Apoptosis

, Volume 17, Issue 10, pp 1039–1049

Expression of the hyperphosphorylated tau attenuates ER stress-induced apoptosis with upregulation of unfolded protein response

Authors

  • Xin-An Liu
    • Pathophysiology Department, Key Laboratory of Neurological Disease of National Education Ministry and Hubei Province, Tongji Medical CollegeHuazhong University of Science and Technology
    • Department of NeuroscienceThe Scripps Research Institute
  • Jie Song
    • Pathophysiology Department, Key Laboratory of Neurological Disease of National Education Ministry and Hubei Province, Tongji Medical CollegeHuazhong University of Science and Technology
  • Qian Jiang
    • Pathophysiology Department, Key Laboratory of Neurological Disease of National Education Ministry and Hubei Province, Tongji Medical CollegeHuazhong University of Science and Technology
  • Qun Wang
    • Pathophysiology Department, Key Laboratory of Neurological Disease of National Education Ministry and Hubei Province, Tongji Medical CollegeHuazhong University of Science and Technology
    • Pathophysiology Department, Key Laboratory of Neurological Disease of National Education Ministry and Hubei Province, Tongji Medical CollegeHuazhong University of Science and Technology
    • Pathophysiology Department, Key Laboratory of Neurological Disease of National Education Ministry and Hubei Province, Tongji Medical CollegeHuazhong University of Science and Technology
Original Paper

DOI: 10.1007/s10495-012-0744-z

Cite this article as:
Liu, X., Song, J., Jiang, Q. et al. Apoptosis (2012) 17: 1039. doi:10.1007/s10495-012-0744-z

Abstract

The neural dysfunction in Alzheimer’s disease (AD) could arise from endoplasmic reticulum (ER) stress and deficits of the unfolded protein response (UPR). To explore whether tau hyperphosphorylation, a hallmark of AD brain pathologies, plays a role in ER stress-induced alterations of cell viability, we established cell lines with stable expression of human tau (HEK293/tau) or the vector (HEK293/vec) and treated the cells with thapsigargin (TG), an ER stress inducer. We observed that the HEK293/tau cells were more resistant than the HEK293/vec cells to the TG-induced apoptosis, importantly, a time dependent increase of tau phosphorylation at Thr205 and Thr231 sites was positively correlated with the inhibition of apoptosis. We also observed that expression of tau upregulated phosphorylation of PERK, eIF2 and IRE1 with an increased cleavage of ATF6 and ATF4. The potentiation of UPR was also detected in HEK293/tau cells treated with other ER stress inducers, including staurosporine, camptothecin and hydrogen peroxide, in which a suppressed apoptosis was also shown. Our data suggest that tau hyperphosphorylation could attenuate the ER stress-induced apoptosis with the mechanism involving upregulation of UPR system.

Keywords

TauPhosphorylationApoptosisER stressUnfolded protein response

Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder represented clinically by a gradual cognitive decline and ultimately leading to dementia. The hallmarker pathologies observed in the AD brains are the formation of numerous senile plaques (SPs) and neurofibrillary tangles (NFTs), which are respectively composed of aggregated Aβ and hyperphosphorylated tau [1, 2]. The general brain atrophy in neurodegeneration may underlie the deterioration of learning and memory abilities. Data from a combined clinical symptom and postmortem pathological observations suggest that amount of the tangles is positively correlated with the degree of dementia in AD patients [3, 4], suggesting a crucial role of tau hyperphosphorylation in the neurodegeneration and cognitive impairments. Currently, it is not fully elucidated regarding the upstream factors leading to tau hyperphosphorylation, and how the phosphorylated tau affects the cell viability.

Endoplasmic reticulum (ER) stress, identified generally by the upregulation of the ER chaperone GRP78, plays a crucial role in cellular protein quality control by degrading proteins that are not correctly folded or assembled into native complexes. This process, known as ER-associated degradation (ERAD), ensures that only properly folded and assembled proteins are transported to their final destinations. Disruption of the ER function elicits an adaptive signaling cascade called the unfolded protein response (UPR). Recent studies suggest that neuronal dysfunctions in the AD brains could arise from ER stress [5, 6] and the failure of the UPR [7, 8]. The aggregated Aβ can cause ER stress with mechanisms involving nuclear translocation of GADD-153 and NF-κB [9]. ER-mediated apoptotic pathway is involved in the toxic effect of Aβ peptides [1013], and lithium inhibits Aβ-induced ER stress in rabbit hippocampus but does not prevent oxidative damage [14]. ER stress induces tau hyperphosphorylation both in vivo and in vitro [1519], but the role of tau phosphorylation in ER stress-induced cell death has not been reported.

It is believed that the protective responses are activated by the UPR to re-establish normal ER functions when ER homeostasis is disrupted. UPR is triggered by activation of three ER transmembrane proteins, i.e., the activating transcription factor-6 (ATF6) [20], inositol-requiring ER-to-nucleus signal kinase-1α (IRE1α) and PRK (RNA-dependent protein kinase)-like ER kinase (PERK), which can be identified by an increased phosphorylation or cleavage of the proteins. Activation of PERK inhibits the translational processes through phosphorylation of initiation factor 2α (eIF2α) and activating transcription factor-4 (ATF4) [21, 22], resulting in downregulation of the biosynthetic load of ER. Primarily, the ER stress should represent a protective response to the cellular insults, while persistent ER stress can also induce a switch in the UPR signaling from pro-survival to pro-apoptotic pathways [13, 23, 24].

We have recently reported that phosphorylation of tau could antagonize cell apoptosis [25], and thus we proposed that the phosphorylated tau-induced abortion of apoptosis may be the origin of the chronic neurodegeneration observed in the AD brains [26]. In the present study, we further investigated whether tau phosphorylation could protect the cells from ER stress-induced apoptosis by treated the HEK293/tau or HEK293/vec cells with thapsigargin (TG), an ER stress inducer. We observed that the cells with overexpression of the hyperphosphorylated human tau were more resistant to the apoptosis initiated by TG and the mechanisms involved upregulation of UPR.

Materials and methods

Chemicals and antibodies

The primary antibodies used in this study are listed in Table 1. Cell culture media were from Gibico (Grand Island, NY). Lipofectamine 2000 was from Invitrogen (Carlsbad, CA). Bicinchoninic acid (BCA) protein detection kit, chemiluminescent substrate kit, and nitrocellulose units were purchased from Pierce Chemical Company (Rockford, IL, USA). The chemical reagents used in this experiment were thapsigargin (TG), staurosporine (STP), camptothecin (CPT) and H2O2. TG was from Alexis Biochemicals Corporation (San Diego, CA, USA). STP, CPT and H2O2 were from Sigma-Aldrich (St Louis, MO, USA). Annexin V–PI staining kit was from KeyGEN biotech Corporation (Nanjing, Jiangsu, China). Cell Death Detection ELISAPLUS was from Roche (Indianapolis, IN, USA).
Table 1

Antibodies employed in the present study

Primary antibody

Recognition site

Property

WBa

IFb

Company

Tau-5

Total tau

mAbc

1:1,000

1:100

NeoMarkers

pS396

Phosphorylated tau at Ser396

pAbd

1:1,000

 

Biosource

pS404

Phosphorylated tau at Ser404

pAb

1:1,000

 

Biosource

pS262

Phosphorylated tau at Ser262

pAb

1:1,000

 

Biosource

pS214

Phosphorylated tau at Ser214

pAb

1:1,000

 

Biosource

pT205

Phosphorylated tau at Thr205

pAb

1:1,000

 

Biosource

pT231

Phosphorylated tau at Thr231

pAb

1:1,000

 

Biosource

Tau-1

Nonphosphorylated tau at Ser198/199/202

mAb

1:30,000

1:3,000

Chemicon

DM1A

α-tubulin

mAb

1:1,000

 

Sigma

PARP

Full length and cleaved PARP

pAb

1:1,000

 

Santa cruz

Cleaved caspase 3 (Asp175)

The large fragment (17/19 kDa) of activated caspase 3 resulting from cleavage adjacent to (Asp175)

pAb

1:1,000

1:100

Cell Signaling

Caspase-12

Propeptide and cleaved Caspase-12

pAb

1:1,000

 

abcam

GRP78

GRP78

pAb

1:1,000

 

abcam

GADD153

GADD153

pAb

1:1,000

 

abcam

PERK

Total PERK

pAb

1:200

 

Santa cruz

p-PERK (Thr981)

Phosphorylated PERK at Thr981

pAb

1:500

 

Santa cruz

eIF2α

Total eIF2α

pAb

1:1,000

 

Santa cruz

p-eIF2α (Ser51)

Phosphorylated eIF2α at Ser51

pAb

1:1,000

 

Cell Signaling

IRE1α

Total IRE1α

pAb

1:1,000

 

Santa cruz

p-IRE1α (Ser724)

Phosphorylated IRE1α at Ser724

pAb

1:1,000

 

abcam

ATF6

Total ATF6α

pAb

1:1,000

 

Santa cruz

GAPDH

Full length GAPDH

pAb

1:5,000

 

abcam

Goat-anti-mouse-peroxidase

  

1:5,000

 

Pierce

Goat-anti-rabbit-peroxidase

  

1:5,000

 

Pierce

IRDye Goat-anti-mouse IgG

  

1:15,000

 

LI-COR Biosciences

IRDye Goat-anti-rabbit IgG

  

1:15,000

 

LI-COR Biosciences

Oregon Green 488 goat anti-mouse IgG (H + L)

   

1:1,000

Molecular Probes

Rhodamine Red-Xgoat anti-mouse IgG (H + L)

   

1:1,000

Molecular Probes

Rhodamine Red-X goat anti-rabbit IgG (H + L)

   

1:1,000

Molecular Probes

aWestern blotting

bImmunofluorescence

cMonoclonal antibody

dPolyclonal antibody

Cell culture, treatment, and lysate preparation

Human embryonic kidney 293 (HEK293/wt) cells (a gift from Dr. H. Xu of the Burnham Institute, San Diego, USA) were cultured in Dulbecco’s modified eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS, Gibico BRL, Gaithersburg, MD, USA). HEK293/wt cells with stably expression of the longest human tau (tau441) (HEK293/tau) or the pcDNA vector (HEK293/vector) [27, 28] were cultured in DMEM containing 10 % FBS and 200 μg/ml G418. The cells were maintained at 37 °C in a humidified atmosphere containing 5 % CO2, cultured for about 24 h after plating and then the culture medium was replaced with serum-free medium before treatment.

To study the effect of ER stress on apoptosis and tau phosphorylation, we treated the cells with TG at various time points. We also treated the cells with 1 μM STP [29], 1 μM CPT [30], or 250 μΜ H2O2 [31] for 6 h in serum-free medium to induce apoptosis.

Cells were rinsed twice in ice-cold phosphate-buffered saline (PBS, pH 7.5) and lysed with buffer containing 50 mM Tris–Cl, pH 8.0, 150 mM sodium chloride, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 0.02 % sodium azide, 100 μg/ml phenylmethylsulfonyl fluoride, and 10 μg/ml protease inhibitors (leupeptin, aprotinin and pepstatin) followed by sonication for 5 s on ice. After centrifugation at 12,000×g for 5 min at 4 °C, supernatants were fetched out and added with equal volume of 2× Laemmli sample buffer (125 mM Tris–HCl, pH 6.8, 8 % SDS, 17 % glycerol, 10 % β-mercaptoethanol, and 0.05 % bromophenol blue). Samples were boiled for 10 min before electrophoresis. Protein concentration was estimated by BCA kit (Pierce, Rockford, IL, USA).

Transient expression of tau441 or the vector

HEK293/wt cells were seeded in six-well plates and grown to 80–90 % confluence, washed twice with 1× D-PBS and then cultured in serum- and antibiotic-free DMEM. Plasmids were transfected on the next day by using Lipofectamine 2000 according to the manufacturer’s instruction. Briefly, Lipofectamine (10 μl) and Tau441-pcDNA, or Tau441-pIRES-EGFP, or the vector (4 μg) were diluted in 250 μl of OPTI-MEM followed by equilibration at room temperature for 10 min after mixing. The Lipofectamine-DNA complex was added to the cells and incubated at 37 °C for further treatment.

Measurement of apoptosis

Apoptosis was assayed by using an Annexin V–PI staining kit by following the manufacturer’s procedure (KeyGen Biotech. Co. Ltd, Nanjing, P.R. China). The apoptotic rate was automatically quantified by flow cytometry with the standardized program of the instrument (FACSCalibur, BD Biosciences, San Jose, CA).

Colorimetric enzyme-linked immunosorbent assay (Cell Death Detection ELISAPLUS) for quantitative determination of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) was performed according to the manufacturer’s instructions (Roche). The results are expressed as relative enrichment of nucleosomes in the cytoplasm of cells.

Western blotting

Western blotting was performed according to methods established previously [32]. Briefly, the cell lysis were mixed with sample buffer containing 50 mM Tris–HCl (pH 7.6), 2 % SDS, 10 % glycerol, 10 mM DTT, and 0.2 % bromophenol blue and boiled for 5 min. The proteins were separated by 10 % SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to PVDF membrane. The membrane was then dried and blocked in 5 % non-fat milk for 1 h at room temperature. Incubations with antibodies to total tau (Tau-5), phosphorylated tau (pS396, pS404, pS262, pS214, pT231 and pT205), unphosphorylated tau (Tau-1), GRP78, caspase-12, PARP, eIF2α, phospho-eIF2α (Ser51), PERK, phospho-PERK(Thr981), IRE1α, phospho-IRE1α (Ser724), ATF6, GAPDH and DM1A were performed at 4 °C overnight. The target proteins were detected using horseradish peroxidase-linked anti-rabbit or anti-mouse IgGs (Pierce Chemical Company, Rockford, IL, USA) at 1:5,000 dilution, then visualized by the enhanced chemiluminescence kit (Pierce Chemical Company, Rockford, IL, USA). Immunoreactive bands were quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak Company, New Haven, CT, USA).

Immunofluorescence and confocal microscopy

The cells were plated at a density of 1.0 × 105 cells/cm2 on glass coverslips for immunocytochemistry. After treatment, cell culture medium was carefully removed. After two rinses in PBS, the cells were fixed in a freshly prepared solution of 4 % paraformaldehyde for 15 min. After two more rinses in PBS, the cells were permeabilized in 1 % Triton X-100 in PBS for 15 min. Then the cells were incubated in 3 % BSA in PBS for 1 h and incubated with primary antibody at 4 °C overnight. The immunoreactivity was probed using Rhodamine Red-X- or Oregon Green 488-conjugated secondary antibodies (1:1,000; Molecular Probes) for 2 h at room temperature. For the triple labeling studies, Hoechst 33258 (1 μg/ml) was used for the nuclear staining. The images were observed by using a confocal laser scanning microscope system (FV500; Olympus, Tokyo, Japan).

Statistical analysis

Data were analyzed using SPSS 12.0 statistical software (SPSS Inc., Chicago, Illinois, USA). The one-way ANOVA procedure followed by LSD’s post hoc tests was used to determine the statistical significance of differences. To analyze the correlations among the variables, Pearson Correlation was computed with bivariate correlations procedure.

Results

TG induces ER stress and cell apoptosis in HEK293 cells

To study the effect of ER stress on cell apoptosis, we treated the HEK293 cells with 1 μM TG [3335] for different time periods and analyzed the ER stress by GRP78 [36] and the cell apoptosis by measuring the cleavage of PARP (c-PARP), an early marker of apoptosis [37, 38]. We observed that the levels of GRP78 and c-PARP started to increase at 3 h after TG treatment (Fig. 1a–c), and a positive correlation of the increased GRP78 and c-PARP was observed (Fig. 1d). These data confirm that TG can induce ER stress and cell apoptosis.
https://static-content.springer.com/image/art%3A10.1007%2Fs10495-012-0744-z/MediaObjects/10495_2012_744_Fig1_HTML.gif
Fig. 1

TG induces ER stress and cell apoptosis in HEK293 cells. ac Wide type HEK293 cells were incubated in serum-free medium in the presence of thapsigargin (TG, 1 μM) for 0–48 h. ER stress and apoptosis were quantitatively analyzed by increased GRP78 (a, b) and the cleaved PARP (c-PARP) (a, c), respectively, and presented as fold changes. DM1A against tubulin was used as a loading control. d Correlation between ER stress (GRP78) and apoptosis (c-PARP) was analyzed by Pearson method. The experiments were repeated at least three times except specially stated and the representative blots were presented. **p < 0.01 versus untreated cultures (mean ± SD)

Expression of the hyperphosphorylated tau attenuates TG-induced cell apoptosis

To investigate the effect of tau on ER stress-induced apoptosis, we treated the HEK293 cells that stably expressing the longest human tau (tau441) (HEK293/tau) or the vector (HEK293/vec) with 1 μM TG for 3, 6, 12 and 24 h, and then analyzed the levels of ER stress, apoptosis and tau phosphorylation by Western blotting. We observed that the GRP78 level was significantly increased with the increased tau phosphorylation at pS396 (6 h), pS262 (6 h), pT205 (6, 12 and 24 h) and pT231(3, 6, 12 and 24 h) site after TG treatment (Fig. 2a–d) [34]. Simultaneously, the levels of c-PARP (Fig. 2a, e) and cleaved caspase-12 (Fig. 2a, f) were lower in HEK293/tau than HEK293/vec cells. The decreased apoptotic rate, reduced enrichment of nucleosomes and reduced cleavage of caspase 3 were also detected by flow cytometry (Fig. 2g), ELISA (Fig. 2h) and immunofluorescence staining (Fig. 2i) in HEK293/tau cells after TG treatment.
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Fig. 2

Expression of the hyperphosphorylated tau attenuates TG-induced cell apoptosis. ah HEK293 cells with stable expression of human tau441 (HEK293/tau) or the pcDNA vector (HEK293/vec) were incubated with thapsigargin (TG, 1 μM) for 0–24 h, then the cell extracts were prepared for analyses of ER stress by GRP78 (a, b), total tau by Tau-5 (a), phosphorylated tau at Thr231, Thr205, Ser396, Ser404, Ser262, Ser214 by pT231, pT205, pS396, pS404, pS262, pS214 and unphosphorylated tau at Ser198/199/202 by Tau-1 (c, d), normalized with total tau probed by Tau-5; and the apoptosis by cleaved PARP (cPARP) (a, e), cleaved caspase-12 (C-12) (a, f), flow cytometry (g), and Cell Death Detection ELISA (h), respectively. DM1A against tubulin was used as a loading control. i Co-immunofluorescence staining of the activated caspase 3 (red) with tau (green) after treatment of 1 μM TG for 24 h. jq The phosphorylation level of tau is negatively correlated with cell apoptosis. Correlation between the increased tau phosphorylation (at Thr231 and Thr205) and the decreased apoptotic rate measured by flow cytometry (j, k), Cell Death Detection ELISA (l, m), cleaved PARP (n, o) and cleaved caspase-12 (p, q) were analyzed by Pearson. Scale bar 20 μm. *p < 0.05, **p < 0.01 versus untreated cultures; #p < 0.05, ##p < 0.01 versus HEK293/vec cells at the same time point (mean ± SD) (Color figure online)

To verify the role of tau phosphorylation in ER-stress-induced cell apoptosis, we did correlative analysis between tau phosphorylation and the cell apoptosis. We found that the increased tau phosphorylation at pT231 and pT205 was positively correlated with the decreased apoptotic rate (Fig. 2j, k), decreased enrichment of nucleosomes (Fig. 2l, m), decreased c-PARP (Fig. 2n, o) and decreased cleavage of caspase-12 (Fig. 2p, q), respectively. These data suggest the hyperphosphorylation of tau at pT205 and pT231 may play a crucial role in its anti-apoptotic function.

To further confirm the role of tau in attenuation of the apoptosis, we transiently expressed tau441 into the HEK293 and measured the apoptosis after 1 μM TG treatment for 24 h. We observed that, with the expression of tau and increase of GRP78 (Fig. 3a–c), the TG-induced cell apoptosis was also reduced, demonstrated by the increase of GADD153 (Fig. 3a, d), decrease of c-PARP (Fig. 3a, e), and decrease of cell death (Fig. 3f).
https://static-content.springer.com/image/art%3A10.1007%2Fs10495-012-0744-z/MediaObjects/10495_2012_744_Fig3_HTML.gif
Fig. 3

Transient expression of tau attenuates ER stress-induced cell apoptosis. ae Wild type HEK293 cells (HEK293/wt) were transiently transfected with human tau441 (HEK293/EGFP-tau) or the vector (HEK293/EGFP-vec) for 24 h, and then treated with 1 μM thapsigargin (TG) for 24 h. The phosphorylation level of tau was measured by pS396 and normalized against total tau (Tau-5) (a, b). ER stress was estimated by the elevated GRP78 (a, c). The cell apoptosis was evaluated by GADD153 (a, d) and cleaved PARP (a, e). DM1A against tubulin was used as a loading control. f Immunofluorescence staining of the activated caspase 3 (red) after treatment with 1 μM TG for 24 h, and the arrows indicate dissociation of tau (green) with activated caspase 3 (red). Scale bar 20 μm. *p < 0.05, **p < 0.01 versus untreated cultures; #p < 0.05, ##p < 0.01 versus HEK293/EGFP-vec cells (mean ± SD) (Color figure online)

Previous studies suggest that GRP78 has anti-apoptotic function [3942], however, we observed that GRP78 levels in HEK293/tau, HEK293/vec and wide type HEK293 cells were similar (Fig. 3c), suggesting that the anti-apoptotic effect of tau in ER stress is not GRP78-dependent.

Expression of tau stimulates UPR

To explore whether the UPR contributes to the anti-apoptotic function of tau, we analyzed the activity-dependent phosphorylation levels of PERK, eIF2α and IRE1α in the cytosolic fraction, and the total levels of ATF6 and ATF4 in the nuclear fraction, the recognized ER stress sensors [43, 44]. We found that the levels of p-PERK, p-eIF2α, ATF4 and ATF6 increased remarkably, whereas the level of p-IRE1α decreased in HEK293/tau cells after TG treatment with a significantly decreased c-PARP (Fig. 4a–h). The purity of the cytosolic and nuclear fractions was confirmed by using the recognized molecular markers PARP and GAPDH, respectively (Fig. 4c). These data together suggest that activation of PERK, eIF2α and ATF-6 but not IRE1α in response to ER stress may contribute to the anti-apoptotic effect of tau [6].
https://static-content.springer.com/image/art%3A10.1007%2Fs10495-012-0744-z/MediaObjects/10495_2012_744_Fig4_HTML.gif
Fig. 4

Expression of tau stimulates UPR system. a, df HEK293 cells with stable expression of tau (HEK293/tau) or the vector (HEK293/vec) were treated with thapsigargin (TG, 1 μM) for 24 h. The phosphorylation levels of the UPS proteins in cell extracts were analyzed by using anti-phospho-PERK (Thr981) (a, d), anti-phospho-eIF2α (Ser51) (a, e), and anti-phospho-IRE1α (Ser724) (a, f) and normalized against total PERK, eIF2α and IRE1α. DM1A against tubulin was used as a cytoplasm loading control. b, g, h The nuclear fraction was prepared for analyses of ATF6 (b, g) and ATF4 (b, h). PARP was used as a nuclear loading control. c The purity of nuclei and cytoplasm extracts was confirmed by anti-PARP (nuclei) and anti-GAPDH (cytoplasm) antibodies, respectively. *p < 0.05, **p < 0.01 versus HEK293/vec cells at the same time point (mean ± SD)

Expression of the hyperphosphorylated tau attenuates the chemically induced apoptosis with stimulating the UPR system

We have reported recently that tau phosphorylation could prevent cell apoptosis induced by STP, CPT, and H2O2 [25]. To further validate the role of UPR in the anti-apoptotic effect of tau, we treated the HEK293/tau and the HEK293/vec cells with 1 μM STP, 1 μM CPT, or 250 μΜ H2O2 and then measured the ER stress and the UPR proteins. We observed that expression of tau antagonized cell apoptosis induced by STP, CPT and H2O2 treatment with an increased GRP78 (Fig. 5a–c). We also observed that the phosphorylation levels of cytosolic PERK, eIF2α, IRE1α and the nuclear cleaved ATF6 and ATF4 in the HEK293/tau cells were much higher than the HEK293/vec cells after the treatment with the chemicals (Fig. 5d–k). These data further support the involvement of UPR in the anti-apoptotic role of tau.
https://static-content.springer.com/image/art%3A10.1007%2Fs10495-012-0744-z/MediaObjects/10495_2012_744_Fig5_HTML.gif
Fig. 5

Expression of the hyperphosphorylated tau attenuates the chemically induced apoptosis with stimulating the UPR system. ac HEK293 cells with stable expression of tau (HEK293/tau) or the vector (HEK293/vec) were treated with 1 μM staurosporine (STP) or 1 μM camptothecin (CPT) or 250 μM H2O2 for 6 h. The expression of tau was confirmed by antibodies Tau-5 and pS396, pS404 (a); and ER stress and apoptosis were analyzed by the increased GRP78 (a, b) and cleaved PARP (a, c), respectively. d, gi The phosphorylation levels of the UPS proteins in cell extracts were analyzed by using anti-phospho-PERK (Thr981) (d, g), anti-phospho-eIF2α (Ser51) (d, h), and anti-phospho-IRE1α (Ser724) (d, i) and normalized against total PERK, eIF2α and IRE1α. DM1A against tubulin was used as a cytoplasm loading control. e, j, k The nuclear fraction was prepared for analyses of ATF6 (e, j) and ATF4 (e, k). PARP was used as a nuclear loading control. f The purity of nuclei and cytoplasm extracts was confirmed by anti-PARP (nuclei) and anti-GAPDH (cytoplasm) antibodies, respectively. *p < 0.05, **p < 0.01 versus HEK293/vec cells with same treatments (mean ± SD)

Discussion

Neurofibrillary degeneration characterized by intracellular accumulation of the hyperphosphorylated tau is the hallmark lesion of AD and the related tauopathies [45, 46]. Caspase activation-mediated events that trigger cytochrome c release and apoptosis have been identified in the affected brain region of AD [47], however, the neurons bearing hyperphosphorylated tau and tangles undergo chronic degeneration rather than acute apoptosis during the development of AD, suggesting at least a temporal neuroprotective effect for tau hyperphosphorylation and the formation of tau filaments [48]. Our recent studies demonstrate that tau phosphorylation rendered the cells more resistant to the apoptosis induced by staurosporine, camptothecin and hydrogen peroxide with the mechanisms involving preservation of β-catenin [25, 26], while tau dephosphorylation potentiates apoptosis [49], which partly explain why the p-tau/tangle-bearing neurons in AD and the related tauopathies do not preferentially die of apoptosis. We speculate that tau phosphorylation may render the cells escape acute apoptosis by preservation of survival factors (such as β-catenin), however, durative hyperphosphorylation detaches tau from microtubule and thus disassembles microtubules [50], causes tau accumulation [51], inhibits proteasome [52], which can eventually lead the neurons to degeneration [26].

Previous studies demonstrate that activation of UPR is an early event of AD that is correlated with tau pathologies [5] and UPR activated by Aβ increases tau phosphorylation [53]. In the present study, we further demonstrated that tau phosphorylation at multiple AD-related sites could protect the cells from ER stress-induced apoptosis, in which the increased phosphorylation of tau at Thr205 and Thr231 were positively correlated with the enhanced ER stress and the attenuated apoptosis. Meanwhile, the enhanced cleavage of ATF6 and phosphorylation of PERK pathway were also detected in the cells with expression of the phosphorylated tau. Our data suggest that tau phosphorylation prevents ER stress-induced cell apoptosis with the mechanisms involving stimulation of UPR systems. To our knowledge, this is the first report demonstrating the protective role of tau phosphorylation in ER stress-related apoptosis and the possible molecular mechanisms.

In our study, we observed that tau expression/phosphorylation by itself also induces ER stress and potentiates UPR. Currently, we do not fully understand how tau phosphorylation may be related to ER stress and UPR activation. It is known that multiple perturbations can cause accumulation of unfolded proteins in the ER and thus activate the UPR in an attempt to re-establish homeostasis of ER. The immediate UPR adaptive responses towards ER stress include activation of IRE1α, PERK and ATF6. These ER stress transducer proteins are inactive when GRP78 is bound to their luminal regulatory domains. GRP78 is released and the UPR is activated when ER homeostasis is disturbed [5, 54]. We speculate that overexpression/hyperphosphorylation of tau may cause ER stress by increasing GRP78, which then potentiates the cleavage of ATF6 and phosphorylation of PERK. The activation of PERK can stimulate subsequently the phosphorylation of eIF2α at Ser51, and thus upregulates ATF4. The combined activation of ATF6 and PERK pathways will lead to activation of CHOP/GADD153, which may eventually leads to an increased cell survival.

While the UPR stimulation should be initially aimed to promote cell survival, the pro-apoptotic potential of the UPR can be triggered by incessant ER stress beyond the limits of adaptation [55], when the compensatory mechanisms fail to restore homeostasis in the ER [56]. It deserves further investigation regarding to what extents the UPR activation is pro-apoptosis or anti-apoptosis. We also observed that tau overexpression significantly decreased the phosphorylation of IRE1α at Ser724, suggesting that IRE1α pathway may not be involved in the anti-apoptotic effects of tau phosphorylation.

ER stress-induced apoptosis is mediated by mitochondria (intrinsic pathway) and/or through activation of death-receptor-mediated pro-apoptotic kinases (extrinsic pathway) [57]. Interestingly, we also observed that upregulation of UPR in HEK293/tau cells was associated with the suppressed apoptosis induced by staurosporine, camptothecin and hydrogen peroxide, implying that the chemicals can induce ER stress, and tau attenuates the chemically-induced apoptosis with the mechanisms also involving enhancement of the UPR signaling.

In order to clarify it is indeed tau proteins in regulating cell viability, we have designed to use human embryonic kidney cells (HEK293), because there is no endogenous expression of tau in this cell line. Although HEK293 has been widely employed for neural-related studies, the role of tau in regulating cell apoptosis has to be confirmed in neuronal system in future studies.

In summary, we found in the present study that tau hyperphosphorylation could attenuate the ER stress-induced cell apoptosis with activation of UPR systems.

Acknowledgments

This work was supported in part by grants from National Natural Science Foundation of China (30971204, 30871035), a grant from Education Ministry of China (NCET050650), and a grant from Alzheimer’s Association (IIRG09133433).

Copyright information

© Springer Science+Business Media, LLC 2012