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Keywords:

  • BiP;
  • cortex;
  • GRP78;
  • sleep;
  • unfolded protein response

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

Little is known about the molecular mechanisms underlying sleep. We show the induction of key regulatory proteins in a cellular protective pathway, the unfolded protein response (UPR), following 6 h of induced wakefulness. Using C57/B6 male mice maintained on a 12:12 light/dark cycle, we examined, in cerebral cortex, the effect of different durations of prolonged wakefulness (0, 3, 6, 9 and 12 h) from the beginning of the lights-on inactivity period, on the protein expression of BiP/GRP78, a chaperone and classical UPR marker. BiP/GRP78 expression is increased with increasing durations of sleep deprivation (6, 9 and 12 h). There is no change in BiP/GRP78 levels in handling control experiments carried out during the lights-off period. PERK, the transmembrane kinase responsible for attenuating protein synthesis, which is negatively regulated by binding to BiP/GRP78, is activated by dissociation from BiP/GRP78 and by autophosphorylation. There is phosphorylation of the elongation initiation factor 2α and alteration in ribosomal function. These changes are first observed after 6 h of induced wakefulness. Thus, prolonging wakefulness beyond a certain duration induces the UPR indicating a physiological limit to wakefulness.

Abbreviations used
BiP

immunoglobulin binding protein

ECL

enhanced chemiluminescence

eIF2α

elongation initiation factor 2α

ER

endoplasmic reticulum

GRP

glucose-regulated protein; NREM, non-rapid eye movement

PET

positron emission topography

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SELDI/MS

surface-enhanced laser desorption/ionization mass spectrometry

UPR

unfolded protein response.

A major question in the field of sleep research is what mechanism establishes the duration of wakefulness that can be sustained without consequences. Studies by Van Dongen and others (e.g. Van Dongen and Dinges 2003) indicate that in humans extended wakefulness beyond 16 h impairs cognition. It is thought that this effect is a result of progressive physiological, biochemical and molecular changes in the brain (Borbely and Tobler 1989). The mechanism(s) for this at a basic molecular level is unknown. Recent studies have shown that extending wakefulness leads to the up-regulation of the transcript of the endoplasmic reticulum (ER) resident protein immunoglobulin binding protein/glucose-regulated protein (BiP/GRP78) in different species, including cerebral cortex in mice (Cirelli and Tononi 2000; Shaw et al. 2000; Terao et al. 2003; Cirelli et al. 2004). Transcription of BiP/GRP78, a molecular chaperone, is a classical marker of the ER stress response/unfolded protein response (UPR) activation in yeast and mammalian cells (Lee 1992; Harding et al. 1999). Thus, it appears that extending wakefulness in the mouse leads to cellular stress and the UPR in mouse cerebral cortex. It is likely that the duration of wakefulness that mice can sustain is less than in humans, potentially explaining their very different sleep/wake patterns, as mice have much less consolidated sleep (Veasey et al. 2000).

The UPR affects the function of the ER, which is the site of secretory and integral membrane folding. The ER has evolved highly specific signaling pathways to ensure that its protein-folding capacity is not overwhelmed. These pathways, collectively termed the UPR, are required if the cell is to survive ER stress that can result from a perturbation in calcium homeostasis or redox status, elevated secretory protein synthesis, misfolded proteins, glucose deprivation or altered glycosylation (Kaufman 2002). The UPR helps restore normal ER function by up-regulating the expression of chaperones to increase the ER's capacity for protein folding, or to promote degradation of misfolded proteins through the process of ER-associated degradation. BiP/GRP78, the major molecular chaperone, binds exposed hydrophobic patches on the surfaces of unfolded proteins preventing the misfolded proteins from aggregating while also helping to properly fold the protein. In a parallel UPR process, translation is attenuated to decrease the protein-folding load. Attenuation of protein synthesis occurs through the activation of the transmembrane kinase PERK (Bertolotti et al. 2000). Because the UPR results in reduced translation of the majority of proteins, this will likely have a profound impact on neuronal function.

We investigated whether the ER stress response is indeed induced in the mouse cerebral cortex during prolonged wakefulness. We studied frontal and parietal cortex because our microarray study, as well that of Cirelli et al. (2004), focused on gene expression changes in the cerebral cortex. Furthermore, a PET study has shown that there is a decrease in glucose uptake in the frontal and parietal cortex following sleep deprivation (Thomas et al. 2000). We did the following: (i) measured the temporal protein expression profile of BiP/GRP78 with increasing durations of wakefulness (sleep deprivation); (ii) carried out BiP/GRP78–PERK interaction studies to see if they became dissociated, as occurs in the UPR; and (iii) measured whether PERK and the elongation initiation factor 2α (eIF2α) were phosphorylated. These proteins are both phosphorylated in the UPR. In addition, we performed polysome analyses to monitor ribosomal function to determine whether protein synthesis was suppressed.

Animal handling and tissue collection

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

Studies were performed on 10-week-old C57B/6J male mice maintained on a 12:12 light/dark cycle. Sleep deprivation was initiated at lights-on (07.00) and deprivation for 3, 6, 9 and 12 h was performed through gentle handling, which included directly observing the animal's motor activity and gently stroking the fur with an artist's brush when no activity was observed. At each time point we had eight control mice (to control for circadian variations), which were left undisturbed until killed, and eight sleep-deprived mice, and used multiple experimental replications. Also, a group of animals was killed at lights-on (07.00) giving us the 0 h time-point. The frontal and parietal cortex of each mouse was rapidly dissected out and flash frozen in liquid nitrogen and stored at −70°C until use. The brain tissue was homogenized, on ice, in a lysis buffer (20 mm Tris–HCl pH 7.5, 1 mm EGTA, 1 mm EDTA, 1% Triton X-100, 10% glycerol) in the presence of protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 2 µg/mL pepstatin and 4 µg/mL aprotinin). The lysate was centrifuged to remove cellular debris and protein was determined by the Pierce micro-BCA assay (Pierce, Rockford, IL, USA). All experiments were performed in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

Temporal expression of BiP/GRP78

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

Individual sleep-deprived and matching control mouse cortex homogenates were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels in triplicate. For each time-point there were eight sets of gels. We used 200 ng of commercial hamster-derived GRP78 standard (Stressgen, Victoria, Canada), in two lanes, on each gel both as a loading control and as an external standard. Following transfer onto nitrocellulose, BiP/GRP78 was detected using the GRP78 antibody from Stressgen. Samples (25 µg protein) representing individual mice were run on SDS–PAGE gels (Bio-Rad, Laboratories, Hercules, CA, USA; 10% Tris–HCl) according to Laemmli (1970), and then transferred to nitrocellulose membranes (Bio-Rad). Blots were incubated with rabbit polyclonal antibody against GRP78 (1 : 1000, Stressgen). After incubation with horseradish peroxidase-conjugated secondary antibody (antirabbit 1 : 3000, Sigma, St. Louis, MO, USA), protein bands were detected and analyzed by enhanced chemiluminescence (ECL; Pierce Supersignal) and quantitative imaging (Fluorochem 8900, Alpha Innotech Corp., San Leandro, CA, USA). All bands were normalized to the external GRP78 standard. Densitometry was performed using the alphaease fc software.

Immunoprecipitation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

One hundred microliters of cortex lysate was pre-incubated and pre-cleared with 10 µL of Protein G–Sepharose beads for 1 h at 4°C. Following centrifugation the supernatant was immunoprecipitated with 1 µL PERK antibody (Santa Cruz Biotechnology, Snata Cruz, CA, USA) overnight at 4°C, and bound to 30 µL Protein-G beads by incubation for 1 h at 4°C. The protein-bound beads were subsequently washed three times in lysis buffer. Bound protein was resolved by 7.5% SDS–PAGE under reducing conditions and transferred to nitrocellulose membranes. Blots were incubated with GRP78 antibody (1 : 1000); signals were observed using horseradish peroxidase-labeled secondary antibody and ECL (Pierce Supersignal). Protein was quantitated as described in the protocol for western analysis. Protein bands on gels were visualized using the Bio-Rad non-colloidal Coomassie Brilliant Blue stain.

Polysome analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

Cortex tissue from individual mice sleep deprived for 6 h and their matching controls was homogenized in 1 mL of polysome extraction buffer (15 mm Tris–HCl pH 7.4, 15 mm MgCl2, 0.3 m NaCl, 1% Triton X-100, 0.1 mg/mL cyclohexamide, 1 mm phenylmethylsulfonyl fluoride, 2 µg/mL pepstatin and 4 µg/mL aprotinin). The lysates were cleared by centrifugation at 12 000 g for 10 min, and then layered on top of a 4 mL 10–50% sucrose gradient prepared in extraction buffer. After spinning for 2 h at 40 000 r.p.m. in a Sorvall AH650 rotor, 200 µL fractions were collected and the absorbance at 254 nm was read.

Statistics

To test the effects of diurnal time and sleep deprivation on BiP protein expression a two-way analysis of variance (anova) was performed using proc.mixed (SAS, Cary, NC, USA) At each time point eight animals were killed and BiP protein expression was assessed by western blotting. Tukey–Kramer adjusted p-values for post-hoc comparisons in protein expression among experimental groups were then calculated.

Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

Microarray data from our group indicate that BiP/GRP78 transcript levels progressively increase with increasing periods of sleep deprivation (Mackiewicz et al. 2003). However, a recent in vitro study suggests that BiP/GRP78 protein expression is regulated on a translational level in unstressed cells and is relatively independent of transcript levels (Gulow et al. 2002). In order to ascertain whether the temporal increase in BiP/GRP78 transcript observed was due to the induction of the ER stress response, we determined the temporal expression profile of BiP/GRP78 protein. Changes in BiP/GRP78 protein expression were determined by western blot analysis using the GRP78 antibody from Stressgen and detected by ECL. Comparisons of the western blots indicate that BiP/GRP78 protein expression levels in cortex did increase progressively with increasing durations of wakefulness (Fig. 1). We assessed variations in BiP/GRP78 protein level using a two-way anova with one factor being experimental group (control, sleep deprived) and the other factor time point (3, 6, 9, 12 h). There was a significant main effect of group (F = 32.74; d.f. 1,56; p < 0.0001) and a significant group by time point interaction (p = 0.0355) indicating that the difference between sleep-deprived and control groups varied by time point. Post-hoc analyses showed significant differences between the two groups at 6, 9, 12 h (Tukey adjustment: p = 0.004, 0.010, 0.026, respectively), but not at 3 h. There are significant differences over time only in the sleep-deprived group (p = 0.040). When we compared data in each group separately with the BiP control level at time 0, we found significant changes from baseline only for the 9 and 12 h sleep-deprived groups (Dunnett adjustment, p = 0.031 and 0.003, respectively).

image

Figure 1. Temporal expression profile of BiP/GRP78 expression as determined by western blot analysis. Western blots were carried out on eight sleep-deprived and eight control mice at each time-point using the Stressgen GRP78 antibody. BiP/GRP78 expression was quantitated by densitometry of the western blots. All bands for sleep-deprived (SD) and control (Con) mice were normalized to 200 ng of external GRP78 standard (STD) from Stressgen. Data represent the means and standard deviation for all mice of an average of three replicates for eight mice in each group at each time point. Tukey-adjusted post-hoc tests indicate significant differences at 6 h (p = 0.004), 9 h (p = 0.010), and 12 h (p = 0.026), respectively, compared with controls.

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BiP/GRP78 is not up-regulated in handled control animals

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

In order to ascertain that our method of sleep deprivation, gentle handling, did not cause general ‘stress’ that led to the up-regulation of BiP/GRP78 in the experimental animals, we carried out a handling control study. We subjected a group of mice (n = 6) to gentle handling, as performed during the sleep-deprivation study, for 6 h from the onset of the lights-out active period. A second control group of six mice was left undisturbed until killed. Both groups of animals were killed at the end of the 6-h period and BiP/GRP78 protein levels in the cortex was determined by western blots as above. Analysis of the blots indicated no difference in BiP/GRP78 protein levels in handled and control animals indicating that gentle handling itself for 6 h does not induce ER stress (Fig. 2).

image

Figure 2. Gentle handling does not lead to an increase in BiP levels. Bar graph showing BiP protein expression levels unchanged in mice handled for 6 h (6 h HC) during their waking period (lights off) compared with un-handled spontaneously wake controls (6 h SW) during the lights-off period. BiP expression levels during the comparable lights-on period are shown for control sleeping (6 h SS) and sleep-deprived (6 h SD) animals. BiP levels quantified by densitometric analysis of western blots, normalized to BiP external standard, n = 6 for the lights off data and n = 8 for the lights on data. Data shown are mean and standard deviation.

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BiP/GRP78 and PERK are dissociated during sleep deprivation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

During the UPR, in parallel with the increase of chaperone function, is an attenuation of translation to decrease the protein-folding load. During ER stress, decrease in protein biosynthesis occurs when the eukaryotic translation initiation factor 2α is phosphorylated on Ser51 by PERK. PERK is an ER resident type I transmembrane protein, whose N-terminal lumen domain is sensitive to the upstream ER stress signal and whose C-terminal cytoplasmic domain directly phosphorylates eIF2α (Harding et al. 1999). PERK is maintained in an inactive state by the binding of the ER chaperone BiP/GRP78 to its lumenal domain. Under conditions of ER stress, BiP/GRP78 dissociates to service the increasing load of ER client proteins; loss of BiP/GRP78 binding correlates with oligomerization, trans-autophosphorylation, and activation of downstream signaling by PERK (Bertolotti et al. 2000). The association of BiP/GRP78 with PERK therefore indicates the state of the cell. We examined the effect of sleep deprivation on the degree to which BiP/GRP78 is associated with PERK. We used an antibody to PERK to co-immunoprecipitate BiP/GRP78 from lysates of 6 h sleep-deprived animals and controls. We focused our study on these additional UPR markers on the first time-point (6 h) that illustrated a significant difference in BiP/GRP78 expression between control and sleep-deprived animals. Furthermore, a study by Terao et al. (2003) indicates that there is differential expression of UPR genes BiP/GRP78 and ERp72 at 6 h of sleep deprivation. Moreover, in our control experiments to assess whether gentle handling itself led to ER stress, we did not find any increase in BiP/GRP78 protein with 6 h of gentle handling in the lights-off period (see above). SDS–PAGE gels stained with Coomassie Brilliant Blue indicated a protein of the same molecular mass as BiP/GRP78 (78 kDa) in both control and sleep-deprived samples; with much more of this protein in the control than in the sleep-deprived samples (Fig. 3a). Using an antibody to BiP/GRP78 for identification, we confirmed that the 78 kDa protein was indeed BiP/GRP78 and that twice as much BiP/GRP78 was associated with PERK in control animals than in sleep-deprived animals (Fig. 3b). Note that with 6 h of sleep deprivation BiP/GRP78 levels are always higher in sleep-deprived samples in straightforward western blots (Fig. 3c). Thus, with 6 h of extended wakefulness, only a small fraction of BiP/GRP78 remains complexed to PERK, and most of the BiP/GRP78 is free to function as a chaperone.

image

Figure 3. (a) BiP and PERK dissociate during sleep deprivation. SDS–PAGE gel of PERK immunoprecipitates of sleep-deprived (SD) and control (Con) samples at 6 h of sleep deprivation. Cortex lysate (100 µg protein) was immunoprecipitated with 1 µL of PERK antibody, bound to Protein–G beads, and resolved by SDS–PAGE. Coomassie Brilliant Blue staining of gel shows BiP in control lysate and not in SD lysate. (b) Immunoprecipitation with PERK antibody indicates more BiP associated with PERK in control (Con) mice than in mice sleep deprived (SD) for 6 h. Western blot shows 200 ng of BiP standard, two control immunoprecipitates (Con) and two sleep-deprived immunoprecipitates (SD) detected with BiP/GRP78 antibody. Bar graph shows quantification of BiP from western blots by densitometry. Data represent an average of 4 mice ± SD (p = 0.01). (c) Overall BiP/GRP78 expression is increased following 6 h of sleep deprivation as shown in straightforward western blots. Shown in the upper panel is a typical western blot of a 6 h SD study. There is a lane for the BiP standard, three control lanes, and three sleep deprived (SD). In the lower part, densitometry quantification of western blots of 6-h sleep-deprived mice (n = 8) (SD) versus control mice (n = 8) (Con) are shown. Experiments were performed in triplicate and average data ± SD are shown. BiP/GRP78 levels in mouse lysates were normalized to an external standard (200 ng purified mouse BiP/GRP78 from Stressgen) (STD). Figure shows increased BiP expression in sleep-deprived samples compared to control.

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PERK and eIF2α are phosphorylated during sleep deprivation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

PERK activation during ER stress correlates with phosphorylation of its cytoplasmic kinase domain. The detection of p-PERK serves as marker for its activation (Harding et al. 1999). We used the p-PERK antibody (Cell Signaling) to detect and quantitate the level of activated phosphorylated-PERK in animals subjected to 6 h of sleep deprivation and their controls. As shown in Fig. 4(a), there is significantly more phosphorylated-PERK, almost 300% more in samples from sleep-deprived mice than in controls (p = 0.001), serving as a further indicator of the UPR occurring in the cortex of these animals.

image

Figure 4. (a) PERK is phosphorylated during sleep deprivation. Animals sleep deprived for 6 h (SD) have more phosphorylated-PERK in cortex than control animals sacrificed at same time point (Con). (Upper) Gel shows p-PERK in control and sleep-deprived samples using the antibody to phospho-PERK; each lane represents a single animal (three control and three sleep deprived, SD). Bar graph below shows p-PERK levels in control and sleep-deprived animals as determined by densitometry of western blots. Data shown are mean ± SD for n = 3. There is substantially more phosphorylated-PERK in the sleep-deprived animals than controls (p = 0.001). (b) eIF2α is phosphorylated in sleep-deprived animals. Western blots showing staining to eIF2α-phospho antibody in a control and in an experimental sample at 6 h of sleep deprivation. Phosphorylated eIF2α is found only in the sleep-deprived sample.

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Activated PERK phosphorylates eIF2α on Ser51, a modification that increases the affinity of eIF2 for eIF2B, a GDP–GTP exchange factor that charges the eIF2-GTP-tRNA-met ternary complex (Kimball et al. 2001). Functioning as a competitive inhibitor of eIF2B, phospho-eIF2α reduces the concentration of the active ternary complex, prevents assembly of the 43S pre-initiation complex and attenuates protein translation. Our data comparing sleep-deprived and control samples at 6 h of sleep deprivation by western blots show that eIF2α is phosphorylated in sleep-deprived and not in control animals (Fig. 4B).

There are changes in the ribosomal profile during sleep deprivation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

Actively translating ribosomes exist as polysomes; during a shutdown or attenuation of translation a large fraction of the ribosomes is disassembled into monosomes (Doutheil et al. 1997; Harding et al. 2000; Gulow et al. 2002). We carried out polysome analyses to further assess whether protein synthesis was being inhibited after 6 h of sustained wakefulness. The microsomal fractions of sleep-deprived and control cortical lysates were separated by sucrose-gradient centrifugation to yield the ribosomal profiles. The ribosomal profile of samples subjected to 6 h of sleep deprivation shows a moderate increase in the monosome peak with a concomitant decrease in the polysome peak, when compared with controls, suggesting an attenuation of translation (Fig. 5).

image

Figure 5. Alteration of the ribosomal profile during sleep deprivation. Ribosomal profile of sample subjected to 6 h of sleep deprivation (6 h SD; broken line) compared with control sacrificed at same time point (6 h; solid line), n = 3. Monosome and polysome fractions are indicated. There is an increase in monosome fraction and decrease in polysome fraction in sleep-deprived compared with control. Similar results were obtained in three additional samples.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

These results indicate that, in mice, prolonged wakefulness over a period of 6 h or more does induce moderate ER stress in the cerebral cortex. We have demonstrated that all of the components of the UPR occur, i.e. increased protein levels of the molecular chaperone BiP/GRP78; dissociation of BiP and the kinase PERK; phosphorylation of PERK and the eIF2α; alteration in ribosomes compatible with attenuation in protein translation.

The concept of the UPR was also suggested by Cirelli et al. (2004) from microarray studies that showed increased cortical mRNA expression of the UPR-associated chaperones BiP and GRP94 in rats during wakefulness. In addition, PERK cerebellar transcript levels were found to be higher in wakefulness than in sleep (Cirelli et al. 2004). In addition, earlier studies by Nakanishi et al. (1997) and Ramm and Smith (1990), which demonstrated that protein synthesis was enhanced during sleep, also lend credence to our findings that sleep deprivation is associated with an attenuation in protein translation with likely increased protein synthesis during recovery sleep. A recent preliminary study by Ding et al. (2004) using surface-enhanced laser desorption/ionization mass spectrometry (SELDI/MS) also indicates that protein expression is decreased with sleep deprivation.

Moreover, immunohistochemical studies indicate that there is an increase in protein levels of ER chaperones GRP94, BiP/GRP78 and ERp72 during sleep deprivation in both the dorsal and lateral cortex of mouse brain (Terao et al. 2003), suggesting induction of the UPR. It seems likely that the UPR with sleep deprivation might occur in other brain regions and whether this is so will need to be addressed in future studies. This effect is also not likely to be unique to mice because rest deprivation in Drosophila, a state analogous to sleep in mammals (Hendricks et al. 2000; Shaw et al. 2000, 2002), also leads to up-regulation of the mRNA for BiP (Shaw et al. 2000, 2002). Little is known, however, about the pathways for UPR in Drosophila and we currently do not know if beyond up-regulation of BiP mRNA the other components of the UPR occur in Drosophila brain when the fly is rest deprived.

In our design, we used mice that were not chronically instrumented for recording of sleep by electroencephalogram and electromyogram We did so because the sleep/wake patterns of C57/B6 mice are well described (Veasey et al. 2000; Franken et al. 2001) and sleep deprivation was obvious by behavioral assessment. The lack of instrumentation avoids the concern that surgical implantation might have contributed to the findings. Others have described increased c-fos expression in brain that is induced solely by the instrumentation itself (Maloney et al. 1999). This strategy does not allow us to assess changes in delta waves that intrude into wakefulness when mice are sleep deprived for more than 6 h (Franken et al. 2001). The studies we report are, however, largely with 6 h of sleep deprivation. A concern with any sleep deprivation study is that the effects observed might not be related to the deprivation of sleep, but rather to non-specific stress effects produced by the methodology used to deprive sleep. To address this, we performed control studies in which mice were handled in the same way as when sleep deprived, but the handling was done during the first 6 h of the lights-off active period, i.e. when mice have their greatest amount of sustained wakefulness. There is, by necessity, the potential confounder of different times of day. Because these control studies did not result in any increase in BiP/GRP78 protein levels, in comparison with 6 h of sleep deprivation during the lights-on period, we conclude that the effects we are describing are specific to prolonging wakefulness and not simply secondary to the gentle handling procedure.

Because the UPR will have a global effect on synthesis of almost all proteins except a few molecular chaperones, this process will almost certainly have a profound effect on cortical function when wakefulness is prolonged to the point that UPR occurs. In humans, it has recently been shown that there is the beginning of impairment in performance when continued wakefulness exceeds 16 h (Van Dongen and Dinges 2003). Beyond this time, wakefulness is much more difficult to sustain and is unstable (Van Dongen and Dinges 2003). There are increasingly frequent performance lapses when wakefulness exceeds this limit (Van Dongen and Dinges 2003). This is important clinically because performance of activities such as driving a vehicle will be impaired. Epidemiological studies show that being awake for more than 20 h is a major risk factor for crashes that result from the driver falling asleep at the wheel (Stutts et al. 2003). Although we do not know whether in humans UPR occurs when wakefulness in prolonged, our data raise this as a hypothesis. In mice, UPR occurs at much shorter duration of wakefulness than 16 h. Mice do not, however, sustain wakefulness for long periods and the maximal wakefulness bout, as determined by EEG, in a C57/B6 mouse is typically of the order of 6 min during lights off and 2 min during lights on (personal communication, RJ Galante).

The precise signal for the ER stress response during sleep deprivation is not known. There are several candidate signals that may contribute to the UPR during extended wakefulness. First, it may be due to increased demand for energy during wakefulness, as a result of enhanced neuronal activity in the cortex (Erecinska and Silver 1989; Fraser et al. 1989). There is evidence that cerebral glucose metabolism is higher in wakefulness than in non-rapid eye movement sleep (NREM) sleep (Maquet 2000). It is known that a decrease in glucose stimulates up-regulation of molecular chaperones like BiP/GRP78 (Lee 2001), whereas depletion of cellular ATP inhibits protein folding (Braakman et al. 1992). It is likely that ATP is depleted during wakefulness because, at least in some brain regions, extracellular adenosine concentration increases with increasing durations of wakefulness (Porkka-Heiskanen et al. 1997). Adenosine acts as sleep-promoting molecule (for reviews, see Basheer et al. 2000; Porkka-Heiskanen et al. 2002). Perhaps, during extended wakefulness maintenance of other cellular processes and energy production occurs at the expense of protein folding. This would result in both reduced folding capacity and greater misfolding thus triggering the UPR and hence ER stress. Other signals for the UPR that may arise during sleep deprivation include altered calcium levels and enhanced glutamate release. Perturbations in calcium levels triggering the UPR are well described in the literature (Lee 1987; Liu et al. 1997; Yu et al. 1999). It has recently been shown that the mRNA of key regulators of Ca2+ release from intracellular stores like calcineurin, FK506 binding protein 12 and inositol 1,4,5-triphosphate receptor, are up-regulated during sleep (Cirelli et al. 2004). Thus, the UPR may be a consequence of depletion from intracellular stores during prolonged wakefulness (Li et al. 1993) with repletion of stores during sleep (Cirelli et al. 2004). A third mechanism by which the UPR may be induced could be an increase in glutamate concentration during prolonged wakefulness. The activity of BiP/GRP78, a key UPR indicator, was shown to be greatly increased in primary hippocampal neurons following exposure to glutamate (Yu et al. 1999). Sleep deprivation has been shown to result in increased levels of glutamate in rat cerebral cortex (Bettendorff et al. 1996). In addition, mRNA differential displays and cDNA microarrays indicate that the glutamate/aspartate transporter GLAST, glutamate NMDA receptor 2A and glutamate AMPA receptor GluR2 and GluR3 are up-regulated during waking and sleep deprivation (Cirelli and Tononi 2000). Thus, there are several potential mechanisms by which UPR occurs when wakefulness is prolonged.

In conclusion, the ER stress response in the mouse occurs after relatively short durations of sleep deprivation, i.e. 6 h. It seems likely that the resultant reduction in protein synthesis will make wakefulness more difficult to maintain since proteins whose translation is reduced will likely include receptors for transmitters that promote wakefulness. Whether the UPR is simply a consequence of sleep deprivation and prolonged wakefulness, or is part of the signaling mechanism for sleep will need to be determined in future studies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References

The microarray data that we reported were conducted in collaboration with Dr John Quackenbush at The Institute for Genomic Research as part of the Programs in Genomic Applications (U01 HL66588). We thank J. Cater for assistance with statistical analyses. The work reported here was supported in part by aging program project grant P01 AG17628, SCOR grant P50 HL60287 and University of Pennsylvania Research Foundation and Sleep Medicine Education Research Foundation grants to N. Naidoo.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animal handling and tissue collection
  5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting
  6. Temporal expression of BiP/GRP78
  7. Immunoprecipitation
  8. Polysome analysis
  9. Results
  10. Protein levels of the UPR marker BiP/GRP78 are increased with progressive durations of sleep deprivation
  11. BiP/GRP78 is not up-regulated in handled control animals
  12. BiP/GRP78 and PERK are dissociated during sleep deprivation
  13. PERK and eIF2α are phosphorylated during sleep deprivation
  14. There are changes in the ribosomal profile during sleep deprivation
  15. Discussion
  16. Acknowledgements
  17. References