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Vol. 17, Issue 8, 3356-3368, August 2006
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*Department of Biochemistry, Neurobiochemistry, Ludwig-Maximilians-Universität München, D-80336 München, Germany; Institut für Physiologische Chemie, Ludwig-Maximilians-Universität München, D-81377 München, Germany; Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom; and Robert-Koch-Institut, D-13353 Berlin, Germany
Submitted January 27, 2006;
Revised April 17, 2006;
Accepted May 10, 2006
Monitoring Editor: Jonathan Weissman
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Prusiner et al., 1998; Collinge, 2001; Chesebro, 2003; Aguzzi and Polymenidou, 2004).
In the majority of prion diseases neurodegeneration is tightly linked to the propagation of infectious prions. However, transgenic mouse models revealed that misfolding or mistargeting of PrPC can induce neuronal cell death in the absence of infectious prions. Vice versa, propagation of infectious prions was observed without inducing neuronal cell death, but only when PrPC is not expressed in neurons (reviewed in Winklhofer and Tatzelt, 2006). Moreover, neuronal expression of a secreted PrP mutant devoid of the glycosylphosphatidylinositol (GPI) anchor sustains propagation of infectious prions in the absence of clinical symptoms (Chesebro et al., 2005).
The biogenesis of PrPC is characterized by a series of co- and posttranslational modifications (reviewed in Tatzelt and Winklhofer, 2004). It involves import of the nascent chain into the endoplasmic reticulum (ER) and the attachment of two N-linked core glycans and a GPI anchor. After processing of the glycans into complex structures in the Golgi compartment, PrPC is targeted to the outer leaflet of the plasma membrane.
Several studies indicated that PrP can acquire a neurotoxic potential when its import into the ER is partially or completely compromised. Lingappa and coworkers demonstrated that during import into the ER, PrP can attain two different transmembrane topologies termed CtmPrP and NtmPrP (Yost et al., 1990). Interestingly, increased synthesis of CtmPrP has been shown to coincide with progressive neurodegeneration both in GSS syndrome patients with an A117V mutation and in transgenic mice carrying a triple mutation within the hydrophobic domain (AV3; Hegde et al., 1998; Stewart et al., 2005). A different transgenic mouse model revealed that preventing the import of PrP into the ER leads to the formation of a neurotoxic PrP species. Mice expressing a PrP mutant with a deleted N-terminal ER targeting signal (cytoPrP) acquired severe ataxia due to cerebellar degeneration and gliosis (Ma et al., 2002). Earlier results already indicated that both wild-type PrP and a PrP mutant associated with an inherited prion disease in humans can be found in the cytosol and are subjected to proteasomal degradation (Ma and Lindquist, 2001; Yedidia et al., 2001). Cytotoxic effects of cytoPrP were also observed in some cell culture models (Ma et al., 2002; Rane et al., 2004), whereas in other studies expression of cytoPrP seemed not to interfere with cellular viability (Roucou et al., 2003; Fioriti et al., 2005). Support for a toxic potential of cytosolically localized PrP was also obtained in a yeast model. During posttranslational targeting of PrP to the ER, PrP was missorted to the cytosol and interfered with yeast growth (Heller et al., 2003).
So far, mutations within the N-terminal signal sequence of PrP, which could affect the efficiency of ER import, have not been identified in patients suffering from prion diseases. The analysis of PrP-W145Stop, however, revealed that a pathogenic PrP mutant linked to GSS syndrome, is found in the cytosolic and nuclear compartment (Zanusso et al., 1999). Further studies including a different pathogenic mutant, PrP-Q160Stop, corroborated these findings and revealed that information in the C-terminal domain of PrP is necessary and sufficient for import into the endoplasmic reticulum (Heske et al., 2004).
In this study, we directed PrP to different cellular compartments and analyzed the impact on cell viability. Although in all compartments analyzed PrP adopted a detergent-insoluble, partially PK-resistant conformation, only cytosolic PrP interfered with cell viability and induced apoptosis. The apoptotic potential of cytosolic PrP was linked to an internal domain of PrP, comprising the hydrophobic domain and helix 1, and correlated with the binding of Bcl-2 to misfolded PrP. Binding of Bcl-2 to PrP, as well as apoptosis, could be prevented by the increased expression of Hsp70 and its cochaperone Hsp40, indicating a protective role of chaperones in PrP-induced toxicity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), anti-PrP 3B5 mAb (Krasemann et al., 1996), anti-PrP antiserum A7 (Winklhofer et al., 2003a), anti-ACTIVE caspase-3 polyclonal antibody (pAb; Promega, Madison, WI), anti-Hsp40 pAb (Stressgen, Victoria, British Columbia, Canada), anti-Bcl-2 mouse mAb (BD Biosciences, San Diego, CA), anti-GAPDH mouse mAb (6C5; Calbiochem, La Jolla, CA), Cy3-conjugated anti-mouse IgG antibody and Cy3-conjugated anti-rabbit IgG antibody (Dianova, Hamburg, Germany), anti-FLAG M2 mAb (Sigma, Deisenhofen, Germany), anti-FLAG pAb (rabbit, Sigma), anti-GFP mAb (Roche, Indianapolis, IN). The following reagents were used: MG132 (Calbiochem), PK (Roche), and staurosporin and valinomycin (Sigma). The mounting medium Mowiol (Calbiochem) was supplemented with 4',6-diamidino-2-phenylindole (DAPI, Sigma).
Plasmids
The following constructs were described previously: wtPrP, PrP
GPI (Winklhofer et al., 2003c), Q160Stop, W145Stop, cytoPrP (Heske et al., 2004), and EYFP-Hsp70, Hsp40 (Kim et al., 2002; Winklhofer et al., 2003b). All amino acid numbers refer to the mouse PrP sequence (GenBank accession number M18070). For the generation of PrP targeted to the nucleus (nucPrP) or mitochondria (mtPrP), the authentic ER signal sequence of PrPC was replaced by the nuclear localization signal of SV40 large T antigen (MDPKKKRKV) or the mitochondrial signal sequence of subunit 1 of the ubiquinol cytochrome C reductase complex (MAASAVCRAACSGTQVLLRTRRSPALLRPALRGTATFA), respectively. Human XIAP with a N-terminal FLAG-tag (MDYKDDDDK) was described earlier (Bartke et al., 2004). FLAG-Bcl-2 was generated by fusing a FLAG-tag (DYKDDDDK) to the C-terminus of human Bcl-2 (GenBank accession number AAA51813). As a mitochondrial marker pEYFP-Mito vector (Clontech, Heidelberg, Germany) was used.
Cell Culture, Transfection, Proteasomal Inhibition, and Proteinase K Digestion
SH-SY5Y cells were cultivated as described earlier (Winklhofer et al., 2003c). Cells cultivated on a 3.5-cm cell culture dish (Nunc, Wiesbaden-Biebrich, Germany) were transfected with a total of 2 µg DNA by a liposome-mediated method using LipofectAMINE Plus reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. To keep the amount of transfected cytoPrP constant, a plasmid expressing EYFP or ECFP under the same promoter was added to a total of 2 µg DNA. The proteasomal inhibitor MG132 was dissolved in dimethyl sulfoxide, added to the cell culture medium (30 µM), and incubated for 3 h at 37°C. For proteolysis experiments, cells were lysed in cold buffer A (0.5% Triton X-100, 0.5% sodium deoxycholate [DOC] in phosphate-buffered saline [PBS]) and incubated for 30 min at 4°C with PK at the concentrations indicated. The reaction was terminated by addition of Pefabloc SC (Roche) and boiling in Laemmli sample buffer. Residual PrP was detected by Western blotting.
Detergent Solubility Assay, Western Blotting, and Sucrose Step Gradient
As described earlier (Tatzelt et al., 1996), cells were washed twice with cold saline buffer (PBS), scraped off the plate, pelleted by centrifugation, and lysed in cold buffer A. After centrifugation the detergent-soluble and -insoluble fractions were analyzed by Western blotting. To examine the secretion of PrP into the cell culture supernatant, cells were cultivated in serum-free medium for 3 h at 37°C. Medium was collected, and proteins precipitated with trichloroacetic acid (TCA) and then analyzed by Western blotting. SDS-PAGE and Western blotting were described previously (Winklhofer and Tatzelt, 2000). For sucrose step gradient analysis, cells were lysed in 150 µl gradient buffer containing 0.1% Triton X-100, 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, and 5% glycerol. The gradient was performed as described earlier (Henn et al., 2005). Ten fractions were collected from the top of the gradient. The proteins were precipitated by TCA and analyzed by Western blotting.
Scrapie Infection and Preparation of Brain Extracts
Intracerebral infections of C57B/6 mice with scrapie-strain 139A were carried out as previously described (Schultz et al., 2004). Brain extracts were prepared in buffer A (10% w/vol) and fractionated by centrifugation into the detergent-soluble and -insoluble fraction as described above. For PK digestion the whole lysate was adjusted to 1% Sarkosyl and incubated with PK (50 µg/ml) for 1 h at 37°C. The reaction was terminated by addition of Pefabloc SC and boiling in Laemmli sample buffer. Residual PrP was detected by Western blotting using the anti-PrP antiserum A7.
In Vitro Translation and Mitochondrial Import Assay
The in vitro translation was carried out by using the TNT T7 Quick Coupled Transcription/Translation System (Promega) by following the manufacturer’s instructions. After translation the samples were used for the mitochondrial import assay as described earlier (Stan et al., 2003). Protein import in yeast mitochondria was performed in SI buffer (3% BSA [w/vol], 0.5 M sorbitol, 50 mM HEPES-KOH, 80 mM KCl, 10 mM MgAc, 2 mM KH2PO4, 2.5 mM EDTA, 2.5 mM MnCl2, 2 mM ATP, 2 mM NADH, pH 7.2). Protease treatment of mitochondria was performed by incubation with PK for 15 min on ice, followed by the addition of 1 mM phenylmethylsulfonyl fluoride for 5 min as described before. Products were analyzed by SDS-PAGE. Gels were impregnated with Amplify (GE Healthcare, Waukesha, WI) and exposed to film.
Metabolic Labeling and Immunoprecipitation
Cells were starved for 30 min in methionine-free minimum Eagle’s medium (Invitrogen) and subsequently labeled for 45 min with 300 µCi/ml Promix [L-35S] in vitro cell label mix (Amersham Biosciences, Braunschweig, Germany; >37 TBq/mmol) in methionine-free minimum Eagle’s medium (pulse). When indicated, the proteasomal inhibitor MG132 was present during starvation, labeling, and chase periods (30 µM final concentration). For the chase, labeling medium was removed, cells were washed twice and then incubated in complete medium for 30 min at 37°C. Immunoprecipitation of PrP was performed as described earlier (Winklhofer et al., 2003c). For the coimmunoprecipitation experiments, low-detergent buffer (0.1% Triton X-100 in PBS) was used for cell lysis and incubation with the antibody. Proteins present in the immunopellet were released by boiling in buffer B (Triton X-100, DOC, Sarkosyl, 0.5% each in PBS) and subjected to a second immunoprecipitation under nonnative conditions.
Co-pullup Experiments
Cells were lysed in low-detergent buffer (0.1% Triton X-100 in PBS) and incubated for 16 h with anti-FLAG M2 mAb at 4°C. To extract FLAG-tagged Bcl-2 proteins, the cell lysate was incubated with magnetic anti-mouse IgG-DYNAbeads (Invitrogen) for 2 h and exposed to a magnetic field for 2 min to pull up bound immunocomplexes. Unbound material was removed and analyzed by Western blotting. After washing, the immunocomplexes were dissolved by boiling in Laemmli sample buffer and analyzed for bound cytoPrP by Western blotting, using the anti-PrP antibody A7. Anti-FLAG M2 mAb was used to detect the Bcl-2 constructs.
Indirect Immunofluorescence and Apoptosis Assay
For indirect immunofluorescence experiments, SH-SY5Y cells were grown on glass coverslips, fixed with 3% PFA for 20 min, and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. The primary antibody was incubated for 45 min at 37°C in PBS containing 1% BSA. After extensive washing with cold PBS, an incubation with the Cy3-conjugated secondary antibody followed at 37°C for 30 min. For detection of apoptosis, cells were fixed and permeabilized as described above. Blocking of the cells was performed with 5% horse serum and 0.1% Tween 20 in PBS for 1 h at room temperature. Cells were then incubated with anti-ACTIVE caspase-3 antibody overnight at 4°C, followed by Cy3-conjugated secondary antibody for 1 h at room temperature. Cells were mounted onto glass slides and examined by fluorescence microscopy using a Zeiss Axiovert 200M microscope (Carl Zeiss, Oberkochen, Germany). To analyze the apoptotic potential of the various PrP constructs, the number of activated caspase-3–positive cells out of 1000 transfected cells was determined. All quantifications were based on at least three independent experiments. Confocal images were obtained on a Leica DM IRE2 confocal microscope (Leica, Heidelberg, Germany) and evaluated with the Leica confocal software version 2.6.1.
Statistical Analysis
Data were expressed as means ± SE. All experiments were performed in duplicates and repeated at least three times. Statistical analysis among groups was performed using student’s t test. P-values are as follows: *p < 0.01, **p < 0.001, and ***p < 0.0001.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The neurotoxic PrP conformer present in the cytosol (cytoPrP) is quite distinct from infectious PrPSc. CytoPrP adopts a misfolded and partially PK-resistant conformation; however, it lacks posttranslational modifications, like the GPI anchor and N-linked carbohydrates; moreover, it does not seem to be infectious. In the present study we addressed the question of whether misfolding of PrP is cytotoxic per se or whether PrP-induced toxicity is linked to the cellular compartment in which PrP adopts a misfolded conformation.
To achieve subcellular targeting of PrP to different compartments, including the nucleus, cytosol, mitochondria and ER, the following constructs were generated: nucPrP with the nuclear localization signal (NLS) of SV40 T antigen, mtPrP with the mitochondrial signal sequence of ubiquinol cytochrome C reductase complex subunit 1, and cytoPrP lacking the authentic ER signal sequence (Figure 1A). To include a PrP conformer that misfolds in the secretory pathway, we used a mutant that contains the N-terminal ER-targeting signal but lacks the C-terminal GPI signal sequence (designated PrPGPI). Targeting to and import into the ER of PrPGPI is efficient, but in the secretory pathway PrPGPI spontaneously adopts a misfolded conformation and is secreted (Rogers et al., 1993; Blochberger et al., 1997; Winklhofer et al., 2003c).
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GPI into the ER was corroborated by the detection of secreted PrPGPI in the cell culture supernatant (Figure 2A). Notably, SH-SY5Y cells do not express significant levels of endogenous PrPC (unpublished data). Thus, only the heterologously expressed PrP constructs are detected in our assays.
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GPI, were mainly insoluble in detergent buffer (Figure 2A) and showed an increased resistance to proteolytic digestion (Figure 2B). In addition, all mutants formed aggregates. Protein extracts of transiently transfected cells were applied on top of a sucrose step gradient. After centrifugation all mutant PrP constructs were found in the bottom fraction, whereas wild-type PrPC remained in the top fractions (Figure 2C). These experiments established that PrP can efficiently be targeted to different cellular compartments. The biochemical properties of the PrP mutants present in the nucleus, cytosol, mitochondria and ER are similar and indicate the formation of misfolded and aggregated conformers.
Apoptosis Is Selectively Induced by Misfolding of PrP in the Cytosol
The Western blot analysis indicated that the steady state level of cytoPrP was considerably lower than that of the other PrP mutants (Figure 2A). The most plausible explanation for this phenomenon was proteasomal degradation of cytoPrP. Indeed, after proteasomal inhibition by MG132 (3 h, 30 µM) the relative levels of cytoPrP and nucPrP were significantly increased, whereas the proteasome inhibitor had no significant effect on the relative amount of PrP imported into the ER or mitochondria (Figure 3A).
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GPI, mtPrP, or nucPrP (Figure 3A and unpublished data). In this context it is interesting to note that a small fraction of mtPrP contained an uncleaved signal peptide, but did not induce apoptosis. It is possible that this precursor, although localized in the cytosol, is already associated with the mitochondria import machinery or bound to cytosolic proteins, like Hsp70, which are involved in targeting to and import into mitochondria. Thus, a toxic potential of misfolded PrP was specifically linked to its cytosolic localization. Cellular viability was not affected by misfolded PrP present in other cellular compartments, be it the ER, the mitochondria, or the nucleus.
Pathogenic Mutations Linked to Inherited Prion Diseases in Humans Are Mistargeted to the Cytosol and Induce Apoptosis
The above experiments demonstrated that mistargeting of PrP to the cytosol can effectively induce apoptosis. The question remained whether such a mistargeting could play a role in the pathogenesis of human prion diseases. Previous studies indicated that upon proteasomal degradation wild-type and mutant D177N PrP accumulates in the cytosol (Ma and Lindquist, 2001; Yedidia et al., 2001). Similarly, we and others showed that Q160Stop and W145Stop (Figure 4A), two pathogenic PrP mutants characterized by large C-terminal deletions, are partially present in the cytosol and are subjected to proteasomal degradation (Zanusso et al., 1999; Heske et al., 2004). This phenomenon is illustrated by an immunoprecipitation analysis of transiently transfected SH-SY5Y cells cultivated for 3 h in the presence of MG132 before the sample preparation (Figure 4B, +MG132). In addition, an indirect immunofluorescence analysis indicated a significant increase of cytosolically localized Q160Stop and W145Stop after transient proteasomal inhibition (Figure 4C). Two scenarios are conceivable to explain this phenomenon. Either the C-terminal deletion mutants are subjected to ER-associated protein degradation (ERAD) or their import into the ER is partially compromised.
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These findings revealed that two pathogenic PrP mutants, linked to inherited prion diseases in humans, are missorted to the cytosol and induce apoptosis. In addition, these experiments indicated that the distal region of the globular C-terminal domain (aa 146–231) is dispensable for the toxic effect of cytosolic PrP.
Cytosolic PrP Binds to and Coaggregates with Bcl-2
Previous in vitro studies showed that PrP can interact with the anti-apoptotic protein Bcl-2 and mapped the interaction site to the carboxy terminus of Bcl-2 (aa 200–236; Kurschner and Morgan, 1995, 1996). Consequently, we tested whether an interaction of cytosolic PrP with Bcl-2 occurs in our cell culture model. First, we analyzed a possible interaction of PrP with Bcl-2 by coimmunoprecipitation experiments. Transiently transfected cells expressing both cytoPrP and FLAG-tagged Bcl-2 were metabolically labeled with [35S]methionine. The cells were then lysed in low-detergent buffer, and PrP was immunoprecipitated with the mAb 3B5 under native conditions. Proteins bound to the immunopellet were subsequently released by boiling in detergent buffer B, and a second immunoprecipitation was performed using the anti-FLAG antibody to isolate FLAG-tagged Bcl-2. Bcl-2 copurified under these conditions, indicating that cytoPrP interacts with Bcl-2 in neuronal cells (Figure 5A, lane 4). Importantly, the same procedure did not purify Bcl-2 from cells that do not express cytoPrP (Figure 5A, lane 3) or from cells expressing wtPrP (unpublished data). Moreover, Bcl-2202–219, a mutant with a deletion in the PrP-interacting domain (Kurschner and Morgan, 1996), did not interact with cytoPrP either (see Figure 7D). In addition, we demonstrated colocalization of cytoPrP with Bcl-2 in intact cells by indirect immunofluorescence (Figure 5B).
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202–219 was not recruited into cytoPrP aggregates (Figure 5C, FLAG-Bcl-2202–219). Coaggregation was not restricted to overexpressed FLAG-Bcl-2 as cytoPrP also induced aggregation of endogenous Bcl-2 (Figure 5D). In this context it is important to note that the cells coexpressing Bcl-2 and cytoPrP were vital and did not undergo apoptosis, due to the overexpression of Bcl-2. Assuming that the aggregation of Bcl-2 could be of pathophysiological relevance to prion diseases, we analyzed Bcl-2 in brain extracts prepared from terminally scrapie-ill mice. In contrast to the uninfected control, Bcl-2 was predominantly detergent-insoluble in the scrapie-diseased brain (Figure 5E, sc). Of note, infection of the brain with scrapie-prions did not cause protein aggregation in general. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytosolic protein, was found in the detergent-soluble fraction in both the control and scrapie-infected brain samples (Figure 5E).
If the interaction of PrP with Bcl-2 correlates with the toxic potential of cytoPrP, coexpression of Bcl-2 should alleviate the apoptotic effects of cytoPrP. To test this possibility, apoptosis was analyzed in cells expressing both Bcl-2 and cytoPrP. Indeed, the coexpression of both Bcl-2 and Bcl-2202–219 interfered with cytoPrP-induced apoptosis (Figure 5F).
In response to apoptotic signals, the release of mitochondrial factors such as cytochrome c leads to the activation of caspase-9 and further on to that of caspase-3. XIAP, a known inhibitor of caspase-9 and -3, suppressed cytoPrP-induced apoptosis when overexpressed in cultured cells. Nonetheless, cytoPrP neither interacted with XIAP in coimmunoprecipitation experiments, nor did it induce the aggregation of XIAP (Figure 6, A and B). This further indicates that cytoPrP does not randomly coaggregate with anti-apoptotic proteins. Notably, the relative levels of cytoPrP were not decreased by the coexpression of Bcl-2 or XIAP (Figure 6C).
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116–173 was comparable to that of cytoPrP (Figure 7C). To provide experimental evidence for a direct association of aggregated Bcl-2 with misfolded PrP, pullup experiments were performed. In this approach Bcl-2 was immunopurified similarly to coimmunoprecipitation described above; however, to avoid centrifugation, magnetic anti-mouse IgG-DYNAbeads were used. The immunocomplexes were pulled up by a magnetic field and analyzed by Western blotting. Again, cyto-PrP copurified under these conditions. However, neither was the nontoxic mutant cyto116–173 pulled up by Bcl-2 nor was cytoPrP bound by Bcl2202–219 (Figure 7D). Further support for the assumption that the formation of Bcl-2/cytoPrP aggregates is linked to apoptosis would be provided if the expression of the nontoxic mutant cyto116–173 does not lead to the aggregation of Bcl-2. Indeed, the sucrose gradient analysis revealed that in contrast to cytoPrP or cyto108–173, cyto116–173 did not induce aggregation of Bcl-2. Notably, the internal deletion did not restore folding of cyto116–173 because the mutant was still found in the bottom fractions of the sucrose gradient (Figure 7E).
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). We therefore analyzed a possible interaction of cytoPrP with the cytosolic chaperones Hsp70 and Hsp40. For coimmunoprecipitation experiments we expressed cytoPrP together with YFP-Hsp70 and Hsp40. Previous experiments established that the chaperone activity of Hsp70 is not compromised by the YFP tag (Kim et al., 2002). After a first immunoprecipitation with an anti-GFP antibody under native conditions, the immunopellet was boiled in detergent buffer B and PrP was immunoprecipitated with the monoclonal anti-PrP antibody 3F4. PrP copurified under these conditions, but only in the presence of Hsp70 (Figure 8A, lane 4). A sucrose step gradient provided further evidence that cytoPrP is bound by the cytosolic chaperones Hsp70 and Hsp40. In the absence of cytoPrP, Hsp70 as well as Hsp40 were exclusively found in the top fractions. When cytoPrP was coexpressed, the chaperones were found in the bottom fraction together with cytoPrP (Figure 8B). Interestingly, the chaperones obviously affected the biochemical properties of cytoPrP. In cells overexpressing Hsp70 and Hsp40, a portion of cytoPrP was found in the top fraction of the sucrose gradient (Figure 8B, compare to Figure 2C). Finally, we performed an indirect immunofluorescence analysis to visualize colocalization of cytoPrP and EYFP-Hsp70 (Figure 8C).
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In summary, these results indicate that misfolded cytoPrP is recognized and bound by Hsp70 and Hsp40. As a consequence, the cytotoxic potential of cytoPrP and the formation of PrP/Bcl2 coaggregates are reduced.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Biogenesis of PrPC is characterized by a series of co- and posttranslational modifications (reviewed in Tatzelt and Winklhofer, 2004). Among those, addition of the GPI anchor seems to be of particular importance. Initial experiments in cell culture revealed that a mutant devoid of the C-terminal GPI anchor signal sequence (PrPGPI) is imported into the ER but remains mainly unglycosylated, spontaneously adopts a misfolded conformation and is efficiently secreted (Rogers et al., 1993; Blochberger et al., 1997; Winklhofer et al., 2003c). The phenotype of anchorless PrP described in cultured cells was recently confirmed in transgenic mice. Notably, expression of PrPGPI did not result in clinical symptoms, at least not during the life span of mice (Chesebro et al., 2005). Biochemically, PrPGPI (named GPI(–)PrPsen in the mouse study) is similar to the PrP mutant targeted to the cytosol (cytoPrP): both mutants lack the C-terminal signal sequence, are unglycosylated, and adopt a misfolded conformation. However, transgenic mice expressing cytoPrP acquired severe ataxia with cerebellar degeneration (Ma et al., 2002). Our cell culture model recapitulates the observations in transgenic mice: cytoPrP efficiently induced apoptosis, whereas expression of PrPGPI had no obvious effect on cellular viability. How can the obvious difference in the neurotoxic potential of PrPGPI and cytoPrP be explained? Possibly, the neurotoxic potential is linked to a specific conformation, and the conformation of PrPGPI and cytoPrP may be different. Alternatively, the cellular compartment in which PrP misfolding occurs is crucial. Our study provides experimental evidence that the cellular compartment might play a significant role. PrP mutants targeted to the cytosol, nucleus, ER, or mitochondria showed similar biochemical features, yet apoptosis was exclusively induced by cytosolic PrP.
Although the pathophysiological role of cytoPrP might be restricted to a subset of prion diseases, there are several ways to generate of cytoPrP in vivo. It was recently shown that the signal sequence of PrP is insufficient for complete protein translocation into the ER, giving rise to a nontranslocated PrP fraction in the cytosol (Rane et al., 2004). In line with these results, the pathogenic mutants Q160Stop and W145Stop are partially mislocalized in the cytosol (Zanusso et al., 1999; Heske et al., 2004), and we could show that the toxic potential is indeed linked to the cytosolic fraction of these mutants. Alternatively, access to the cytosol is possible via retrograde translocation of PrP out of the ER; this route has been demonstrated for wild-type PrP and another pathogenic PrP mutant, D177N (Ma and Lindquist, 2001; Yedidia et al., 2001). Finally, the generation of transmembrane forms of PrP, which is favored by some pathogenic mutations, leads to the partial exposure of PrP to the cytosol (reviewed in Hegde et al., 1999). Notably, a quantitative study of the ultrastructural localization of PrP in mouse brain revealed that PrP is present in the cytosol in subpopulations of neurons (Mironov et al., 2003).
What might be the mechanism underlying the toxic potential of cytoPrP? Recruitment of anti-apoptotic Bcl-2 to misfolded PrP provides a plausible explanation for the observed effects. An interaction of PrP and Bcl-2 was described previously for recombinantly expressed proteins (Kurschner and Morgan, 1995, 1996). We now show for the first time that such an interaction can occur in neuronal cells and seems to be of pathophysiological relevance. First, only PrP mutants with a toxic potential induced aggregation of Bcl-2. Bcl-2 did not aggregate in cells expressing mitochondrially localized PrP or the nontoxic cytosolic PrP mutant cytoPrP116–173. Second, overexpression of Bcl-2 interfered with cytotoxicity induced by cytoPrP. Third, Bcl-2202–219, a functionally active mutant with a deletion in the PrP-interacting domain, did not coaggregate with cytoPrP, but did interfere with cytoPrP-induced apoptosis. Fourth, the protective effect of Hsp70/Hsp40 in cytoPrP-induced toxicity correlated with their potential to interfere with the recruitment of Bcl-2 into cytoPrP aggregates. Finally, Bcl-2 adopted a detergent-insoluble conformation in scrapie-diseased mouse brain. This interesting observation might provide a link between prion propagation and intracellular apoptotic signaling pathways. Support for the possibility that PrPSc itself could induce apoptosis through caspase activation was provided previously in a cell culture model (Hetz et al., 2003). In addition, aggresome formation in scrapie-infected cells was shown to induce caspase-3 activation and apoptosis (Kristiansen et al., 2005). In this context it is also interesting to note that an interaction of PrP with NRAGE, another protein involved in neuronal apoptosis, was reported recently (Bragason and Palsdottir, 2005).
The exact mechanism by which Bcl-2 exerts its anti-apoptotic function is not fully understood, but binding to proapoptotic members of the Bcl-2 family seems to be crucial. Regarding the role of Bcl-2 in cytoPrP-mediated toxicity, two scenarios are conceivable. Bcl-2 may be inactivated by its sequestration into cytoPrP aggregates. In support of this possibility, a recent study revealed that aggregation of Bcl-2 can be of pathophysiological relevance for another neurodegenerative disease. It was shown that the amyotrophic lateral sclerosis (ALS)-associated SOD1 mutant proteins bind to and aggregate with Bcl-2 (Pasinelli et al., 2004). Alternatively, binding of Bcl-2 to cytoPrP may induce a conformational change of Bcl-2, converting it to a proapoptotic protein. Evidence for opposing phenotypes of Bcl-2 was provided by Lin et al. (2004), who showed that the interaction with Nurr77 converts Bcl-2 from a protector to a killer protein.
Our study clearly revealed that PrP-induced toxicity can be modulated by various cellular factors. Obviously, proteasomal degradation and chaperone activity have a major impact on the formation of toxic PrP conformers. To disclose the cytotoxic potential of cytoPrP, it was necessary to transiently inhibit the proteasome. In untreated cells cytoPrP was hardly detectable by Western blotting, suggesting that the main effect of MG132 was to elevate the amount of cytoPrP. However, it is also conceivable that cells stressed by proteasomal impairment are sensitized to cytoPrP. Similarly, the relative level of Bcl-2 determines whether or not apoptosis is induced by cytoPrP. Efficiency of the ubiquitin-proteasome system as well as chaperone activity decrease with aging, supporting the notion that aged neurons are particularly vulnerable to the accumulation of misfolded proteins. In this context, inefficiencies in the signal-sequence–mediated translocation of PrP into the ER might further promote the generation and accumulation of cytosollically localized PrP conformers. This multifaceted influence on the formation and clearance of cytoPrP, which is highly dependent on the cellular homeostasis, also provides a plausible explanation for the discrepancy between different reports on the toxic potential of cytosolic PrP (Ma et al., 2002; Roucou et al., 2003; Rane et al., 2004; Fioriti et al., 2005).
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ACKNOWLEDGMENTS |
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Footnotes |
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|| These authors contributed equally to this work. 
Address correspondence to: Jörg Tatzelt ( Joerg.Tatzelt{at}med.uni-muenchen.de)
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REFERENCES |
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, alpha helix; 
, beta strand; GPI-SS, GPI anchor signal sequence; mt-SS, mitochondrial signal sequence; NLS, nuclear localization signal. All mutants are devoid of the GPI-SS (aa 231–253). (B) SH-SY5Y cells were transiently transfected with plasmids encoding wild-type PrP or the mutant prion proteins and analyzed by indirect immunofluorescence using the anti-PrP mAb 3F4 (
