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Protection against gp120-induced neurotoxicity by an array of estrogenic steroids
Brain Research 958 (2002) 272–276 www.elsevier.com / locate / brainres
Research report
Protection against gp120-induced neurotoxicity by an array of estrogenic steroids Ilona Zemlyak, Sheila M. Brooke, Robert M. Sapolsky* Departments of Biological Sciences and Neurology and Neurological Sciences, Gilbert Laboratory MC 5020, Stanford University, Stanford, CA 94305 -5020, USA Accepted 28 August 2002
Abstract gp120, the coat protein of HIV, can be neurotoxic and is thought to contribute to AIDS-related dementia complex. Such toxicity involves activation of glutamate receptors, mobilization of free cytosolic calcium, and generation of oxygen radicals. We have previously shown that the estrogen 17b-estradiol, in concentrations of 100 nM or higher, lessens the neurotoxicity of gp120 in hippocampal and cortical cultures, blunts gp120-induced calcium mobilization, and lessens the oxidative consequences. In this study, we examined the protective potential of other estrogens. We found gp120 neurotoxicity in hippocampal cultures to be significantly lessened by estrone, equilin and estriol, although with an order of magnitude less potent than 17b-estradiol. We also found all four estrogens to blunt gp120-induced calcium mobilization, with estriol being more efficacious than the other three estrogens. These findings give insight both into the mechanisms of estrogenic protection (e.g. receptor-dependent versus independent actions) as well as into the potential therapeutic use of estrogens against AIDS-related dementia complex. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: gp120; HIV; ADC (AIDS-related dementia complex); 17b-Estradiol; Equilin; Estrone; Estriol; Hippocampus
1. Introduction About 20% of cases of HIV infection involve AIDSrelated dementia complex (ADC). ADC develops in the later stages of AIDS and involves neurologic and neuropsychologic impairments, including loss of memory, confusion, motor deficits, and varied behavioral abnormalities [1]. A possible contributor to AIDS-related dementia complex is the HIV envelope glycoprotein gp120. It may play a role in ADC through directly toxic actions on neurons, via chemokine receptors, or indirectly by inducing macrophages or microglia to secrete glutamatergic excitotoxins and neurotoxic cytokines [4,17]. This then leads to neuron death via the cascade of activation of glutamate receptors, *Corresponding author. Tel.: 11-650-723-2649; fax: 11-650-7255356. E-mail address: [email protected] (R.M. Sapolsky).
mobilization of free cytosolic calcium, and generation of oxygen radicals. The glycoprotein, however, is not toxic to glia. With this understanding of the mechanisms of gp120 neurotoxicity has come an appreciation of some factors that can modulate such toxicity. As one example, glucocorticoids, the adrenal steroids secreted during stress, can exacerbate gp120 neurotoxicity in hippocampal, cortical and striatal cultures [5,6,11,13]. In contrast, the estrogen, 17b-estradiol, has opposite effects, decreasing gp120-induced neurotoxicity, cytosolic calcium mobilization and oxygen radical generation [5,12]. These protective effects are commensurate with an emerging literature showing that estrogens are neuroprotective against a number of excitotoxic and oxidative insults to the nervous system [19,24]. The term ‘estrogen’ refers to a family of steroid hormones and, to date, only 17b-estradiol has been tested for its protective effects against gp120. In this report, we
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03558-8
I. Zemlyak et al. / Brain Research 958 (2002) 272–276
expand on this prior finding, demonstrating the protective effects of the additional and common estrogens equilin, estrone and estriol.
2. Materials and methods
2.1. Primary cultures Hippocampal tissues were removed from 18-day-old fetal Sprague–Dawley rats. Cells were dissociated with papain, filtered through an 80 mm cell strainer and resuspended in a modified MEM (Univ. California, San Francisco, Tissue Culture Facility) containing 30 mM glucose and 10% horse serum (HyClone, Logan, UT). Cells were plated at a density of 187,500 cells / cm 2 on 96-well plates for neurotoxicity studies or on 12 mm glass coverslips (Fisher Scientific, Pittsburgh, PA) at a density of 150,000 cells / cm 2 for calcium studies. The cells were used between days 10 and 12 in culture. These cultures are typically 20–30% neuronal. Media contains Phenol Red, which has known estrogenic activity; however, the concentrations were in a range generally considered negligible [22].
273
mM glucose (HBSSh) for 30 min. Coverslips were placed on the stage with continuous perfusion of HBSSh with or without 200 pM of gp120. Five initial readings prior to gp120 exposure were followed by 60 readings at 20 s intervals afterward. Calcium concentrations in individual cells were determined from the ratio of light intensity at 340 and 380 nm excitation using standard imaging techniques and analysis with METAFLUOR software from Universal Imaging (West Chester, PA). The amount of calcium mobilization was determined by calculating the area under the curve of the calcium concentrations versus time over the 20 min of gp120 exposure. This calculation was done by subtracting the average pre-insult calcium concentration and using an in-house macro program for Excel.
2.4. Statistical analysis All results are expressed as percentage control with the error term indicating S.E.M. One-way analysis of variance (ANOVA), followed by the Tukey test to determine significance between individual experimental conditions was performed on the data. n5the number of wells / group; all data were derived from a minimum of three separate weeks of cultures.
2.2. Toxicity studies Cultures were incubated for 72 h in MEM (but not medium) containing 200 pM gp120 with or without indicated concentrations of equilin, estrone, estriol or 17bestradiol (Sigma, St Louis, MO). Solutions were made just prior to each experiment by diluting appropriate amounts of stock gp120 (made in PBS) and estrogens (made in ethanol) in MEM to the required concentration. Controls used the same vehicles, minus the gp120 and / or estrogen. Cultures were exposed to gp120 and / or estrogens after complete removal of prior medium. After 72 h, medium was removed and cells were fixed for 24 h in 48 C methanol. Neurotoxicity was determined by immunocytochemistry with a primary antibody against neuronal MAP-2 (Sigma). A biotinylated secondary antibody (Vector, Burlingame, CA) was then presented to the primary. After addition of avidin-linked horseradish peroxidase, ABTS (both from Vector) was added. Cleavage resulted in a color change and absorbance at 405 nm was measured on a plate reader. Cold media was added to kill neurons in specific wells that served as blanks [7].
2.3. Calcium imaging Cultures were treated for 24 h with or without 1 mM of equilin, estrone, estriol or 17b-estradiol. Media was then removed and coverslips treated with 4 mM Fura-2AM and 0.005% pluronic F-127 (Molecular Probes, Eugene, OR) dissolved in Hanks balanced salt solution (Gibco, Grand Island, NY) with 50 mM HEPES Buffer at pH 7.4 and 5
3. Results As reported previously [5,6,11,12], 200 pM gp120 caused a loss of approximately 25% of cultured hippocampal neurons, with no toxic effects upon glia (Fig. 1). In agreement with our prior finding, such toxicity was blocked by 100 nM or higher of 17b-estradiol (and with a non-significant trend towards protection at 10 nM) (Fig. 1A). Protection was also afforded by the other estrogens tested. However, equilin (Fig. 1B), estrone (Fig. 1C) and estriol (Fig. 1D) were each an order of magnitude less potent than 17b-estradiol. We then examined the effects of these various estrogens on cytosolic calcium concentrations. In the absence of gp120, they had significant effects on initial basal calcium concentrations (Table 1), with estrone causing a significant increase, and estriol a significant decrease. As reported in numerous previous studies [4,15], gp120 caused a significant mobilization of free cytosolic calcium (Fig. 2). 17b-estradiol, equilin and estrone all significantly decreased this mobilization by approximately 25%, whereas estriol decreased it approximately 45%.
4. Discussion gp120 damages neurons both through direct actions and, of probably greater relevance, via stimulating macrophages and microglia to release excitotoxins and cytokines. We
274
I. Zemlyak et al. / Brain Research 958 (2002) 272–276
Fig. 1. The effect of estrogens on gp120-induced neurotoxicity in hippocampal cultures. 200 pM gp120 was significantly toxic ( [[[ P,0.001) as compared to control. Significant protection was provided by 17b-estradiol, equilin, estrone and estriol. **, ***P,0.01, 0.001, respectively, as compared to gp120.
previously demonstrated that 17b-estradiol reduces gp120 neurotoxicity in hippocampal and cortical cultures [5,12]. This fits into a broad literature showing estrogenic protection against various insults (reviewed in Refs. [19,24]), as well as reports of protection against a neurodegenerative disorder (i.e. Alzheimer’s disease) [10]. In this paper, we replicate the protective effects of 17b-estradiol concen-
trations at or above 100 nM, and protection, albeit with lesser efficacy, with estrone, estriol and equilin. In agreement with our prior studies [5], 17b-estradiol
Table 1 Effects of various estrogens on basal calcium concentrations in hippocampal cultures Treatment
Calcium concentration (nM)
Control 17b-estradiol Equiline Estrone Estriol
21365 205613 20666 25366*** 8862***
n550–116 / group. ***P,0.001, as compared to control, by post-hoc test following one-way ANOVA.
Fig. 2. The effect of estrogens on gp120-induced mobilization of free cytosolic calcium. Cultures were treated with 200 pM gp120. **, ***P, 0.01, 0.001, respectively, as compared with gp120 alone.
I. Zemlyak et al. / Brain Research 958 (2002) 272–276
also reduced gp120-induced free cytosolic calcium mobilization in neurons, as did the other three steroids. As noted, gp120 releases glutamatergic excitotoxins from microglia and macrophages, and the subsequent glutamate receptor stimulation will mobilize cytosolic calcium. Moreover, gp120 can directly stimulate calcium mobilization in neurons via interactions with chemokine receptors [4,17]. Such calcium excess leads to generation of oxygen radicals and cytoskeletal degradation, and is central to the resulting neuron death. Thus, it is plausible that the neuroprotective effects of these estrogens should arise directly from their ability to reduce the calcium mobilization. Our data support such a relationship, in that the orders of potency for decreasing calcium mobilization and blocking neurotoxicity (with 1 mM steroid) were identical, namely estriol.estrone5equilin.17b-estradiol. A next question is how the profile of estrogenic protection against gp120 compares to estrogenic protection against other insults. Protection against gp120 was particularly potent, requiring lower concentrations of 17bestradiol than against hemoglobin (in cortical cultures [23]), and lower concentrations of 17b-estradiol, equilin and estrone than against kainic acid, NMDA, sodium azide (cortical cultures [23]), or b-amyloid (in cortical cultures or in a hippocampal cell line [2]). The greater potency of 17b-estradiol against gp120 as compared to the other three estrogen does not generalize to other insults (Table 2); 17b-estradiol was significantly more potent in only two of the nine studies. These heterogeneous profiles of potency among the various estrogens raises the issue of the mechanisms of estrogenic protection. As steroid hormones, estrogens have their classical effects via intracellular receptors which, when binding hormone, act as transcriptional regulators. The ERa estrogen receptor has a greater affinity for 17bestradiol than for the other three estrogens [3,15,18]. Superficially, this appears consonant with our finding of
Table 2 Summary of the neuroprotective effects of different estrogens across varied necrotic insults Insult Cortical culture
Pattern of protection Kainic acid NMDA Hemaglobin Sodium azide b-amyloid H2O2
17b-estradiol5estrone5equilin [23] 17b-estradiol5estrone [14] 17b-estradiol.equilin5estrone [23] 17b-estradiol5equilin5estrone [23] equiline5estrone [23] 17b-estradiol5estriol.estrone [2] 17b-estradiol5estriol5estrone [2]
Hipp cultures
gp120
17b-estradiol.estriol5estrone5equilin (the present study)
Hipp cell line
b-amyloid
17b-estradiol5estriol5estrone [2]
Hippocampus
Kainic acid
estrone.estriol [8]
Numbers in brackets refer to citations
275
greater protection against gp120 with 17b-estradiol. However, we have previously observed that the estrogenic protection is receptor-independent, occurring in the presence of an ERa antagonist and mimicked with 17aestradiol, which does not bind to the ERa [12]. The rapidly expanding literature concerning estrogenic neuroprotection contains precedents for receptor-independent actions (reviewed in Refs. [19,24]), although numerous instances of protection are clearly receptor-mediated [9]. There is some understanding of which downstream estrogenic actions are neuroprotective. Within the realm of receptor-mediated effects, estrogens induce expression of neurotrophins, and increase the ratio of anti- to pro-apoptotic molecules [19,24]. Best documented of the receptorindependent effects are the anti-oxidant actions of estrogens. This arises in part from the ability of estrogen to intercalate into membranes, protecting them against peroxidative attack. Estrogens are considerably more potent at this than are other steroids [20], and this is thought to be due to its distinctive phenolic ring structure [21]. gp120 generates oxygen radicals and oxidative damage [4,17], and this is blocked by 17b-estradiol [12]. Just as the neuroprotective potency of the four estrogens does not closely parallel their affinity for ERa, the potency does not closely parallel the antioxidant potency of these estrogens (estriol.17b-estradiol..estrone) [16]. In conclusion, we observe that a variety of estrogens decrease the toxicity of gp120 to hippocampal neurons. Data from this and other studies suggest that the relative protective potency among these estrogens is not a simple consequence of their affinity for the estrogen receptor, or their efficacy as an antioxidant. Prospective trials are now underway testing whether estrogen administration can decelerate the neuropsychological consequences of Alzheimer’s disease; potentially, estrogen could be of therapeutic use against ADC. An understanding of the mechanisms of estrogenic protection against gp120 would aid selecting the particular estrogen which would optimize protection while minimizing some of the (typically, receptor-mediated) cancer risks of estrogen therapy.
Acknowledgements Support was provided by NIH RO1 MH53814 to RS.
References [1] H. Bacellar, A. Munoz, E. Miller, Temporal trends in the incidence of HIV-1-related neurologic diseases: Multicenter AIDS Cohort Study, 1985-1992, Neurology 44 (1994) 1892–1900. [2] C. Behl, T. Skutella, F. Lezoualc’h, A. Post, M. Widmann, C. Newton, F. Holsboer, Neuroprotection against oxidative stress by estrogens: structure–activity relationships, Mol. Pharmacol. 51 (1997) 535–541. [3] B. Bhavnani, C. Woolever, Interaction of ring B unsaturated
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I. Zemlyak et al. / Brain Research 958 (2002) 272–276 estrogens with estrogen receptors of human endometrium and rat uterus, Steroids 56 (1991) 201–210. S. Brooke, R. Sapolsky, The effects of steroid hormones in HIVrelated neurotoxicity: a mini review, Biol. Psychiatry 48 (2000) 881–888. S. Brooke, R. Chan, S. Howard, R. Sapolsky, Endocrine modulation of the neurotoxicity of gp120: implications for AIDS-related dementia complex, Proc. Natl. Acad. Sci. USA 94 (1997) 9457–9462. S. Brooke, S. Howard, R. Sapolsky, Energy dependency of glucocorticoid exacerbation of gp120 neurotoxicity, J. Neurochem. 71 (1998) 1187–1193. S. Brooke, T. Bliss, L. Franklin, R. Sapolsky, Quantification of neuron survival in monolayer cultures using an ELISA approach, rather than by cell counting, Neurosci. Lett. 267 (1999) 21–28. B. Budziszewska, M. Leskiewicz, M. Kubera, L. Jaworska-Feil, M. Kajta, W. Lason, Estrone, but not 17b-estradiol, attenuates kainateinduced seizures and toxicity in male mice, Exp. Clin. Endocrinol. Diabetes 109 (2001) 168–173. D. Dubal, H. Zhu, J. Yu, S. Au, P. Shughrue, S. Merchenthaler, M. Kindy, P. Wise, Estrogen receptor alpha, not beta, is a critical link in estradiol-mediated protection against brain injury, Proc. Natl. Acad. Sci. USA 98 (2001) 1952–1955. V. Henderson, A. Paganini-Hill, C. Emanuel, M. Dunn, J. Buckwalter, Estrogen replacement therapy in older women. Comparisons between Alzheimer’s disease cases and nondemented control subjects, Arch. Neurol. 51 (1994) 896–900. S. Howard, A. Nakayama, S. Brooke, R. Sapolsky, Glucocorticoid modulation of gp120-induced effects on calcium-dependent degenerative events in primary hippocampal and cortical cultures, Exp. Neurol. 158 (1999) 164–170. S. Howard, S. Brooke, R. Sapolsky, Mechanisms of estrogenic protection against gp120-induced neurotoxicity, Exp. Neurol. 168 (2001) 385–391. A. Iyer, S. Brooke, R. Sapolsky, Glucocorticoids interact with gp120
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Research report
Protection against gp120-induced neurotoxicity by an array of estrogenic steroids Ilona Zemlyak, Sheila M. Brooke, Robert M. Sapolsky* Departments of Biological Sciences and Neurology and Neurological Sciences, Gilbert Laboratory MC 5020, Stanford University, Stanford, CA 94305 -5020, USA Accepted 28 August 2002
Abstract gp120, the coat protein of HIV, can be neurotoxic and is thought to contribute to AIDS-related dementia complex. Such toxicity involves activation of glutamate receptors, mobilization of free cytosolic calcium, and generation of oxygen radicals. We have previously shown that the estrogen 17b-estradiol, in concentrations of 100 nM or higher, lessens the neurotoxicity of gp120 in hippocampal and cortical cultures, blunts gp120-induced calcium mobilization, and lessens the oxidative consequences. In this study, we examined the protective potential of other estrogens. We found gp120 neurotoxicity in hippocampal cultures to be significantly lessened by estrone, equilin and estriol, although with an order of magnitude less potent than 17b-estradiol. We also found all four estrogens to blunt gp120-induced calcium mobilization, with estriol being more efficacious than the other three estrogens. These findings give insight both into the mechanisms of estrogenic protection (e.g. receptor-dependent versus independent actions) as well as into the potential therapeutic use of estrogens against AIDS-related dementia complex. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: gp120; HIV; ADC (AIDS-related dementia complex); 17b-Estradiol; Equilin; Estrone; Estriol; Hippocampus
1. Introduction About 20% of cases of HIV infection involve AIDSrelated dementia complex (ADC). ADC develops in the later stages of AIDS and involves neurologic and neuropsychologic impairments, including loss of memory, confusion, motor deficits, and varied behavioral abnormalities [1]. A possible contributor to AIDS-related dementia complex is the HIV envelope glycoprotein gp120. It may play a role in ADC through directly toxic actions on neurons, via chemokine receptors, or indirectly by inducing macrophages or microglia to secrete glutamatergic excitotoxins and neurotoxic cytokines [4,17]. This then leads to neuron death via the cascade of activation of glutamate receptors, *Corresponding author. Tel.: 11-650-723-2649; fax: 11-650-7255356. E-mail address: [email protected] (R.M. Sapolsky).
mobilization of free cytosolic calcium, and generation of oxygen radicals. The glycoprotein, however, is not toxic to glia. With this understanding of the mechanisms of gp120 neurotoxicity has come an appreciation of some factors that can modulate such toxicity. As one example, glucocorticoids, the adrenal steroids secreted during stress, can exacerbate gp120 neurotoxicity in hippocampal, cortical and striatal cultures [5,6,11,13]. In contrast, the estrogen, 17b-estradiol, has opposite effects, decreasing gp120-induced neurotoxicity, cytosolic calcium mobilization and oxygen radical generation [5,12]. These protective effects are commensurate with an emerging literature showing that estrogens are neuroprotective against a number of excitotoxic and oxidative insults to the nervous system [19,24]. The term ‘estrogen’ refers to a family of steroid hormones and, to date, only 17b-estradiol has been tested for its protective effects against gp120. In this report, we
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03558-8
I. Zemlyak et al. / Brain Research 958 (2002) 272–276
expand on this prior finding, demonstrating the protective effects of the additional and common estrogens equilin, estrone and estriol.
2. Materials and methods
2.1. Primary cultures Hippocampal tissues were removed from 18-day-old fetal Sprague–Dawley rats. Cells were dissociated with papain, filtered through an 80 mm cell strainer and resuspended in a modified MEM (Univ. California, San Francisco, Tissue Culture Facility) containing 30 mM glucose and 10% horse serum (HyClone, Logan, UT). Cells were plated at a density of 187,500 cells / cm 2 on 96-well plates for neurotoxicity studies or on 12 mm glass coverslips (Fisher Scientific, Pittsburgh, PA) at a density of 150,000 cells / cm 2 for calcium studies. The cells were used between days 10 and 12 in culture. These cultures are typically 20–30% neuronal. Media contains Phenol Red, which has known estrogenic activity; however, the concentrations were in a range generally considered negligible [22].
273
mM glucose (HBSSh) for 30 min. Coverslips were placed on the stage with continuous perfusion of HBSSh with or without 200 pM of gp120. Five initial readings prior to gp120 exposure were followed by 60 readings at 20 s intervals afterward. Calcium concentrations in individual cells were determined from the ratio of light intensity at 340 and 380 nm excitation using standard imaging techniques and analysis with METAFLUOR software from Universal Imaging (West Chester, PA). The amount of calcium mobilization was determined by calculating the area under the curve of the calcium concentrations versus time over the 20 min of gp120 exposure. This calculation was done by subtracting the average pre-insult calcium concentration and using an in-house macro program for Excel.
2.4. Statistical analysis All results are expressed as percentage control with the error term indicating S.E.M. One-way analysis of variance (ANOVA), followed by the Tukey test to determine significance between individual experimental conditions was performed on the data. n5the number of wells / group; all data were derived from a minimum of three separate weeks of cultures.
2.2. Toxicity studies Cultures were incubated for 72 h in MEM (but not medium) containing 200 pM gp120 with or without indicated concentrations of equilin, estrone, estriol or 17bestradiol (Sigma, St Louis, MO). Solutions were made just prior to each experiment by diluting appropriate amounts of stock gp120 (made in PBS) and estrogens (made in ethanol) in MEM to the required concentration. Controls used the same vehicles, minus the gp120 and / or estrogen. Cultures were exposed to gp120 and / or estrogens after complete removal of prior medium. After 72 h, medium was removed and cells were fixed for 24 h in 48 C methanol. Neurotoxicity was determined by immunocytochemistry with a primary antibody against neuronal MAP-2 (Sigma). A biotinylated secondary antibody (Vector, Burlingame, CA) was then presented to the primary. After addition of avidin-linked horseradish peroxidase, ABTS (both from Vector) was added. Cleavage resulted in a color change and absorbance at 405 nm was measured on a plate reader. Cold media was added to kill neurons in specific wells that served as blanks [7].
2.3. Calcium imaging Cultures were treated for 24 h with or without 1 mM of equilin, estrone, estriol or 17b-estradiol. Media was then removed and coverslips treated with 4 mM Fura-2AM and 0.005% pluronic F-127 (Molecular Probes, Eugene, OR) dissolved in Hanks balanced salt solution (Gibco, Grand Island, NY) with 50 mM HEPES Buffer at pH 7.4 and 5
3. Results As reported previously [5,6,11,12], 200 pM gp120 caused a loss of approximately 25% of cultured hippocampal neurons, with no toxic effects upon glia (Fig. 1). In agreement with our prior finding, such toxicity was blocked by 100 nM or higher of 17b-estradiol (and with a non-significant trend towards protection at 10 nM) (Fig. 1A). Protection was also afforded by the other estrogens tested. However, equilin (Fig. 1B), estrone (Fig. 1C) and estriol (Fig. 1D) were each an order of magnitude less potent than 17b-estradiol. We then examined the effects of these various estrogens on cytosolic calcium concentrations. In the absence of gp120, they had significant effects on initial basal calcium concentrations (Table 1), with estrone causing a significant increase, and estriol a significant decrease. As reported in numerous previous studies [4,15], gp120 caused a significant mobilization of free cytosolic calcium (Fig. 2). 17b-estradiol, equilin and estrone all significantly decreased this mobilization by approximately 25%, whereas estriol decreased it approximately 45%.
4. Discussion gp120 damages neurons both through direct actions and, of probably greater relevance, via stimulating macrophages and microglia to release excitotoxins and cytokines. We
274
I. Zemlyak et al. / Brain Research 958 (2002) 272–276
Fig. 1. The effect of estrogens on gp120-induced neurotoxicity in hippocampal cultures. 200 pM gp120 was significantly toxic ( [[[ P,0.001) as compared to control. Significant protection was provided by 17b-estradiol, equilin, estrone and estriol. **, ***P,0.01, 0.001, respectively, as compared to gp120.
previously demonstrated that 17b-estradiol reduces gp120 neurotoxicity in hippocampal and cortical cultures [5,12]. This fits into a broad literature showing estrogenic protection against various insults (reviewed in Refs. [19,24]), as well as reports of protection against a neurodegenerative disorder (i.e. Alzheimer’s disease) [10]. In this paper, we replicate the protective effects of 17b-estradiol concen-
trations at or above 100 nM, and protection, albeit with lesser efficacy, with estrone, estriol and equilin. In agreement with our prior studies [5], 17b-estradiol
Table 1 Effects of various estrogens on basal calcium concentrations in hippocampal cultures Treatment
Calcium concentration (nM)
Control 17b-estradiol Equiline Estrone Estriol
21365 205613 20666 25366*** 8862***
n550–116 / group. ***P,0.001, as compared to control, by post-hoc test following one-way ANOVA.
Fig. 2. The effect of estrogens on gp120-induced mobilization of free cytosolic calcium. Cultures were treated with 200 pM gp120. **, ***P, 0.01, 0.001, respectively, as compared with gp120 alone.
I. Zemlyak et al. / Brain Research 958 (2002) 272–276
also reduced gp120-induced free cytosolic calcium mobilization in neurons, as did the other three steroids. As noted, gp120 releases glutamatergic excitotoxins from microglia and macrophages, and the subsequent glutamate receptor stimulation will mobilize cytosolic calcium. Moreover, gp120 can directly stimulate calcium mobilization in neurons via interactions with chemokine receptors [4,17]. Such calcium excess leads to generation of oxygen radicals and cytoskeletal degradation, and is central to the resulting neuron death. Thus, it is plausible that the neuroprotective effects of these estrogens should arise directly from their ability to reduce the calcium mobilization. Our data support such a relationship, in that the orders of potency for decreasing calcium mobilization and blocking neurotoxicity (with 1 mM steroid) were identical, namely estriol.estrone5equilin.17b-estradiol. A next question is how the profile of estrogenic protection against gp120 compares to estrogenic protection against other insults. Protection against gp120 was particularly potent, requiring lower concentrations of 17bestradiol than against hemoglobin (in cortical cultures [23]), and lower concentrations of 17b-estradiol, equilin and estrone than against kainic acid, NMDA, sodium azide (cortical cultures [23]), or b-amyloid (in cortical cultures or in a hippocampal cell line [2]). The greater potency of 17b-estradiol against gp120 as compared to the other three estrogen does not generalize to other insults (Table 2); 17b-estradiol was significantly more potent in only two of the nine studies. These heterogeneous profiles of potency among the various estrogens raises the issue of the mechanisms of estrogenic protection. As steroid hormones, estrogens have their classical effects via intracellular receptors which, when binding hormone, act as transcriptional regulators. The ERa estrogen receptor has a greater affinity for 17bestradiol than for the other three estrogens [3,15,18]. Superficially, this appears consonant with our finding of
Table 2 Summary of the neuroprotective effects of different estrogens across varied necrotic insults Insult Cortical culture
Pattern of protection Kainic acid NMDA Hemaglobin Sodium azide b-amyloid H2O2
17b-estradiol5estrone5equilin [23] 17b-estradiol5estrone [14] 17b-estradiol.equilin5estrone [23] 17b-estradiol5equilin5estrone [23] equiline5estrone [23] 17b-estradiol5estriol.estrone [2] 17b-estradiol5estriol5estrone [2]
Hipp cultures
gp120
17b-estradiol.estriol5estrone5equilin (the present study)
Hipp cell line
b-amyloid
17b-estradiol5estriol5estrone [2]
Hippocampus
Kainic acid
estrone.estriol [8]
Numbers in brackets refer to citations
275
greater protection against gp120 with 17b-estradiol. However, we have previously observed that the estrogenic protection is receptor-independent, occurring in the presence of an ERa antagonist and mimicked with 17aestradiol, which does not bind to the ERa [12]. The rapidly expanding literature concerning estrogenic neuroprotection contains precedents for receptor-independent actions (reviewed in Refs. [19,24]), although numerous instances of protection are clearly receptor-mediated [9]. There is some understanding of which downstream estrogenic actions are neuroprotective. Within the realm of receptor-mediated effects, estrogens induce expression of neurotrophins, and increase the ratio of anti- to pro-apoptotic molecules [19,24]. Best documented of the receptorindependent effects are the anti-oxidant actions of estrogens. This arises in part from the ability of estrogen to intercalate into membranes, protecting them against peroxidative attack. Estrogens are considerably more potent at this than are other steroids [20], and this is thought to be due to its distinctive phenolic ring structure [21]. gp120 generates oxygen radicals and oxidative damage [4,17], and this is blocked by 17b-estradiol [12]. Just as the neuroprotective potency of the four estrogens does not closely parallel their affinity for ERa, the potency does not closely parallel the antioxidant potency of these estrogens (estriol.17b-estradiol..estrone) [16]. In conclusion, we observe that a variety of estrogens decrease the toxicity of gp120 to hippocampal neurons. Data from this and other studies suggest that the relative protective potency among these estrogens is not a simple consequence of their affinity for the estrogen receptor, or their efficacy as an antioxidant. Prospective trials are now underway testing whether estrogen administration can decelerate the neuropsychological consequences of Alzheimer’s disease; potentially, estrogen could be of therapeutic use against ADC. An understanding of the mechanisms of estrogenic protection against gp120 would aid selecting the particular estrogen which would optimize protection while minimizing some of the (typically, receptor-mediated) cancer risks of estrogen therapy.
Acknowledgements Support was provided by NIH RO1 MH53814 to RS.
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