Brendan Molloy, Hilary E. M. McMahon
‹UCD School of Biomolecular and Biomedical Science, Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland
Prion disorders are associated with the accumulation of a misfolded form (PrPSc) of the normal prion protein, PrPC. Here,
we show that estrogen acts as a regulator of the processes of both prion infection and prion maintenance. Estrogen was found to be cell biased in its effect; it protected cells against prion infection in a prevention mode and enabled prion main-tenance in a treatment mode. These processes were regulated by the estrogen receptor subtypes Er␣and Er. By using spe-cific receptor agonists, Er␣was found to be the main receptor active in slowing prion infection, whereas in chronically in-fected cells, although Er␣allowed partial maintenance of PrPSclevels, Erwas the main receptor involved in maintaining PrPScin a treatment paradigm. A cell-biased effect of estrogen has been reported for other neurodegenerative disorders,
including Alzheimer’s disease. Estrogen’s effect is dependent on the cell’s health status, which impacts the use of estrogen. This work also identified that by targeting the estrogen receptors with the selective estrogen receptor modulators tamox-ifen (Tam) and 4-hydroxy-tamoxtamox-ifen (OHT), PrPSccould be cleared from prion-infected cell culture. Tam and OHT had half-maximal inhibitory concentrations for clearance of PrPScof 0.47M and 0.14 nM, respectively. This work identifies
further factors involved in the prion disease process, and through antagonism of the estrogen system, we demonstrate that the estrogen system is a target for controlling PrPSclevels.
P
rion disorders are generally associated with the accumulation in the brain of an abnormal, partially protease-resistant iso-form (PrPSc) of the normal endogenous prion protein (PrPC).PrPChas been linked to female-specific neuroprotective effects (1), but in terms of the estrogen system, a role for estrogen and its receptors in prion diseases has yet to be shown. The ability of estrogen to be neuroprotective is complex, as its action is depen-dent on a “window of opportunity” outside which estrogen has been shown to enhance neurological demise (2). Chen et al., (3) proposed this to be the “healthy-cell bias of estrogen’s action,” a process that is highly dependent on the expression of the estrogen receptor (Er) subtypes Er␣and Er. Here, we identify a role for estrogen in prion diseases that is cell biased in its effect and depen-dent on the estrogen receptors Er␣and Er.
Estrogen is known for its role in the development and mainte-nance of reproductive function (4), but it is also critical in brain development (5). It is produced by the enzyme cytochrome P450 aromatase, and during brain injury, this enzyme enables local in-creases in estrogen levels (reviewed by Nilsson and Gustafsson [6]). The ovaries are the main site of estrogen production in women, whereas in men and postmenopausal women, extragonal sites are involved, which include a number of sites in the brain (7). Once estrogen interacts with Er␣or Er, a cascade of events is induced, which allows interactions to occur with estrogen re-sponse elements (EREs) in the promoter regions of reporter genes, enabling transcription stimulation or inhibition (8,9). The recep-tors can be differentiated by their regulation of transcription (10), but they are both brain associated (6,11) and are linked to estro-gen-induced neuronal survival.
Studies have looked into the potential role of androgen re-ceptors and Er␣in prion disease development (12). Castration and androgen receptor deficiency in male C57/BL6N mice were found to prolong the incubation period of prion disease in animals intraperitoneally (i.p.) inoculated with the ME-7 scrapie strain. The study by Loeuillet et al. (12) supported a potential role for androgens in prion disease development
when inoculation occurred at a peripheral site. In the same study, neither ovariectomy nor knockout of Er␣affected the prion incubation period in female mice (12). However, paren-tal female C57/BL6N mice were observed to have a shorter prion incubation period than their male counterparts (12). Fe-male-specific prion-linked neuroprotection was reported in a study with Zrch IPrnp0/0 mice; in this study, ischemia insult led to high neuronal damage in the CA1 region, but only in the male mouse model (1). Estradiol (E2) failed to protect against ischemia insult in the male models (1), which is intriguing, as estrogen is a known neuroprotector: it alters ischemia across animal species in stroke models (13–15), it acts as a mediator of gender differences in stroke, and it has been found to have protective functions in male stroke models (15–17).
Estrogen’s “healthy-cell bias of action” has been used to ex-plain the divergent effects of estrogen in the treatment of neuro-degenerative conditions, including Alzheimer’s disease (AD). In AD, clinical support demonstrated a potential for estrogen in the treatment of AD; however, this has not been the case in all studies. The efficiency of estrogen treatment has been found to change with increasing age and treatment duration (18). The conditional effect of estrogen therapy was particularly highlighted in the Women’s Health Initiative Memory Study (WHIMS) clinical trial (19). It has emerged that estrogen’s action is affected by neurolog-ical health: estrogen has a positive protective effect in healthy cells, whereas it exacerbates conditions in diseased, unhealthy, or aged cells (20). Chen et al. (3) observed that estrogen protected against A1-42-induced neurodegeneration in hippocampal neurons
Received8 October 2013Accepted5 November 2013
Published ahead of print13 November 2013
Address correspondence to Hilary E. M. McMahon, [email protected].
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.02936-13
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when used in prevention mode, whereas when used postexposure to A1-42, neurodegeneration was enhanced. The molecular basis of the healthy-cell bias of action or window of opportunity is un-clear, but it is becoming clear that it relates to the expression of functional forms of Er␣and Er(21,22).
Here, we show for the first time a role for estrogen in the infec-tion and maintenance processes of the prion agent. Estrogen was cell biased in its effect. It afforded partial protection against prion infection in a prevention mode, while it enabled the maintenance of PrPScin persistently infected cells in a treatment mode. The cell-biased effect was dependent on the estrogen receptors. When activated with specific selective agonists, Er␣was more protective in the prevention mode, and Erwas most active in the mainte-nance of PrPScin the treatment mode. This work identifies a dual role for estrogen in the prion disease process, which is dependent on its time of use.
MATERIALS AND METHODS
Reagents and antibodies.Dulbecco’s modified Eagle medium (DMEM),
fetal calf serum (FCS), horse serum (HS), penicillin-streptomycin, and
L-glutamine were obtained from Gibco products supplied by Biosciences. All other reagents were from Sigma-Aldrich. The antibody 7A12, which recognize epitope 175 to 185 of mouse PrP, was kindly provided by Man-Sun Sy (Case Western Reserve University School of Medicine). The anti-body has been previously described by Pham et al. (23). Antibodies to actin (1 to 19) (sc-161), Er␣(MC20), and Er(H-70) were from Santa Cruz. Secondary horseradish peroxidase rabbit anti-mouse antibody was from Calbiochem, goat anti-rabbit was from Upstate-Millipore, and don-key anti-goat was from Santa Cruz. Immortalized hypothalamic neuronal GT-1 cells infected with scrapie strain 22L (GT22L) and isolate Chandler (K) (GTK) (24) and the prion-transfected N2a cell line N2a58 (24) were kindly provided by S. Lehmann (Montpelier, France).
Cell culture.The neuroblastoma cell line infected with the 22L scrapie
strain (N2a22L20) was reported previously (25). N2a22L20 and N2a58 cells were grown in DMEM supplemented with 10% FCS, 10 mM peni-cillin-streptomycin, and 300g/ml Geneticin. GT1 cells infected with the Chandler scrapie isolate (GTK) and the 22L scrapie strain (GT22L) (24) were maintained in DMEM supplemented with 5% FCS, 5% HS, 1 mM sodium pyruvate, and penicillin-streptomycin. The cells were maintained at 37°C in an atmosphere of 5% CO2and 95% air.
Where indicated, to avoid estrogenic effects from the medium, cells were grown in an estrogen-free “stripped medium,” which is phenol red-free DMEM supplemented with 10% stripped serum and appropriate antibiotics. Phenol red is used in medium as a pH indicator; however, it has estrogenic properties, enabling it to bind to estrogen receptors (26). Likewise, to avoid the estrogen effects of serum, estrogen was removed from the serum using the method of Heringa et al. (27). To strip serum, a dextran-charcoal (DC) slurry was prepared; a slurry of 0.5% charcoal and 0.05% dextran in 50 mM Tris-HCl (pH 7.4) was prepared and left to stir overnight at 4°C. A volume of DC slurry similar to the volume of serum to be stripped was centrifuged for 10 min at 675⫻g. The supernatant was discarded, and the serum was added to the remaining pellet. This slurry was gently shaken for 45 min in a water bath at 45°C and subsequently centrifuged for 20 min at 675⫻g. The supernatant serum was then trans-ferred to fresh DC pellets, and the stripping procedure was repeated. The resulting supernatant serum was filtered through a 0.1-m filter.
Cell growth trypan blue exclusion assay.Cells were passaged from
normal growth medium into the medium indicated, and they were then grown for the indicated time, after which they were trypsinized and the cell suspension was mixed with an equal volume of 0.4% trypan blue solution. Viable cells (unstained cells) were counted with a hemocytom-eter.
MTT cell viability.The MTT (3,[4,5 dimethylthiazol-2-yl]-2,5
diphe-nyltetrazolium bromide) assay is widely used for quantifying
metaboli-cally active cells and analyzing cell viability (28). Cells were seeded at 8,000 per well in a 96-well plate and grown for 24 h in the medium, which was then replaced with fresh medium containing the compound at the con-centrations indicated below. After a further 24 h, cell survival was deter-mined with MTT. The medium was removed from the cells, 110l MTT (0.5 mg/ml in medium) was added to each well of a 96-well plate, and the cells were then cultured for 2 h at 37°C. Dimethyl sulfoxide was then added to release the formazan product, which was measured at 570 nm. Relative survival was determined in comparison to untreated controls.
Cell-to-cell prion infection.Cells from 7 60-cm2confluent N2a22L20
flasks were collected under sterile conditions and resuspended in 100l cold phosphate-buffered saline with 5% glucose. The suspension was freeze-thawed 4 times and then homogenized by passing the suspension through a 27-gauge needle several times. Homogenate (10l or 25l) was added to the cells at the time of passage in 60-cm2flasks, and the cells were passaged every 3 days and examined for the presence of PrPSc.
Western blotting.For standard analysis of protein, cells were lysed in
cold lysis buffer (LB) (0.5% [vol/vol] Triton X-100, 0.5% [wt/vol] sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1g/ml pepstatin, 1g/ml leupeptin, 2 mM EDTA). The total protein concentration was measured using a bicinchoninic acid protein assay kit (Sigma). For PrPSc analysis, lysates were digested with 16g of proteinase K (pK)/mg of protein at 37°C for 15 min; the reaction was then stopped with 1 mM Pefabloc. Samples in loading buffer were boiled for 5 min, loaded onto 12% SDS-PAGE, and analyzed by Western blotting, employing standard techniques. For PrPCand/or total PrP analysis, lysates were not pK di-gested. Protein was detected by incubating immunoblots with the indi-cated antibodies, followed by a horseradish peroxidase secondary anti-body, and developed by enhanced chemiluminescence (ECL).
For the determination of protein insolubility, samples in lysis buffer were centrifuged at 264,909⫻gfor 40 min at 4°C, and the soluble portion (supernatant) was then separated from the insoluble portion (pellet). The supernatant was solvent precipitated, and the insoluble fraction was di-rectly resuspended in sample loading buffer.
RESULTS
Effect of targeting estrogen receptors on PrPScmaintenance. The objective of this study was to examine whether estrogen and its receptors are involved in the maintenance of prion infection. In a first approach to examine possible involvement of the estrogen system in prion maintenance, we looked at the effects of selective Er modulators (SERMs) on PrPSc. The estrogen receptors can be
activated/inactivated by SERMs, such as tamoxifen (Tam) and 4-hydroxy-tamoxifen (OHT), which are among the most studied SERMs. Tam is a triphenylethylene mixed estrogen receptor ago-nist and antagoago-nist, and OHT is a metabolite of Tam but is more active as a SERM than Tam (29,30). To investigate the effects of these SERMs on PrPScmaintenance, N2a cells infected with the
22L strain of scrapie (N2a22L20) were treated with both Tam and OHT at 5M and 5 nM final concentrations, respectively (Fig. 1A). After 2 weeks of treatment, both compounds cleared PrPSc from the cells, while the vector ethanol had no effect. This ability of Tam and OHT to reduce PrPSclevels suggested a potential role for the Ers in prion maintenance. As a result, we decided to exam-ine the effect of removing preexisting estrogen, which is present in standard cell culture media, on PrPScmaintenance. Both the pH
indicator phenol red, which is used in growth media, and serum itself are known to contribute to cellular estrogenic activity (26, 27). To remove their effects, cells were grown in stripped medium that lacked phenol red and contained serum that had been stripped of estrogen with dextran and activated charcoal (27). Growth of N2a22L20 cells in stripped medium resulted in the clearance of PrPScfrom the cells (Fig. 1B, lane 2). To confirm that
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estrogen contributed to the stripped medium’s effect, the stripped medium was supplemented with Tam, OHT, or estrogen (E2). Of the supplements, only E2 maintained PrPSclevels (Fig. 1B, lane 5), further suggesting a role for E2 in the maintenance of PrPSc. The
effects seen with OHT and Tam on PrPScin normal media were not due to toxic effects on the N2a22L20 cells, as neither com-pound was toxic at the concentrations employed (Fig. 1C).
It has been suggested that the growth rate can influence the levels of PrPScseen in cells, and if an antiprion compound affects
the cellular growth rate, growth modulation itself may play a role in the clearance of the prion agent (31). We examined whether the N2a22L20 cell growth rate was affected by OHT or Tam over a 96-hour period (Fig. 1D). No significant difference was seen be-tween cells grown in normal medium and in medium
supple-Control
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FIG 1Effects of SERMs on PrPScin N2a22L20 cells. (A and B) N2a22L20 cells grown in normal (A) and hormone-free [indicated by Strip and (S)] (B) media were treated with OHT (5 nM), Tam (5M), E2 (5M), and the vector ethanol for 2 weeks, as indicated. The cells were lysed, and 50g of the lysate was treated with proteinase K for detection of PrPSc. The digests were analyzed by 12% SDS-PAGE and immunoblotting with 7A12 antibody. Protein size (in kDa) is indicated on the left of the blots. (C to F) Each compound, at the concentrations indicated, was examined for its effect on cell viability with the MTT assay in normal medium (C) or stripped medium (E). Cell growth in the presence of each compound was also examined over a 96-h period in normal medium (D) or stripped medium (F). The results are representative of three independent experiments. The data represent the means and standard deviations (SD) between experiments. *,P⬍0.05 (Student’sttest).
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[image:3.585.79.501.66.552.2]mented with the compounds. A lowered cell level, although not significant in effect, was noted from 72 h onward with Tam but was not seen with OHT. This could indicate that Tam, but not OHT, may have an effect on the growth of the cells. It is also important to note that these cells would not normally be left to grow past 48 to 72 h without passaging. Effects on toxicity and growth rates were also examined when cells were grown in stripped medium (Fig. 1EandF). At the concentrations used to clear PrPSc, 5M Tam and 5M E2, the SERMs had no effect on
cell toxicity in stripped medium (Fig. 1E). With OHT, we tested concentrations higher than that used to clear PrPSc, and at 1M
OHT, no toxic effects were seen (Fig. 1E). When growth rates were examined, it was observed that cell growth in stripped medium mirrored that seen in normal medium (Fig. 1F). E2 and OHT did not affect the growth of the cells in stripped medium, but a differ-ence was seen with Tam. Of the compounds, only E2 blocked the stripped medium’s effect on PrPScclearance, but this effect does
not appear to be related to an effect on the cell growth rate. Of the SERMs OHT and Tam, OHT has the highest antiestro-gen activity; it is 30- to 100-fold more active than Tam (29,30). As a result, if Tam and OHT were acting at the estrogen receptors to clear PrPSc, OHT would be expected to have higher antiprion ac-tivity than Tam. To examine this, the half-maximal inhibitory concentrations (IC50s) for PrPScclearance by Tam and OHT were
determined in normal medium. Tam and OHT at concentrations up to 1M and 1 nM, respectively, were added to N2a22L20 cells for 2 weeks, and PrPSclevels were then monitored. Tam cleared
PrPScwith an IC50of 0.47M (Fig. 2AandB), while OHT was
significantly more active, with an IC50of 0.14 nM (Fig. 2CandD).
The significantly higher activity of OHT than Tam would suggest that these SERMs work through the estrogen receptor system.
We also examined the effect of targeting the estrogen system in the GT1-7 cell line infected with 22L and Chandler (K). GT22L and GTK cells were grown in normal medium supplemented with Tam or OHT or in stripped medium supplemented with E2 for 2 weeks at the concentrations indicated (Fig. 3). Both Tam and OHT significantly reduced PrPSclevels in both the GT22L and
GTK cells. When these cell lines were grown in stripped medium, PrPScwas cleared within 2 weeks (Fig. 3AandB, lanes 5), and this
effect was blocked by E2 (Fig. 3AandB, lanes 6). These results indicate that similar estrogen pathways of control of PrPSc
main-tenance appear to exist across the GT and N2a infected cell lines. Effects of E2 on PrPScand PrPC.To further examine E2’s abil-ity to maintain PrPSclevels, we exposed N2a22L20 cells to increas-ing concentrations of E2 (0.05 to 5M) in normal or stripped medium (Fig. 4A,B, andC). In normal medium, 1M E2 showed an ability to increase PrPSclevels over control levels (Fig. 4Aand
C). As before, when cells were grown in stripped medium, PrPSc was cleared after 2 weeks (Fig. 4B, lane 2); this effect was partially blocked at 0.1M E2, and complete maintenance of PrPScwas seen with 5M E2 (Fig. 4B, lanes 3 to 8, andC). We were inter-ested in whether E2 allowed this maintenance of PrPScby inducing PrPCto develop abnormal prion properties as a route to PrPSc
production. To examine this, N2a22L20 cells that had been per-manently cleared of PrPScby treatment with Congo red (Cr22L20
cells) were treated with 5M E2 for 2 weeks (Fig. 4D). E2 did not affect the total PrPClevels (Fig. 4D, compare lanes 5 and 8), nor
did it induce PrPCto develop the abnormal insolubility properties that are characteristic of PrPSc(Fig. 4D, compare lanes 4 and 7;
lane 2 shows insoluble PrP in N2a22L20 cells). Insoluble PrP was
seen only in the control N2a22L20 cells, and PrP in these cells was seen to partition between soluble and insoluble phases (Fig. 4D, lanes 1 and 2). In N2a22L20 cells, PrPSc is found in truncated forms, allowing the lower-molecular-weight PrP species to be ob-served in the insoluble phase without pK digestion (Fig. 4D, lane FIG 2Determination of the IC50s for Tam and OHT in N2a22L20 cells. (A and C) N2a22L20 cells were incubated with increasing concentrations of Tam (A) and OHT (C) for 2 weeks, and the cells were then lysed and analyzed for PrPScusing antibody 7A12. Protein size (in kDa) is indicated on the left of the blots. (B and D) Representative bands from panels A and C were quantified by densitometry and expressed as percentages of their respective controls. The results are representative of three independent experiments. The graphic data represent the means⫾SD between experiments.
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[image:4.585.300.532.61.573.2]2). These truncated PrP species can be seen without pK diges-tion in the 22L20 cells, whereas they are not present in Cr22L20 cells (Fig. 4E, compare lanes 1 and 3). pK-resistant PrP was observed only in the N2a22L20 cells (Fig. 4E, lane 4), and the truncated PrP species seen after pK digestion are similar in size to the truncated PrP observed without pK digestion (Fig. 4E, compare lanes 3 and 4).
Effect of targeting specific estrogen receptors, Er␣and Er, on PrPScmaintenance.In examining the potential role of Er␣in prion disease development, Loeuillet et al. (12) knocked out the Er␣in C57/BL6N mice, but they saw no effect of Er␣on prion disease development. As a result, we examined which of the Er receptors was responsible for the estrogen response seen here. The
effects of estrogen may be mediated by the receptors Er␣and/or Er, and to target these receptors specifically, we used the Er sub-type-selective agonists 4,4=,4⬙-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT) (selective for Er␣) and 2,3-bis(4-hydroxy-phe-FIG 3Effects of targeting estrogen receptors in GT22L and GTK cells on
PrPSc. (A and B) GT22L (A) and GTK (B) cells grown in normal and hormone-free [stripped (S)] media were treated with OHT (10 nM), Tam (5M), and E2 (5M) for 2 weeks. The cells were lysed, and 50g was treated with proteinase K for detection of PrPSc. The digests were analyzed by 12% SDS-PAGE and immunoblotting with 7A12 antibody. Protein size (in kDa) is indicated on the left of the blots. (C) Representative bands from panels A and B were quantified by densitometry and expressed as a percentage of the control (untreated cells grown in normal medium). The results are representative of three indepen-dent experiments. The graphic data represent the means and SD between ex-periments. *,P⬍0.05; **,P⬍0.005 [Student’sttest, relative to control or control (S)].
FIG 4Effects of increasing E2 on PrPScand PrPC. (A and B) N2a22L20 cells grown in normal (A) and hormone-free/stripped (B) media were incubated with increasing concentrations of E2 for 2 weeks. In panel B, the control is N2a22L20 cells grown in normal medium. The cells were lysed after 2 weeks, and 50g was analyzed for PrPScusing 7A12 antibody. (C) Representative bands from A and B were quantified by densitometry and expressed as percentages of their respective controls. The graphic data represent the means and SD between experiments. (D) Cr22L20 cells were grown in the presence and absence of E2 (5M) for 2 weeks. The cells, including N2a22L20 cells, were lysed and processed for insolubility de-termination (S, soluble; I, insoluble). Where 18-g samples are loaded, they were not processed for insolubility. (E) Untreated cells digested (⫹) (50g) or not (⫺) (18g) with pK. Samples were analyzed by 12% SDS-PAGE and immunoblotting with 7A12 antibody. The results are representative of three independent experi-ments. Protein size (in kDa) is indicated on the left of the blots.
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[image:5.585.300.541.65.524.2] [image:5.585.72.253.69.444.2]nyl)-propionitrile (DPN) (Er selective). These agonists are highly specific and have been widely used in Er functional analysis experiments (reviewed by Weiser et al. [32]). DPN has a 70-fold-greater binding affinity and 170-fold-70-fold-greater relative potency for Erthan for Er␣(33,34), and PPT has a 400-fold-greater binding affinity for Er␣than for Er(35). GTK, GT22L, and N2a22L20 cells were grown in stripped medium in the presence and absence of E2 (5M), PPT (10 to 1,000 nM), and DPN (10 to 1,000 nM). Growth in stripped medium cleared PrPScfrom each of the cell
types within 2 weeks (Fig. 5A,B, andC, compare lanes 1 and 2, and D,E, andF), and this effect was blocked by the presence of 5M E2 (Fig. 5A,B, andC, lanes 3). Although PPT at increasing con-centrations partially blocked the effect of growth in stripped me-dium (Fig. 5A,B, andC, lanes 4 to 6), the greatest maintenance of PrPSclevels was seen with the Er-selective agonist DPN (Fig. 5A,
B, andC, lanes 7 to 9). This suggests a stronger role for Erin the estrogen response observed. When we examined the growth of cells in stripped medium in comparison with medium supple-mented with PPT or DPN, neither PPT nor DPN affected the growth of the cells, indicating that their effects on PrPScin stripped medium are not through modulation of the cellular growth rate (Fig. 5G).
The actions of Erand Er␣in signaling processes within the cell are complex. The receptors are known to interact: Erhas been shown to inhibit Er␣response to estrogen (35,36), and the Er/Er␣ ratio is thought to be significant for the estrogen re-sponse. Each of the cell types, N2a22L20, GTK, and GT22L, ex-pressed both Erand Er␣(Fig. 6). When we examined the effects of E2, PPT, and DPN on the levels of these receptors in cells, the compounds did not show a significant effect on the expression of the receptors. The Er␣and Erlevels in a given cell can determine the cell’s response to estrogen (6), and although these results do not show variability in the receptors in terms of protein levels, the activities of the receptors may still be altered.
Effect of targeting estrogen receptors on the prion infection process.The theory of the healthy-cell bias action of estrogen in AD proposes that estrogen’s action is dependent on the health status of the cell (3). In this work, cells persistently infected with the prion agent responded in a negative manner to estrogen, as it acted as a causative agent for prion maintenance in stripped me-dium. Estrogen is known to enhance factors for neurological de-mise when the health status of the cell is already comprode-mised (3). In contrast, if cells are healthy at the time of estrogen exposure, estrogen has been found to be beneficial for neurological survival (3). We wanted to examine whether the estrogen prevention par-adigm also applied to prion disorders. To investigate this, we used a prion cell-to-cellex vivotransmission assay with the highly in-fectable cell line N2a58 (37). These cells, in a 60-cm2dish, were infected with 10l N2a22L20 homogenate, and at the time of infection, the cells either were left untreated; were treated once at the time of infection with E2 (5M), PPT (1M), or DPN (1M) and then subsequently passaged into normal medium lacking drugs; or were treated with each compound at each passage (Fig. 7).
Minute—almost undetectable—levels of PrPSc were seen in
the cells two passages postinfection (P2), it took three passages (P3) for PrPScto reach quantifiable levels. Relative to the infection
control at P3 (Fig. 7A, lane 3, P3), none of the drugs had a signif-icant effect on the level of infection when given once, except E2, which lowered the efficiency of infection slightly (Fig. 7A,
com-pare lanes 4, 6, and 8 with lane 3). The infection process was significantly reduced when E2 and PPT were given at each passage (Fig. 7A, compare lanes 5 and 7 with lane 3, andB), but DPN had no significant effect (lane 9, P3). At P5, a similar profile was seen: cells treated once with DPN and PPT were found to have similar levels of PrPScrelative to the control (Fig. 7, compare lanes 6 and 8 with lane 3, P5, andB), whereas by P5 the single E2 treatment reduced PrPScproduction by 37% (Fig. 7A, lane 4, P5). Again, with continuous treatment, by P5, E2 and PPT both significantly reduced infection efficiency by 77% and 58.7%, respectively, whereas DPN had a limited effect.
The efficiency of inhibition of the infection process was greatly enhanced by continuous treatment with the compounds, and be-cause of this, we were interested in examining whether the load of infectious agent used affected E2’s protective ability. To examine this, cells were exposed to 25l of infectious lysate, and the infection process was monitored up to passage 5 (Fig. 8). Relative to infected cells at passage 5, Inf P5 (Fig. 8A, lane 5), the level of PrPScrose as expected from P3 to P5 in the absence of the compounds (Fig. 8A, lanes 3, 4, and 5, andB). With continuous E2 treatment, prion infec-tion was again significantly reduced at each passage, irrespective of the higher load of PrPScemployed (Fig. 8A, lanes 6, 7, and 8, andB). Both DPN and PPT have been shown to upregulate the activities of their respective receptors in N2a cells (38). In the prevention mode, PPT was more effective than DPN in reducing infection efficiency when cells were infected with 10l infectious lysate (Fig. 7), and their actions were also compared when the infection load was increased to 25l (Fig. 8CandD). Relative to DPN, infection was significantly less efficient when PPT was used (Fig. 8CandD). From this work, it appears that estrogen has a healthy-cell bias of action in the prion system, as it protects against prion infection and yet maintains infec-tion in established prion cell lines. Surprisingly, while DPN was more active than PPT at maintaining infection in the 22L20 cell line, the reverse was seen in the prevention paradigm, suggesting varying roles for Er␣and Erin prion infection and prion maintenance.
DISCUSSION
Estrogen has been reported to have a wide range of cellular effects (39) that can be induced through genomic pathways, which have long reactive responses, or nongenomic pathways, which respond within seconds (40,41). Er␣and Eraffect how a cell responds to estrogen; Er␣’s response is proposed to be neuroprotective, whereas that of Erhas been reported to induce neuronal apop-tosis (42). Estrogen acts as a regulator in a number of neuronal systems (43,44), and it is significant in age-associated neuronal degenerative diseases (45,46). The timing and the health status of the cell are both key in dictating whether estrogen has a positive or negative influence in a system (4,47). This cell-biased effect of estrogen has been used to explain the diverging effects of estrogen in neurodegenerative models, including AD. Here, we show for the first time that estrogen has a role in the maintenance of PrPSc,
and as in the AD systems, it is biased in its effect: it reduces infec-tion efficiency in a preveninfec-tion paradigm and maintains PrPSc
lev-els in chronically infected cells, a treatment paradigm. The effect of estrogen on PrPScis related to the response of its receptors, Er␣
and Er. Er, which was more responsive than Er␣in maintaining PrPScin chronically infected cells, was not as protective as Er␣in
the prevention paradigm.
In line with targeting the estrogen system to regulate PrPSc,
both of the SERMS Tam and OHT lowered PrPSclevels in all of
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1µM 1µM
FIG 5Targeting specific estrogen receptors. (A to C) GTK (A), GT22L (B), and N2a22L20 (C) cells were grown for 2 weeks in the presence and absence of E2 (5M), PPT (10 to 1,000 nM), and DPN (10 to 1,000 nM). The cells were lysed, and 50g was treated with proteinase K for detection of PrPSc. The digests were analyzed by 12% SDS-PAGE and immunoblotting with 7A12 antibody. Protein size (in kDa) is indicated on the left of the blots. (D, E, and F) Representative bands from panels A, B, and C were quantified by densitometry and expressed as percentages of the control (untreated cells grown in normal medium). (G) N2a22L20 cells were grown in the indicated media, and cell counts were determined at the time points shown. The results are representative of three independent experiments. The graphic data represent the means and SD between experiments. *,P⬍0.05; **,P⬍0.005 [Student’sttest, relative to control (S)].
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[image:7.585.110.475.59.641.2]the infected cell lines tested here. The promoter context of the Ers can dictate the actions of SERMs. Er␣ and Ershare a number of transcriptional pathways; however, their protein sequences differ, and this leads to the unique mechanisms of action of the receptors in response to SERMs. The estrogen receptors are made up of two activation function (AF) do-mains, AF1 and AF2, which are found in the N terminus and C terminus, respectively; a DNA-binding domain (DBD); and a ligand-binding domain. The lowest homology between Er␣ and Erlies in the AF-1 and N-terminal domains (48). These differences in the receptors affect how SERMs work. Tam has been reported to act as both agonist and antagonist through Er␣, but it is only antagonistic at Er(49,50). In keeping with OHT being more antiestrogenic than Tam, OHT had an IC50of
0.14 nM in N2a22L20 cells, which is significantly higher than
that of Tam (0.47M). The effects of the SERMs and estrogen on PrPSc could indicate that blocking the Ers is a potential
therapeutic channel for prion disorders. Indeed, growth of scrapie-infected cells in stripped medium, which has lowered estrogenic activity, cleared PrPScfrom the infected cells. Estro-gen’s effect on maintaining PrPScwas not due to an effect on
the insolubility properties of PrPC; it does not cause PrPCto be-come PrPSc-like in terms of insolubility characteristics.
How-ever, this does not rule out a potential effect of estrogen on the trafficking of PrPC, which could affect its availability as a
sub-strate for conversion.
The results with DPN and PPT indicate that although the Er␣ -responsive pathway is partially effective at maintaining PrPSc lev-els, PrPScmaintenance is strongest through the activation of Er.
This is also in line with the neurodegenerative pathways proposed FIG 6Estrogen receptor levels are not affected by E2, PPT, and DPN. (A to C) GTK (A), GT22L (B), and N2a22L20 (C) cells were grown for 72 h in the presence or absence of E2 (5M), PPT (10 to 1,000 nM), and DPN (10 to 1,000 nM). The cells were lysed, and 50g lysate was analyzed by 12% SDS-PAGE and immunoblotting for Erand then stripped and analyzed for Er␣and the corresponding actin. (D, E, and F) Representative bands from panels A, B, and C were quantified relative to their respective actins by densitometry and were expressed as percentages of the control (lanes 1). Black bars, Er␣; gray bars, Er. The results are representative of three independent experiments. The graphic data represent the means and SD between experiments.
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[image:8.585.94.492.64.487.2]for Er(42). The distinction in the effects of the estrogen recep-tors on PrPScmay lie in the diverging mechanisms of action of the
receptors. The estrogen receptors have 60% homology in their hormone-binding regions, so although both receptors bind estro-gen, the receptors can vary functionally. Both receptors are ex-pressed in the brain, either together or independently (51,52), and are required for spatial learning, but their significance for brain function varies. It has been shown that knockout of Er␣impairs reproductive functions in mice; however, deficiency of Erresults in abnormalities linked with defects in neuronal migration (non-reproductive brain function) (46,53,54). As reports indicate that knockout of Erleads to significant neuronal degeneration, this could indicate that Eris more significant than Er␣in its role in
neurodegenerative disease progression (5) and potentially in prion disease maintenance.
The estrogen response is complex; the diverging effects of es-trogen in the treatment of neurodegenerative diseases have led to FIG 7Effect of estrogen receptors on infection. (A) N2a58 cells were infected
with 10l 22L20 lysate; they were then grown in the presence or absence of E2 (5M), PPT (1M), or DPN (1M). The cells were either treated with drugs once at the time of infection (-1) or at each passage (-E). The cells were lysed at the passage times indicated, and 200-g samples were proteinase K digested for detection of PrPSc. The digests were analyzed by 12% SDS-PAGE and immunoblotted with antibody 7A12. Lane 22L20 shows PrPScin the infectious lysate; lane 58 is 200g of noninfected pK-digested N2a58 lysate. Protein size (in kDa) is indicated on the left of the blots. (B) Representative bands from panel A (P3 and P5) were quantified by densitometry and were expressed as percentages of the control (untreated infected [Inf], lanes 3). The results are representative of three independent experiments. The graphic data represent the means and SD between experiments. *,P⬍0.05; **,P⬍0.005 (Student’s ttest relative to Inf).
FIG 8Effect of an increasing infection load on the estrogen receptor reg-ulator response. (A) N2a58 cells were infected with 25l 22L20 lysate and grown in the absence (Inf) or presence of E2 (5M) up to 5 passages. Cells were lysed at the passage indicated, and 200-g samples were proteinase K digested for detection of PrPSc. The digests were analyzed by 12% SDS-PAGE and immunoblotted with antibody 7A12. (B) Bands from panel A were quantified and expressed as percentages of Inf P5. (C) N2a58 cells infected with 25l 22L20 lysate were also grown in the presence of 1M PPT or DPN up to 5 passages. (D) Cells were lysed and analyzed as for panel A, and bands from panel C were quantified and expressed as percentages of lanes Inf⫹DPN P5. Lanes 22L20 show PrPScin the infectious lysate, and lanes 58 are 200g of noninfected pK-digested N2a58 lysate. The results are representative of three independent experiments. The graphic data represent the means and SD between experiments. Protein size (in kDa) is indicated on the left of the blots. *,P⬍0.05; **,P⬍0.005 (Student’sttest, relative to P3, P4, or P5 in panels B and D).
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[image:9.585.42.279.66.431.2] [image:9.585.321.514.74.511.2]reassessments of how best to use estrogen. In the prevention mode, we have found that estrogen is protective against prion infection. In this paradigm, unlike the treatment paradigm, Er␣is the main responsive receptor. There is a “window in time” in regard to how models respond to estrogen treatment, and this is affected by which receptor is responsive. In female rat models, aging leads to a reduction in both Er␣and Er(55,56), but of the receptors, only Erremains responsive to estrogen treatment in aged rats (56). Leon et al. (57) recently observed estradiol to be neuroprotective in the postischemia rat brain, but only in young adult rats, whereas it worsened conditions in the aged rat model. The critical period for a beneficial effect of E2 is reflected in the treatment of aged animals, where E2 results in 16% increased mortality (21). Er␣is significant for the cell-biased effect of estro-gen. Synaptic Er␣is known to decrease in the CA1 region of the aged rat, a region that is significant for cognitive performance (58). Zhang et al. (21) recently showed that Er␣is critical to the cell-biased effect of estrogen. In their study, long-term estrogen deprivation (LTED) resulted in decreased Er␣in the CA1 region, which was linked to a failure of estrogen to be neuroprotective. Interestingly, LTED had no effect on uterus Er␣levels, and al-though the neuronal system was not responsive to estrogen, the uterotropic action of E2 remained (21). The positive action of estrogen in our prion models and the involvement of Er␣in this process correspond to the most recent understanding of E2’s cell-biased action.
In the work of Loeuillet et al. (12) on hormonal aspects of prion diseases, a potential role for the androgen receptor in the devel-opment of prion diseases was identified. They found that castra-tion affected the prion incubacastra-tion period in male mice, and then only when i.p. inoculation was used; they saw no effect in ovari-ectomized female models. In their work with female models, nei-ther ovariectomy nor knockout of Er␣affected the prion incuba-tion period. In terms of the healthy-cell-biased acincuba-tion that we have seen, Er␣plays the strongest role in protection against prion in-fection. It could be imagined, then, that knockout of Er␣in the Loeuillet et al. study (12) either would have had a limited effect on prion infection in animal models, which occurred, or could en-hance the process, as Erremains. In terms of the lack of effect of ovariectomy on prion infection, past studies have shown that ovariectomy and long-term ovarian hormone deprivation signif-icantly affect hippocampal Er␣levels without any effect on Er (59), which is a driver of prion maintenance.
As reviewed by Zhao et al. (47), the health status of the neuron affects the estrogen response; in diseased or dysfunctional neu-rons, estrogen may exacerbate the disease process. Estrogen ther-apy has been shown to lower the risk of development of Alzhei-mer’s disease (47,60,61); however, in aged rat models, estrogen treatment postneurodegeneration worsened the degeneration process (19,62,63). With this in mind, estrogen application post-prion infection, particularly in an aged rat model, may have a negative impact on the disease process. This is a significant con-sideration for late-onset prion diseases, especially if the response of the human estrogen receptors mirrors that seen in aged rat models, in which only Erremains responsive to estrogen. For familial prion disease, where the age of onset is quite variable (64), the estrogen prevention paradigm may provide a treatment ave-nue. This work shows for the first time a cell-biased effect of es-trogen in a prion model (Fig. 9). It allows greater understanding of
the receptors that play a role in prion diseases and the dual role that estrogen receptors play in the process.
ACKNOWLEDGMENT
We thank Regina O’Sullivan for critically reading the manuscript.
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![FIG 3 Effects of targeting estrogen receptors in GT22L and GTK cells on(5free [stripped (S)] media were treated with OHT (10 nM), Tam (5K for detection of PrPPrPSc](https://thumb-us.123doks.com/thumbv2/123dok_us/147638.24782/5.585.300.541.65.524/effects-targeting-estrogen-receptors-stripped-treated-detection-prpprpsc.webp)
