Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Jan 4;105(2):757–762. doi: 10.1073/pnas.0711094105

Unfolded protein response transcription factor XBP-1 does not influence prion replication or pathogenesis

Claudio Hetz *,†,‡, Ann-Hwee Lee *, Dennisse Gonzalez-Romero §, Peter Thielen *, Joaquín Castilla §, Claudio Soto §, Laurie H Glimcher *,¶,‡
PMCID: PMC2206609  PMID: 18178615

Abstract

The unfolded protein response (UPR) is a conserved adaptive reaction that increases cell survival under endoplasmic reticulum (ER) stress conditions. X-box-binding protein-1 (XBP-1) is a key transcriptional regulator of the UPR that activates genes involved in protein folding, secretion, and degradation to restore ER function. The occurrence of chronic ER stress has been extensively described in neurodegenerative conditions linked to protein misfolding and aggregation. However, the role of the UPR in the CNS has not been addressed directly. Here we describe the generation of a brain-specific XBP-1 conditional KO strain (XBP-1Nes−/−). XBP-1Nes−/− mice are viable and do not develop any spontaneous neurological dysfunction, although ER stress signaling in XBP-1Nes−/− primary neuronal cell cultures was impaired. To assess the function of XBP-1 in pathological conditions involving protein misfolding and ER stress, we infected XBP-1Nes−/− mice with murine prions. To our surprise, the activation of stress responses triggered by prion replication was not influenced by XBP-1 deficiency. Neither prion aggregation, neuronal loss, nor animal survival was affected. Hence, this most highly conserved arm of the UPR may not contribute to the occurrence or pathology of neurodegenerative conditions associated with prion protein misfolding despite predictions that such diseases are related to ER stress and irreversible neuronal damage.

Keywords: ER stress, neurodegenerative disease, prion infection, protein misfolding disorders, UPR

The endoplasmic reticulum (ER) can be thought of as a sophisticated machine for protein folding and secretion that employs an efficient system of chaperones to promote folding and to prevent abnormal aggregation or misfolding of proteins (1). The ER is responsible for executing many posttranslational modifications; it is the major calcium store and the site of cholesterol and lipid biosynthesis. A number of stress conditions can interfere with the function of this organelle, leading to abnormal protein folding in the ER lumen, resulting in a cellular condition referred to as ER stress (see reviews in refs. 1 and 2). To alleviate ER stress, cells activate an intracellular-signaling pathway known as the unfolded protein response (UPR), which aims to reestablish homeostasis by transmitting information to the nucleus about the protein-folding status in the ER lumen, triggering adaptive responses. In higher eukaryotes, ER stress stimulates three distinct UPR-signaling pathways through sensors that include inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α), protein kinase-like ER kinase (PERK), and activation of transcription factor 6 (ATF6). IRE1α is a serine-threonine protein kinase and endoribonuclease that, upon activation, initiates the unconventional splicing of the mRNA encoding X-box-binding protein-1 (XBP-1) (3–5). Spliced XBP-1 is a potent transcriptional activator that increases the expression of a subset of UPR-related genes (6, 7).

The first insights about the function of XBP-1 in the nervous system came from genetic studies of human patients affected with bipolar disorders (8, 9). A polymorphism in the XBP-1 promoter was shown to be a risk factor for bipolar disorder, sensitivity to lithium (10), the occurrence of particular personality types in females (11), and schizophrenia in the Japanese population (12). Developmental studies in Xenopus demonstrated that XBP-1 is a negative regulator of neuronal tissue formation/differentiation during early morphogenesis (13). Expression of XBP-1 in Caenorhabditis elegans is increased during neuronal development and is a crucial factor for the assembly and secretion of the glutamate receptor (14). Similarly, in Drosophila, XBP-1 mediates the retinal neuronal degeneration present in models of autosomal-dominant retinitis pigmentosa (15). Accumulating evidence suggests that ER stress is particularly relevant to a variety of neurological disorders involving the misfolding and deposition of abnormal protein aggregates in the brain. Engagement of the IRE1α–XBP-1 pathway occurs in animal models of brain ischemia injury (16, 17), brain trauma (18), behavioral stress (19), retinal cell death (20), spinal cord injury (21) and trauma (22), and brain viral infections (23). Moreover, the accumulation of mutant protein aggregates in cellular and animal models of Huntington's disease (24), Parkinson's disease (25), and ALS (26, 27) correlates with IRE1α activation, suggesting that the UPR is a primary response against neurodegeneration.

Prion diseases (PrDs) are classic examples of diseases involving protein misfolding. PrDs are fatal neurodegenerative diseases characterized by spongiform degeneration of the brain, accompanied by extensive neuronal loss (28) and accumulation of a misfolded and protease-resistant form of the normal prion protein (PrPC), PrPres (29). The conversion of PrPC into PrPres involves a conformational change in which the α-helical content of the protein is reduced while the β-sheet content is increased (29). It is proposed that prions replicate in a cyclic manner by interaction of the incoming PrPres with the host PrPC, leading to the transformation of the normal protein into the pathological form (30). We and others have previously shown an engagement of ER stress responses in PrD models, where the up-regulation of UPR-responsive chaperones, such as Grp78/BiP, Grp94, and Grp58, is observed in the brains of patients affected with Creutzfeldt–Jacob disease (31, 32) and in mouse models of scrapie (31, 33, 34). In addition, recent evidence indicates that the activation of IRE1α and XBP-1 determines the rate of PrPC aggregation in vitro under stress conditions (35, 36) and in yeast models (37), suggesting that the UPR has an active role in preventing neurodegeneration. Despite these strong correlations between UPR activation and protein aggregation in PrDs, the role of this pathway in the disease process has not been addressed directly and remains speculative.

XBP-1 deficiency leads to embryonic lethality due to impairment of liver function (38). To circumvent the lethal liver phenotype of XBP-1−/− mice, we targeted an XBP-1 transgene back to liver by using a liver-specific promoter (39). However, these mice died shortly after birth from severe developmental abnormalities and dysfunction of the exocrine organs, preventing the study of XBP-1 function in the CNS. To bypass this lethality, here we generated a conditional KO allele of XBP-1. XBP-1flox/flox mice were crossed with mice expressing Cre recombinase under control of the Nestin promoter (referred to as XBP-1Nes−/−) to achieve deletion of XBP-1 in the CNS. To our surprise, XBP-1Nes−/− mice developed normally and did not show qualitative signs of spontaneous neurological impairment. To test the susceptibility of XBP-1Nes−/− mice to perturbation of ER function associated with neurodegeneration, we infected XBP-1Nes−/− mice with scrapie prions (hereafter referred to as “prion”). The extent of prion replication, neuronal loss, up-regulation of apoptosis markers, and animal survival in XBP-1Nes−/− mice was indistinguishable from that of control mice. Our results indicate that ablation of the xbp-1 gene does not drastically affect neuronal function and prion pathogenesis in vivo, suggesting that this arm of the UPR is not sufficient to confer protection against neurodegeneration.

Results

Generation of a Brain-Specific XBP-1-Deficient Mouse.

Here we describe the generation of a XBP-1 conditionally deficient strain specific for the CNS (Fig. 1A). XBP-1flox/flox mice were crossed with mice expressing Cre recombinase under the control of the Nestin promoter to achieve deletion of xbp-1 in the CNS (XBP-1Nes−/−). XBP-1Nes−/− animals were viable and were born in a Mendelian ratio. To determine the efficiency of XBP-1 deletion in the CNS, we performed Southern blotting of genomic DNA samples obtained from various brain regions and the spinal cord; >95% deletion of the xbp-1-floxed allele was observed in all samples analyzed, indicating a successful targeting strategy (Fig. 1B). No spontaneous neurological dysfunctions were observed in XBP-1Nes−/− mice, as measured by the occurrence of abnormal limb clasping, tremors, wobbly gait, ruffled fur, hunched posture, reduced mobility, loss of muscle strength, changes in body weight, or signs of paralysis [supporting information (SI) Fig. 5 and data not shown]. As a positive control for all of these parameters, we followed disease progression in a mouse model of ALS, the SOD1G86R-transgenic strain. Animals were followed for >12 months.

Fig. 1.

Fig. 1.

Open in a new tab

Generation of XBP-1Nes−/− mice. (A) Schematic representation of xbp-1 gene-targeting strategy. F, frontal; P, posterior. (B) Southern blot analysis of genomic DNA from different brain regions and spinal cord to address the efficiency of xbp-1 deletion. (C) In vitro differentiation of primary cortical neurons from E16.5 mouse embryos transduced with lentiviral vectors to express EGFP to visualize the projection of axons and dendrites. (D) Primary neurons were treated with 5 μg/ml Tm for the indicated time points, and the levels of XBP-1, ATF4, and CHOP were analyzed by Western blot in total protein extracts. The asterisk indicates a nonspecific band. (E) XBP-1 mRNA splicing in primary cortical neurons treated with the indicated concentrations of Tm for 4 h. (F) Quantification of mRNA levels of UPR target genes in primary cortical neurons undergoing ER stress. Primary cortical neurons were treated with 5 μg/ml Tm for 8 h, and mRNA levels of grp58, pdi, wfs-1, herp, chop, and bip were analyzed in total cDNA by real-time PCR.

ER Stress Responses of XBP-1-Deficient Primary Cortical Neurons.

To define the functional effects of XBP-1 deletion in neurons, we prepared primary neuronal cultures from embryonic day 16.5 (E16.5) embryos of control and XBP-1Nes−/− mice. To monitor morphological changes associated with differentiation, primary cultures were transduced with EGFP-expressing lentiviruses (Fig. 1C). Axonal outgrowth was qualitatively normal in XBP-1Nes−/− neuronal cultures, compared with control cultures. In addition, the levels of neuronal differentiation markers, such as Gap43, Cdkn1b, CyclinD1, Map2, and Tau, were not significantly altered in XBP-1Nes−/− primary neurons or mouse brain embryos (SI Fig. 6 A and B). We next examined levels of XBP-1 protein in primary cortical neurons undergoing ER stress. Treatment of cells with tunicamycin (Tm) triggered XBP-1 mRNA splicing in both control and XBP-1Nes−/− neurons, where the truncated–deleted mRNA contains the splicing site, allowing us to monitor IRE1α activity in the absence of XBP-1 expression (Fig. 1E). Quantitative RT-PCR analysis revealed that a complete deletion of the floxed allele was achieved in XBP-1Nes−/− primary neuronal cultures (SI Fig. 6C). Consistent with this result, no XBP-1 protein was detected in XBP-1Nes−/− neurons treated with Tm (Fig. 1D), but the expression of XBP-1-independent UPR genes, such as ATF4 and CHOP, was normally up-regulated in XBP-1-deficient neurons (Fig. 1E).

XBP-1 target genes have been previously defined in murine embryonic fibroblasts (MEFs) (6) and B cells (7), and they constitute a subset of UPR-related genes. Some defined XBP-1 target genes in MEFs include erdj4, edem, and sec61, which are involved in folding, ER-associated degradation (ERAD), and protein translocation into the ER, respectively. However, recent evidence suggests that the universe of XBP-1-regulated genes contains both unique and overlapping sets depending on the cell type analyzed (40, 41). Thus, we compared the expression levels of different UPR-related genes in control and XBP-1Nes−/− primary neurons. To our surprise, treatment of primary cortical neurons with Tm did not induce significant changes in the mRNA levels of ERdj4, EDEM, or Sec61, compared with XBP-1−/− MEFs (SI Fig. 7). However, the induction of other UPR genes, such as herp, pdi, and grp58, was significantly reduced in XBP-1Nes−/− neurons, demonstrating that the subset of target genes regulated by XBP-1 in neurons may differ from its targets in other cell types (Fig. 1F). In addition, we corroborated the dependency of the Wolfram Syndrome gene WSF-1 induction on XBP-1 expression (40). The expression of XBP-1-independent UPR genes, such as CHOP and BiP, was not altered in XBP-1Nes−/− neurons (Fig. 1F), suggesting that ER function and integrity were not drastically affected in this cell type by the absence of XBP-1, compared with the phenotype described in other tissues, such as liver (38), pancreas, and salivary gland (39).

Stress Signaling in XBP-1Nes−/− Mice Infected with Prions.

As mentioned, the activation of the IRE1α–XBP-1 pathway is observed in several animal models of neurodegeneration, as well as in brain damage due to ischemia, trauma, and viral infections. Thus, we decided to address the possible role of XBP-1 in neuropathological conditions triggered by protein misfolding and aggregation.

We and others previously described a strong engagement of ER stress responses in PrD mouse models (28). PrD, also known as transmissible spongiform encephalopathies, are a group of diseases that affect humans and animals characterized by neurological dysfunction, including dementia, ataxia, and psychological disturbances (29). In infectious forms of the disease, such as scrapie, the formation of PrPres from wild-type PrPC is initiated by the exposure to exogenous infectious agents, promoting a conformational change that results in the accumulation of PrPres. Although the nature of the infectious agent has not been completely elucidated, PrPres seems to be the main constituent (30, 42). In agreement with previous findings, we were able to detect a significant induction of XBP-1 mRNA splicing in the brain of symptomatic animals infected with three different murine prion strains (Fig. 2A). Based on this result, we analyzed the susceptibility of XBP-1Nes−/− mice to experimental PrD. Infection of mice with 139A prions triggered the up-regulation of the UPR-responsive genes, Grp58 and PDI, but not Hsp90 (Fig. 2B). Similarly, prominent phosphorylation of the stress kinases, JNK and ERK, was observed, events that have been shown to be mediated by IRE1α under ER stress conditions (43–45). To our surprise, none of these five stress markers were significantly affected in XBP-1Nes−/− prion-infected mice (Fig. 2B), suggesting that XBP-1 is dispensable for this reaction. Our results indicate that the stress responses triggered by prion replication are not enhanced in the absence of XBP-1, which could be expected if a higher basal stress or ER malfunction occurs.

Fig. 2.

Fig. 2.

Open in a new tab

Stress responses in prion-infected XBP-1Nes−/− mice. (A) XBP-1 mRNA splicing was performed in cDNA obtained from the brain samples of control animals or mice infected with the murine scrapie prion strains M7, 139A, and RML. Spliced and nonspliced XBP-1 PCR fragments are indicated. As a positive control, mRNA from tunicamycin (Tm)-treated NSC34 cells is shown. (B) The expression levels of Grp58, PDI, and Hsp90 and the phosphorylation of JNK and ERK were analyzed by Western blot in brain extracts from control and XBP-1Nes−/− mice not infected or infected with 139A prions at the late stage of the disease. As a control, total JNK and ERK levels are shown. Two and three control and prion-infected animals are shown to depict experimental reproducibility. (C) The expression levels of phospho-eIF2α, total eIF2α, ATF4, and CHOP were analyzed by Western blot in brain extracts from samples described in B. As a positive control, protein extracts from NSC34 cells were treated with Tm for 6 h or left untreated (NT). A longer exposure for CHOP Western blot is shown. (D) In parallel, the levels of processed ATF6 were measured by Western blot. As a positive control, liver extracts from mice injected with Tm for 12 h (Tm) or left untreated (NT) are shown. The asterisk indicates a nonspecific band.

We also analyzed the activation of UPR events that are IRE1α-independent. As previously described (33, 46), we found that PERK-downstream targets, such as eIF2α phosphorylation or the up-regulation of ATF4 and CHOP, were not induced by prion infection (Fig. 2C). Only a slight up-regulation of CHOP and ATF4 was observed in XBP-1Nes−/− prion-infected mice (Fig. 2C), implying minor compensatory effects of the PERK pathway in the absence of XBP-1. Similarly, no ATF6 processing was observed in prion-infected brains at the late stage of the disease (Fig. 2D). Thus, this PrD model offers a unique condition where XBP-1 splicing is preferentially activated similarly to the phenotype described in XBP-1-deficient B lymphocytes, when immunoglobulin secretion was stimulated with LPS or other stimuli (47, 48).

Neuronal apoptosis is a characteristic feature of PrDs. Thus, we quantified the levels of several proapoptotic genes in the brains of prion-infected animals. We observed a significant up-regulation of the proapoptotic BCL-2 family members BIM and PUMA in addition to pro-caspase-12 processing (Fig. 3A). These events have been linked to ER stress-mediated apoptosis (49–51), and they occurred when spongiform degeneration of the brain or inflammatory events (Fig. 3B and SI Fig. 8) were present, as revealed by histological analysis. In agreement with the lack of effect on ER stress signaling, apoptosis-related features were not altered in XBP-1Nes−/− prion-infected mice.

Fig. 3.

Fig. 3.

Open in a new tab

Neuronal loss in prion-infected XBP-1Nes−/− mice. (A) The expression levels of BIM, PUMA, and pro-caspase-12 processing were analyzed in brain extracts from control (XBP-1WT) and XBP-1Nes−/− mice not infected or infected with 139A prions at the late stage of the disease. (B) Spongiform degeneration of the brain was analyzed in parallel after H&E staining in different brain regions.

XBP-1 Deletion Does Not Affect Prion Pathogenesis.

Prion pathogenesis is mediated by the structural replication of PrPres, leading to the progressive accumulation of PrP aggregates in the brain. Based on the known role of XBP-1 in fomenting protein folding and protein quality control, we determined the extent of PrP misfolding in XBP-1Nes−/− prion-infected mice. As shown in Fig. 4 A and B, neither the appearance of detergent-insoluble PrP oligomers nor the accumulation of PrP in the brain of symptomatic animals was affected by XBP-1 deletion. We then quantified the degree of protease-resistant PrP in this disease model, a hallmark of PrD. No differences were observed in the sensitivity of PrP to proteinase K (PK) treatment, even at high concentrations of PK (Fig. 4C), indicating that prion replication and misfolding were not altered in XBP-1Nes−/− mice. Finally, we determined the survival of XBP-1Nes−/− mice after 139A prion infection. In all, 23–28 animals per group were used in these experiments to provide confidence in the results of the survival curve (control, n = 28; XBP-1Nes−/−, n = 23). As shown in Fig. 4D, the survival curve of XBP-1Nes−/− mice was virtually indistinguishable from control mice, with a median survival of 159 and 157 days after birth, respectively (the P value is nonsignificant).

Fig. 4.

Fig. 4.

Open in a new tab

Prion pathogenesis in XBP-1Nes−/− mice. (A) Total levels of PrP were analyzed by Western blot in control and XBP-1Nes−/− mice infected with prions or left uninfected. Of note, accumulation of total PrPC and oligomeric PrP species is observed in prion-infected mice. (B) PrP deposition was assessed by histological analysis (PrP olig.). (C) (Upper) PrPres was determined in brain homogenates from prion infection after treatment with indicated concentrations of PK and Western blot analysis. (Lower) Quantification of five experiments as percentage of PrPres with 125 μg/ml PK. (D) Animal survival was followed after 139A prion infection of control (n = 28) and XBP-1Nes−/− (n = 23) mice.

Discussion

XBP-1 is an essential regulator of the UPR and constitutes the most conserved ER stress pathway in evolution (1). Characterization of IRE1α–XBP-1 UPR-responsive genes revealed that this pathway controls adaptive processes involving global changes of ER function, including ERAD, ER and Golgi biogenesis, protein folding, and translocation into the ER (6). For example, the ectopic expression of XBP-1 in vitro induced the expression of multiple genes related to the secretory pathway, increased cell size, expanded the ER, and elevated total protein synthesis (7). Thus, XBP-1 enforces changes in cellular structure and function consistent with the requirements of professional secretory cells.

Attempts to define the role of XBP-1 in vivo resulted in striking phenotypes in specialized secretory organs where XBP-1 was ablated. Initial studies in RAG1−/− mice whose lymphoid system was reconstituted with XBP-1−/− stem cells showed profound defects in Ig secretion, explained by the failure of B cells to differentiate to plasma cells (47, 52, 53). To circumvent the lethal liver phenotype of XBP-1−/− mice (38), we targeted an XBP-1 transgene back to liver by using a liver-specific promoter (39). This rescued mouse died shortly after birth from a severe impairment in the production of pancreatic digestive enzymes, leading to hypoglycemia and death (39). XBP-1 deficiency resulted in severe disorganization of the ER structure in pancreatic exocrine secretory cells and salivary gland acinar cells. Thus, XBP-1 is essential for the development and function of highly secretory cells.

The exact role of XBP-1 in the CNS is unknown. A genetic link is observed between an XBP-1 promoter polymorphism and the occurrence of bipolar disorders (8), schizophrenia (54), and certain personality types in the Japanese population (55). Models of behavioral stress, such as inescapable electric foot shock, trigger XBP-1 mRNA splicing in the hippocampus (19), suggesting a connection with neuronal function. Extensive studies indicate a strong association between accumulation of protein aggregates and ER stress induction in several important neurodegenerative conditions, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, PrD, and many others (28, 56). Direct evidence indicating that perturbation of ER function could result in neurodegeneration came from the characterization of the Woozy mutant mice, where disruption of a BiP cochaperone triggers neuronal dysfunction associated with spontaneous protein aggregation in the brain (57). However, most of the evidence supporting the involvement of ER stress in neurodegeneration is correlative, and manipulation of the UPR in vivo was required to define the actual contribution of the pathway to the disease process.

Here we describe the generation of a brain-specific XBP-1-deficient mouse model. To our surprise, XBP-1 deficiency did not result in spontaneous neurological dysfunction, as measured by the absence of signs of trembling, paralysis, body weight loss, breeding abnormalities, spontaneous death, muscle strength, or motor performance. We hypothesized, however, that the IRE1α–XBP-1 pathway might contribute to conditions of chronic stress, rather than basal neuronal function. Thus, we challenged XBP-1Nes−/− mice with murine prions. This disease model evokes several central features of diverse neurodegenerative diseases, such as the accumulation of misfolded protein aggregates, neuronal loss, and progressive appearance of neurological-disease signs leading to death of the animal. More importantly, disease progression in prion-infected mice correlates with an ER stress response involving the up-regulation of ER chaperones and other UPR-related genes (28). Prion infection led to XBP-1 mRNA splicing and the up-regulation of ER chaperones, such as PDI and Grp58; phosphorylation of the stress kinases, JNK and ERK; and the induction of proapoptotic genes PUMA, BIM, and caspase-12 processing. Unexpectedly, infection of XBP-1Nes−/− with prions did not affect the appearance of any of these ER stress markers, nor did it affect prion misfolding, replication, or animal survival. Similarly, we recently described that genetic manipulation of the apoptosis program does not affect prion pathogenesis (58). Based on the known role of XBP-1 as a survival factor against ER stress in other experimental systems, we predicted an enhancement of PrD pathogenesis in the absence of XBP-1 expression in the CNS. Our results suggest that the occurrence of ER stress in neurological disorders may be an epiphenomenon associated with irreversible cellular damage and not causative of the disease. Alternatively, one may speculate that the up-regulation of ER chaperones is due to specific regulation of their promoters and not UPR activation. In mammals, the UPR is not restricted to the IRE1α–XBP-1 pathway, and activation of other UPR pathways may compensate XBP-1 deficiency in our PrD model. However, our data suggest that this may not be the case because we did not observe any significant alteration in the activation of ATF6 or the PERK pathway, as measured by processing of ATF6, eIF2α phosphorylation, or the expression of ATF4 and CHOP at late stages of the disease. The possible effects of ATF6 and PERK on prion pathogenesis in vivo by using genetic manipulation remain to be determined.

Our data suggest that XBP-1 may be dispensable for certain functions of the CNS under basal and pathological conditions. XBP-1 expression is relevant to ERAD, a pathway that mediates the degradation of abnormally folded proteins spontaneously generated during the protein-folding process at the ER. Disruption of other protein-clearance pathways in the CNS, such as autophagy, results in spontaneous neurodegeneration due to the accumulation of ubiquitinated intracellular protein aggregates, leading to neuronal apoptosis, motor dysfunction, and early animal death. Spontaneous disease was not observed in XBP-1Nes−/− mice, suggesting that ERAD may not be drastically altered in this model. In summary, our study presents the unexpected finding that XBP-1, the most conserved arm of the UPR-signaling pathway, does not influence the occurrence of a neurodegenerative condition associated with PrP misfolding and aggregation despite predictions that such diseases are related to ER stress and irreversible neuronal damage.

Materials and Methods

Generation of Brain-Specific XBP-1 Conditional KO Mice.

We designed the targeting vector so that exon 2 is flanked by two loxP sites (see SI Materials and Methods). XBP-1flox/flox mice were crossed with mice expressing Cre recombinase under the control of the Nestin promoter (NesCre/+) to achieve deletion of XBP-1 in the CNS (XBP-1Nes−/−). To determine the efficiency of XBP-1 deletion in the CNS, genomic DNA was isolated from different brain regions and the spinal cord and was analyzed by Southern blotting. Near total deletion of xbp-1 in this tissue was observed (Fig. 1B).

Analysis of Neurological Functions.

Several assays were used to monitor XBP-1Nes−/− performance, including rotarod, grip strength, and the inverted grill. Detailed methods are described in SI Materials and Methods.

In Vivo Scrapie Prion Infection.

Animals were injected stereotaxically in the right hippocampus with 1 μl of RML-infected brain homogenate as previously described (30). Brains and other tissues were extracted and analyzed histologically.

SDS/PAGE, Western Blot, and PrP Analysis.

Cell lysates or tissue homogenates were prepared in RIPA buffer [0.15 mM NaCl.,05 mM Tris·HCl (pH 7.2), 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS] as previously described (33). Western blotting was performed as previously described (33). The profile of PK sensitivity for in vivo-generated PrPres was studied by subjecting samples to incubation for 60 min at 45°C with different concentrations of PK ranging from 0 to 1,500 μg/ml. The digestion was stopped by adding an electrophoresis sample buffer. Complete methodological details are described in SI Materials and Methods.

Supplementary Material

Supporting Information
pnas_0711094105_index.html (13.5KB, html)

ACKNOWLEDGMENTS.

We thank Dr. Fabio Martinon for helpful discussion and Jennifer Donovan for technical assistance. This work was supported by the V. Harold and Leila Y. Mathers Charitable Foundation (L.H.G.), Fondo Nacional de Desarrollo Científico y Tecnológico Grant 1070444, Fondo de Financiamiento de Centros de Excelencia en Investigación Grant 15010006, the High Q Foundation (C.H.), and National Institutes of Health Grant NS050349 (to C.S.).

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0711094105/DC1.

References

  • 1.Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 2007;8:519–529. doi: 10.1038/nrm2199. [DOI] [PubMed] [Google Scholar]
  • 2.Schroder M, Kaufman R-J. The mammalian unfolded protein response. Annu Rev Biochem. 2005;74:739–789. doi: 10.1146/annurev.biochem.73.011303.074134. [DOI] [PubMed] [Google Scholar]
  • 3.Calfon M, et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature. 2002;415:92–96. doi: 10.1038/415092a. [DOI] [PubMed] [Google Scholar]
  • 4.Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell. 2001;107:881–891. doi: 10.1016/s0092-8674(01)00611-0. [DOI] [PubMed] [Google Scholar]
  • 5.Lee K, et al. IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response. Genes Dev. 2002;16:452–466. doi: 10.1101/gad.964702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.LeeA H, Iwakoshi N-N, Glimcher L-H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol. 2003;23:7448–7459. doi: 10.1128/MCB.23.21.7448-7459.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Shaffer A-L, et al. XBP1, downstream of Blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation. Immunity. 2004;21:81–93. doi: 10.1016/j.immuni.2004.06.010. [DOI] [PubMed] [Google Scholar]
  • 8.Kakiuchi C, et al. Impaired feedback regulation of XBP1 as a genetic risk factor for bipolar disorder. Nat Genet. 2003;35:171–175. doi: 10.1038/ng1235. [DOI] [PubMed] [Google Scholar]
  • 9.Kato T, Kuratomi G, Kato N. Genetics of bipolar disorder. Drugs Today (Barcelona) 2005;41:335–344. doi: 10.1358/dot.2005.41.5.893616. [DOI] [PubMed] [Google Scholar]
  • 10.Kakiuchi C, Kato T. Lithium response and −116C/G polymorphism of XBP1 in Japanese patients with bipolar disorder. Int J Neuropsychopharmacol. 2005;8:631–632. doi: 10.1017/S146114570500533X. [DOI] [PubMed] [Google Scholar]
  • 11.Kato C, et al. XBP1 gene polymorphism (-116C/G) and personality. Am J Med Genet B Neuropsychiatr Genet. 2005;136:103–105. doi: 10.1002/ajmg.b.30098. [DOI] [PubMed] [Google Scholar]
  • 12.Kakiuchi C, et al. Association of the XBP1–116C/G polymorphism with schizophrenia in the Japanese population. Psychiatry Clin Neurosci. 2004;58:438–440. doi: 10.1111/j.1440-1819.2004.01280.x. [DOI] [PubMed] [Google Scholar]
  • 13.Cao Y, et al. XBP1 forms a regulatory loop with BMP-4 and suppresses mesodermal and neural differentiation in Xenopus embryos. Mech Dev. 2006;123:84–96. doi: 10.1016/j.mod.2005.09.003. [DOI] [PubMed] [Google Scholar]
  • 14.Shim J, Umemura T, Nothstein E, Rongo C. The unfolded protein response regulates glutamate receptor export from the endoplasmic reticulum. Mol Biol Cell. 2004;15:4818–4828. doi: 10.1091/mbc.E04-02-0108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ryoo H-D, Domingos P-M, Kang M-J, Steller H. Unfolded protein response in a Drosophila model for retinal degeneration. EMBO J. 2007;26:242–252. doi: 10.1038/sj.emboj.7601477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Paschen W, Aufenberg C, Hotop S, Mengesdorf T. Transient cerebral ischemia activates processing of xbp1 messenger RNA indicative of endoplasmic reticulum stress. J Cereb Blood Flow Metab. 2003;23:449–461. doi: 10.1097/01.WCB.0000054216.21675.AC. [DOI] [PubMed] [Google Scholar]
  • 17.Morimoto N, et al. Involvement of endoplasmic reticulum stress after middle cerebral artery occlusion in mice. Neuroscience. 2007;147:957–967. doi: 10.1016/j.neuroscience.2007.04.017. [DOI] [PubMed] [Google Scholar]
  • 18.Paschen W, Yatsiv I, Shoham S, Shohami E. Brain trauma induces X-box protein 1 processing indicative of activation of the endoplasmic reticulum unfolded protein response. J Neurochem. 2004;88:983–992. doi: 10.1046/j.1471-4159.2003.02218.x. [DOI] [PubMed] [Google Scholar]
  • 19.Toda H, et al. Behavioral stress and activated serotonergic neurotransmission induce XBP-1 splicing in the rat brain. Brain Res. 2006;1112:26–32. doi: 10.1016/j.brainres.2006.07.008. [DOI] [PubMed] [Google Scholar]
  • 20.Shimazawa M, et al. Involvement of ER stress in retinal cell death. Mol Vis. 2007;13:578–587. [PMC free article] [PubMed] [Google Scholar]
  • 21.Penas C, et al. Spinal cord injury induces endoplasmic reticulum stress with different cell-type dependent response. J Neurochem. 2007;102:1242–1255. doi: 10.1111/j.1471-4159.2007.04671.x. [DOI] [PubMed] [Google Scholar]
  • 22.Aufenberg C, Wenkel S, Mautes A, Paschen W. Short communication: Spinal cord trauma activates processing of xbp1 mRNA indicative of endoplasmic reticulum dysfunction. J Neurotrauma. 2005;22:1018–1024. doi: 10.1089/neu.2005.22.1018. [DOI] [PubMed] [Google Scholar]
  • 23.Williams BL, Lipkin WI. Endoplasmic reticulum stress and neurodegeneration in rats neonatally infected with borna disease virus. J Virol. 2006;80:8613–8626. doi: 10.1128/JVI.00836-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Nishitoh H, et al. ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev. 2002;16:1345–1355. doi: 10.1101/gad.992302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Holtz W-A, O'Malley K-L. Parkinsonian mimetics induce aspects of unfolded protein response in death of dopaminergic neurons. J Biol Chem. 2003;278:19367–19377. doi: 10.1074/jbc.M211821200. [DOI] [PubMed] [Google Scholar]
  • 26.Atkin J-D, et al. Induction of the unfolded protein response in familial amyotrophic lateral sclerosis and association of protein-disulfide isomerase with superoxide dismutase 1. J Biol Chem. 2006;281:30152–30165. doi: 10.1074/jbc.M603393200. [DOI] [PubMed] [Google Scholar]
  • 27.Kikuchi H, et al. Spinal cord endoplasmic reticulum stress associated with a microsomal accumulation of mutant superoxide dismutase-1 in an ALS model. Proc Natl Acad Sci USA. 2006;103:6025–6030. doi: 10.1073/pnas.0509227103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Hetz C, Soto C. Stressing out the ER: A role of the unfolded protein response in prion-related disorders. Curr Mol Med. 2006;6:37–43. doi: 10.2174/156652406775574578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Prusiner S-B. Prions. Proc Natl Acad Sci USA. 1998;95:13363–13383. doi: 10.1073/pnas.95.23.13363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Castilla J, Saa P, Hetz C, Soto C. In vitro generation of infectious scrapie prions. Cell. 2005;121:195–206. doi: 10.1016/j.cell.2005.02.011. [DOI] [PubMed] [Google Scholar]
  • 31.Hetz C, Russelakis-Carneiro M, Maundrell K, Castilla J, Soto C. Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein. EMBO J. 2003;22:5435–5445. doi: 10.1093/emboj/cdg537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Yoo B-C, et al. Overexpressed protein disulfide isomerase in brains of patients with sporadic Creutzfeldt-Jakob disease. Neurosci Lett. 2002;334:196–200. doi: 10.1016/s0304-3940(02)01071-6. [DOI] [PubMed] [Google Scholar]
  • 33.Hetz C, et al. The disulfide isomerase Grp58 is a protective factor against prion neurotoxicity. J Neurosci. 2005;25:2793–2802. doi: 10.1523/JNEUROSCI.4090-04.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Brown A-R, et al. Gene expression profiling of the preclinical scrapie-infected hippocampus. Biochem Biophys Res Commun. 2005;334:86–95. doi: 10.1016/j.bbrc.2005.06.060. [DOI] [PubMed] [Google Scholar]
  • 35.Hetz C, Castilla J, Soto C. Perturbation of endoplasmic reticulum homeostasis facilitates prion replication. J Biol Chem. 2007;282:12725–12733. doi: 10.1074/jbc.M611909200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Orsi A, Fioriti L, Chiesa R, Sitia R. Conditions of endoplasmic reticulum stress favor the accumulation of cytosolic prion protein. J Biol Chem. 2006;281:30431–30438. doi: 10.1074/jbc.M605320200. [DOI] [PubMed] [Google Scholar]
  • 37.Apodaca J, Kim I, Rao H. Cellular tolerance of prion protein PrP in yeast involves proteolysis and the unfolded protein response. Biochem Biophys Res Commun. 2006;347:319–326. doi: 10.1016/j.bbrc.2006.06.078. [DOI] [PubMed] [Google Scholar]
  • 38.Reimold A-M, et al. An essential role in liver development for transcription factor XBP-1. Genes Dev. 2000;14:152–157. [PMC free article] [PubMed] [Google Scholar]
  • 39.Lee A-H, Chu G-C, Iwakoshi N-N, Glimcher L-H. XBP-1 is required for biogenesis of cellular secretory machinery of exocrine glands. EMBO J. 2005;24:4368–4380. doi: 10.1038/sj.emboj.7600903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Kakiuchi C, Ishiwata M, Hayashi A, Kato T. XBP1 induces WFS1 through an endoplasmic reticulum stress response element-like motif in SH-SY5Y cells. J Neurochem. 2006;97:545–555. doi: 10.1111/j.1471-4159.2006.03772.x. [DOI] [PubMed] [Google Scholar]
  • 41.Carrasco D-R, et al. The differentiation and stress response factor XBP-1 drives multiple myeloma pathogenesis. Cancer Cell. 2007;11:349–360. doi: 10.1016/j.ccr.2007.02.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Legname G, et al. Synthetic mammalian prions. Science. 2004;305:673–676. doi: 10.1126/science.1100195. [DOI] [PubMed] [Google Scholar]
  • 43.Urano F, et al. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science. 2000;287:664–666. doi: 10.1126/science.287.5453.664. [DOI] [PubMed] [Google Scholar]
  • 44.Nguyen D-T, et al. Nck-dependent activation of extracellular signal-regulated kinase-1 and regulation of cell survival during endoplasmic reticulum stress. Mol Biol Cell. 2004;15:4248–4260. doi: 10.1091/mbc.E03-11-0851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Hetz C, et al. Proapoptotic BAX, BAK modulate the unfolded protein response by a direct interaction with IRE1alpha. Science. 2006;312:572–576. doi: 10.1126/science.1123480. [DOI] [PubMed] [Google Scholar]
  • 46.Unterberger U, et al. Endoplasmic reticulum stress features are prominent in Alzheimer disease but not in prion diseases in vivo. J Neuropathol Exp Neurol. 2006;65:348–357. doi: 10.1097/01.jnen.0000218445.30535.6f. [DOI] [PubMed] [Google Scholar]
  • 47.Iwakoshi N-N, et al. Plasma cell differentiation and the unfolded protein response intersect at the transcription factor XBP-1. Nat Immunol. 2003;4:321–329. doi: 10.1038/ni907. [DOI] [PubMed] [Google Scholar]
  • 48.Gass J-N, Jiang H-Y, Wek R-C, Brewer J-W. The unfolded protein response of B-lymphocytes: PERK-independent development of antibody-secreting cells. Mol Immunol. 2007;45:1035–1043. doi: 10.1016/j.molimm.2007.07.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Li J, Lee B, Lee A-S. Endoplasmic reticulum stress-induced apoptosis: Multiple pathways and activation of p53-up-regulated modulator of apoptosis (PUMA) and NOXA by p53. J Biol Chem. 2006;281:7260–7270. doi: 10.1074/jbc.M509868200. [DOI] [PubMed] [Google Scholar]
  • 50.Nakagawa T, et al. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature. 2000;403:98–103. doi: 10.1038/47513. [DOI] [PubMed] [Google Scholar]
  • 51.Puthalakath H, et al. ER stress triggers apoptosis by activating BH3-only protein bim. Cell. 2007;129:1337–1349. doi: 10.1016/j.cell.2007.04.027. [DOI] [PubMed] [Google Scholar]
  • 52.Reimold A-M, et al. Plasma cell differentiation requires the transcription factor XBP-1. Nature. 2001;412:300–307. doi: 10.1038/35085509. [DOI] [PubMed] [Google Scholar]
  • 53.Tirosh B, Iwakoshi N-N, Glimcher L-H, Ploegh H-L. XBP-1 specifically promotes IgM synthesis and secretion, but is dispensable for degradation of glycoproteins in primary B cells. J Exp Med. 2005;202:505–516. doi: 10.1084/jem.20050575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Chen W, et al. A case-control study provides evidence of association for a functional polymorphism −197C/G in XBP1 to schizophrenia and suggests a sex-dependent effect. Biochem Biophys Res Commun. 2004;319:866–870. doi: 10.1016/j.bbrc.2004.05.060. [DOI] [PubMed] [Google Scholar]
  • 55.Kusumi I, et al. Relationship between XBP1 genotype and personality traits assessed by TCI, NEO-FFI. Neurosci Lett. 2005;391:7–10. doi: 10.1016/j.neulet.2005.08.023. [DOI] [PubMed] [Google Scholar]
  • 56.Lindholm D, Wootz H, Korhonen L. ER stress and neurodegenerative diseases. Cell Death Differ. 2006;13:385–392. doi: 10.1038/sj.cdd.4401778. [DOI] [PubMed] [Google Scholar]
  • 57.Zhao L, Longo-Guess C, Harris B-S, Lee J-W, Ackerman S-L. Protein accumulation and neurodegeneration in the woozy mutant mouse is caused by disruption of SIL1, a cochaperone of BiP. Nat Genet. 2005;37:974–979. doi: 10.1038/ng1620. [DOI] [PubMed] [Google Scholar]
  • 58.Steele A-D, et al. Diminishing apoptosis by deletion of Bax or overexpression of Bcl-2 does not protect against infectious prion toxicity in vivo. J Neurosci. 2007;27:13022–13027. doi: 10.1523/JNEUROSCI.3290-07.2007. [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

Supporting Information
pnas_0711094105_index.html (13.5KB, html)
pnas_0711094105_1.pdf (23.2KB, pdf)
pnas_0711094105_2.pdf (23.9KB, pdf)
pnas_0711094105_3.pdf (16.7KB, pdf)
pnas_0711094105_4.pdf (1.2MB, pdf)

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES