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Originally published In Press as doi:10.1074/jbc.M605320200 on August 14, 2006

J. Biol. Chem., Vol. 281, Issue 41, 30431-30438, October 13, 2006
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Conditions of Endoplasmic Reticulum Stress Favor the Accumulation of Cytosolic Prion Protein*

Andrea Orsi{ddagger}, Luana Fioriti§1, Roberto Chiesa§2, and Roberto Sitia{ddagger}3

From the {ddagger}Università Vita-Salute San Raffaele, DiBiT Istituto Scientifico San Raffaele, Via Olgettina 58, 20132 Milano, Italy, and the §Dulbecco Telethon Institute and Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy

Received for publication, June 2, 2006 , and in revised form, August 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
After signal sequence-dependent targeting to the endoplasmic reticulum (ER), prion protein (PrP) undergoes several post-translational modifications, including glycosylation, disulfide bond formation, and the addition of a glycosylphosphatidylinositol anchor. As a result, multiple isoforms are generated. Because of the intrinsic weakness of the PrP signal sequence, a fraction of newly synthesized molecules fails to translocate and localizes to the cytosol. The physiopathologic role of this cytosolic isoform is still being debated. Here we have shown that, in both cultured cell lines and primary neurons, ER stress conditions weaken PrP co-translational translocation, favoring accumulation of aggregation-prone cytosolic species, which retain the signal sequence but lack N-glycans and disulfides. Inhibition of proteasomes further increases the levels of cytosolic PrP. Overexpression of spliced XBP1 facilitates ER translocation, suggesting that downstream elements of the Ire1-XBP1 pathway are involved in PrP targeting. These studies reveal a link between ER stress and the formation of cytosolic PrP isoforms potentially endowed with novel signaling or cytotoxic functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prion diseases are neurodegenerative disorders involving spongiosis and neuronal loss in the central nervous system. Compelling evidence points to a pathogenic role of the prion protein (PrP)4 (1). PrP is a glycosylphosphatidylinositol (GPI)-anchored membrane protein expressed mainly in the central nervous system, the normal function of which is still not clear. The key event in the pathogenesis is the conversion of normal cellular PrP (PrPC) into the scrapie conformation (PrPSc), which can catalyze further conversion of PrPC (2, 3). Despite the assessed role of PrPSc in the infectious process, many aspects remain unclear (4, 5). What is the cytotoxic molecule? How is the first PrPSc molecule generated in inherited or sporadic diseases?

PrP is an eccentric protein existing in a variety of different isoforms depending on post-translational modifications. The different utilization of two glycosylation sites gives rise to three glycoforms carrying no, one, or two N-glycans (6). These glycoforms are extensively processed during intracellular transport, yielding hundreds of glycotypes (7–9) often with cell type-specific patterns (10). Additional variability can arise from the formation of a disulfide bond between the two cysteines (an event that may control N-glycosylation (11)), appendage of a GPI anchor, and from the differential topologies it can adopt. PrP has a conserved N-terminal signal peptide (SP) and a putative internal transmembrane domain (12). Because of the intrinsic weakness of the SP, some molecules are not co-translationally inserted into the ER and end up in the cytosol (13, 14). Utilization of the internal transmembrane region can generate additional topological variants, with the N or C termini protruding into the cytosol (CtmPrP, NtmPrP) (15).

The intrinsically inefficient translocation of PrP into ER is puzzling, especially when we consider that the SP is quite conserved (12). What is the role of these topological variants? Cytosolic PrP appears to be toxic for certain neuronal cells (16–18), although there are also reports of a protective role (19, 20), perhaps suggesting cell type-specific interactions of mislocalized PrP.

The efficiency of co-translational translocation may depend on recognition of the SP by the signal recognition particle complex and transport across the ER membrane, an event that, in turn, can depend on interactions with ER-resident chaperones and enzymes (21). As soon as newly synthesized polypeptides are inserted into the ER, chaperones bind to them and promote their folding. At the same time, chaperones prevent backward movements of the growing chains, ratcheting them in the ER lumen. For efficient protein folding to take place in the ER, chaperones must outnumber substrates (22).

ER-resident chaperones may become insufficient during the differentiation of specialized secretory cells or when folding efficiency is perturbed by drugs that prevent glycosylation (tunicamycin) or disulfide bond formation (dithiothreitol (DTT)) or alter calcium homeostasis (thapsigargin or cyclopiazonic acid) (23). In these conditions, cells mount a tripartite response collectively called the unfolded protein response (UPR), the main purpose of which is to alleviate stress and restore proper folding efficiency within the ER (24). To do so, cells transiently inhibit translation through the activation of the ER-associated kinase PERK (PKR-like ER kinase) and the consequent phosphorylation of eIF2{alpha}. Concomitantly, they activate the transcription of ER chaperones and folding assistants through ATF6, a transcription factor that binds to regulatory elements in target genes. Meanwhile, phosphorylation of Ire1{alpha} activates non-spliceosomal processing of XBP1 transcripts, which allows the synthesis of a transcription factor, sXbp1, that increases the efficiency of ER folding, secretion, and ER-associated degradation (25, 26). Although operationally distinct, the three arms of UPR are thought to be activated by the lower levels of unbound BiP when misfolded proteins accumulate in the lumen (27, 28), although it has been recently proposed that Ire1{alpha} directly recognizes aberrant proteins (29).

In view of the physiopathological interest of topological PrP variants, we investigated whether the UPR affects the biogenesis of cytosolic PrP molecules. Here we have shown that ER stress conditions result in a decrease in the translocation efficiency of PrP, as evidenced by the accumulation of cytosolic molecules with an uncleaved SP. Our data suggest that factor(s) involved in PrP translocation become limiting during the UPR. Overexpression of spliced XBP1 partly rescued this phenotype, indicating an important role for this arm of the pathway. Taken together, these findings reveal a link between the accumulation of cytosolic PrP and ER stress conditions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Transfection—HeLa cells, maintained in Dulbecco's modified Eagle's medium, 5% fetal calf serum, and penicillin/streptomycin were transiently transfected with Lipofectin (Invitrogen) and 1 µg/ml plasmid (unless differently indicated) 48 h before metabolic labeling. The pCDNA 3 vector (Invitrogen) containing the coding sequence for murine PrP epitope-tagged for 3F4 antibody has been described previously (19, 30). The plasmid for expression of sXBP1 (31) was a kind gift from Dr. K. Mori (Kyoto University). Neuroblastoma N2a cells and stable transfectants expressing PrP were maintained as described in Ref. 19.

UPR Induction and Metabolic Labeling—Cells at ~80% confluency were incubated for 3.5–4 h with 2.5 µg/ml thapsigargin, 10 µM MG132, 2.5 mM DTT, and 2.5 µg/ml tunicamycin or 1 µg/ml brefeldin A, washed and detached with phosphate-buffered saline/EDTA, and resuspended at 1–2 x 107 cells/ml in cysteine/methionine-free medium. After 15 min at 37 °C, 1 x 106 cells were incubated with 30 µCi ProMix (Amersham Biosciences) for 10 min at 37 °C. When indicated, 10 µM MG132 or 2 µM PS-341 (Millenium Pharmaceuticals, Cambridge, MA) was added during both starvation and pulse. Labeling was stopped by washing twice with ice-cold phosphate-buffered saline containing 10 mM N-ethylmaleimide, and the cells were lysed in radioimmune precipitation assay buffer (0.2% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 10 mM Tris-HCl, pH 7.6) containing protease inhibitors and 10 mM N-ethylmaleimide.

Immunoprecipitation and SDS-PAGE—Lysates from 106 labeled cells were precleared on fetal calf serum-Sepharose, incubated with antibodies specific for the 3F4 epitope (3F4), the N-terminal PrP original peptide ({alpha}-SP) or the C-terminal peptide that is removed during GPI addition ({alpha}-GP) (kindly provided by Drs. R. Kascsak (Institute for Basic Research in Developmental Disabilities, Staten Island, NY) and D. A. Harris (Washington University, St. Louis, MO)) (32, 33) and recovered on protein A-Sepharose. To remove N-linked glycans, beads were treated with endoglycosidase H or PGNaseF (New England Biolabs) according to the manufacturer's instructions. After three washes with 10 mM Tris-HCl, pH 7, 0.25% Nonidet P-40, and 150 mM NaCl, the immunoprecipitates were eluted at 95 °C in sample buffer with or without 200 mM DTT. Before loading on 12% polyacrylamide SDS gels, the samples were alkylated with 400 mM iodoacetamide. The gels were dried and revealed by autoradiography. Bands were quantified with ImageQuant (Molecular Dynamics) software.

Western Blotting—After SDS-PAGE, proteins were blotted onto nitrocellulose membrane and immunodetected with one of the following antibodies: 3F4 (monoclonal anti-PrP) (32); P45–66 (rabbit polyclonal raised against a synthetic peptide encompassing residues 45–66 of mouse PrP (30)); {alpha}-SP (rabbit polyclonal {alpha}-SP-PrP) (34); Stressgene SPA-827 ({alpha}-KDEL); and Santa Cruz Biotechnology sc-7160 ({alpha}-XBP1). Immunodetection was followed by incubation with the relative horseradish peroxidase-conjugated secondary antibody.

In Vitro Translation—Plasmids encoding for the 3F4-tagged murine PrP were transcribed and translated by the T7 TNT® system (Promega).

XBP1 Splicing—Total RNA was extracted with the TRIzol method (Invitrogen). RNA samples were reverse-transcribed with Superscript II reverse transcriptase (Invitrogen) by priming with oligo(dT). Splicing of XBP1 mRNA was amplified with primers flanking the 26b intron (5'-GGAGTGGAGTAAGGCTGGTG and 5'-CCAGAATGCCCAAAAGGATA) and PCR products resolved on 2.5% agarose gels.

Primary Neurons—Cortices were dissected from transgenic mice expressing 3F4-tagged mouse PrP (27) at postnatal day 2, sliced into ~1-mm pieces, and incubated in Hanks' balance salt solution (Invitrogen) containing 20 units/ml trypsin (Sigma) at 34 °C for 30 min. Trypsin inhibitor (Sigma) was added to a final concentration of 0.5 mg/ml, and the tissue was mechanically dissociated by passing through a flame-polished Pasteur pipette. Cells were plated at 3–4 x 105 cells/cm2 on poly-L-lysine-coated (0.1 mg/ml) plates and maintained in Neurobasal basal medium (Invitrogen) supplemented with B27 (Invitrogen), penicillin/streptomycin, and glutamine at 2 mM. To reduce the number of non-neuronal cells, aphidicolin (3.3 µg/ml, Sigma) was added to the medium 48 h after plating.

Cortical neurons were exposed to proteasome inhibitors (MG132, epoxomycin, 5 µM) or tunicamycin (5 µg/ml) 2 weeks after plating and then lysed in 10 mM Tris, pH 7.5, 100 mM NaCl, 0.5% sodium deoxycholate, and 0.5% Nonidet P-40 containing protease inhibitors and 2 mM EDTA. Lysates corresponding to 300 µg of protein were centrifuged at 186,000 x g for 40 min in a Beckman Optima Max-E ultracentrifuge. PrP in the pellet and supernatant was analyzed by Western blotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Multiple PrP Isoforms Are Generated by Differential N-Glycosylation, GPI Addition, and Removal of the Signal Sequence—To avoid the heterogeneity caused by N-glycan modifications in the Golgi apparatus, transiently transfected HeLa cells were labeled for 10 min. In these conditions, only PrP molecules with endoglycosidase H-sensitive glycans are precipitated by antibodies against the central portion of PrP (3F4) (13). Nonetheless, seven discrete bands were detectable (Fig. 1A). These were identified based on size, immunoreactivity with antibodies directed to the signal peptide ({alpha}-SP) or the C-terminal sequence that is removed before the addition of the GPI anchor ({alpha}-GP), sensitivity to endoglycosidases, and the presence or absence of disulfide bonds (Figs. 1 and 2).


Figure 1
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  		FIGURE 1.
Characterization of newly synthesized PrP isoforms. HeLa transfectants expressing PrP were pulse-labeled for 10 min with 35S-labeled amino acids. A, lysates were immunoprecipitated with antibodies directed against the C-terminal peptide that is removed before the addition of the GPI anchor ({alpha}-GP, lane 1), the central region of PrP (3F4, lane 2), or the N-terminal signal peptide ({alpha}-SP, lane 3). The scheme on the right summarizes the identification of the seven PrP isoforms. White boxes indicate the C-terminal pro-peptide, which is replaced by a GPI anchor (dark circles) after synthesis. The striped box indicates the signal peptide. N-Glycans are marked above the common region in black. B, lysates from PrP (P) or mock (m) transfectants were immunoprecipitated (IP) with different batches of {alpha}-SP. Note that the bands recognized by {alpha}-SP co-migrated with in vitro translated full-length PrP (ivt). nil, no IP.

 
After a 10-min pulse, most of the PrP was translocated into the ER, as demonstrated by the removal of the SP and the addition of N-glycans (Figs. 1A and 2A). The addition of the GPI anchor was not complete, and four bands were precipitated by {alpha}-GP. Three of them (isoforms 2, 5, and 7) did not react with {alpha}-SP antibodies and thus corresponded to ER-localized proteins with two, one, or no N-glycans, which had not yet undergone GPI processing. The {alpha}-SP antibody recognized a single band (isoform 3), which was absent from mock-transfected cells. As expected, this band was immunoprecipitated also by {alpha}-GP. To confirm the above results, several batches of {alpha}-SP were tested. All precipitated isoform 3 with different efficiency and backgrounds (Fig. 1B). In agreement with both the N- and C-terminal sequences being present, isoform 3 co-migrated with a full-length PrP translated in a cell-free system in the absence of membranes (see Fig. 1B, ivt). For simplicity, we will refer to this isoform as SP-PrP.


Figure 2
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  		FIGURE 2.
Cytosolic SP-PrP lacks N-glycans and intrachain disulfide bonds. HeLa transfectants expressing PrP were pulse-labeled as in Fig. 1. A, cell lysates were immunoprecipitated with 3F4 or {alpha}-SP antibodies and treated with or without endoglycosidase H (EH) as indicated. In both panels, the band indicated by an asterisk corresponds to SP-PrP and comigrated with in vitro translated PrP (not shown, see Fig. 1B). B, immunoprecipitates were resolved under reducing (R) or non-reducing (NR) conditions. The increased mobility of PrP in NR conditions reflects the formation of the intrachain disulfide bond in molecules that entered the ER.

 
Consistent with its cytosolic localization, neither N-glycans nor intrachain disulfide bonds were present in SP-PrP (Fig. 2). Its mobility was not influenced by endoglycosidase H (Fig. 2A, EH), which instead efficiently cleaved the two main glycoforms recognized by the 3F4 antibody. Likewise, a clear mobility shift was observed when 3F4 immunoprecipitates were resolved under reducing (R) or non-reducing (NR) conditions (Fig. 2B, left two lanes), consistent with the presence of an intrachain disulfide bond. In contrast, no differences were detected in SP-PrP. The concomitant presence of both N- and C-terminal propeptides and the absence of N-glycans and a disulfide bond suggest that SP-PrP never entered the ER.

Also, a topological variant of PrP called CtmPrP has an uncleaved SP. This isoform uses a downstream hydrophobic region to span the membrane, which leaves the C-terminal portion within the ER lumen. This species contains N-glycans and a functional GPI anchor and thus differs from SP-PrP (33, 35). We did not detect bands consistent with CtmPrP in our HeLa transfectants.

Thapsigargin Alters PrP Processing and Induces a Cytosolic SP-PrP—Next, we analyzed the translocation and processing of PrP under conditions of ER stress. First, we used thapsigargin (Fig. 3, A and B; Th), a drug that lowers the luminal Ca2+ levels by inhibiting the sarcoplasmic/ER calcium pumps (36) and thus impairs ER folding. UPR induction was demonstrated by the increased levels of the ER chaperone BiP and the splicing of XBP1 transcripts (Fig. 3A, upper and middle panel), an event implying Ire1{alpha} dimerization and phosphorylation (31, 37). Interestingly, a band recognized by {alpha}-SP antibody was selectively induced (Fig. 3A, lower panel).

To compensate for the concomitant translational attenuation in ER-stressed cells, we quantified cytosolic PrP as the ratio of {alpha}-SP-reactive to total 3F4-reactive PrP synthesized during the pulse. The values were then normalized to the ratio observed in untreated cells and expressed as fold induction (Fig. 3B). The decrease in the total amount of PrP synthesized by ER-stressed cells probably reflects PERK-dependent translational attenuation. It also argues against the possibility that the high levels of synthesis obtained with transient transfection are responsible for PrP mistranslocation. To rule out this possibility, we transfected HeLa cells with increasing amounts of plasmid. The ratio between translocated and non-translocated PrP was similar in all of the transfection conditions (Fig. 3, C and D). These data excluded that the increase in SP-PrP observed in stressed HeLa transfectants was a consequence of protein overexpression. Indeed, SP-PrP was detected in stressed neuroblastoma cells and primary neurons (see Figs. 6B and 7, respectively, and "Discussion").


Figure 3
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  		FIGURE 3.
Thapsigargin induces ER stress and favors appearance of SP-PrP. HeLa transfectants expressing PrP were treated with (Th) or without (NT) thapsigargin for 4 h. A, UPR induction was monitored by comparing the amounts of intracellular BiP. Aliquots of cell lysates normalized for protein content were resolved and assayed by Western blot (WB) with a specific antibody (top panel). The extent of XBP1 splicing was also analyzed, as determined by the appearance of rapidly migrating spliced XBP1 transcripts in thapsigargin-treated cells (middle panel). The arrows point to unspliced (u) and spliced (s) transcripts. Untreated cells contain only the former (lane 1) in variable amounts. ER stress causes the accumulation of sXBP1 (see also Fig. 4A). Note the increased synthesis of SP-PrP in thapsigargin-treated cells (lower panel). IP, immunoprecipitated. B, to account for the decreased translation rates in ER-stressed cells, SP-PrP was expressed as the percent of signal precipitated by {alpha}-SP relative to 3F4 antibodies. The results from at least three separate experiments are quantified as fold increase ± S.E., considering untreated cells (NT) as 1. C, HeLa cells were transfected with different amounts of plasmid encoding PrP as indicated (µg of plasmid/ml of transfection mix). Lysates from pulse-labeled cells were immunoprecipitated with 3F4 or {alpha}-SP antibodies. D, the relative abundance of SP-PrP species to total PrP in C were quantified and expressed as percentages.

 
ER Stress Induces Mistranslocation of PrP—Next, we tested other drugs (i.e. DTT, the proteasome inhibitor MG132, tunicamycin, and brefeldin A) that induce ER stress by acting on different pathways (disulfide bond formation, ER-associated degradation, N-glycosylation, and ER-Golgi transport) (23). Detection of spliced XBP1 mRNA confirmed UPR activation in all conditions (Fig. 4A). Some unspliced XBP1 transcripts persisted in brefeldin A- and MG132-treated cells. In the latter case, incomplete splicing could reflect additional roles of proteasome inhibitors, as proposed by Lee et al. (38). The simultaneous presence of thapsigargin and DTT yielded a robust UPR, which was also evident by translational attenuation (Fig. 4B, lane 3). All treatments increased the relative amount of SP-PrP (Fig. 4, C and D). It is thus clear that various ER stresses similarly inhibit the co-translational insertion of PrP, causing an increase of cytosolic species that retain the SP. In most experiments, the synthesis of SP-PrP increased also in absolute amounts upon ER stress induction.


Figure 4
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  		FIGURE 4.
Different ER stress conditions cause SP-PrP accumulation. A, HeLa transfectants expressing PrP were treated with thapsigargin (Th), dithiothreitol (DTT), MG132 (MG), tunicamycin (Tm) or brefeldin A (BFA) for 4 h before lysis and reverse transcription-PCR analysis. XBP1 splicing was assessed as described in the legend to Fig. 3. In this experiment, the appearance of spliced XBP1 mRNAs is paralleled by an overall increase in XBP1 transcripts in most samples. However, this assay is mostly qualitative. Aliquots of cells treated as above were pulse-labeled and immunoprecipitated with 3F4 (B) or {alpha}-SP (C) antibodies and resolved by SDS gels. Most ER stressors reduced the total amount of total PrP synthesized, very likely reflecting translational attenuation. Nonetheless, the absolute amount of SP-PrP was more abundant with most if not all stressors (C). D, the graph shows the relative amounts of SP-PrP, quantified as described in the legend to Fig. 3B relative to the total PrP synthesized during the pulse.

 
Besides their effects on ER targeting, certain drugs affected the PrP glycosylation patterns. As expected, tunicamycin led to the accumulation of non-glycosylated molecules. Most of these lacked the SP and hence negotiated entry into the ER, suggesting that glycosylation per se played a minor role in translocation. Consistent with previous results (11),5 DTT favored the accumulation of diglycosylated species. Thapsigargin had different effects, causing the fraction of non-glycosylated species to increase. The thapsigargin effects seemed to be dominant, as non-glycosylated molecules were abundant in cells treated with both DTT and thapsigargin. These findings suggest complex interplays between oxidative folding, glycosylation, and calcium homeostasis, the mechanisms of which will be described in a separate study.6

SP-PrP Is Degraded by the Proteasome—Cytosolic PrP is an aggregation-prone molecule, which is degraded by proteasomes (13, 19, 39–41). Therefore, the accumulation of SP-PrP in the cytosol of ER-stressed cells could reflect reduced efficiency in translocation and/or reduced proteasomal degradation, which also seems to be affected by the UPR (42).


Figure 5
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  		FIGURE 5.
In both ER-stressed and control cells, SP-PrP is degraded by proteasomes. A, HeLa transfectants expressing PrP were treated with (Th) or without (NT) thapsigargin for 4 h and pulse-labeled for 10 min in the presence or absence of 10 µM MG132 (MG) to prevent proteasomal degradation. Lysates were immunoprecipitated with 3F4 (not shown) or {alpha}-SP antibodies. Also in this experiment, thapsigargin induced a 3-fold increase in the fraction of PrP that retained its SP. To allow better visualization of the relevant bands, twice as much was loaded for the NT samples. B, SP-PrP/total PrP ratio from three independent experiments was calculated by densitometry as described in the legend to Fig. 3B and expressed as fold increase, considering cells not treated with MG132 (–) as 1. Also in this experiment, previous exposure to thapsigargin increased the synthesis of SP-PrP, despite an overall translational attenuation. The presence of proteasome inhibitors during the pulse caused a further increase in SP-PrP, demonstrating that this species is rapidly degraded by proteasomes in both untreated or ER-stressed cells.

 
To clarify the effects on translocation and degradation, cells were pretreated with or without thapsigargin and then pulse-labeled in the presence or absence of MG132. Although the differences between thapsigargin-treated and untreated cells reflect increased SP-PrP production, the difference between the two labeling conditions provide an indication of the fraction of molecules that are rapidly degraded. As expected, the presence of MG132 during the pulse stabilized SP-PrP (Fig. 5A). Also in this experiment, thapsigargin increased the fraction of PrP that retained its SP of ~3-fold. To better appreciate the effects of degradation, however, we considered the initial amount of SP-PrP as one in both ER stress and normal conditions, and we expressed the variations due to MG132 as fold increase (Fig. 5B). In both untreated and thapsigargin-treated cells, the proteasome inhibitor approximately doubled the amount of SP-PrP recovered. In contrast, ER stress increased the fraction of mistargeted molecules at least 3-fold (see Figs. 3B and 4D).

Because MG132 may also inhibit calpain, which has been shown to cleave PrP (43), we repeated the experiment using the more specific inhibitor PS-341. The presence of PS-341 during the pulse stabilized SP-PrP, even if slightly less than MG132 (not shown). We conclude that the proteasomes contribute to most of the degradation of SP-PrP.


Figure 6
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  		FIGURE 6.
Accumulation of exogenous and endogenous SP-PrP in HeLa and N2a cells. A, HeLa transfectants expressing PrP were treated with 2.5 µg/ml thapsigargin (Th) and 10 µM MG132 (MG) for 4 h as indicated and then incubated for a further 30 min with or without MG. The same blot was sequentially probed with {alpha}-SP and 3F4 antibodies. Actin served as a loading control. B, N2a clones stably expressing 3F4-tagged murine PrP (lanes 1–3) and parental N2A cells (lanes 4–6) were left untreated (NT) or treated with thapsigargin and MG132 for 16 h as indicated. All cells were incubated for the last hour with 10 µM MG132 and then lysed. The blot was sequentially probed with {alpha}-SP and P45–66 antibodies. In this experiment, we utilized a N2a subline displaying higher resistance to proteasome inhibitors than the one employed in previous experiments (19). This allowed us to detect endogenous SP-PrP. Note that non-transfected N2a expresses PrP under the endogenous promoter, thus excluding the possible effects of proteasome inhibitors on viral regulatory elements.

 
These data suggest that the increase in SP-PrP is primarily because of impaired translocation and is further exaggerated by the inhibition of proteasomal degradation (see also Figs. 6A and 8).

Accumulation of SP-PrP upon ER Stress—The labeling experiments described so far indicated that, upon ER stress, a higher fraction of PrP does not enter the ER and retains its SP. To see whether ER stress could actually lead to an accumulation of SP-PrP in time, PrP-expressing cells were treated with thapsigargin for 4 h and then incubated with MG132 for the last 30 min. Total PrP was analyzed by Western blotting with 3F4 and {alpha}-SP antibodies. Although in pulse-labeling experiments only immature forms of PrP (molecular mass from 27 to 34 kDa) were detected, mature glycoforms of PrP (molecular mass up to 45 kDa) also became evident in Western blotting. Incubation with thapsigargin alone for 4 h caused a modest accumulation of SP-PrP (Fig. 6A, lane 4), likely because newly made SP-PrP molecules were promptly degraded by the proteasome. Indeed, when cells were incubated for the last 30 min with MG132, abundant SP-PrP accumulated (Fig. 6A, lane 3). Note that MG132 alone had no relevant effects on the accumulation of SP-PrP (Fig. 6A, lanes 1 and 2) unless cells were previously incubated with thapsigargin to induce ER stress. Because the amount of SP-PrP detected in Fig. 6A, lane 3, represents the protein produced and stabilized in the last 30 min, we concluded that, upon thapsigargin pretreatment, a considerable fraction of the total PrP produced in this time interval was being mistranslocated. The continuous presence of both thapsigargin and MG132 further increased the accumulation of SP-PrP (Fig. 6A, lane 5).

Next, we used N2a cells to examine the effects of thapsigargin in a neuronal cell context. The glycosylation pattern of endogenous and overexpressed PrP was similar to the one observed in HeLa transfectants (Fig. 6B, middle panel). Upon 16 h of treatment with thapsigargin, we observed a significant increase in SP-PrP (Fig. 6B, lanes 2 and 5). A more pronounced effect was obtained with MG132 and both endogenous and transfected SP-PrP (Fig. 6B, lanes 3 and 6). This is consistent with the notion that prolonged proteasomal blockade induces the UPR (see also Fig. 4) and that the resulting SP-PrP undergoes proteasomal degradation. Very similar results were obtained in both parental N2a cells and transfectants stably overexpressing PrP, confirming that mistranslocation of PrP is not due to its overexpression nor to effects on the viral regulatory sequences.


Figure 7
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  		FIGURE 7.
Effects of the Xbp1 pathway on the accumulation of SP-PrP. A, HeLa cells were transiently transfected with PrP alone (mock) or with a vector driving the expression of sXbp1. After lysis in 2% SDS, proteins were resolved by SDS-PAGE and blotted with anti-Xbp1 antibodies. WB, Western blot. B, HeLa transfectants were incubated for 4 h with (+) or without (–) thapsigargin and then pulse-labeled for 10 min. Lysates were immunoprecipitated (IP) with 3F4 or {alpha}-SP. The proportion of SP-PrP precipitated with {alpha}-SP was calculated relative to the total signal generated with 3F4 antibodies (gel not shown). The results are expressed as fold increase considering 1 as the ratio obtained in untreated cells expressing PrP alone.

 
Altogether, these data confirmed that ER stress induces PrP mistranslocation and that proteasomes degrade ectopically localized SP-PrP in both neuronal and non-neuronal cells. They also showed that, in the presence of an ER stress, when the activity of the proteasome is compromised, a considerable amount of SP-PrP accumulates in the cytosol (Fig. 6).

A Role for sXBP1 in PrP Translocation—The overexpression of spliced XBP1 (sXBP1) causes an increase in cell volume that seems primarily because of expansion of the secretory pathway (44, 45). sXBP1 also seems to regulate the translation and release of secretory µ-chains in differentiating B cells (46). In view of its multiple effects, we analyzed the sXBP1 role in ER targeting of nascent PrP. Plasmids encoding sXBP1 were transiently transfected in HeLa cells together with PrP. Having confirmed the efficiency of sXBP1 expression (Fig. 7A), the cells were incubated with thapsigargin for 4 h and analyzed by metabolic labeling and immunoprecipitation (Fig. 7B, upper panel). Quantification of the data (Fig. 7B, lower panel) confirmed that the synthesis of sXBP1 protected HeLa cells from stress-induced PrP mistargeting. These findings suggest that elements of the XBP1 pathway also control the efficiency of co-translational translocation.

ER Stress Causes SP-PrP Accumulation in Primary Neurons—The above results indicate that ER stress causes mistargeting of PrP in cultured cell lines, probably saturating element(s) of the Ire1{alpha} -Xbp-1 pathway. As these observations could have implications for the pathogenesis of sporadic prion diseases, we sought to confirm a link between ER stress and PrP targeting in primary neurons. Exposure of murine cortical neurons to proteasome inhibitors (MG132 or epoxomycin) for 24 h led to the accumulation of several PrP isoforms in the detergent-insoluble fraction, the most abundant of which consisted of SP-PrP based on its size and immunoreactivity with the {alpha}-SP antibody (Fig. 8A, lane 4; and B, lane 2). Accumulation of detergent-insoluble SP-PrP was also observed upon treatment with tunicamycin for 24 h (Fig. 8B, lane 3). The presence of MG132 during the last 4 h of a 24-h treatment with tunicamycin slightly increased the intensity of the SP-PrP band (Fig. 8C, lanes 3 and 4). These results confirmed that ER stress disturbs PrP targeting also in primary neurons. In the absence of previous ER stress, very little SP-PrP was detectable after a 4-h exposure to proteasome inhibitors (Fig. 8C, lane 2). In primary neurons too, therefore, SP-PrP accumulation depends more on inefficient translocation induced by ER stress than on reduced proteolysis.


Figure 8
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  		FIGURE 8.
Accumulation of SP-PrP in primary cortical neurons. Murine cortical neurons were incubated with or without (NT)5 µM MG132 (A), 5 µM epoxomycin (Ep) or 5 µg/ml tunicamycin (Tm) for 24 h (B) or with tunicamycin and/or MG132 for the times indicated (C). After lysis in Triton X-100, detergent-soluble (SN) and -insoluble (P) fractions were resolved and blotted with 3F4 or {alpha}-SP, as indicated. B, only pellet fractions are shown. Tunicamycin-treated cells were exposed to epoxomycin for the last 4 h to improve the detectability of short-lived SP-PrP. As in the case of MG (C, lane 2), exposure to epoxomycin for 4 h was not sufficient to cause detectable accumulation of SP-PrP. The asterisk indicates SP-PrP. Molecular mass markers are in kDa. It is important to note that primary neurons express PrP under the endogenous promoter, thus excluding the possible effects of proteasome inhibitors on viral regulatory elements.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Because normal proteins can become toxic when delivered to the wrong compartment (22), the targeting of macromolecules in living cells needs to be efficient and precise. In view of the different ionic redox and chaperone compositions of cytosol and ER, the two main folding compartments of the cell, targeting is particularly important for newly made proteins. Yet an increasing number of proteins are found in multiple compartments. As with plasminogen activator inhibitor (47) and US2 (48), inefficient co-translational translocation generates a pool of cytosolic PrP that retains the hydrophobic signal peptide (13). The conservation of the latter in evolution suggests these ectopically localized molecules may play functional role(s). Some reports indicate that cytosolic PrP is neurotoxic (16–18), whereas others suggest a protective role against Bax-mediated apoptosis (20, 49). These differences may well reflect the existence of cell type-specific control pathways. There is little doubt, however, that the presence of a hydrophobic peptide and the fact that the protein finds itself in an unsuitable folding compartment make cytosolic SP-PrP prone to aggregate (40) and potentially capable of generating protease-resistant PrPSc-like seeds (41). Regardless of their ultimate function, therefore, it is important to know more about the mechanisms that control protein targeting, particularly for molecules that can adopt different topologies.


Figure 9
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  		FIGURE 9.
Schematic representation of PrP traffic and localization. Most nascent PrP co-translationally translocate into the ER lumen (thick gray arrow), where they undergo oxidative folding, N-glycosylation, and C-terminal addition of a GPI anchor. These modifications generate at least six distinct isoforms (see Fig. 1A). ER chaperones and other translocation accessory factors (TrAFs) likely favor translocation. In view of the intrinsic weakness of the PrP signal peptide, some molecules do not complete their translocation and end up with an uncleaved signal sequence in the cytosol (thin gray arrow) where they are degraded by proteasomes. In both HeLa and neuronal cells, ER stress impairs translocation, and more SP-PrP localizes in the cytosol. Proteasome inhibitors favor the accumulation of aggregation-prone SP-PrP molecules in the cytosol. Possibly, depending on the cell type, these may cause cytotoxicity or engage in signaling circuits.

 
The main finding of our studies is that conditions of ER stress favor the accumulation of cytosolic PrP. Consistent with their localization, these molecules retain their N- and C-terminal pro-peptides and lack N-glycans and intrachain disulfide bonds. We used different drugs that inhibit folding of ER proteins through diverse mechanisms to show that the accumulation of cytosolic PrP is a consequence of ER stress. In the cytosol, SP-PrP is degraded by the proteasome. This explains why proteasome inhibitors (which induce a robust ER stress and inhibit clearance of SP-PrP) induced its accumulation the most efficiently (Fig. 9).

Why does the UPR favor PrP mislocalization? The progression of nascent proteins through the translocon is thought to be facilitated by interactions with luminal chaperones (50, 51). These welcome the growing polypeptides in the ER, likely assisting their vectorial movement from the cytosol to the lumen. This is well established for post-translational insertion in yeast. Interactions with ER residents and post-translational modifications (i.e. folding, assembly, and glycosylation) likely ratchet the nascent protein in the lumen, preventing or at least reducing backward movement to the cytosol (52). One possible explanation, therefore, could be that aberrant proteins accumulating in the lumen under diverse pharmacological treatments sequester chaperones, thus reducing the availability of pullers and/or retainers. As a result, some nascent polypeptides may fall back into the cytosol before the signal peptide is removed. N-Glycosylation seems to play a minor role, because most unglycosylated PrP synthesized in the presence of tunicamycin accumulated in the ER with intrachain disulfides.

It is also possible that newly made chaperones and enzymes entering the ER during the UPR compete with cargo proteins for a limiting number of signal recognition particles. In principle, the inefficient translocation of certain proteins might quantitatively and qualitatively control ER load after translation resumes.

HeLa transfectants overexpressing sXbp1 translocate better PrP during ER stress (Fig. 6), suggesting a role for the Ire1{alpha}-sXbp1 pathway in controlling ER targeting. sXbp1-dependent factors may include TRAP (translocon-associated protein) (53) and other luminal or membrane ER proteins involved in targeting, importing, and ratcheting incoming proteins.

Because proteasome inhibitors can cause PrP accumulation by increasing transcription of certain viral promoters (13), it was important to verify that PrP also driven by its own promoter undergoes defective translocation in N2a cells and primary neurons. Mislocalization was independent from gene dosage, as it was similar in HeLa cells transfected with different amounts of PrP-driving vector. Therefore, inefficient PrP translocation does not depend on mere competition but rather reflects an intrinsic feature of the PrP targeting sequence (12) that is exaggerated by ER stress.

The intrinsic weakness of PrP SP suggests that this effect could be specific for PrP. Indeed our previous studies have never revealed any effect of UPR on the translocation of other secretory proteins such as Ero1{alpha} or µchain.7

If cytosolic PrP were indeed toxic, these results may have important implications in the pathogenesis of PrP-related diseases. There are many instances in which cells can experience a UPR, related either to pathology (synthesis of mutant proteins with impaired folding, virus infections, lack of cofactors, etc.) (54–56) or to normal development (production of abundant secretory proteins, such as in the endocrine pancreas or plasma cells) (57, 58). These might promote accumulation of aggregation-prone SP-PrP in the cytosol, which in turn could seed further precipitation (41; see also Refs. 13 and 19). Accumulation of stresses and impaired proteasome function could concur in increasing the susceptibility of aged cells. In cells where SP-PrP exerts protective effects, cytosolic PrP might be a mechanism to retard stress-induced and Bax-mediated apoptosis (20, 49), whereas the UPR and XBP1 substrates in particular try to solve the problems that generated ER stress.

Whatever the function of PrP and SP-PrP, our findings identify in protein translocation a novel level at which the UPR exerts its regulatory effects.


   FOOTNOTES
 
* This work was supported in part through grants from the Associazione Italiana per la Ricerca sul Cancro, Fondazione Cariplo, Ministero della Sanità, Ministero dell'Istruzione Universitá e Ricerca and Telethon-Italy (S00083 to R. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a fellowship from the Fondazione Monzino. Back

2 An Assistant Telethon Scientist (Dulbecco Telethon Institute Foundation). Back

3 To whom correspondence should be addressed: Università Vita-Salute San Raffaele, Via Olgettina 58, 20132 Milano, Italy. Tel.: 39-02-26434763; Fax: 39-02-26434723; E-mail: r.sitia{at}hsr.it.

4 The abbreviations and trivial names used are: PrP, prion protein; SP, signal peptide; ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol; DTT, dithiothreitol; UPR, unfolded protein response. Back

5 A. Orsi and R. Sitia, unpublished data. Back

6 A. Orsi and R. Sitia, manuscript in preparation. Back

7 M. Otsu, C. Fagioli, and R. Sitia, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Neil J. Bulleid, Gianluigi Forloni, Laurie Glimcher, David A. Harris, Rick Kascsak, Hidde Ploegh, Fabrizio Tagliavini and Boaz Tirosh for providing excellent reagents, suggestions, and helpful discussions; Ana Fella and Raffaella Brambati for secretarial assistance; and Claudio Fagioli and Elena Pasqualetto for technical help.



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