Skip to main content
PMC home page
Scientific Reports logoLink to Scientific Reports
. 2017 Mar 23;7:44723. doi: 10.1038/srep44723

The Endoplasmic Reticulum Chaperone GRP78/BiP Modulates Prion Propagation in vitro and in vivo

Kyung-Won Park 1, Gyoung Eun Kim 2, Rodrigo Morales 1, Fabio Moda 1, Ines Moreno-Gonzalez 1, Luis Concha-Marambio 1,3, Amy S Lee 4, Claudio Hetz 5,6,7,8, Claudio Soto 1,3,a
PMCID: PMC5363067  PMID: 28333162

Abstract

Prion diseases are fatal neurodegenerative disorders affecting several mammalian species, characterized by the accumulation of the misfolded form of the prion protein, which is followed by the induction of endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). GRP78, also called BiP, is a master regulator of the UPR, reducing ER stress levels and apoptosis due to an enhancement of the cellular folding capacity. Here, we studied the role of GRP78 in prion diseases using several in vivo and in vitro approaches. Our results show that a reduction in the expression of this molecular chaperone accelerates prion pathogenesis in vivo. In addition, we observed that prion replication in cell culture was inversely related to the levels of expression of GRP78 and that both proteins interact in the cellular context. Finally, incubation of PrPSc with recombinant GRP78 led to the dose-dependent reduction of protease-resistant PrPSc in vitro. Our results uncover a novel role of GRP78 in reducing prion pathogenesis, suggesting that modulating its levels/activity may offer a novel opportunity for designing therapeutic approaches for these diseases. These findings may also have implications for other diseases involving the accumulation of misfolded proteins.

Prion diseases are fatal and transmissible neurodegenerative disorders that affect humans and animals1. They are characterized by spongiform brain degeneration, neuronal loss, and the accumulation of a pathogenic and infectious form of the prion protein (PrPSc) generated at expenses of the normal/cellular prion protein (PrPC) (Ref. 2). Accumulation of misfolded prions is thought to exert their deleterious effects in cells by different signaling cascades. Compelling evidence indicates that endoplasmic reticulum (ER) stress is a hallmark event in prion diseases3,4.

The ER is an essential organelle that plays a key role in the maintenance of calcium homeostasis, lipid synthesis, as well as the synthesis and folding of secreted and membrane bound proteins5. Perturbation of physiological conditions in cells can induce ER stress, which triggers an adaptive reaction known as the unfolded protein response (UPR), aimed to restore cellular homeostasis or trigger apoptosis in irreversibly damaged cells6,7. The UPR controls the expression of a variety of genes involved in protein folding secretion and quality control7. ER stress has been reported in many different animal and cellular models of prion diseases3, in addition to patients affected with Creutzfeldt-Jakob disease8. ER stress has been proposed to have two main consequences on the progression of prion diseases: (i) it may contribute to neurological impairment due to repression of the synthesis of a cluster of synaptic proteins9, and (ii) it may operate as a signal to trigger neuronal loss8,10. Importantly, accumulating evidence indicates that the contribution of the UPR to neurodegeneration is complex and largely depends on the UPR signaling branch affected and the disease context11.

The ER chaperone GRP78 (78-kDa glucose regulated protein), also referred to as BiP (Binding immunoglobulin protein) or HSPA5 (heat shock protein family A, member 5), is considered an essential ER chaperone and a master regulator of ER homeostasis12. GRP78 facilitates folding and assembly of nascent polypeptides, prevents their misfolding and aggregation, targets misfolded proteins for proteasome degradation, and controls the signaling for the initiation of the various arms of the UPR13,14. GRP78 operates as a repressor of UPR stress sensors through direct binding to them7. GRP78 has also been shown to have a chaperone activity by directly binding and preventing the aggregation of misfolded proteins in the ER15,16. In addition, GRP78 has multiple functions in cell signaling beyond its role in protein folding and has been found in various subcellular locations besides of the ER (reviewed in ref. 17). Considering its multiple functions, GRP78 has been described to actively participate in a wide variety of physiological and pathological processes18,19.

With respect to prion induced pathology, we and others showed that GRP78 levels were significantly increased in cells treated with PrPSc (refs 8 and 20), as well as transiently in prion infected mice21. Importantly, brains from patients affected by sporadic and variant Creutzfeldt-Jakob disease also showed increased levels of this particular chaperone8. GRP78 has been reported to physically interact with mutant PrP and mediate its degradation by the proteasome15, providing evidence that GRP78 is chaperoning the folding of PrP. Until now, only in vitro evidence and correlative studies in mouse models suggest a possible role of GRP78 in prion diseases. However, its actual contribution to prion pathogenesis remains unexplored. In this study, we examined the impact of targeting GRP78 in prion-induced pathology in animal models, as well as in genetically modified cell cultures. Our data shows that the reduction of GRP78 in vivo accelerates prion replication, thus resulting in a decreased incubation time of the disease. Additionally, we show that GRP78 over-expression reduces PrPSc levels in CAD5 cells infected with scrapie prions, whereas knocking down GRP78 by treatment with siRNA significantly increases prion replication. Immunocytochemistry and co-immunoprecipitation studies suggest that GRP78 and PrPC directly interact in cells. Moreover, in vitro experiments using recombinant GRP78 show that this chaperon is able to disassembly PrPSc in a dose-dependent manner. Our findings indicate that GRP78 plays a key protective role in preventing the propagation of infectious prions, suggesting that the ER proteostasis network is implicated in prion diseases.

Results

In vivo reduction of GRP78 expression accelerates prion disease

To study the possible involvement of GRP78 in prion disease in vivo, we used a mice model heterozygous for GRP78 (GRP78+/−). Complete knock out mice of this important chaperone are not viable, whereas heterozygous mice are viable and develop normally22, although they have been shown to suffer from haplo-insufficient phenotypes under pathological conditions such as cancer23. GRP78+/− and control (GRP78+/+) mice of the same strain were inoculated with the Rocky Mountain Laboratory (RML) strain of murine adapted scrapie prions. We directly injected these animals with RML prions into the brain and monitored disease progression and animal survival. Remarkably, GRP78+/− mice were highly susceptible to prion infection, resulting on an acceleration of the disease when compared with GRP78+/+ littermate controls (Fig. 1A). The median survival time for GRP78+/− mice was 144 days-post-inoculation (d.p.i), whereas GRP78+/+ mice survived for 175 d.p.i (Fig. 1B). Differences between both groups were statistically significant (p < 0.001). These data indicate that a reduction in GRP78 levels substantially accelerates the pathogenesis of prion diseases, suggesting an important protective role of this chaperone in this pathology.

Figure 1. Decreased levels of GRP78 accelerate prion disease in mice.

Figure 1

Open in a new tab

(A) Survival curve of GRP78 heterozygous (GRP78+/−) and wild type (GRP78+/+) mice intra-cerebrally inoculated with RML prions. Differences in animal survival were analyzed by the Log-rank (Mantel Cox) test and found highly significant (P = 0.0018). (B) Average incubation periods of the different groups showed in panel A. Data is expressed as averages ± standard errors. Differences among the groups were analyzed by student’s t-test. ***P < 0.001.

End-point prion disease characteristics are not altered by lower levels of GRP78 expression

We then characterized different histopathological features of prion disease on our RML-infected mice. We analyzed the brain vacuolation profile in each group, as previously described24,25. Brain coronal sections stained with hematoxylin/eosin (H&E) showed the characteristic lesions expected for RML prions in thalamus and frontal cortex (Fig. 2A). The average lesion profile in various brain regions for both GRP78+/+ and GRP78+/− mice was indistinguishable (Fig. 2B). We observed that the degree and proportion of vacuolation in both animal groups was the same at the end disease stage, regardless of the different incubation times observed. These results suggest that the faster appearance of clinical signs in GRP78+/− mice was not due to a differential targeting of prions to specific brain structures. Additionally, we did not observe differences in PrPSc accumulation in the brain of GRP78+/− and GRP78+/+ animals as assessed by immunostaining at the terminal stage of the disease (data not shown).

Figure 2. GRP78 expression does not alter the vacuolation profile of terminally ill prion infected mice.

Figure 2

Open in a new tab

(A) Thalamus and frontal cortex sections of brains from RML-symptomatic GRP78 heterozygous (Grp78+/−) and wild type (Grp78+/+) mice were analyzed histologically for spongiform degeneration after hematoxylin-eosin staining. Bar in the lower right panel depict 100 μm and is representative of all pictures in this panel. (B) The vacuolation lesion profiles were determined on H&E stained sections from 5 different animals in each group. Degree of vacuolation was analyzed by scoring midbrain; hypothalamus; thalamus; hippocampus and motor cortex.

Expression levels of ER chaperones and stress proteins in prion infected GRP78 +/− and GRP78 +/+ mice

Next, we analyzed the expression of chaperones, ER-stress targets and proteins involved in the UPR in prion infected GRP78+/− and GRP78+/+ mice to investigate whether the reduction of GRP78 exacerbates ER stress levels in prion-infected mice. As expected, we found that GRP78 protein levels in GRP78+/− brains were reduced by about 50% when compared to those of control littermates (Fig. 3A). On the contrary, the expression levels of GRP94 and calreticulin were increased by ~1.5 and ~1.7 fold, respectively, in GRP78+/− mice, suggesting compensatory changes to reduce protein folding stress (Fig. 3B). Other ER chaperones, such as calnexin and PDI did not show any significant differences between GRP78+/− and GRP78+/+ infected mice (Fig. 3). Although the expression levels of other UPR targets, such as PERK, IRE1, eIF2α and CHOP, were not altered in GRP78+/− mice brains when compared to the GRP78+/+ counterparts, the levels of PERK-P and eIF2α-P were increased by ~3 and ~1.4 fold, respectively, in GRP78+/− mice (Fig. 3B). These results suggest that reduction of GRP78 during prion infection does not generate a global alteration in the ER proteostasis network, but might induce changes on specific UPR targets, particularly PERK-P and eIF2α-P, which may lead to significantly reduced survival of prion infected mice. This conclusion is consistent with recent reports indicating a key role of PERK-P and eIF2α-P in prion-induced neurodegeneration9,26.

Figure 3. Expression levels of ER stress-responsive proteins in prion infected GRP78+/+ and GRP78+/− mice.

Figure 3

Open in a new tab

(A) Protein expression levels of UPR signaling proteins and ER chaperones from brains of GRP78+/+ and GRP78+/− mice infected with prions were examined by Western blot. Proteins analyzed included GRP78, GRP94, PDI, IRE1, CHOP, PERK, eIF2α, calnexin, calreticulin as well as the phosphorylated forms of PERK and eIF2α. PrPSc content was assessed after treating brain extracts with PK as explained in Experimental Procedures. β-actin is shown as a loading control. Numbers at the top of the panel represent samples from three different animals in each group. For all samples, the same amount of total protein was loaded in each lane. For space constrains blots were cropped, but all samples were run using the same conditions and in the same gel. (B) Quantifications of Western blot signals from 3 replicates of the experiment shown in panel A are represented as averages ± standard errors. Statistical differences were analyzed by using student’s t-test. *P < 0.05, ***P < 0.001.

We also monitored the levels of PrPSc at the terminal stage of the disease using proteinase K (PK) assays followed by Western blot analysis. The levels of PrPSc were similar in all mice regardless of the incubation periods (Fig. 3A), suggesting that although misfolded prion accumulation was accelerated, the end levels of the abnormal protein were not altered by reduced GRP78 expression.

GRP78 interacts with PrPC

Since PrPC is synthesized and modified in the ER (including disulfide bond formation, N-linked glycosylation, and GPI-anchor addition), we examined whether GRP78 may directly bind to this protein. We first performed immunocytochemistry experiments in primary cultures of wild type, non-infected, mouse fibroblasts. PrP was stained by using the 6H4 monoclonal antibody, followed by secondary antibody labeled with Alexa488 (in green). Staining was seen in the cytoplasm, the perinuclear compartment, and the cell surface (Fig. 4A, top left panel). GRP78 was stained by a specific antibody against this protein followed by the respective secondary antibody labeled with Alexa568 (in red) and showed a similar sub-cellular localization as PrP (Fig. 4A, top right panel). When the double labeling of both the anti-PrP and anti-GRP78 antibodies was examined simultaneously, there was a substantial blending of the immuno-reactivity merge, suggesting co-localization of both proteins (Fig. 4A, bottom panels). Co-localization analysis was performed to quantify the pixel co-distribution of 6H4 and anti-GRP78 antibodies using images obtained in a confocal microscope (Fig. 4B). The Pearson correlation coefficient (0.509 ± 0.037) demonstrated a good co-localization between GRP78 and PrP (1 = perfect correlation, 0 = no correlation, and −1 = perfect inverse correlation). In addition, Mander’s overlap coefficient (0.838 ± 0.044) also indicated that the 6H4 and GRP78 signals co-localize in the cell. The two-dimensional histogram for the distribution of pixel intensities for 6H4 and GRP78 reveals a positive spatial correlation (Fig. 4B).

Figure 4. GRP78 interacts with PrP.

Figure 4

Open in a new tab

(A) Primary cultures of mouse fibroblasts were doubly labeled with antibodies against PrP and GRP78 proteins. Top left panel represents cells that have been labeled with the 6H4 anti-PrP antibody and detected with Alexa488 secondary antibody (green). Top right panel represents cells that have been stained with anti-GRP78/BiP and detected with Alexa568 secondary antibody (red). Bottom left panel represents the merge between the two staining. Bottom right panel is a zoomed picture of one cell of the merged pictures (depicted in the dotted box in the bottom left panel). Samples were visualized by a confocal microscope. Scale bar: 50 μm or 25 μm. (B) Representative fluorogram indicating the signal intensity for both stainings and the colocalization of 6H4 (Alexa 488) and GRP78 (Alexa 568) obtained from confocal images. (C) Wild type mouse brain homogenates were immunoprecipitated with the anti-GRP78 antibody. Samples were analyzed by Western blot using an anti-PrP antibody (6D11). Lane 1 represents untreated brain homogenates used as a control, lane 2 corresponds to precipitation done with uncoated beads (without anti-GRP78 antibody), and lane 3 represents the immunoprecipitation with anti-GRP78 antibody. (D) Wild type mouse brain homogenates were immunoprecipated with the 6D11 anti-PrP antibody and samples analyzed by Western blot with anti-GRP78 antibody. First lane corresponds to the immoprecipitation with the 6D11 antibody, whereas the second line is the precipitation with the beads alone. Third lane depicts recombinant GRP78. Numbers on the left side of the gels correspond to the molecular weight standards. Separation line in the right blot indicate gel splicing to remove some irrelevant lines, even though all the samples were run in the same gel.

To further study a possible interaction between PrPC and GRP78, co-immunoprecipitation experiments were done with brain homogenates prepared from wild type mice. PrPC was efficiently precipitated with the anti-GRP78 antibody (Fig. 4C, lane 3), whereas no signal was detected after incubation with anti-rabbit IgG Dynabeads alone (Fig. 4C, lane 2). Similarly, GRP78 was co-immunoprecipitated with anti-PrP antibodies, but not with beads alone (Fig. 4C). Altogether, these results indicate that PrPC and GRP78 directly interact inside cells.

GRP78 expression modifies PrPSc replication in prion infected cells

To further study the functional role of GRP78 in prion replication, we performed studies with a CAD5 cell line chronically infected with mouse prions27. GRP78 expression was experimentally altered in these cells in order to investigate whether this protein modulates the formation of PrP aggregates. We performed gain- and loss-of function studies by overexpressing the GRP78 gene, or by silencing its expression using siRNAs. Modified cells were lysed and GRP78 and PrPSc levels monitored by western blot analysis. Remarkably, immunoblotting revealed that reduction of GRP78 using siRNA significantly increased PrPSc accumulation in CAD5 cells (Fig. 5A), whereas overexpression of GRP78 decreased the accumulation of the disease associated prion isoform (Fig. 5B). These results suggest that PrPSc replication is altered by changes in GRP78 expression, confirming the in vivo results shown above. The effect of GRP78 was not at the level of PrPC expression, since non-infected cells treated with siRNA to reduce GRP78 expression showed no differences in the levels of PrPC (Supplementary Figure 1).

Figure 5. Expression levels of GRP78 modify prion replication in chronically infected CAD5 cells.

Figure 5

Open in a new tab

(A) Prion-infected CAD5 cells transfected with GRP78 siRNA or control siRNA were harvested and lysed. The expression of GRP78, actin (loading control), and PrPSc was analyzed by Western blotting. Left blot shows the staining with GRP78 antibody. Right blot corresponds to the staining with anti-PrP antibody. The graph shows the densitometric analysis of the levels of PrPSc in cells treated with control or GRP78 siRNA. (B) Prion-infected CAD5 cells transfected with GRP78 overexpressing plasmid or control plasmid were harvested and lysed. The expression of GRP78, actin, and PrPSc was analyzed by Western blotting. Left blot depicts the staining for GRP78. Right blot corresponds to the staining for PrP. In this panel, the vertical line indicates gel splicing to remove some irrelevant lanes, but samples were run in the same gel and were developed with the same exposition. The graph shows the densitometric analysis of PrPSc levels in cells expressing endogenous amounts of GRP78 (control) or over-expressing this protein. In both panels A and B, NBH: normal brain homogenate, not treated with PK, used as a marker of PrPC migration. SBH: RML-infected brain homogenate treated with PK, used as a marker of protease-resistant PrPSc migration. For space constrains some blots were cropped, but all samples were run using the same conditions and in the same gel. Statistical differences were analyzed by using student’s t-test. **P < 0.01, ***P < 0.001.

GRP78 reduces protease-resistance of PrPSc in cell-free systems

Besides its role as a master regulator of the UPR, GRP78 is a chaperone protein that binds hydrophobic residues in unfolded or partially folded proteins28. GRP78 was shown before to physically associate with mutant PrP15. To examine whether GRP78 may directly affect PrPSc, brain homogenates of RML-infected mice containing large quantities of pre-formed PrPSc aggregates were incubated with purified recombinant GRP78. After incubation, we monitored the levels of PrPSc by employing an assay that takes advantage of the classical property of PrPSc of being highly resistant to proteolytic degradation. We observed a dose-dependent reduction in the levels of protease-resistant PrPSc in extracts incubated with different concentrations of recombinant GRP78 (Fig. 6A). Although these results suggest a direct effect of GRP78 on PrPSc, an indirect activity of GRP78 over other putative molecules supporting the PrPSc structure cannot be discarded. To study the possible effects of GRP78 over PrPSc in a more direct manner, highly purified PrPSc was incubated with different concentrations of recombinant GRP78. We found that 8 μM of recombinant GRP78 almost completely eliminated protease-resistance of purified PrPSc species under our experimental conditions (Fig. 6B). As a control, we added the same concentration of an unrelated protein (Bovine Serum Albumin, BSA) and the results did not show any effect on PrPSc (Fig. 6B). A similar, albeit less pronounced effect, was observed with PrPSc associated to other murine prion strains, such as 79A and 301 C (Supplementary Figure 2). To determine whether the effect of GRP78 over PrPSc was time-dependent, 8 μM of the recombinant chaperone was incubated with purified PrPSc preparations for various times (Fig. 6C). We observed a time-dependent reduction of PrPSc signal in PK-resistant assays. However, GRP78 activity was optimum only after prolonged incubation. Our results suggest that GRP78 has a direct effect on preventing PrPSc replication through altering the biochemical/structural properties of PrPSc into conformations that are sensitive to PK.

Figure 6. GRP78 reduces the amount of protease-resistant PrPSc in vitro.

Figure 6

Open in a new tab

(A) RML infected brain homogenates were incubated with different concentrations of purified recombinant GRP78 for 20 h (1200 minutes), and the amount of PrPSc remaining resistant to PK digestion was assessed by Western blotting (top panel). Numbers at the top represent GRP78 concentrations, expressed as μM. (B) Highly purified PrPSc preparations were incubated with different concentrations of GRP78 for 20 h, and reactions were analyzed by Western blotting. As a control, BSA (bovine serum albumin) was used at the same concentrations. Numbers at top of each gel represent μM protein concentration supplemented in each case. Graphs below each blot represent the densitometric analysis of 3 replicates for each respective experiment. Values correspond to the average ± standard error and differences were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. (C) Purified PrPSc preparations were incubated with 8 μM of GRP78 for different time points ranging from 30 to 1200 minutes. “Control” represents a purified PrPSc aliquot without any treatment. The graph below represents the densitometric analysis of 3 replicates showed as the average ± standard error. Differences were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. All samples used to evaluate PrPSc signal were first treated with PK. Immunoblot was used to assess remaining protease-resistant PrPSc in each case. *P < 0.05; ***P < 0.001.

Discussion

Many neurodegenerative diseases are associated with the accumulation of misfolded proteins in the brain29,30. Compelling evidence has shown that disease-related proteins alter distinct aspects of the secretory pathway, triggering as a common event ER stress. PrPSc accumulation in prion diseases has been shown to cause an imbalance in ER homeostasis and activation of the UPR in different experimental systems, possibly due to altered calcium homeostasis3,31. GRP78 is a master regulator of ER protein folding, in addition to fine-tuning the threshold to activate the UPR. Under physiological conditions, GRP78 binds the luminal domains of the three main UPR stress sensors including ATF6, PERK and IRE1 in order to keep them in an inactive state7. When misfolded proteins accumulate in the ER, GRP78 preferentially assists folding releasing ER stress sensors, triggering a global UPR response to restore proteostasis14.

It has been proposed that chronic ER stress produced by the persistent and progressive accumulation of misfolded proteins leads to massive perturbation of GRP78 and its signaling targets, triggering apoptosis and neurodegeneration18. Depletion of GRP78 in Purkinje cells accelerates neuronal degeneration and retards growth in young mice32. Similarly, mutant knock-in mice for GRP78 undergo age-dependent neurodegeneration33. In other studies, reduction of GRP78 expression resulted in increased aggregation and toxicity of proteins harboring poly-glutamine repeats34. Dysfunction of GRP78 also deteriorated retinal damage and induced apoptosis in the retinal tubular injury35. The disruption of the GRP78 co-chaperone, SIL1, caused ER stress and accumulation of abnormally folded proteins, leading to neurodegeneration linked to apoptosis and autophagy36. Similarly, SIL1 has recently been shown to mediate neuroprotection in models of amyotrophic lateral sclerosis37. GRP78 associates with caspase-7 and −12 in the ER compartment and inhibits caspase activation and caspase-mediated cell death38. Deletion or missense mutation in the ATP binding domain of GRP78 fail to bind caspase-7, leading to loss of its protective effects39. GRP78 also exerts an anti-apoptotic effect through suppressing other ER localized apoptotic machinery40. These findings provide evidence that the expression level and activity of GRP78 are essential for neuroprotection, to prevent protein aggregation and to regulate proper UPR signaling.

Although ER stress and the UPR appear to be heavily involved in prion disease pathogenesis3,4, the exact mechanisms are presently unclear. Early studies from our group and others suggested a role for ER chaperones on prion diseases, particularly the disulfide isomerase ERp57/Grp58 which is highly induced in prion infected mice and patients affected with CJD8,21,41. We previously reported that ERp57/Grp58 has a neuroprotective role against PrPSc toxicity and also physically interacts with PrPC (Ref. 21). ERp57/Grp58 is part of the calnexin and controls the steady state of PrPC levels in vitro and in vivo42. Recent findings suggest that over-activation of PERK signaling lead to sustained eIF2α phosphorylation with concomitant reduction in protein synthesis of a cluster of synaptic proteins9. Furthermore, treatment of scrapie infected mice with a PERK inhibitor significantly delayed disease progression26. In contrast, we reported that targeting XBP1, the downstream transcription factor of IRE1 does not alter prion pathogenesis in vivo43. Similarly, caspase-12 deficient animals develop prion disease in the same way as wild type animals44. These data suggest that the contribution of the UPR to prion diseases is complex and may depend on the signaling branch affected11.

GRP78 plays a dual role, binding hydrophobic fragments of misfolded or unfolded proteins to regulate their folding and prevent their aggregation, and modulating the UPR in acute stress conditions18. We hypothesize that GRP78 may have a direct effect in PrPSc replication and their disease associated phenotypes due to a direct inhibitory activity on PrP misfolding. Our in vivo data support this hypothesis, since animals expressing lower levels of GRP78 developed prion disease at a significantly shorter time. From the in vivo results, however, it is not possible to distinguish whether GRP78 directly alters prion replication or the effect observed in mice was due to an indirect activation of signaling pathways.

It has been shown that GRP78 can bind disease-associated misfolded proteins and prevent their pathological consequences16. For example, over-expressed GRP78 formed a complex with α-synuclein, resulting in reduced toxicity and increased survival of nigral dopaminergic neurons45. To gain more detail information about the mechanism by which GRP78 is protective in prion diseases, we performed various studies using prion infected cells as well as cell-free in vitro experiments. Reduction of GRP78 in cells led to a significant increase on PrPSc replication. Conversely, overexpression of GRP78 reduced PrPSc formation in cells. Interestingly, incubation of PrPSc with purified recombinant GRP78 resulted in a significant and dose-dependent decrease of protease-resistant PrPSc. Indeed, after prolonged incubation with an optimal concentration of GRP78, protease-resistant PrPSc virtually disappeared from the sample. This data suggests that GRP78 may modulate the misfolding and aggregation of PrPSc by direct interaction with prion proteins. However, given the multiple and important functions of GRP78 we cannot rule out that in vivo GRP78 might be acting through various simultaneous mechanisms to attempt preventing prion replication.

Our results may have implications for the development of novel therapeutic approaches against prion diseases. Strategies to enhance the activity of GRP78 have already been tested in other models with successful results46,47. For example, gene therapy to deliver GRP78 into dopaminergic neurons resulted in reduced α-synuclein aggregation and delayed disease progression in genetic models of Parkinson´s disease45. In addition, a small molecule termed BiP protein Inducer X (BIX) increases GRP78 expression in vivo and protects neurons in models of brain ischemia48,49, retinal damage50, and photoreceptor death35. Furthermore, a series of small molecules and other gene therapy strategies have been developed to target the UPR and reduce ER stress levels on a variety of preclinical models of disease51. Altogether, our results indicate that GRP78 may play an important role in the defense against prion propagation. Our findings suggest that modulation of GRP78 activity might provide a novel strategy to design therapies directed to combat prion diseases and maybe other neurodegenerative diseases associated to the accumulation of misfolded protein aggregates.

Methods

Infectivity studies

In vivo infectivity studies were done in GRP78+/− heterozygous (n = 5) or GRP78+/+ (WT, n = 5) female mice both with C57Bl6 background. GRP78+/− mice were previously generated and characterized as described22. Animals were 90 days old at the time of prion inoculation. Mice were injected stereotaxically into the right hippocampus with 2 μL of a 10% w/v brain extract obtained from a symptomatic RML-infected mouse. Animals were checked for appearance of prion-associated clinical signs as previously described52. Symptomatic animals were sacrificed at the terminal stage of the disease and brains collected for biochemical and histological analyses. Surgical procedures were performed under isoflurane anesthesia. All animal manipulations were carried out in accordance to NIH regulations and approved by the Animal Welfare Committee of the University of Texas Health Science Center at Houston.

Histopathological studies

Brain tissue was fixed in Carnoy fixative, dehydrated, and embedded in paraffin. Serial sections (7 μm thick) from each block were deparaffinized, hydrated and stained with hematoxylin-eosin (H&E). Then, samples were dehydrated, cleared in xylene and coverslipped with DPX. Samples were visualized with a DMI6000B Leica® microscope. Brain vacuolation profile was determined on H&E stained sections by scoring the extent of vacuolation in midbrain, hypothalamus, thalamus, hippocampus and motor cortex as previously described24,25.

PrPSc purification from infected brains

PrPSc was purified from the brain of clinically ill mice infected with RML prions, as previously described53,54. Briefly, brain tissue was homogenized at 10% w/v in PBS. After a low speed centrifugation to remove debris, samples were mixed with 1 volume of 20% sarkosyl and subjected to a series of differential centrifugations employing a Beckman TL-100 ultracentrifuge (OptimaMAX Ultracentrifuge, Beckman-Coulter) with the final step consisting of a sucrose gradient. The resulting material was treated with proteinase K (PK) (100 μg/ml) for 2 h at 37 °C followed by ultracentrifugation to precipitate PrPSc. The purity of PrPSc was confirmed by silver staining and estimated to be >95%. PrPSc concentration was measured by micro BCA protein assay reagent (Pierce).

Proteinase K digestion

Cell lysates or 10% w/v brain homogenates were prepared in PBS supplemented with a cocktail of protease inhibitors as previously described55. Debris-cleared aliquots were digested with 50 μg/mL PK with shaking (450 rpm in an Eppendorf thermomixer) at 37 °C for 1 h. PK resistant PrPSc was then detected by Western blot.

Western blot

Cell lysates and brain homogenates were prepared in PBS supplemented by a cocktail of protease inhibitors. Protein concentration was determined with the Pierce BCA Protein Assay kit (Thermo Scientific). Proteins from cell lysates or brain homogenates were fractionated by electrophoresis using 4–12% SDS-polyacrylamide gels (SDS-PAGE), transferred into nitrocellulose membranes, and probed with the following antibodies: anti-prion 6D11 antibody (1:5000, Sigma), anti-calreticulin (1:1000, Cell Signaling), anti-GRP94 (1:1000, Cell Signaling), anti-protein disulphide isomerase (anti-PDI, 1:1000, Cell Signaling), anti-protein kinase RNA-like endoplasmic reticulum kinase (PERK) (1:1000, Cell Signaling), anti-phospho-PERK (1:1000, Cell Signaling), anti-inositol-requiring 1 protein (IRE1) (1:1000, Cell Signaling), anti-eIF2α (1:1000, Cell Signaling), anti-phospho-eIF2α (1:1000, Cell Signaling), anti-calnexin (1:1000, Cell Signaling), anti-CCAAT/-enhancer-binding protein homologous protein (CHOP) (1:1000, Cell Signaling), and anti-β-actin (1:1000, Cell Signaling). The immunoreactive bands were analyzed using the Quantify One (4.6.7) software (BioRad®).

Cell culture

Prion infected CAD5 cells (a generous gift from Dr. Charles Weissmann, Scripps Institute, Jupiter, FL) were cultured in Optimem supplemented with 10% fetal calf serum and antibiotics. Cells were maintained in DMEM (N2) or Optimem (CAD5) and split 1:10 at confluence. Mouse embryonic fibroblasts (MEFs) were isolated following a previously described protocol56. Briefly, 14 day-old wild type mice embryos were obtained from the uterine horns from pregnant mothers. Head and red organs were removed and the rest was digested in presence of trypsin and DNase, followed by pipette dissociation. Thereafter, trypsin activity was inactivated with MEFs culture medium (DMEM, containing 10% fetal bovine serum, L-glutamine, and penicillin-streptomycin). Cells were centrifuged at low speed (300 × g for 5 minutes) and resuspended in MEFs culture medium. Cells were plated onto flask coated with 0.2% gelatine for 2 h. The fibroblasts are the only cells that have the ability to attach to the gelatine-coated flasks. Cells were grown in MEFs culture medium until they reached 70% confluence.

Plasmids and siRNA

GFP DNA fragments were removed from pEGFP (Clontech®), resulting in pE vector. GRP78 PCR fragments were subcloned into pE vector to create pE-GRP78. Scrambled siRNA as a negative control and siRNA targeting GRP78 were designed and purchased from Thermo Scientific/Dharmacon. For siRNA transfection, cultures of CAD5 at 70% confluence were transfected with 25 nmol/L scrambled siRNA as a negative control or siRNA targeting GRP78 using the Lipofectamine® 2000 Reagent (Life technologies).

Expression and purification of recombinant GRP78

E. coli cells [BL21 (DE3)] containing pQE80-GRP78 were grown overnight in 50 mL of LB medium with carbenicillin (100 μg/ml). The overnight culture was used to inoculate 1 L of LB containing carbenicillin (100 μg/mL) at 37 °C with shaking (250 rpm). After 3 h, 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was added to induce protein expression for 4 h. GRP78 in frame with the N-terminal 6 × His tag sequence was purified under native conditions using Ni-nitrilotriacetic acid (NTA) Superflow resin (Qiagen) using manufacturer’s suggested protocol. After this procedure the protein was highly pure as analyzed by coomassie staining and western blot (Supplementary Figure 3).

Immunocytochemistry

For double staining of PrP and GRP78, fibroblasts were fixed for 30 minutes in 10% formalin solution. Cells were first pre-treated with sodium citrate and then sequentially incubated with mouse anti-6H4 (1:100) primary antibody and then rabbit anti-GRP78 (1:200) primary antibody overnight, followed by the corresponding Alexa488 anti-mouse secondary antibody (1:500) and Alexa568 anti-rabbit (1:500). Sections were cover-slipped with mounting medium containing DAPI and examined under a confocal laser microscope (Nikon A1R). The degree of overlapping pixels of 6H4 and GRP78 signal was quantified using the NIS-Elements software (Image Analysis System). The intensity of a given pixel in the green image was used as the x-coordinate of the scatter plot and the intensity of the corresponding pixel in the red image as the y-coordinate. Results were displayed in a pixel distribution scatterplot or fluorogram and Pearson’s correlation and Mander’s overlap coefficients were determined.

Coimmunoprecipitation

Wild type brain homogenates were prepared in PBS at 10% w/v, supplemented by a cocktail of protease inhibitors. Immunoprecipitation was performed with the anti-GRP78 antibody and anti-rabbit IgG Dynabeads or anti-PrP (6D11) and anti-mouse IgG Dynabeds, following the recommendations of the manufacturer (Life Technologies). The presence of PrP or GRP78 in the precipitated material was evaluated by Western blots, as described above. Controls were done using Dynabeds conjugated with the secondary antibody without the primary antibody.

Statistical Analysis

Means are presented with their standard errors and compared by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test or by two-tailed unpaired t-test with Welch’s correction. For infectivity experiments, Log-rank (Mantel-Cox) test was used to determine differences among the groups. Data was analyzed using the GraphPad prism software, version 5.0. Statistical differences were considered significant for values of P < 0.05.

Additional Information

How to cite this article: Park, K.-W. et al. The Endoplasmic Reticulum Chaperone GRP78/BiP Modulates Prion Propagation in vitro and in vivo. Sci. Rep. 7, 44723; doi: 10.1038/srep44723 (2017).

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Material

Supplementary Figures
srep44723-s1.pdf (169.3KB, pdf)

Acknowledgments

We thank Andrea Flores-Ramirez for technical help in maintaining and examining prion infected animals, Karina Cuanalo-Contreras for providing fibroblasts primary cultures for our studies and Dr. Charles Mays for critical editing of the manuscript. This work was funded by FONDAP program 15150012, Office of Naval Research-Global (ONR-G) N62909-16-1-2003, Millennium Institute P09-015-F, FONDEF ID16I10223, US Air Force of Scientific Research FA9550-16-1-0384 and CONICYT-Brazil 441921/2016-7 to CH and NIH grants NS04973 to CS and CA-027607 to ASL.

Footnotes

The authors declare no competing financial interests.

Author Contributions K.-W.P. designed the studies, carried out the majority of the experiments, analyzed the results and prepared the final version of the figures. G.E.K. collaborated with the cellular studies. R.M. participated in the in vivo infectivity studies and writing of the manuscript. I.M.-G. carried out the immunocytochemistry studies. F.M. performed the histological studies. L.C.-M. collaborated on the immunoprecipitation studies and A.S.L. and C.H. provided the GRP78 transgenic mice and provide expert guidance in some of the studies. C.S. is the principal investigator on the project and was responsible for coordinating research activity, analyzing the data, funding, writing the manuscript and producing the final version of the article.

References

  1. Prusiner S. B. Prions. Proc. Natl. Acad. Sci. USA 95, 13363–13383 (1998). [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Soto C. & Saborio G. P. Prions: disease propagation and disease therapy by conformational transmission. Trends Mol. Med. 7, 109–114 (2001). [DOI] [PubMed] [Google Scholar]
  3. Hetz C. A. & Soto C. Stressing Out the ER: A Role of the Unfolded Protein Response in Prion-Related Disorders. 6, 37–43 (2006). [DOI] [PMC free article] [PubMed]
  4. Halliday M. & Mallucci G. R. Targeting the unfolded protein response in neurodegeneration: A new approach to therapy. 76 Pt A, 169–174 (2014). [DOI] [PubMed]
  5. Malhotra J. D. & Kaufman R. J. The endoplasmic reticulum and the unfolded protein response. Semin. Cell Dev. Biol. 18, 716–731 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kim I., Xu W. & Reed J. C. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat. Rev. Drug Discov. 7, 1013–1030 (2008). [DOI] [PubMed] [Google Scholar]
  7. Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat. Rev. Mol. Cell Biol. 13, 89–102 (2012). [DOI] [PubMed] [Google Scholar]
  8. Hetz C. et al. Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein. EMBO J. 22, 5435–5445 (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Moreno J. A. et al. Sustained translational repression by eIF2alpha-P mediates prion neurodegeneration. Nature 485, 507–511 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mukherjee A. & Soto C. Role of calcineurin in neurodegeneration produced by misfolded proteins and endoplasmic reticulum stress. Curr. Opin. Cell Biol. 23, 223–230 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hetz C. & Mollereau B. Disturbance of endoplasmic reticulum proteostasis in neurodegenerative diseases. Nat. Rev. Neurosci. 15, 233–249 (2014). [DOI] [PubMed] [Google Scholar]
  12. Ni M. & Lee A. S. ER chaperones in mammalian development and human diseases. FEBS Lett. 581, 3641–3651 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lee A. S. The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem. Sci. 26, 504–510 (2001). [DOI] [PubMed] [Google Scholar]
  14. Hendershot L. M. The ER function BiP is a master regulator of ER function. Mt. Sinai J. Med. 71, 289–297 (2004). [PubMed] [Google Scholar]
  15. Jin T. et al. The chaperone protein BiP binds to a mutant prion protein and mediates its degradation by the proteasome. J. Biol. Chem. 275, 38699–38704 (2000). [DOI] [PubMed] [Google Scholar]
  16. Morris J. A. et al. Immunoglobulin binding protein (BiP) function is required to protect cells from endoplasmic reticulum stress but is not required for the secretion of selective proteins. J. Biol. Chem. 272, 4327–4334 (1997). [DOI] [PubMed] [Google Scholar]
  17. Ni M., Zhang Y. & Lee A. S. Beyond the endoplasmic reticulum: atypical GRP78 in cell viability, signalling and therapeutic targeting. Biochem. J. 434, 181–188 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wang M. et al. Role of the unfolded protein response regulator GRP78/BiP in development, cancer, and neurological disorders. Antioxid. Redox. Signal. 11, 2307–2316 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee A. S. Glucose-regulated proteins in cancer: molecular mechanisms and therapeutic potential. Nat. Rev. Cancer 14, 263–276 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Torres M. et al. Prion protein misfolding affects calcium homeostasis and sensitizes cells to endoplasmic reticulum stress. PLoS One. 5, e15658 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hetz C. et al. The disulfide isomerase Grp58 is a protective factor against prion neurotoxicity. J. Neurosci. 25, 2793–2802 (2005). [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Luo S. et al. GRP78/BiP is required for cell proliferation and protecting the inner cell mass from apoptosis during early mouse embryonic development. Mol. Cell Biol. 26, 5688–5697 (2006). [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Dong D. et al. Critical role of the stress chaperone GRP78/BiP in tumor proliferation, survival, and tumor angiogenesis in transgene-induced mammary tumor development. Cancer Res. 68, 498–505 (2008). [DOI] [PubMed] [Google Scholar]
  24. Fraser H. & Dickinson A. G. The sequential development of the brain lesion of scrapie in three strains of mice. J. Comp Pathol. 78, 301–311 (1968). [DOI] [PubMed] [Google Scholar]
  25. Moda F. et al. Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice. Prion. 6, 383–390 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moreno J. A. et al. Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice. Sci. Transl. Med. 5, 206ra138 (2013). [DOI] [PubMed] [Google Scholar]
  27. Mahal S. P. et al. Prion strain discrimination in cell culture: the cell panel assay. Proc. Natl. Acad. Sci. USA 104, 20908–20913 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moore K. A. & Hollien J. The unfolded protein response in secretory cell function. Annu. Rev. Genet. 46, 165–183 (2012). [DOI] [PubMed] [Google Scholar]
  29. Moreno-Gonzalez I. & Soto C. Misfolded protein aggregates: mechanisms, structures and potential for disease transmission. Semin. Cell Dev. Biol. 22, 482–487 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Soto C. Transmissible Proteins: Expanding the Prion Heresy. Cell 149, 968–977 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Torres M. et al. Abnormal calcium homeostasis and protein folding stress at the ER: A common factor in familial and infectious prion disorders. Commun. Integr. Biol. 4, 258–261 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang M. et al. Essential role of the unfolded protein response regulator GRP78/BiP in protection from neuronal apoptosis. Cell Death. Differ. 17, 488–498 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Jin H. et al. Late-onset of spinal neurodegeneration in knock-in mice expressing a mutant BiP. PLoS One. 9, e112837 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yamagishi N. et al. Endoplasmic reticulum chaperone GRP78 suppresses the aggregation of proteins containing expanded polyglutamine tract. Biochem. Biophys. Res. Commun. 422, 527–533 (2012). [DOI] [PubMed] [Google Scholar]
  35. Nakanishi T. et al. Role of endoplasmic reticulum stress in light-induced photoreceptor degeneration in mice. J. Neurochem. 125, 111–124 (2013). [DOI] [PubMed] [Google Scholar]
  36. Zhao L. et al. Protein accumulation and neurodegeneration in the woozy mutant mouse is caused by disruption of SIL1, a cochaperone of BiP. Nat. Genet. 37, 974–979 (2005). [DOI] [PubMed] [Google Scholar]
  37. Filezac de L. E. et al. Marinesco-Sjogren syndrome protein SIL1 regulates motor neuron subtype-selective ER stress in ALS. Nat. Neurosci. 18, 227–238 (2015). [DOI] [PubMed] [Google Scholar]
  38. Rao R. V. et al. Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78. FEBS Lett. 514, 122–128 (2002). [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reddy R. K. et al. Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors - Role of ATP binding site in suppression of caspase-7 activation. J. Biol. Chem. 278, 20915–20924 (2003). [DOI] [PubMed] [Google Scholar]
  40. Zhou H. et al. Novel mechanism of anti-apoptotic function of 78-kDa glucose-regulated protein (GRP78): endocrine resistance factor in breast cancer, through release of B-cell lymphoma 2 (BCL-2) from BCL-2-interacting killer (BIK). J. Biol. Chem. 286, 25687–25696 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yoo B. C. et al. Overexpressed protein disulfide isomerase in brains of patients with sporadic Creutzfeldt-Jakob disease. Neurosci. Lett. 334, 196–200 (2002). [DOI] [PubMed] [Google Scholar]
  42. Torres M. et al. The Protein Disulfide Isomerase ERp57 Regulates the Steady-State Levels of the Prion Protein. J. Biol. Chem. (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Hetz C. et al. Unfolded protein response transcription factor XBP-1 does not influence prion replication or pathogenesis. Proc. Natl. Acad. Sci. USA 105, 757–762 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Steele A. D. et al. Prion pathogenesis is independent of caspase-12. 1, 243–247 (2007). [DOI] [PMC free article] [PubMed]
  45. Gorbatyuk M. S. et al. Glucose regulated protein 78 diminishes alpha-synuclein neurotoxicity in a rat model of Parkinson disease. Mol. Ther. 20, 1327–1337 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Gorbatyuk M. S. & Gorbatyuk O. S. The Molecular Chaperone GRP78/BiP as a Therapeutic Target for Neurodegenerative Disorders: A Mini Review. J. Genet. Syndr. Gene Ther. 4 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Baumeister P. et al. Transcriptional induction of GRP78/BiP by histone deacetylase inhibitors and resistance to histone deacetylase inhibitor-induced apoptosis. Mol. Cancer Ther. 8, 1086–1094 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Kudo T. et al. A molecular chaperone inducer protects neurons from ER stress. Cell Death. Differ. 15, 364–375 (2008). [DOI] [PubMed] [Google Scholar]
  49. Oida Y. et al. Induction of BiP, an ER-resident protein, prevents the neuronal death induced by transient forebrain ischemia in gerbil. Brain Res. 1208, 217–224 (2008). [DOI] [PubMed] [Google Scholar]
  50. Inokuchi Y. et al. Effect of an inducer of BiP, a molecular chaperone, on endoplasmic reticulum (ER) stress-induced retinal cell death. Invest Ophthalmol. Vis. Sci. 50, 334–344 (2009). [DOI] [PubMed] [Google Scholar]
  51. Hetz C., Chevet E. & Harding H. P. Targeting the unfolded protein response in disease. Nat. Rev. Drug Discov. 12, 703–719 (2013). [DOI] [PubMed] [Google Scholar]
  52. Castilla J. et al. Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions. Cell 134, 757–768 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Abid K., Morales R. & Soto C. Cellular factors implicated in prion replication. FEBS Lett. 584, 2409–2414 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Urayama A. et al. Initial fate of prions upon peripheral infection: half-life, distribution, clearance, and tissue uptake. FASEB J. 25, 2792–2803 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Morales R. et al. Lack of prion transmission by sexual or parental routes in experimentally infected hamsters. Prion. 7, 412–419 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Jozefczuk J., Drews K. & Adjaye J. Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells. J. Vis. Exp. (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figures
srep44723-s1.pdf (169.3KB, pdf)

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group

RESOURCES