease gp120 toxicity. We see here that estradiol decreased gp120-induced lipid peroxidation to levels of control. To this end, estradiol may decrease the generation of radicals or prevent their destructive

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behavior by protecting cell membranes or by converting radicals to harmless intermediates. We further demonstrate that estradiol decreased extracellular superoxide accumulation induced by gp120, which may partially account for the decrease in lipid peroxidation. Notably, in our data, the magnitudes with which lipid peroxidation and superoxide accumulation increased differed by 10%. This may be due to examining different time points postinsult, or may indicate that there is not a one-to-one correlation between amount of superoxide and amount of lipid peroxidation. Indeed, oxygen radicals are thought to start a cascade of peroxidation within lipid membranes (9). In conclusion, we have further characterized gp120 neurotoxicity and the mechanism of estrogenic protection against gp120. Importantly, we have shown that estrogenic protection is not mediated by the classical estrogen receptor, but could be due to decreases in lipid peroxidation and in superoxide accumulation. Further insight into these protective effects could pave the way for prospective use of estrogens to attenuate facets of ADC, much as is now being pioneered with Alzheimer’s disease. ACKNOWLEDGMENTS Support was provided by NIH RO1 MH53814 to R.M.S. We thank Dr. Rona Giffard for the generous use of the Perkin-Elmer LS 50B spectrometer and Dr. Pat Jones for the use of the plate reader.

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