Ovarian steroids reduce apoptosis induced by trophic insufficiency in nerve growth factor-differentiated pc12 cells and axotomized rat facial motoneurons

Neuroscience 118 (2003) 741–754

OVARIAN STEROIDS REDUCE APOPTOSIS INDUCED BY TROPHIC INSUFFICIENCY IN NERVE GROWTH FACTOR-DIFFERENTIATED PC12 CELLS AND AXOTOMIZED RAT FACIAL MOTONEURONS N. J. MACLUSKY,a* R. CHALMERS-REDMAN,b G. KAY,b W. JU,b I. S. NETHRAPALLIa AND W. G. TATTONb

antiprogestin, phaeochromocytoma, neuroprotection.

a

Center for Reproductive Sciences, Department of Obstetrics and Gynecology, Columbia-Presbyterian Medical Center, 622 West 168th Street, New York, NY 10032-3702, USA

Neurodegenerative disorders do not occur with the same frequency in men and women (Jorm and Jolley, 1998; Bower et al., 2000). The mechanisms responsible for this sex difference remain poorly understood, but effects of the sex hormones probably represent a contributory factor. Sex steroids affect normal cognitive function as well as the deficits resulting from injury to the brain (Sherwin 1997; Schmidt et al., 1996; Simpkins et al., 1997; Roof and Hall, 2000). Estrogen replacement has been reported to reduce the extent of the damage caused by cerebrovascular incidents, such as small strokes (Schmidt et al., 1996). Women with Parkinson’s disease (PD) on estrogen therapy exhibit lower levels of dementia (Marder et al., 1998) and motor disability (Saunders-Pullman et al., 1999) than postmenopausal PD patients who do not receive estrogen. The prevalence of Alzheimer’s disease (AD) is higher in older women than in men (Jorm and Jolley, 1998) and the risk of AD conferred by the apolipoprotein E ⑀-4 allele appears to be greater for postmenopausal women than for men (Bretsky et al., 1999). Consistent with the hypothesis that the loss of ovarian steroids contributes to the higher incidence of AD in aging women, the incidence and rate of progression of AD appear to be reduced in postmenopausal women who take hormone replacement therapy (HRT) (Yaffe et al., 1998; Tang et al., 1996). The ovaries secrete two principal steroid hormones, estradiol (E2) and progesterone (P). Both of these hormones are usually replaced in HRT. The relative contributions of estrogenic and progestational effects to the neuroprotective effects of HRT, however, remain uncertain. A considerable body of work has shown that estrogen is neuroprotective in animal models and cell culture systems (Simpkins et al., 1997; Green et al., 1997, 1998; Bonnefont et al., 1998; Moosmann and Behl, 1999; Dubal et al., 1999; Veliskova et al., 2000; Wise et al., 2001). The possibility of protection by the progestin component has received relatively less attention and remains controversial. A number of studies have demonstrated inhibition of estrogen responses by progestins, in the brain as well as the reproductive tract (Brown and MacLusky 1994; Okulicz et al., 1981). However, P is also cytoprotective and has been shown to significantly reduce glutamate- or ␤ amyloidinduced toxicity in dispersed primary cultures of rat hippocampal neurons (Goodman et al., 1996). This effect may be progestin-specific: P and 19-norprogesterone have been reported to protect hippocampal neurons against

b Department of Neurology, Mount Sinai School of Medicine New York, NY, 10029-6574, USA

Abstract—Previous studies have demonstrated that ovarian steroids exert neuroprotective effects in a variety of in vitro and in vivo systems. The mechanisms underlying these effects remain poorly understood. In the present study, the neuroprotective effects of estradiol (E2) and progesterone (P) were examined in two models of apoptosis induced by growth factor insufficiency: partially nerve growth factor (NGF)-differentiated PC12 cells, after serum and NGF withdrawal; and axotomized immature rat facial motor motoneurons. E2 and P both increased the survival of trophically withdrawn NGF-differentiated PC12 cells, at physiologically relevant concentrations. However, neither steroid had a significant effect on the survival of PC12 cells that had not been NGF treated. Exposure to NGF had no effect on the expression of estrogen receptor (ER)␤, but markedly increased the levels of ER␣ and altered the expression of the progesterone receptor (PR) from predominantly PR-B in NGF naive cells, to predominantly PR-A after NGF. The survival promoting effects of E2 and P were blocked by the specific steroid receptor antagonists Faslodex (ICI 182780) and onapristone (ZK98299), respectively. Inhibitors of RNA (actinomycin D) or protein (cycloheximide) synthesis also abrogated the protective effects of both steroids. In immature rats, E2 and P both significantly increased the numbers of surviving facial motor neurons at 21 days after axotomy. These data demonstrate significant protective effects of E2 and P in two well-characterized models of apoptosis induced by trophic withdrawal and suggest that, at least in PC12 cells, the effects of the steroids are mediated via interaction with nuclear steroid receptor systems. The lack of steroid responsiveness in NGF-naive PC12 cells despite the presence of abundant ER␤ and PR-B are consistent with the view that ER␣ and PR-A may be particularly important as mediators of the neuroprotective effects of their corresponding hormonal ligands. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key

words:

17␤-estradiol,

progesterone,

antiestrogen,

*Corresponding author. Tel: ⫹1-212-305-2304; fax: ⫹1-212-3053869. E-mail address: [email protected] (N. J. MacLusky). Abbreviations: AD, Alzheimer’s disease; ChAT, choline acetyl transferase; E2, estradiol; ER, estrogen receptor; HRT, hormone replacement therapy; LCSM, laser confocal scanning microscopy; MEM, minimum essential medium; M/O, MEM only; M/S, MEM with serum; M/S⫹N, MEM with serum and NGF; NGF, nerve growth factor; P, progesterone; PD, Parkinson’s disease; PR, progesterone receptor.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00940-5

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glutamate toxicity, while the widely used synthetic progestin medroxyprogesterone acetate is ineffective by itself, and antagonizes neuroprotective effects of E2 (Nilsen and Brinton, 2002). In animals, P (Roof and Hall, 2000), like E2 (Simpkins et al., 1997), exerts significant protective effects in models of experimental stroke. The potential significance of neuroprotective responses to P is augmented by recent data indicating that this steroid is not derived exclusively from the gonads, but is also synthesized within the brain itself (Jung-Testas et al., 1999; Tsutsui et al., 1999). Several different mechanisms may contribute the neuroprotective effects of the ovarian steroids. A considerable amount of work has focused on the ability of the steroids to act as chemical antioxidants, in concert with other cellular antioxidant and free radical scavenger mechanisms (Moosmann and Behl, 1999; Green et al., 1997, 1998). Protection against sub-necrotic oxidative damage, however, may represent only one component of the spectrum of activity of the ovarian steroids that contributes to their neuroprotective properties (Wise et al., 2001). Apoptosis resulting from insufficient trophic support is a normal feature of CNS development (Burek and Oppenheim, 1996). Damage to the CNS is followed by alterations in growth factor synthesis and increased growth factor levels can limit the extent of cell death associated with neurotoxic insults (Han and Holtzman, 2000; Houle and Ye, 1999; Oyesiku et al., 1999). The cellular pathways activated by growth factors and the ovarian steroids converge on common signaling elements, raising the possibility that sex steroids may either substitute directly for growth factors, or potentiate the effects of these factors under conditions of injury (Toran-Allerand et al., 1999). However, relatively little is known about the effects of gonadal steroids on apoptosis induced by trophic insufficiency. Thus, the extent of the contribution from trophic responses to the neuroprotective effects of the steroids remains uncertain. The present studies were performed to explore the neuroprotective effects of ovarian steroids on apoptosis induced by growth factor insufficiency. Two models of trophic insufficiency were utilized: an in vitro model involving withdrawal of serum and nerve growth factor (NGF) from partially NGF-differentiated PC12 rat phaeochromocytoma cells and in vivo model involving axotomy of immature rat facial motor motoneurons by facial nerve transection (Ju et al., 1994; Ansari et al., 1993; Carlile et al., 2000; Tatton et al., 1994). These models have been previously shown to respond to a variety of neuroprotective agents (Ju et al., 1994; Ansari et al., 1993; Carlile et al., 2000; Tatton et al., 1994; Ferrari et al., 1995), including drugs that are effective in slowing the clinical progress of AD and PD (Birks and Flicker, 2000; Larsen et al., 1999).

EXPERIMENTAL PROCEDURES PC12 cell culture, treatment and counting PC12 cells (American Tissue Culture Collection [ATCC] Manassas, MD, USA) were propagated in minimum essential medium (MEM) containing 10% horse serum, 5% fetal bovine serum, 2-mM L-glutamine, 50 units/ml penicillin, and 50 ␮g/ml streptomycin (MEM with serum, abbreviated as M/S), all purchased from

Life Technologies (Rockville, MD, USA). The cells were grown on 24-well plates (8⫻104 cells/well) for counting of intact nuclei as an estimate of survival, poly-L-lysine-treated coverslips (1⫻104 cells/ coverslip) for imaging with laser confocal scanning microscopy (LCSM) or 100 mm dishes (1⫻106 cells/plate) for protein chemistry. The cells were differentiated for up to 6 days in M/S supplemented with 100 ng/ml 7S NGF (Upstate Biotechnology, Lake Placid, NY, USA). MEM with serum and NGF is abbreviated as M/S⫹N (see Tatton et al., 1994; Wadia et al., 1998; Carlile et al., 2000; Tatton et al., 2002 for further details of culture and treatment). Following incubation for 6 days in M/S⫹N, cells underwent three successive washes in Hanks’ balanced salt solution (Life Technologies, Rockville, MD, USA) to remove NGF and serumborne trophic agents and then were replaced into M/S⫹N for controls or into MEM only (abbreviated as M/O) to induce apoptosis by serum and NGF withdrawal. Varying concentrations of E2 (Sigma-Aldrich, St. Louis, MO, USA), P (Sigma-Aldrich), mifepristone (Ru486, a gift from Roussel-UCLAF, Romaineville, France) Faslodex (ICI 182780; a gift from A. E. Wakeling, AstraZeneca Pharmaceuticals, Macclesfield, Cheshire, UK) and onapristone (ZK98299, a gift from Dr. J. D. Blaustein, University of Massachusetts, Amherst, MA, USA), actinomycin D or cycloheximide (all from Sigma-Aldrich) were added to the M/S⫹N or M/O cultures at varying times to block E2 or P receptors or to inhibit new RNA and protein synthesis. NGF-naive undifferentiated PC12 cells were maintained in M/S on plates, wells or coverslips for 6 days prior to washing to induce apoptosis by serum withdrawal. Washed cells were replaced into M/S or M/O with or without identical additives to those used with the partially NGF-differentiated PC12 cells. Cell survival and the percentages of cells with evidence of apoptotic nuclear degradation were assessed for all treatments. To estimate survival, the cells were seeded at a density of 8⫻104 cells/well in 24-well plates. Cells were harvested 24 h after treatment and lysed. Cells were harvested from each well by trituration and centrifuged at 500⫻g for 5 min. The supernatant was removed and the pelleted cells were lysed with 200 ␮l of lysing buffer (Zap-o-globin II, Coulter Electronics, Hialeah, FL, USA). Intact nuclei were counted in a hemocytometer by a “blinded” observer as previously described (see Tatton et al., 1994, 2002; Wadia et al., 1998; Carlile et al., 2000 for details of harvesting and counting methods). Percentages of cells with apoptotic nuclei were determined for cells grown on poly-L-lysine-treated coverslips (density 1⫻104/ coverslip). At 12 h after treatment, the cells were stained with the DNA binding dye YOYO-1 (Molecular Probes, Eugene, OR, USA) to reveal chromatin condensation as a marker of apoptotic nuclear degradation (Tatton et al., 1994, 2002; Wadia et al., 1998; Carlile et al., 2000). Cells on coverslips were washed three times in PBS followed by 100% methanol incubation at ⫺20 °C for 30 s. The methanol was then replaced with YOYO-1 (1.5 ␮M in PBS) for 30 min at room temperature. After three PBS washes, the cells on coverslips were mounted in Aquamount (Gurr EM Industries, Cincinnati, OH, USA) for LCSM imaging. The total number of YOYO1-stained nuclei with chromatin condensation were counted on 25 40⫻ fields for each coverslip, each field chosen by pairs of randomly generated x–y coordinates. The proportion of nuclei with chromatin condensation was expressed as a percentage of the total number of cells in each field. The values were pooled for three coverslips for each treatment and time point.

Western blot analysis PC12 cell cultures were harvested into protease inhibitor- and phosphatase inhibitor-containing lysis buffer and prepared for electrophoresis on 8% polyacrylamide gels, as described previously (Singh et al., 2000). For comparison purposes, some blots also included a sample of adult rat uterine lysate. After electrophoretic separation, proteins were transferred onto polyvinylidene

N. J. MacLusky et al. / Neuroscience 118 (2003) 741–754 difluoride membranes (0.22-␮m pore size; Bio-Rad, Hercules, CA, USA), blocked overnight with 5% Carnation non-fat dried milk in Tris-buffered saline containing 0.2% Tween-20, and probed with antibodies raised against estrogen receptor (ER)␣ (6F11; Novacastra Laboratories Ltd., Newcastle upon Tyne, UK), ER␤ (Zymed Laboratories, Inc., San Francisco, CA, USA) or the P receptor (C19; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Detection of antibody binding to protein was detected, using a secondary antibody conjugated to horseradish peroxidase (1:40,000, Pierce) and visualized autoradiographically on film, using enzyme-linked chemiluminescence (Amersham), as previously described (Singh et al., 2000).

Axotomized facial motoneuron preparation Studies of ovarian steroid effects in rats were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize both the suffering and number of rats used, and all procedures were pre-approved by the relevant institutional animal care and use committees. Facial nerve axotomy was performed as previously described (Salo and Tatton, 1992; Ansari et al., 1993; Ju et al., 1994; Zhang et al., 1995). Briefly, 14-day postnatal rats (Charles River Sprague–Dawley strain) were anesthetized with halothane (4% for induction; 1–1.5% for maintenance) and nitrous oxide (70%⫹30% oxygen) at a flow rate of 4 l/min. The right facial nerve was exposed and transected immediately distal to its exit from the stylomastoid foramen, removing a 4-mm section of the nerve to preclude reinnervation. After surgery, the animals were randomly assigned to three experimental groups: 1) a control group receiving daily s.c. corn oil (100 ␮l; vehicle control) injections; 2) a group injected with 5 ␮g/day E2 s.c. daily in corn oil; and 3) a group injected with 500 ␮g P s.c. daily in corn oil. A fourth group of animals was sham operated by exposing the facial motor nerve under anesthesia, then immediately closing the incision. Animals were killed by anesthetic overdose followed by perfusion with 4% paraformaldehyde in phosphate buffer on postnatal day 35, 21 days after surgery. Brains were removed and blocks containing the pons and medulla were cryopreserved in 20% sucrose and frozen in ⫺70 °C methyl butane. Serial 10-␮m sections were cut through the length of the facial motor nucleus and mounted on three different sets of slides depending on serial order: the first section of each group of three was immunoreacted for an antibody against choline acetyl transferase (ChAT; Ansari et al., 1993), each second section was Nissl stained (see Ansari et al., 1993; Ju et al., 1994 for details of methods) and each third section was retained for subsequent immunocytological studies. The sections for ChAT immunoreaction were incubated overnight with a polyclonal antibody (Chemicon International, Temecula, CA, USA) at a dilution of 1/1000 and a temperature of 4 °C. The sections were then incubated in biotinylated goat antirabbit IgG and followed by avidin– horseradish peroxidase. Visualization of ChAT immunoreaction was performed in a 0.05% solution of diaminobenzidine in 0.01% H2O2. Two randomly chosen control sections were examined from each brainstem using non-immune serum rather than the primary ChAT antibody. Numbers of surviving facial motoneurons were estimated in the right and left facial nuclei by two different methods of counting Nissl-stained somata. The ChAT-immunoreacted sections were first used to determine the full extent of the facial nucleus but were not counted since we and others have previously shown that a proportion of motoneurons show reduced or absent ChAT immunoreactivity after axotomy (Hoover and Hancock, 1985; Ansari et al., 1993). Similar to our previous studies, Nissl-stained facial motoneuron somata containing a nucleolus and meeting the nuclear size criteria of Oppenheim (Oppenheim, 1986) were counted under interference contrast optics for each Nissl section within the domain of sections demonstrating ChAT-immunopositive somata.

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The correction factor developed by Abercrombie was applied to the counts to compensate for the splitting or aproportion of neuronal profiles caused by sectioning (see Williams and Rakic, 1988 for details) The same sections were also counted using stereological methods (see Pakkenberg and Gundersen, 1995 for rationale of the optical disector method and West et al. 1991 for the procedures that were employed).

DNA electrophoresis Additional groups of sham-operated and facial nerve-transected animals were killed and processed for studies of DNA cleavage in the facial motor nucleus at 18 and 24 h after surgery. The ipsilateral and contralateral facial nucleus was microdissected from 400 ␮m-thick sections for each animal and the respective facial nuclei tissue pooled for 12 animals from each treatment group and time point. Soluble DNA was extracted by the method of Hockenbery et al. (1990) and resuspended in TE buffer (10-nM Tris, 1-mM EDTA). The samples were then incubated with 50 mg/ml DNAsefree RNAse (Boehringer Mannheim Corporation, Indianapolis, IN, USA) at 37 °C for 30 min. The recovered soluble DNA was electrophoresed on a 1.2% agarose gel and blotted onto Gene Screen Plus membrane (Dupont, Boston, MA, USA). Blots were probed with total genomic facial nuclear DNA digested with Sau 3A (Boerhinger Mannheim). 33P-labeled probe was prepared by the random priming reaction and hybridization and washings were performed according to the manufacturer’s protocol.

Statistical analysis The individual counts for PC12 cell and motoneuron survival or nuclear chromatin condensation from different treatment groups were analyzed using Statistica software (StatSoft). Levene’s testing for homogeneity of variances showed that most pairs of samples were not homogeneous and chi-square evaluation of the distributions showed that most did not fit a normal distribution (see Siegel, 1956) so that parametric methods might not provide valid results. The data were therefore rank ordered and compared in a pairwise fashion using Statistica software to perform non-parametric Mann Whitney U testing or Kruskal-Wallis non-parametric ANOVA (Siegel, 1956). A probability value of P⬍0.05 was considered statistically significant.

RESULTS Expression of ovarian steroid receptors in partially differentiated PC12 cells Two previous studies have suggested that ER␣ levels in PC12 cells are up-regulated during NGF-induced differentiation (Sohrabji et al., 1994; Nilsen et al., 1998). To determine whether this was also the case for the PC12 cells used in the present study, cells grown in either M/S, or M/S⫹N were analyzed by Western blotting using antibodies specific for ER␣ and ER␤. Six days of NGF exposure resulted in marked changes in ER expression in the PC12 cells. After culture in medium with serum alone, ER␣ was not detectable, while multiple bands were observed in blots stained for ER␤, corresponding to the molecular variants of this receptor protein (Price et al. 2000, 2001; Fig. 1A, B). Following 6 days of treatment with NGF, a single band at 67 kDa corresponding to ER␣ was seen, while the relative intensity of the bands corresponding to ER␤ decreased (Fig. 1A, B). The blots were stripped and re-probed with an antibody specific for the progestin receptor (PR). In cells cultured in

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Fig. 1. Western blot analysis of ER and PR receptor expression in PC12 cells grown in the presence (⫹) or absence (⫺) of 100 ng/ml NGF. (A) Representative blot labeled with a mouse monoclonal antibody (6F11; Novacastra) against ER␣. In the absence of NGF, immunoreactivity for ER␣ was undetectable (middle lane). For comparison purposes, a sample of rat uterine tissue lysate was run in the same gel: a strong immunoreactive band was observed migrating at the expected position (67 kDa) of authentic ER␣. In NGF-treated PC12 cells, an immunoreactive band was observed at 67 kDa, considerably less intense than in the uterine lysate, but clearly above background. (B) With a polyclonal antibody against ER␤ (Zymed), in lysates from both naive and NGF-treated cells, multiple immunoreactive bands were observed at approximately 45 kDa, 56 kDa, 60 kDa (the expected position of full-length ER␤) and 65 kDa. (C) Upper panel: results from three sets of cells with and without NGF treatment separated on the same polyacrylamide gel and labeled with a polyclonal antibody against PR (Santa Cruz, C19). In all lysates, two distinct immunoreactive bands were observed migrating at approximately 110 kDa (the expected position for PR-B) and approximately 90 kDa (PR-A). In cells grown without NGF, the intensity of the PR-B band was consistently higher than that for PR-A. Following NGF treatment, this ratio was reversed. Lower panel: densitometric analysis of the PR Western data. Densitometric measurements are expressed relative to the results for PR-B in NGF-naive cells (arbitrarily set to 100%). Each histogram bar represents the mean (⫾S.E.M.) of data from the three individual samples shown in the top panel.

medium without NGF, a single band corresponding to the full-length PR-B was observed at 109 kD, with smaller amounts of labeling at 90 kD in the expected position of the N-terminal truncated PR-A. After NGF-induced differentiation, PR expression changed dramatically. While the intensity of the PR-B band declined, in NGF treated cells PR-A labeling increased dramatically (Fig. 1C). Ovarian steroids reduce apoptosis in partially differentiated PC12 cells after serum and NGF withdrawal Paralleling the changes in steroid receptor expression, NGF dramatically altered the capacity of the ovarian steroids to inhibit cell death induced by trophic withdrawal. As shown in Fig. 2, placement of PC12 cells into medium without serum or growth factor support for 24 h resulted in substantial cell loss, whether or not the cells were preconditioned with NGF. Transfer of cells grown in M/S for 6 days (NGF-naive cells) into serum-free media induced an approximately 50% reduction in cell counts by 24 h, while under the same conditions a slightly greater proportion

(60%) of partially NGF differentiated (NGF⫹serum for 6 days) cells was lost. Neither E2 nor P increased the survival of NGF naive cells at any concentration over a 10⫺5 10⫺13-M range (all points represent means⫾S.E.M. for 12–18 replicate wells; in all cases P⬎0.21, in comparison to untreated cells shown by the open triangle). By contrast, both steroids increased the survival of the partially NGF differentiated cells in a dose-dependent fashion (as shown by closed circles in comparison to the closed triangle). E2 was most effective in increasing the survival of the partially NGF-differentiated cell at 10⫺9 M (P⫽0.014), increasing cell counts by about 80%, while P was most effective at 10⫺7 M (P⫽0.018), increasing survival by about 70%. Previous studies have shown that the loss of both NGF-naive and NGF-differentiated PC12 cells after trophic withdrawal results from apoptosis (Tatton et al., 1994, 2002; Wadia et al., 1998; Carlile et al., 2000). Cell survival is a cumulative measurement while cells with nuclear chromatin condensation as a marker of apoptotic nuclear degradation are only transiently present. We have previously reported that the maximum numbers of cells with nuclear

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Fig. 2. Effect of E2 and P on survival of PC12 cells after withdrawal of trophic support, using percent of intact nuclei as a measure of cell survival. M/S⫹N: cells differentiated in MEM with NGF and serum for 6 days, washed repeatedly and then replaced in MEM with serum and NGF as a control. M/O: cells were maintained in NGF and serum for 6 days, then washed repeatedly and placed in MEM only in order to withdraw NGF and serum-borne trophic support. M/S: cells grown in MEM with serum for 6 days, washed and replaced in MEM with serum. Serum withdrawn: same as M/S, except cells were replaced after washing in MEM medium without serum. All filled symbols are for cells differentiated in serum and NGF, while open symbols are for cells exposed to serum but not NGF. The cell counts are normalized against the values obtained for the corresponding controls (either M/O or serum withdrawn, set to 100%). These plots, as well as those in subsequent figures, present means⫾S.E.M. for 12–18 replicate wells in each case. Withdrawal of growth factors resulted in significant cell loss, in both the naive (approximately 50% loss, compare open diamonds and open triangles) and NGF-treated cells (⬎60%, compare closed diamonds and closed triangles). At 10⫺9 M and 10⫺7 M respectively, E2 and P increased cell survival in cells withdrawn from both serum and NGF, by about 70% compared with M/O cells maintained in the absence of steroid (compare closed circles to closed triangle). By contrast, neither steroid had a significant effect on survival of serum-withdrawn NGF-naive cells (compare open circles to open triangle).

chromatin condensation are present at 12 h after serum and NGF or serum withdrawal (Wadia et al., 1998; Carlile et al., 2000). Accordingly, we carried out nuclear counts using the nucleic acid binding dye YOYO-1 (for details see Carlile et al., 2000 and Tatton et al., 2002) to determine whether E2 and P reduced cell loss following trophic withdrawal by inhibiting apoptosis. Fig. 3 presents typical examples of LCSM images of nuclear chromatin condensation shown with YOYO-1 staining. Normal nuclei show a reticulated pattern of YOYO-1 fluorescence while nuclei with chromatin condensation are markedly shrunken compared with normal nuclei and show bright YOYO-1 fluorescence. The difference in fluorescence brightness is evident upon comparison of the normal nuclei to a single nucleus with chromatin condensation in Fig. 3A2 imaged for NGFnaive cells at 12 h after serum withdrawal. Each pair of images in Fig. 3 (3A1 and 3A2, 3B1 and 3B2, 3C1 and 3C2) present an interference contrast image and a YOYO-1 fluorescence image taken for an identical image field. The marked shrinkage of the nuclei with chromatin

condensation compared with the two normal nuclei is evident in Fig. 3A1. Furthermore, the interference contrast image in Fig. 3A1 also shows the cytoplasmic remnants surrounding the shrunken nucleus that are typical of apoptotic degradation. Figs. 3B1 and 3B2 present lower-power LCSM images of typical partially NGF-differentiated cells at 12 h after washing and replacement into media with serum and NGF while Figs. 3C1 and 3C2 present typical lower-power images of the partially NGF-differentiated cells at 12 h after withdrawal of serum and NGF. Figs. 3D1–D4 present a range of examples of the forms that YOYO-1 fluorescence can reveal for nuclear chromatin condensation in the cells after serum withdrawal or serum and NGF withdrawal. The concentrations of P and E2 that were maximally effective in increasing cell survival (10⫺7 and 10⫺9 M) were studied for their effects on nuclear YOYO-1 fluorescence as a marker of apoptotic chromatin condensation. As shown in Fig. 3E, E2 and P both reduced the percentages of cells with nuclear chromatin condensation in the partially

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Fig. 3. Examples of LCSM images of nuclear chromatin condensation shown with YOYO-1 staining. Each pair of images (3A1 and 3A2, 3B1 and 3B2, and 3C1 and 3C2) presents an interference contrast image and a YOYO-1 fluorescence image taken for an identical image field. 3A1 and 3A2: NGF-naive cells at 12 h after serum withdrawal. 3B1 and 3B2: lower-power LCSM images of typical partially NGF-differentiated cells at 12 h after washing and replacement into medium with serum containing NGF. 3C1 and 3C2: images of partially NGF-differentiated cells at 12 h after withdrawal of serum and NGF. 3D1-D4: higher-magnification examples of the different forms that YOYO-1 fluorescence can take for nuclear chromatin condensation in apoptotic PC12 cell nuclei. Calibration bars⫽10 ␮M. 3E: effects of P and E2 (10⫺7 and 10⫺9 M) on the percentages of cells with nuclear chromatin condensation in partially NGF-differentiated cells at 12 h after serum and NGF withdrawal. Each value represents the mean⫾S.E.M. for 12 identically treated coverslips. For key to symbols and treatment groups, see Fig. 2.

NGF-differentiated cells at 12 h after serum and NGF withdrawal (vehicle, open circles, versus steroid-treated, closed circles: E2 10⫺9M P⫽0.008, E2 10⫺7M P⫽0.012; P 10⫺9M P⫽0.031, P 10⫺7M P⫽0.038; n⫽12 coverslips for all points). Neither ovarian steroid reduced the percentages of cells with nuclear chromatin condensation in NGF naive cells after serum withdrawal at the same time point (E2 10⫺9M P⫽0.411, E2 10⫺7M P⫽0.381, P 10⫺9M P⫽0.21, P 10⫺7M P⫽0.26). These data are consistent with the hypothesis that P and E2 reduce cell death in trophically withdrawn partially NGF-differentiated PC12 cells, at least in part by inhibiting apoptosis. However, the same steroid treatments do not affect survival or apoptosis induced by serum withdrawal in NGF-naive cells. Effects of steroid receptor antagonists The preceding data suggested that E2 and P exerted their protective effects on PC12 cells via steroid receptor-mediated mechanisms, since these responses were observed only after partial NGF-induced differentiation, when ER␣ and PR mRNA levels were up-regulated compared with control cells grown in serum without NGF. To further test the role of steroid receptor activation, we examined the capacity of E2 and P to increase survival

of the partially NGF-differentiated PC12 cells in the presence of specific estrogen and PR antagonists (Fig. 4). The increased survival of partially NGF-differentiated cells induced by E2 at 10⫺7–10⫺11 M was blocked by addition of the anti-estrogen Faslodex (ICI in Fig. 4) at 10⫺7 M (Fig. 4A2, Ps⫽0.048 – 0.009 for E2 alone compared M/O and 0.43– 0.37 for E2⫹ICI compared with M/O). Faslodex did not affect the increased survival provided by P (Fig. 4A3). Mifepristone (Ru486), an anti-progestin with some anti-estrogenic activity (Nedvidkova et al., 1997), at 10⫺7 M blocked the capacity of both E2 and P to increase survival after serum and NGF withdrawal (data not shown). The more specific PR antagonist onapristone (ZK in Fig. 4) at 10⫺7 M had no effect on the increased survival induced by E2 (Fig. 4B2) while it completely blocked the increased survival provided by P at 10⫺7–10⫺11 M (Fig. 4B3, Ps⫽0.042– 0.0087 for P alone compared with P⫹ZK). Dependence of ovarian steroid responses on new protein synthesis Apoptosis induced by serum and NGF withdrawal in PC12 cells partially differentiated by NGF for 6 days is independent of new protein synthesis, so that protein synthesis inhibition does not impair apoptosis induced by serum and

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Fig. 4. The ER antagonist Faslodex (ICI182,780, abbreviated to IC) and PR antagonist onapristone (ZK98299; abbreviated to ZK) block the capacity of E2 (panels A2 and B2) and P (panels A3 and B3), respectively, to increase the survival of NGF-differentiated PC12 cells after serum and NGF withdrawal. Faslodex had no effect on responses to P (A3) while onapristone was without effect on responses to E2 (B2). Each value represents the mean⫾S.E.M. for 12–18 identically treated wells taken from three experiments.

NGF withdrawal (Tatton et al., 2002). Thus, inhibitors of transcription (actinomycin D) and translation (cycloheximide) can be used to determine whether cytoprotective effects in partially NGF-differentiated PC12 cells require new protein synthesis. Actinomycin D (2.5 ␮g/ml) or cycloheximide (10 ␮g/ml) was added to cultured PC12 cells together with E2 (10⫺7, 10⫺9 or 10⫺11 M). We have previously reported based on metabolic labeling studies that these concentrations of actinomycin D and cycloheximide reduce new protein synthesis by more than 94% in partially NGF-differentiated PC12 cells (see detailed experiments and results in Tatton et al., 2002). As shown in Fig. 5, the inhibitors did not reduce survival of serum- and NGFsupported cells (left panel, points labeled M/S⫹N Ps⫽0.174 and 0.231 versus vehicle control for actinomycin D and cycloheximide, respectively) or the survival of the cells after serum and NGF withdrawal (points labeled M/0, Ps⫽0.094 and 0.181, respectively). They did, however, markedly inhibit the survival-promoting effects of E2 and P at all steroid concentrations tested (Ps⫽0.012– 0.009). Ovarian steroids promote survival in axotomized facial motor neurons The completeness of motoneuron axon transection and the absence of re-innervation of facial musculature were

monitored by daily examination of pinna movements and whisker movements in the anterior–posterior direction. Each animal was placed on a black card to view the whisker movements. In all animals, pinna and whisker movement was absent ipsilateral to the facial nerve transection, but remained robust on the contralateral side. The interference contrast micrographs of Nissl-stained facial motoneurons at 21 days after facial nerve transection show the prominent nucleoli in the motoneurons in Fig. 6A. Previous studies of the immature motoneurons using in situ end labeling of nuclear DNA have suggested that maximum rates of nuclear DNA cleavage occur at approximately 1 day after facial nerve transection (de Bilbao and Dubois-Dauphin, 1996) antecedent to the maximum rates of facial motoneuron loss at between 2 and 5 days after nerve transection (Ju et al., 1994). In accord with these findings, we found prominent “laddering” on DNA electrophoresis gels for facial nuclei ipsilateral but not contralateral to the facial nerve transactions at 18 and 24 h after transection (Fig. 6B). Surviving neurons in the facial motor nuclei ipsilateral and contralateral to axotomy were counted at 35 days of age, 21 days after unilateral facial nerve transection. The nucleolus-dependent method with the Abercrombie correction provided similar values to the disector method as shown on Fig. 6C. Similar to our previous reports (Salo and

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Fig. 5. Effects of cycloheximide and actinomycin on PC12 cell survival in M/S⫹N or M/O (left panel), M/O with addition of varying concentrations of E2 (middle panel) or M/O in the presence of varying concentrations of P (right panel). Both inhibitors blocked increases in survival induced by either steroid. The sex steroids and the inhibitors were added together at the time of washing the cells free from serum and NGF. Each value represents the mean⫾S.E.M. for eight to 12 identically treated wells taken from three experiments.

Tatton, 1992; Ansari et al., 1993; Ju et al., 1994; Zhang et al., 1995), both methods showed 85– 87% motoneuron survival in the contralateral facial nucleus (Ps⫽0.042 and 0.047, respectively, for facial nerve transection compared with sham operation) and 29 –33% survival in ipsilateral nucleus (Ps⫽0.014 and 0.019 for facial nerve transection compared with sham operation). E2 and P increased the contralateral survival to 88 –91% and 91–92% respectively, neither of which was statistically significant (Ps⫽ 0.073– 0.057 for facial nerve transection with saline compared with facial nerve transection with steroid treatment). Both steroids also increased ipsilateral survival, to 50 – 55% and 43– 47% respectively, which were statistically significant (Ps⫽0.023– 0.035 for facial nerve transection with saline compared with facial nerve transection with steroid treatment).

DISCUSSION Studies from several laboratories have established that estrogen stimulates growth and differentiation (for reviews see Toran-Allerand et al., 1999; McEwen et al., 1995), and inhibits programmed cell death (Gollapudi and Oblinger, 1999; Pike, 1999; Sawada et al., 1998; Honda et al., 2000), in a variety of in vivo and in vitro experimental systems.

The extent to which ovarian steroids can protect neurons against apoptosis induced by withdrawal of growth factor support has not, however, been extensively studied. The present data indicate that E2 and P inhibit cell death in two models of apoptosis induced by trophic withdrawal: NGFdifferentiated PC12 cells transferred to serum-free medium without NGF; and axotomized facial motor neurons. In the PC12 cell model, they also suggest that the protective effects of the steroids are mediated via either nuclear E2 and P receptors, or signal transduction systems with ligand specificities that closely resemble those of the corresponding nuclear receptor proteins. PC12 cells have been widely used in studies of the effects of growth factors, as well as in work on the mechanisms involved in apoptosis triggered by exposure to a variety of cellular insults (Ferrari et al., 1995; Sohrabji et al., 1994; Carlile et al., 2000; Chalmers-Redman et al., 1999). These cells do not exhibit protective or growth responses to estrogen in their undifferentiated state, unless they are transfected with a vector expressing ER␣ (Gollapudi and Oblinger, 1999). After NGF-induced differentiation, however, endogenous ER␣ synthesis increases. This response has been observed in several laboratories, under a variety of different culture conditions. Nilsen et al.

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Fig. 6. E2 and P reduce apoptosis of immature rat facial motoneurons initiated by peripheral axon transection. (A) Interference contrast micrographs of Nissl-stained facial motoneurons at 35 days of age, 21 days after facial nerve transection. (B) “Laddering” on DNA electrophoresis gels for facial nuclei ipsilateral but not contralateral to the facial nerve transections at 18 and 24 h. (C) Surviving neurons in the facial motor nuclei ipsilateral and contralateral to axotomy 21 days after unilateral facial nerve transection. Both steroids significantly increased ipsilateral survival. The nucleolus dependent method with the Abercrombie correction provided similar values to the disector method. Results represent means⫾S.E.M. for eight independent observations in each case (** P⬍0.01; * P⬍0.05 versus saline-treated controls, Mann-Whitney U test).

(Nilsen et al., 1998) reported induction of ER␣ synthesis after 14 days of NGF treatment in PC12 cells obtained from ATCC and grown in MEM⫹10% horse serum. Sohrabji et al. (1994) reported a six-fold induction of ER levels measured using a binding assay in PC12 cells obtained from Dr. Lloyd Greene (Columbia University) after 10 days of NGF exposure in RPMI medium⫹10% gelded horse serum. The present data, also using PC12 cells originally derived from ATCC and grown in MEM with 5% fetal bovine serum and 5% horse serum, are consistent with these previous observations. In NGF-naive cells, immunoreactivity for ER␤ is observed, while ER␣ remains undetectable. After 6 days of NGF-induced differentiation, ER␤ immunoreactivity remains essentially unchanged, while ER␣ levels increase (Fig. 1). Concomitant to the increase in ER␣ expression, the cells acquire the capacity to exhibit and anti-apoptotic response to estrogen. Thus, in PC12 cells grown without NGF, E2 has no significant protective effect against apoptosis induced by transfer to medium without serum and growth factors. After 6 days of NGF exposure, however, protective responses to E2 are observed (Fig. 2). While there has been considerable interest in the antioxidant and neuroprotective effects of E2 (Green et al., 1997, 1998; Moosmann and Behl, 1999; Roof and Hall, 2000), relatively little work has been done on the possible contributions from P. This is despite the fact that HRT regimens usually incorporate a progestin component (Casper et al., 1996); and P has long been included in tissue culture media used to study effects of potential neurotoxicants in vitro, either via addition of fetal bovine serum, or as a component of the supplements used in serum-free media (e.g. N1 supplement, Bottenstein et al.,

1980). Previous work on neuroprotective and growth-promoting effects of ovarian steroids in PC12 cells has focused exclusively on the effects of estrogen and androgen (Calderon et al., 1999; Gollapudi and Oblinger, 1999; Bonnefont et al., 1998; Nilsen et al., 1998; Mattson et al., 1997; Lustig, 1994). The present studies demonstrate for the first time that undifferentiated and NGF-differentiated PC12 cells both contain measurable PR. In undifferentiated cells, the majority of the PR present appears to be full-length PR-B. After 6 days of NGF-induced differentiation, the pattern of PR expression changes dramatically, so that the ratio of PR-B: PR-A is reversed. The induction of PR-A is accompanied by a change in the response of the cells to P administration. In undifferentiated cells, serum withdrawal induces apoptosis in the majority of cells, a response that is not significantly inhibited by P. After NGF, however, P exerts a dose-dependent inhibitory effect on apoptosis induced by trophic withdrawal. Three lines of evidence suggest that the protective effects of E2 and P on PC12 cells are mediated via ER and PR, rather than a consequence of non-specific membrane stabilization or chemical antioxidant mechanisms. First, the effects are observed at physiological steroid concentrations. Maximal responses to E2 are achieved between 10 and 11 M and 10⫺9 M, within the range of serum E2 levels observed in female mammals during the follicular phase of the ovulatory cycle (Freeman, 1988; Knobil and Hotchkiss, 1988). Likewise, maximal P responses are observed at a concentration of 10⫺7 M, within the range of P concentrations observed during the post-ovulatory phase of the reproductive cycle (Freeman, 1988; Knobil and Hotchkiss, 1988) as well as during pregnancy (Hodgen and Itskovitz, 1988). The steroid concentrations required to

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elicit maximal responses are close to those required to saturate the ER and PR ligand-binding domains, based on the known equilibrium dissociation constants of these receptors for binding to E2 and P, respectively (Ginsburg et al., 1974; MacLusky and McEwen, 1980). By contrast, chemical antioxidant effects increase in magnitude as steroid concentrations rise beyond the normal physiological range (Green et al., 1998). Second, the effects of the steroids are blocked by specific steroid receptor antagonists. Faslodex, a highly specific steroidal estrogen receptor antagonist that is devoid of intrinsic estrogenic activity (Wakeling, 1995), completely blocked the E2 effect without affecting responses to P. Onapristone, a specific steroidal antiprogestin and antiglucocorticoid (D’Souza et al., 1994), blocked the effects of P but did not inhibit responses to E2. Mifepristone, a less specific progestin antagonist with weak antiestrogenic activity (Nedvidkova et al., 1997) blocked the effects of both E2 and P on PC12 survival. Third, the effects of E2 and P are abrogated by inhibitors of RNA or protein synthesis. Actinomycin D and cycloheximide, at concentrations that did not themselves have overt cytotoxic effects, blocked the protective effects of E2 and P (Fig. 5). In ovarian steroid target organs, responses to ER and PR activation are believed to be mediated primarily through effects on gene transcription (DeMayo et al., 2002). Although it seems likely that ER and PR mediate the protective effects of E2 and P on PC12 survival, the mechanisms underlying acquisition of E2 and P sensitivity during NGF-induced differentiation remain unclear. ER␤ and PR-B are both present in undifferentiated PC12 cells, yet neither E2 nor P significantly inhibited apoptosis induced by serum withdrawal. During NGF-induced differentiation, expression of ER␣ and PR-A increases dramatically and the cells become sensitive to the anti-apoptotic effects of E2 and P. While it is possible that the effects of NGF involve responses downstream of the receptors (e.g. induction of receptor coactivator proteins), changes in the steroid receptors themselves may play a critical role. As reported by Nilsen et al. (1998) and confirmed in the present study (Fig. 1), undifferentiated PC12 cells express only ER␤ with no detectable ER␣. Studies in SV40-transformed neuronal cell lines have suggested that expression of ER␣ may mediate anti-apoptotic effects of E2 while ER␤ exerts the opposite effect (Nilsen et al., 2000), consistent with the lack of anti-apoptotic response to E2 in undifferentiated PC12 cells. In contrast to the observations of Nilsen et al. (1998) our data further suggest that the ER␤ present in undifferentiated cells is heterogeneous, with only a minority of the immunoreactive ER␤ protein migrating at 60 kDa, the expected molecular weight of full-length ER␤. RT-PCR studies have demonstrated that multiple ER␤ mRNA species are expressed in the rat CNS, corresponding to full-length ER␤ as well as receptor splice variants in which part of the DNA binding domain corresponding to exon 3 is deleted (ER␤␦3) and/or an 18-amino acid sequence is inserted (ER␤2) into the ligand binding domain (Petersen et al., 1998; Price et al., 2001). A fifth variant (ER␤␦4) results from deletion of exon 4 (Price et

al., 2000). The exon 4 deletion variant is incapable of binding estrogen (Price et al., 2000), while the affinity of ER␤ containing the 18-amino acid binding domain insert is dramatically reduced compared with ER␤ itself (Petersen et al., 1998). ER␤2 has been shown to act as a mixed agonist/antagonist of estrogen-activated transcriptional responses (Petersen et al., 1998). While we cannot definitively identify the immunoreactive proteins observed in PC12 cells on the basis of their apparent molecular weights alone, a reasonable assumption is that they may at least in part represent ER␤ variants in which ligand binding is impaired or abolished. If so, in both undifferentiated and differentiated PC12 cells the majority of the immunoreactive ER␤ present may be incapable of activating responses to physiological concentrations of E2. Consistent with this view, previous studies using ligand-binding assays have detected very little estrogen binding in undifferentiated PC12 cells (Sohrabji et al., 1994) suggesting that most of the immunoreactive ER␤ present does not contain a high-affinity ligand-binding domain. Following NGF, however, there is a dramatic increase in nuclear estrogen binding (Sohrabji et al., 1994) which probably reflects induction of ER␣ (Fig. 1). Although the changes in PR expression after NGF are different from those for ER, their functional consequences appear to be analogous in the terms of the steroid responsiveness of the cells. PR-A and PR-B are expressed from a single gene, PR-B representing the full-length protein while PR-A lacks 164 amino acids at the N-terminal of the molecule. During development, the rat brain initially synthesizes predominantly PR-B mRNA, levels of PR-A mRNA increasing as the brain matures (Kato et al., 1994). A number of studies have demonstrated that PR-B and PR-A differ significantly in their molecular properties, as well as their transcriptional efficiencies for activation of different genes (Gao et al., 2000; Mulac-Jericevic et al., 2000; Clemm et al., 2000; Leonhardt et al., 1998; Carbajo et al., 1996). In undifferentiated PC12 cells, virtually all of the PR-immunoreactive protein migrates at 109 kDa, consistent with the hypothesis that it represents full-length PR-B. During NGF-induced differentiation, there is a dramatic increase in the expression of a band in the expected position of PR-A (90 kDa), while PR-B levels decline. The gene products required for P-mediated inhibition of apoptosis may be selectively induced by activation of PR-A (or heterodimeric receptor complexes containing PR-A and PR-B; Leonhardt et al., 1998), but not by PR-B alone. The apparent dependence of the anti-apoptotic effects of E2 and P on ER- and PR-mediated responses in the present study contrasts with a number of previous studies which have suggested that the neuroprotective actions of E2 and P may be related primarily to their properties as antioxidants or membrane-stabilizing agents (Moosmann and Behl, 1999; Green et al., 1997, 1998; Roof and Hall, 2000). In the estrogen-responsive SK-N-SH human neuroblastoma cell line, for example, E2 inhibits cell death induced by trophic withdrawal (serum deprivation) as it does in PC12 cells; but this effect seems to be largely independent of activation of nuclear ER. Thus, the protec-

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tion afforded by E2 is only weakly inhibited by the estrogen antagonist, tamoxifen (Green et al., 1997); and is reproduced by 17␣ E2, which binds ER␣ with less than 10% of the affinity of the natural 17␤ isomer (Ginsburg et al., 1977). These findings are not necessarily discordant with those of the present study. Estrogens regulate multiple cellular signaling pathways, only some of which are dependent on transcriptional responses mediated via nuclear ER (Green and Simpkins, 2000). The estrogen-sensitive MAP kinase pathway, in particular, appears to respond to both 17␣ and 17␤ E2, in normal developing brain (Singh et al., 2000) as well as in rat-2 cells transfected with ER (Wade et al., 2001). The extent to which different pathways contribute to the neuroprotective effects of estrogen may vary greatly between experimental systems, which could explain why these effects seem to exhibit somewhat variable pharmacological properties. In normal brain tissue, the available evidence suggests that receptor-dependent and receptor-independent effects probably both contribute to the neuroprotective actions of estrogen. In explant cultures of developing rat cerebral cortex, Wilson et al. (2000) reported that E2 reduces the extent of cell death resulting from exposure to either kainic acid or a combination of potassium cyanide and 2-deoxy glucose. This response was not produced by 17␣ E2, while the effects of 17␤ E2 were blocked by treatment with Faslodex (Wilson et al., 2000), consistent with the effect being mediated via activation of estrogen receptors. In rats, Simpkins and coworkers reported that treatment with E2 either before or shortly after occlusion of the middle cerebral artery dramatically reduces the size of the resultant cerebral infarct, a response that is also elicited by 17␣ E2, suggesting that it is not nuclear receptor-mediated (Zhang et al., 1998; Simpkins et al., 1997). By contrast, Dubal et al. (2001) recently reported that the protective effects of long-term (7 day) E2 treatment against occlusion of the anterior cerebral artery are abrogated in mice with homozygous disruption of the ER␣ gene, consistent with the hypothesis that the effects of sustained physiological E2 exposure require ER␣-mediated responses. The present data suggest that in partially NGF-differentiated PC12 cells, E2 may protect against apoptosis induced by trophic withdrawal via a similar mechanism: the response is observed only after NGF treatment, which induces ER␣ expression, and it is inhibited by agents that are known to block ER␣ action. To determine whether E2 and P can also inhibit trophic withdrawal-induced apoptosis in vivo, we utilized the axotomized immature rat facial motor nerve preparation. This preparation has been used in numerous studies of neuronal death after axotomy. Axotomy of immature facial motoneurons is thought to deprive the motoneurons of trophic support from Schwann cells on the distal portions of the nerve and from the muscle fibers that they innervate. The majority of motor neuron loss occurs within the first few days after axotomy, via a process involving apoptotic cell death (Ju et al., 1994; and Fig. 6). When axotomy is performed at postnatal day 14, approximately 75% of the motor neurons ipsilateral to the nerve transection are lost

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within the first 7–14 days of surgery. In the present study, E2 and P both significantly increased the number of surviving motor neurons, by 71% and 50%, respectively. The mechanisms underlying these responses remain to be determined. The increased survival does not reflect regrowth of axons across the ablated section of nerve: in all cases, complete loss of motor function on the side of the operation remained at 21 days after surgery, as evidenced by a lack of whisker movements ipsilateral to the transection. During the first and second postnatal weeks of life, the ventromedial subdivision of the rat facial motor nucleus transiently expresses ER␣ (Yokosuka and Hayashi 1992), suggesting that during early postnatal life there may be direct effects of physiological estrogen levels on this nucleus. These “transient” receptors may be responsible for the protective effects of estrogen observed in the present study. It is also possible that effects of estrogen on neuronal survival could involve other pathways, including membrane estrogen receptor systems (Wise et al., 2001; Toran-Allerand et al., 1999). Previous studies have suggested that effects of estrogen on recovery after facial motor nerve damage do not require nuclear ER. In adult hamsters, which do not express detectable levels of nuclear ER in the facial motor nucleus, E2 has previously been shown to enhance facial motor neuron regeneration (Tanzer et al., 1999). This response may not be comparable to the effects reported here, however, since supraphysiological doses of E2 were used in these previous studies; and supraphysiological concentrations of E2 can exert effects on the CNS via activation of androgen (Tanzer et al., 1999) as well as progestin (Parsons et al., 1984) receptors. With respect to P, it is not known whether PRs are expressed in the facial motor nucleus, either during development or in adulthood. The present data are consistent with previous reports that P significantly protects facial motor neurons after axotomy in the adult rat (Yu, 1989), suggesting that sensitivity to the neuroprotective effects of P is still expressed in adulthood. In summary, these data demonstrate that E2 and P reduce the extent of cell death in two well-characterized models of apoptosis induced by trophic withdrawal: growth factor-deprived NGF-differentiated PC12 cells; and axotomized immature rat facial motor neurons. In view of the importance of trophic withdrawal-induced apoptosis in both normal development and neurodegenerative diseases, these experimental systems should prove valuable in future studies of the mechanisms of E2- and P-mediated neuroprotection. The majority of previous in vitro work on the neuroprotective effects of ovarian steroids has utilized either mixed primary cell cultures, or cell lines that do not express detectable levels of ER (Moosmann and Behl, 1999; Green et al., 1998; Green and Simpkins, 2000). There has been limited progress in defining the mechanisms by which effects mediated via nuclear steroid receptor systems may contribute to cell survival, despite the fact that these effects are clearly important for the neuroprotective effects of E2 in normal brain tissue (Wilson et al., 2000; Dubal et al., 2001). In PC12 cells, protective responses to physiological concentrations of E2 and P are

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augmented by NGF differentiation, concomitant to the induction of ER␣ and PR-A synthesis. The protective effects of the steroids appear to be dependent on ER- and PRmediated responses, in that they are blocked by either ER and PR antagonists, or inhibitors of RNA and protein synthesis. NGF-differentiated PC12 cells may therefore provide a valuable system in which to investigate the cellular and molecular mechanisms responsible for E2- and P receptor-mediated neuroprotection. Acknowledgements—The authors would like to thank Molly Mammen for her invaluable technical assistance with these studies. We are indebted to Shaila Mani for advice regarding procedures for Western blot analysis of progesterone receptor expression and to C. Dominique Toran-Allerand for support and advice with respect to Western blot procedures for detection of ER␣ and ER␤. Financial support was provided by US Army grant 98222053 and the Lowenstein Foundation (to W.G.T.). I.S.N. was supported by NIH grant AG 15092 (to C. D. Toran-Allerand).

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(Accepted 21 November 2002)