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Published as doi: 10.1096/fj.08-115360.
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(The FASEB Journal. 2009;23:613-622.)
© 2009 FASEB

Opposing effects of ERK and p38-JNK MAP kinase pathways on formation of prions in GT1-1 cells

Elin Nordström1, Gilberto Fisone and Krister Kristensson

Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

1Correspondence: Department of Neuroscience, Retzius väg 8, Karolinska Institutet, Stockholm, SE-171 77 Sweden. E-mail: elin.nordstrom{at}ki.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Brain-derived neurotrophic factor, which activates the extracellular regulated kinase (ERK) pathway, increases formation of prions in scrapie-infected gonadotropin-releasing hormone (GT1-1) cells. This indicates that conversion of the cellular prion protein PrPC to its pathogenic isoform, PrPSc, can be regulated by physiological stimuli acting on specific signal transduction pathways. In the present study, we examined the involvement of different mitogen-activated protein (MAP) kinase cascades and the cAMP-PKA pathway in formation of proteinase K-resistant PrPSc (rPrPSc). Long-term depolarization of GT1-1 cells infected with the Rocky Mountain Laboratory strain of scrapie increased the formation of rPrPSc. This effect was associated to ERK activation and was blocked by the MAPK/ERK kinase (MEK) inhibitor U0126. Treatment with forskolin caused a similar increase in rPrPSc formation that was prevented by the protein kinase A (PKA) inhibitor H89. Both depolarization and forskolin treatment were accompanied by increased phosphorylation of the S6 ribosomal protein, while phosphorylation of histone H3 occurred only after forskolin treatment. Inhibitors of p38- and c-Jun NH2-terminal kinase (JNK) promoted the formation of rPrPSc, in contrast to the clearance of rPrPSc produced by inhibitors of the ERK pathway. Thus, the ERK and the p38-JNK MAP kinase pathways appear to exert opposing effects on rPrPSc formation, suggesting that balances between these intracellular signaling cascades may regulate replication of prions.—Nordström, E., Fisone, G., Kristensson, K. Opposing effects of ERK and p38-JNK MAP kinase pathways on formation of prions in GT1-1 cells.


Key Words: scrapie • neuron • neurodegeneration • protein misfolding • PKA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PRION DISEASES, OR TRANSMISSIBLE spongiform encephalopathies, are invariably fatal disorders, characterized by spongiform neurodegeneration, neuronal loss, and astrocytic reaction in the brain. They are associated with the conversion of a normal plasma membrane protein, the cellular prion protein (PrPC), to a misfolded isoform of the protein (PrPSc), which accumulates in the infected brain. In detergents PrPSc comprise aggregates of a continuum of sizes (1) . The larger aggregates are partially resistant to treatment with proteinase K (1 2 3) and are designated rPrPSc, while the smaller aggregates may be sensitive to the treatment and are designated sPrPSc. PrPC is a glycolipid-anchored protein that is sorted into cholesterol-rich detergent-resistant domains (lipid rafts) in the plasma membrane. Sorting of PrPC to lipid rafts is mandatory for the conversion to PrPSc (4 , 5) , but the mechanisms behind this process and the causes of neurodegeneration remain to be clarified. Although a number of compounds can interfere with the conversion process (e.g., by shielding of PrPC or PrPSc or by destabilizing lipid rafts [6 , 7 ]), and a still unidentified conversion cofactor has been suggested (8 9 10) , little is known about the implication of specific signaling cascades in prion formation.

We have previously shown that the brain-derived neurotrophic factor (BDNF) can promote the formation of rPrPSc in immortalized hypothalamic gonadotropin-releasing hormone neurons (GT1-1 cells) infected with the Rocky Mountain Laboratory (RML) strain of scrapie, and that inhibition of the BDNF-activated ERK pathway can efficiently clear the cells from rPrPSc (11) . The ERK (extracellular signal-regulated kinase) pathway belongs to a group of intracellular signaling pathways that operates via sequential activation of protein kinases, called mitogen-activated protein (MAP) kinases. The ERK pathway is activated by a variety of extracellular ligands, such as growth factors, hormones, and neurotransmitters, and it plays a role in cell differentiation and neuroprotection (for review see ref. 12 ). In contrast, stimulation of other MAP kinase pathways, such as the p38 and JNK (c-JUN NH2-terminal protein kinase) cascades, can have opposing effects and induce nerve cell death (13) .

In the present study, we employed high potassium-induced depolarization and selective inhibition of MAP kinase pathways to examine the involvement of ERK, p38, and JNK in the formation of rPrPSc. Our data indicate that p38 and JNK inhibit, whereas ERK promotes, formation of rPrPSc; these MAP kinase pathways thus exert opposing effects on prion formation. We also examined the involvement of cAMP-dependent signaling in rPrPSc formation and show that activation of cAMP-dependent protein kinase (PKA) leads to accumulation of rPrPSc in the cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
GT1-1 cells and scrapie infection
The GT1-1 cell line is derived from immortalized mouse hypothalamic gonadotropin-releasing hormone neurons and was a generous gift from Prof. Pamela Mellon (Department of Reproductive Medicine, University of California, San Diego, CA, USA). The cells were cultivated in Dulbecco’s modified Eagle’s medium 4.5 g/L glucose with Glutamax I (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 5% heat-inactivated horse serum (HS), and 50 U/ml penicillin-streptomycin (PEST; all obtained from Gibco BRL, Paisley, UK). The cells were split at a ratio of 1:5 once a week using 1x trypsin-EDTA (Gibco BRL). Before treatment of cells with various compounds, they were seeded on 35 mm cell culture dishes (Corning Inc., Corning, NY, USA) in DMEM containing 10% serum (HS and FBS; ratio 1:1, used in all experiments).

Infection of GT1-1 cells with scrapie was performed in 24-well culture clusters (Corning Inc.). The cells were grown in supplemented DMEM to ~75% confluence and then incubated at 37°C with a 0.1% homogenate of mouse brains infected with the RML strain of scrapie, a kind gift from Prof. Stanley B. Prusiner (Institute for Neurodegenerative Diseases and Department of Neurology, University of California, San Francisco, CA, USA). After 4 d of exposure, the medium was changed. The presence of rPrPSc after 6 passages was confirmed by Western blotting after treatment with proteinase K (PK; Boehringer Mannheim, Mannheim, Germany) (see below). These infected cells are referred to as ScGT1-1 cells.

Western immunoblot
The cells were lysed on ice in lysis buffer (10 mM Tris-HCl, pH 8; 150 mM NaCl; 0.5% sodium deoxycholate; 0.5% Triton X-100), and debris were removed by centrifugation for 1 min at 16,000 g. The protein concentration was determined using the Bio-Rad Bradford protein assay (Bio-Rad, Hercules, CA, USA) and spectrophotometry (Ultrospec plus, Pharmacia LKB, Cambridge, UK) at 595 nm according to the manufacturer’s instructions, and all samples were standardized to contain the same amount of protein. The lysates were split into 2 parts. One part was treated with PK (20 µg/ml) at 37°C for 30 min, and the reaction was stopped by incubation with 5 mM phenylmethyl sulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 10 min. The remaining part of the lysate was not PK-treated. All samples were boiled for 10 min in sodium dodecyl sulfate (SDS) sample buffer.

Electrophoresis was performed using Criterion XT Bis-Tris gels (Bio-Rad) with NuPAGE® MOPS SDS Running Buffer (Invitrogen Corporation, Carlsbad, CA, USA). Proteins were transferred to Immobilon-PSQ Transfer membranes (Millipore, Bedford, MA, USA) at 16 V for 40 min using tris-glycin buffer with 20% methanol in a Trans-Blot SD cell (Bio-Rad) and blocked in 5% bovine serum albumin (BSA; Sigma-Aldrich) for 30 min. PrP was labeled with recombinant Fab HuM-D13 (InPro Biotechnology Inc., San Francisco, CA, USA), 1 µg/ml, followed by a secondary goat anti-human F(ab)2-peroxidase-conjugated antibody (Pierce, Rockford, IL, USA), 0.16 µg/ml. Labeling was detected with ECLplus (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) and scanned in a StormTM 860 Gel and Blot Imaging System (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Optical densities of the bands were determined using the software ImageQuant 5.0 (GE Healthcare Bio-Sciences AB). All samples were normalized to mean densities of the controls. Statistics used were Student’s t test and one-way ANOVA in combination with Dunnett’s multiple comparison test or Bonferroni’s post hoc test in Graph Pad Prism (Graph Pad software, San Diego, CA, USA).

For studies of phosphoproteins, the following modifications were made: cells were lysed on ice in Cell Lysis Buffer (Cell Signaling Technology Inc., Danvers, MA, USA) and sonicated briefly in a water bath. Centrifugation was performed at 4°C for 10 min at 14,000 g, and protein concentration was determined as described above. Samples were boiled for 5 min. All steps were performed at 4°C or on ice. Phospho-ERK1/2 and total ERK1/2 were detected using antibodies from Cell Signaling (#4370 and #4695) and a secondary goat anti-rabbit-HRP (Dako Denmark A/S, Glostrup, Denmark).

Immunofluorescence
Cells grown on 35-mm cell culture dishes (Corning Inc.) were fixed in 10% formalin (Merck KGaA, Darmstadt, Germany), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS, and treated with 3 M guanidinium thiocyanate (GdnSCN; Merck KGaA) for 5 min to detect PrPSc (14) . After blocking with 5% BSA for 20 min, the cells were incubated overnight at 4°C with the primary antibodies diluted in PBS containing 1% BSA. The monoclonal primary antibodies used were Fab HuM-D13 (3.5 µg/ml), phospho-S6 ribosomal protein (Ser-235/236) antibody (1:100; Cell Signaling), and anti-phospho (Ser-10)-acetyl-(Lys14)-histone H3 (1:100; Upstate, Lake Placid, NY, USA). The cells were then incubated with secondary antibodies for 40 min at room temperature. The secondary antibodies used were Cy3-conjugated donkey anti-human immunoglobulin G (IgG)and Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The cells were rinsed in PBS with 1% NH4Cl (Sigma-Aldrich), mounted in glycerol with 2.5% 1,4-diazabicyclo[2.2.2]octane (Sigma-Aldrich), and coverslipped.

Treatment of cells with various compounds
The following substances were used: forskolin (1 µM; Sigma-Aldrich Chemie, Steinhem, Germany), KCl (35 mM; Sigma-Aldrich), the p38 inhibitor 4-(4-fluorophenyl)-2- (4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB202190; 1 µM; Sigma-Aldrich), the JNK inhibitor 1,9-pyrazoloanthrone anthrapyrazolone (SP600125; 2.5 µM; Sigma-Aldrich), the MEK1/2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene ethanolate (U0126; 1–2 µM; Promega Corp., Madison, WI, USA), the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89; 1 µM; Sigma-Aldrich), leupeptin hydrochloride (leupeptin; 15 µM; Sigma-Aldrich), and pentosan polysulfate (PPS; 5 µg/ml; Sigma-Aldrich). Cells grown on 35 mm cell culture dishes were treated for 5 d in DMEM containing 1–10% serum and 50 U/ml PEST. KCl was dissolved in cell culture media, forskolin, U0126, SB202190, and SP600125 in dimethyl sulfoxide (Sigma-Aldrich), and leupeptin and PPS in PBS, while H89 was dissolved in H2O.

Cell counts and cell death markers
Concentrations of signaling pathway inhibitors/stimulators that caused no cell death were chosen for the experiments. To further control for this, the number of cells both floating in the medium (which was not replaced during the 4 d incubation periods) and attached to the culture dish (after their detachment with trypsin-EDTA) were counted in a Bürker chamber.

In addition, cultures were incubated with Hoechst 33342 stain (5 µg/ml; Sigma-Aldrich) and propidium iodide (0.5 µg/ml; Sigma-Aldrich), and thereafter fixed in 10% formalin to determine the proportion of apoptotic and necrotic cells, respectively, in cultures exposed to the various treatments. The proportion of dead to living cells was estimated by counting cells in defined areas of the dishes (~150–250 cells in each area), 4 dishes/treatment. Statistics used were one-way ANOVA and Dunnett’s multiple comparison test in Graph Pad Prism.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Forskolin treatment and long-term KCl depolarization increase the levels of PrPSc in ScGT1-1 cells
We first studied whether activation of the ERK cascade by other stimuli than BDNF can affect prion formation. We used the diterpene forskolin, which activates ERK in slices and primary cultures of the hippocampus as well as in PC12 cells (15 16 17) , and high [KCl], which stimulates ERK phosphorylation in neurons (18) .

ScGT1-1 cells exposed to forskolin (1 µM) or to high [KCl] (35 mM) for 5 d showed increased intensity of rPrPSc bands in the immunoblots (Fig. 1A-C ). Quantification showed a 1.5-fold increase in rPrPSc on forskolin treatment (Fig. 1B ; 1.5±0.05; P<0.0001; Student’s t test) and a 1.9-fold increase in rPrPSc on depolarization with KCl (Fig. 1C ; 1.9±0.11; P<0.0001; Student’s t test). All samples were normalized to the mean of the densities of the control samples for each experiment (5 experiments with duplicate cultures for each treatment). The treatments had no effects on the level of PrPC in the cells (Fig. 1D-F ), which shows that the increased levels of rPrPSc did not reflect an increase in PrPC available for conversion.


Figure 1
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  		Figure 1. Effects on PrP levels after treatment with forskolin (forsko; 1 µM) and KCl (35 mM). A) Western blot showing levels of rPrPSc in ScGT1-1 cells treated with forskolin and high [KCl]. B, C) Quantification of rPrPSc bands after treatment with forskolin (B) and high [KCl] (C). All samples are normalized to the mean of the control samples for each experiment and analyzed by Student’s t test. Boxes and error bars represent means ± SE of 10 samples from 5 sets of experiments; ***P < 0.0001. D) Western blots showing levels of PrPC in uninfected GT1-1 cells treated with forskolin and high [KCl]. E, F) Quantification of PrPC-specific bands after treatment of uninfected GT1-1 cells with forskolin (E) and high [KCl] (F). Boxes and error bars represent means ± SE of 6–8 samples from 3–4 sets of experiments. ns, nonsignificant (P>0.05; Student’s t test). All samples are normalized to the mean of the control samples for each experiment. Cells were treated for 5 d.

We then asked whether the increase in rPrPSc following treatment with forskolin and high [KCl] was paralleled by activation of ERK1/2 and found that phosphorylation of ERK1/2 was not affected by forskolin treatment but was enhanced by depolarization with high [KCl]; a 1.9-fold and a 2.4-fold increase in phospho-ERK after 20 and 60 min high [KCl] treatment, respectively (Fig. 2 ; P<0.001; one-way ANOVA and Dunnett’s multiple comparison test). All samples were normalized to the mean of the densities of the control levels of ERK for each experiment (3 experiments with duplicate cultures at each time point and treatment). Increased ERK phosphorylation was still observed after 5 d of treatment with high [KCl] (data not shown).


Figure 2
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  		Figure 2. Effects of forskolin and high [KCl] on ERK1/2 levels in ScGT1-1 cells. A) Western blots showing levels of phospho-ERK1/2 and total-ERK1/2 in untreated cells (C, control) or cells treated with forskolin (F; 1 µM) or KCl (35 mM) for 20 or 60 min. B) Quantification of ERK-specific bands after treatment with forskolin and high [KCl]. Boxes and error bars represent means ± SE of 6–7 samples from 3 sets of experiments; ***P < 0.001; one-way ANOVA and Dunnett’s multiple comparison test. All samples are normalized to the mean of the control samples for each experiment.

The lack of ERK-mediated effects by forskolin on rPrPSc levels was confirmed by combining forskolin treatment with the MEK1/2 inhibitor U0126. This inhibitor decreased the levels of rPrPSc in the GT1-1 cells, as described previously (11) , but could not block the forskolin-induced increase in rPrPSc (Fig. 3A ). The high [KCl]-induced rPrPSc could, on the other hand, be inhibited by U0126 (Fig. 3B ). Forskolin is also known to activate the cAMP-PKA pathway, and treatment of the cells with the PKA inhibitor H89 blocked the forskolin-induced, but not the high [KCl]-induced, rPrPSc (Fig. 3C, D ). rPrPSc bands were quantified and analyzed by one-way ANOVA and Bonferroni’s post hoc test (Fig. 3 ). These data show that high [KCl] and forskolin promote formation of rPrPSc via 2 different pathways: namely, through activation of the ERK pathway and through the PKA pathway, respectively.


Figure 3
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  		Figure 3. Effects of forskolin (forsko, 1 µM) or KCl (35 mM) treatment in combination with MEK inhibitor U0126 (1µM) or PKA inhibitor H89 (1µM) on rPrPSc in ScGT1-1 cells. A) Western blot showing levels of rPrPSc in cells treated with forskolin or U0126 or both in combination and quantification of rPrPSc bands after these treatments. B) Western blot showing levels of rPrPSc in cells treated with high [KCl] or U0126 or both in combination and quantification of rPrPSc bands after these treatments. C) Western blot showing levels of rPrPSc in cells treated with forskolin, or H89 or both in combination and quantification of rPrPSc bands after these treatments. D) Western blot showing levels of rPrPSc in cells treated with high [KCl] or H89 or both in combination and quantification of rPrPSc bands after these treatments. Cells were treated for 5 d. All samples are normalized to the mean of the control samples for each experiment; means are indicated in figures. *P < 0.05, **P < 0.01, ***P <0.001; one-way ANOVA and Bonferroni’s post hoc test. ns, nonsignificant.

Since inhibition of ERK (via MEK inhibitors) blocks, and activation of ERK (via depolarization) increases the formation of PrPSc, there must be molecules downstream of ERK1/2 affecting the conversion process. ERK1/2 acts on several targets involved in the regulation of protein expression. In this study, we focused on activation of the cytoplasmatic S6 ribosomal protein, whose phosphorylation promotes mRNA translation (19) , and the nuclear protein histone H3, whose modification may lead to chromatin rearrangement and changes in transcriptional activity (20) . Phosphorylation of S6 at Ser-235/236 is regulated by ERK via activation of the p90 ribosomal S6 kinase (RSK) (19) , whereas phosphorylation of histone H3 is achieved via ERK-dependent activation of the mitogen and stress-activated protein kinases 1 and 2 (MSK1/2) (21) .

By immunocytochemistry, ScGT1-1 cells exposed to forskolin or to high [KCl] showed an increase in PrPSc displayed as intracellular granules (Fig. 4A-C ). In the treated cells, a large cluster of granules was often seen in addition to the more widely scattered granules. These cells were not PK treated, which may explain why the increases in PrPSc appear to be stronger than those of rPrPSc seen in the PK-treated specimens used in immunoblots (Fig. 1 ). This immunocytochemical increase in PrPSc was paralleled by an increase in the activity of the S6 ribosomal protein (Fig. 4D-F ). Interestingly, forskolin, which did not affect ERK phosphorylation, also promoted phosphorylation of S6 ribosomal protein (Fig. 4E ). These results indicate that the ERK and PKA pathways can exert independent regulation at this downstream level. Forskolin produced a large increase in the number of ScGT1-1 cells positive for acetyl-Lys14-phospho-Ser10-histone H3, but depolarization did not affect histone H3 activation (Fig. 4G-I ).


Figure 4
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  		Figure 4. ScGT1-1cells treated with forskolin (1 µM) or KCl (35 mM). A–C) Immunofluorescence showing labeling of non-PK-treated PrPSc. A) Untreated cells. B) Cells treated with forskolin. C) Cells treated with high [KCl]. Cells were treated for 5 d and were exposed to guanidine thiocyanate prior to immunolabeling. D–F) Immunofluorescence showing labeling of phospho-S6 ribosomal protein. D) Untreated cells. E) Cells treated with forskolin. F) Cells treated with high [KCl]. Cells were treated for 60 min. G–I) Immunofluorescence showing labeling of phosphorylated and acetylated histone H3. G) Untreated cells. H) Cells treated with forskolin. I) Cells treated with high [KCl]. Cells were treated for 60 min. Scale bars = 25 µm.

Inhibition of p38 and JNK increase the levels of PrPSc in ScGT1-1 cells
Since inhibition of the ERK pathway can clear GT1-1 cells infected with the scrapie RML strain from PrPSc (11) , we then studied the effects of inhibition of the p38 and JNK cascades. Treatment with either SB202190 (inhibitor of p38) or SP600125 (inhibitor of JNK) increased the density of the rPrPSc bands (Fig. 5A-C ) and numbers of PrPSc immunopositive granules in the cells (Fig. 5D ). The effect was more marked after treatment with SB202190 than with SP600125: a 1.9-fold (Fig. 5B ; 1.9±0.12; P<0.0001; Student’s t test; 6 experiments with duplicate cultures) and a 1.6-fold increase in rPrPSc (Fig. 5C ; 1.6±0.096; P<0.0001; Student’s t test; 4 experiments with duplicate cultures), respectively. All samples were normalized to the mean intensity of the controls for each experiment. The inhibitors had no effects on the level of PrPC as seen in immunoblots (Fig. 5E-G ).


Figure 5
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  		Figure 5. Effects on PrP levels of treatments with inhibitors of p38 (SB, SB202190; 1µM) and JNK (SP, SP600125; 2.5 µM). A) Western blot showing levels of rPrPSc in ScGT1-1 cells treated with SB or SP. B, C) Quantification of rPrPSc bands after treatment with SB (B) or SP (C). Boxes and error bars represent means ± SE of 8–12 samples from 4–6 different experiments; ***P < 0.0001; Student’s t test. All samples are normalized to the mean of the control samples for each experiment. D) Immunofluorescence showing labeling of non-PK-treated PrPSc in ScGT1-1 cells treated with SB and SP. Cells were exposed to guanidine thiocyanate prior to immunolabeling. Scale bars = 25 µm. E) Western blot showing levels of PrPC in uninfected GT1-1 cells treated with SB or SP. F, G) Quantification of PrPC-specific bands after treatment of uninfected GT1-1 cells with SB (F) or SP (G). Boxes and error bars represent means ± SE of 6 samples from 3 sets of experiments. ns, nonsignificant (Student’s t test). All samples are normalized to the mean of the control samples for each experiment. H) Western blot showing levels of rPrPSc in ScGT1-1 cells treated with SB, leupeptin (Leu; 15 µM), or pentosan polysulfate (PPS; 5 µg/ml), or in the indicated combinations, and quantification of PrPSc-specific bands. All samples are normalized to the mean of the control samples (mean indicated in figure).

By studying the effects of the ERK1/2 inhibitor U0126 on cellular rPrPSc levels following inhibition of its formation with pentosan polysulfate (22) or its degradation with leupeptin (23) , we have previously shown that U0126 blocks the formation rather than promotes the degradation of rPrPSc (11) . We similarly analyzed whether the increase in rPrPSc, which was most marked after inhibition of the p38 pathway, reflected an increased formation or a decreased degradation of the misfolded protein. When degradation was inhibited with leupeptin, the treatment with SB202190 still caused enhanced intensity of the rPrPSc bands, while inhibition of prion formation by pentosan polysulfate prevented the effect of the inhibitor (Fig. 5F ). We conclude that inhibition of p38 causes an increase in formation, and not a reduced degradation, of rPrPSc and therefore has an opposing effect of ERK inhibition. Inhibition of p38 and JNK did not affect activity of the S6 ribosomal protein or histone H3, as seen by immunofluorescence (data not shown).

Treatment with forskolin or high [KCl] and inhibition of p38 or JNK do not affect survival of ScGT1-1 cells
Since inhibition of stress-activated MAP kinase pathways can affect cell survival, nontoxic doses were chosen as described above, and the number of cells attached to the culture dishes, as well as free-floating cells in the culture medium, was counted. The number of attached and free-floating cells did not differ between control cultures and cultures treated with high [KCl] and forskolin or with the inhibitors SB202190 and SP600125 (P>0.05). Neither did the index of apoptotic (pyknotic or fragmented nuclei as seen by Hoechst staining) cells change following any of the treatments (P>0.05), as analyzed by one-way ANOVA and Dunnett’s multiple comparison test (data not shown). Necrosis (as seen by propidium iodide staining) was not evident in any of the cultures.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have observed that 4 intracellular signaling pathways are involved in regulating the formation of RML prions in ScGT1-1 cells. Concisely, increased conversion of PrPC to PrPSc was seen in the cells after treatment with high [KCl] and forskolin, which activated the ERK and cAMP-PKA pathways, respectively, and with inhibitors of the p38 and JNK pathways (summary of the results is presented in Fig. 6 ).


Figure 6
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  		Figure 6. Summary of the results showing the opposing effects of intracellular signaling pathways on conversion of PrPC to PrPSc. The ERK and PKA pathways stimulate, while the p38 and JNK pathways suppress this process. Thereby, formation of prions can be increased by stimulation of the former pathways with high [KCl] or forskolin or by inhibition of the latter pathways by the inhibitors SB202190 or SP6001215.

High [KCl] stimulates ERK phosphorylation by Ca2+, which enters neurons through voltage-dependent Ca2+-channels after depolarization (18) . The activation of ERK produced by depolarization did not result in concomitant phosphorylation of the nuclear target histone H3, which instead was activated by incubation with forskolin. On the other hand, our results show that depolarization causes increased phosphorylation of the S6 ribosomal protein at Ser-235/236, 2 sites that are regulated by ERK via activation of the p90 ribosomal S6 kinase (RSK) (19) . Consequently, in GT1-1 cells, the effects of ERK, including the effects on PrPSc formation, may be restricted to the cytoplasm. A similar, albeit less pronounced, effect on S6 phosphorylation was observed following activation of the cAMP cascade, achieved with forskolin. Taken together, these observations suggest that depolarization- and forskolin-induced PrPSc formation may require translational rather than transcriptional activity in the cell. Analyses of downstream effects of the S6 ribosomal protein may in the future disclose a link between activation of the S6 ribosomal protein and promotion of prion formation as well as the topological site of this process in the cell.

In contrast to the findings that blockers of the ERK pathway could inhibit rPrPSc formation, we observed that inhibitors of the JNK or p38 pathways instead caused an increase in rPrPSc accumulation in the infected cells. This increase in rPrPSc accumulation did not reflect increased levels of PrPC available for conversion or prolonged cell survival. The effect of the p38 inhibitor SB202190 was on rPrPSc formation rather than degradation, since an increase in rPrPSc was still evident when degradation of rPrPSc was inhibited by leupeptin, but was abolished when the conversion of PrPC to PrPSc was blocked by pentosan polysulfate. We therefore conclude that inhibition of the ERK and the p38 pathways has opposing effects on the formation of prions in a cell, whereby inhibition of the former clears rPrPSc from the cells while inhibition of the latter promotes rPrPSc formation.

Xia et al. (13) , studying NGF-deprived PC-12 cells, have shown that a dynamic balance between activation of the ERK and the p38-JNK pathways is important in determining whether a cell survives or undergo apoptosis. A balance between these pathways has also been demonstrated to regulate survival in other cell types, such as cardiac myocytes, HeLa cells, and gastric epithelial cells (24 25 26) . The ERK pathway can also be negatively regulated by the p38-JNK pathways to promote apoptosis, which implies that there is a crosstalk between the different MAP kinase pathways (for review, see ref. 27 ). Changes in MAP kinase activation associated with prion infections have previously been described in mouse and hamster brains as well as in cell lines (28 29 30 31) . Thereby, PrPSc and phospho-ERK increases over time in neurons and astrocytes in scrapie-infected hamster brains (31) . Such an increase was also observed in mouse brains and was suggested to accelerate neurodegeneration in this experimental paradigm (30) . In addition to prion diseases, chronic activation of ERK has been associated with both Alzheimer’s and Parkinson’s disease, and inhibition of ERK may be neuroprotective in animal models of these diseases (32 33 34 35 36 37) . Activation of the ERK pathway in neurons therefore in prion infections may have a dual function: on the one hand, it may be involved in regulating survival or death of the neurons, but the on the other hand, it may promote PrPSc formation.

Note that the ERK pathway also plays a role in the spread and replication of other infectious agents. For instance, the MEK inhibitor U0126 can block spread of Borna disease virus in cell cultures (38) and the virus seems to use the cellular ERK pathway to increase its replication (39) . This indicates that pathogens of such diverse natures as prions and viruses may develop interactions with the ERK intracellular signaling system to promote their formation.

The observation that depolarization of the infected cells by high [KCl] caused an increase in PrPSc levels could be of pathobiological relevance. GT1-1 cells are derived from the hypothalamus and release GABA and gonadotropin-releasing hormone in response to depolarization with high [KCl] (40 , 41) . They have membrane characteristics of neurons, and when infected with the RML strain of scrapie they show reduced responses of the N-type, but not the L-type, voltage-gated Ca2+ channels to high [KCl], indicating that the infection can cause disturbances in synaptic vesicle release in these cells (42) . Experimental prion infections in hamsters are associated with decreased evoked [3H]-GABA release from synaptosomes, suggesting that a reduced release of inhibitory GABA transmitters may be one mechanism contributing to increased neuronal activities (43) , which could be manifested in the seizure-like activities common in both humans (44) and experimental animals infected with prions (45 46 47 48) . The observed increase in prion formation following depolarization of scrapie-infected GT1-1 cells could imply a vicious circle whereby the prion infection causes an increased excitability in nervous tissues that in turn promotes formation of more prions.

It should be emphasized that our data relate only to formation of rPrPSc of the RML strain of scrapie. Different prion strains can target different areas in the brain and cause distinct clinical signs of disease. It will therefore be important to study whether the different MAP kinase cascades are involved also in regulating the formation of other prion strains. Moreover, this may be of interest for therapeutic considerations of the disease. A number of cellular factors and drugs are known to interfere with prion formation. For instance, the laminin receptor and heparan sulfate proteoglycans may facilitate prion formation (49 , 50) , and synthetic heparan mimics may inhibit the binding of PrPSc to heparan sulfate and effectively block prion formation (51) . Polyanions such as pentosan polysulfate and dextran sulfate can interact directly with PrPSc to shield it from binding to endogenous sulfated glucosaminoglycans (22 , 52) . Molecules that interfere with the composition of lipid rafts and the endosomal/lysosomal system can also affect the amount of prions in a cell (6 , 7) . Although efficient in cell culture, none of these treatments have been successful in in vivo situations. Our observation that rPrPSc formation can be regulated by the MAP kinase signaling pathways may therefore point to a novel therapeutic strategy based on interference with intracellular signaling pathways.

In conclusion, we show here that intracellular signaling can regulate the formation of prions whereby different pathways can have opposing effects. It has recently been hypothesized that the cytoplasmic polyadenylation element binding protein in Aplysia can be converted into a prion-like state during synaptic stimulation to play a role in long-term synaptic changes (53) . Our data suggest that prions, analogous to the prion-like proteins, may be under control by neuronal activity and physiological signals in the nervous system.


   ACKNOWLEDGMENTS
 
This study was supported by a grant from EC FP6-2004-FOOD-3-B-023183 StrainBarrier, LSHB-CT 2006-019090 AntePrion, and Stiftelsen Golje Minne.

Received for publication June 17, 2008. Accepted for publication September 4, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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