SB216763

SIL1 Rescued Bip Elevation-Related Tau Hyperphosphorylation in ER Stress

Abstract Endoplasmic reticulum (ER) stress has been indi- cated in the early stage of Alzheimer’s disease (AD), in which tau hyperphosphorylation is one major pathological alteration. The elevation of binding immunoglobulin protein (Bip), an important ER chaperon, was reported in AD brain. It is im- portant to study the roles of ER-related chaperons in tau hyperphosphorylation. In this research, increased Bip was found in the brains of the AD model mice (Tg2576) compared to the age-matched control mice. Meanwhile, deficiency of SIL1, an important co-chaperon of Bip, was observed in brains of Tg2576 mice and in ER stress both in vivo and in vitro. Then, we transfected Bip-EGFP plasmid into HEK293 cells stably expressing the longest human tau (HEK293/tau) or N2a cells and found that increased Bip induced tau hyperphosphorylation via activating glycogen synthase kinase-3β (GSK-3β), an important tau kinase, and increased the association with tau and GSK-3β. When we overexpressed SIL1 in Bip-transfected HEK293/tau cells and thapsigargin-treated HEK293/tau cells, significantly reduced tau hyperphosphorylation and GSK-3β activation were ob- served. These results suggested the important roles of ER- related chaperons, Bip and SIL1, in AD-like tau hyperphosphorylation.

Keywords : Alzheimer’s disease . Bip . SIL1 . GSK-3β . Tau

Introduction

Binding immunoglobulin protein (Bip), also known as the 78- kDa glucose-regulated protein 78 (GRP78) [1, 2], is an abun- dant endoplasmic reticulum (ER) chaperon protein that is rapidly increased responsive to ER stress. The multifunctional roles of Bip in protein folding, ER calcium binding, and as a receptor on the cell surface display its major involvement in physiological and numerous pathological conditions [3]. Neu- rons are vulnerable to different genetic and environmental insults that affect the homeostasis of ER function [4]. There- fore, a number of reports have demonstrated that ER stress and aggregation of misfolded protein are present in several neuro- degenerative diseases [5]. Recently, increased Bip was shown in the cortex and hippocampus of patients with Alzheimer’s disease (AD) [6–8] and in brains of AD model [9]. Binding of substrates to Bip and subsequent release from Bip are con- trolled by a continuous cycle of ATP hydrolysis and exchange of ATP for ADP. One important co-chaperon of Bip, nucleo- tide exchange factor SIL1, encoded by the SIL1 gene [10] and also known as Bip-associated protein (BAP), regulates the ATPase activity of Bip [11, 12]. A mutation of SIL1 causes ataxia and neurodegeneration [13]. Therefore, Bip and its co- chaperon SIL1 are important to the normality of neurons.

Formation of intracellular neurofibrillary tangles (NFTs) is one of the most prominent histopathological features in AD. Abnormally hyperphosphorylated microtubule-associated protein tau is the main component of NFTs [14, 15], which is positively correlated with the decline of learning and memory in the AD patients [16]. Therefore, proper modulation of tau phosphorylation may be a steering step in arresting AD [17]. However, the effectors leading to tau hyperphosphorylation are still not fully understood. Upregulation of Bip in neurons of temporal cortex and hippocampus from Braak stage III indicates its early involvement in the tau pathology of AD [7]. Previously, we found ER stress inducers, thapsigargin (TG) and tunicamycin (TM), could induce tau hyperphosphorylation with the increased Bip both in vivo and in vitro [18, 19]. However, the roles of Bip and its co-chaperon in tau hyperphosphorylation need further understanding.
In this study, we found Bip was increased while SIL1 was reduced in the brains of AD model mice compared with the age-matched control mice. Then, we constructed Bip-EGFP plasmid according to Qian Y’s delineation [20] and transfected the plasmid into Human embryonic kidney 293 stably expressing the longest human tau (tau441) cDNA (HEK293/tau) or Neuro2A (N2a) cells. It was found that overexpression of Bip induced tau hyperphosphorylation via activating glycogen synthase kinase-3β (GSK-3β), an impor- tant tau kinase in AD brain, and increased the association with tau and GSK-3β. Compared with increased Bip, deficiency of SIL1 was observed in ER stress both in vivo and in vitro. Overexpressing SIL1 significantly reduced Bip overexpres- sion and TG-induced tau hyperphosphorylation and GSK-3β activation, and reduced the association of Bip with tau and GSK-3β. All these data suggested that increased Bip compared with deficiency of SIL1 participated in AD- like tau hyperphosphorylation. Overexpressing SIL1 is one important measure to prevent ER stress-induced tau hyperphosphorylation.

Materials and Methods

Reagents and Antibodies

The antibodies used in this study are listed in Table 1. TG (Sigma, USA) and TM (Sigma, USA) could induce ER stress. SB216763 (SB), a specific GSK-3β inhibitor, was from Tocris Bioscience (Bristol, UK) and freshly dissolved in dimethyl sulfoxide (DMSO) from light before use. Bip-EGFP plasmid was constructed according to delineation of Qian Y [20]. cDNA (1.9 kb) of rat Bip (GeneBank M14050) was cloned into a pEGFP-N1 vector (GenChem) carrying EGFP cDNA at HindIII/BamHI restriction enzyme sites to construct chimeric Bip-EGFP cDNA for the expression of Bip-EGFP fusion protein. SIL1 plasmids were gifts from Dr. Linda M. Hendershot at the University of Tennessee Health Science Center (Memphis, TN).

Animals and Brain Injection

Tg2576 mice (Tg), age-matched C57/BL6 mice (C57), and 4- month-old male Sprague Dawley (SD) rats were supplied by the Experimental Animal Central of Tongji Medical College. All experimental procedures were approved by the Animal Care and Use Committee at the Huazhong University of Science and Technology and were performed in compliance with National Institutes of Health guidelines on the ethical use of animals. Mice were kept in cages under a 12-h light/12-h dark (L/D) cycle with the light on from 7:00 a.m. to 7:00 p.m. Four-month-old (280±20 g) male SD rats (n=6 for each group) were anesthetized with 6 % chloral hydrate (400 mg/kg) and placed in a stereotaxic instrument (RWD Life Science Co. Ltd, China). After the scalp was incised (5– 8 mm), the skull was cleaned and a hole (diameter 1.0 mm) was made with a dental drill for the infusion. For the lateral ventricular infusion, the coordinate of AP-0.8, L-1.5, V-4.0 (in mm from bregma and dura, flat skull) was selected according to the stereotaxic atlas of Franklin and Paxinos. A sterilized needle connected to a Hamilton syringe was used to deliver TM into the lateral ventricle (10 μl, 50 μM). An equal volume of DMSO with normal saline was infused as a vehicle control.

The control group received no treatment. Cell Culture and Plasmid Transfection

HEK293 cells stably transfected with the longest human tau (tau441) cDNA (HEK293/tau) were cultured in 90 % DMEM supplemented with 10 % fetal bovine serum (FBS) and 200 μg/ml G418 (FBS, v/v) [21, 22] at 37 °C in 5 % CO2.N2a cells were cultured in Dulbecco’s modified Eagle’s medi- um (DMEM) and OptiMEM I reduced serum medium (1:1; v/v) with5% FBS (Gibco, NY, USA) at 37 °C in5% CO2 [21]. For studying the role of Bip, Bip-EGFP plasmids were transfected into HEK293/tau cell and N2a using Lipofectamine 2000 according to the manufacturer’s instruction. To validate the role of GSK-3β in Bip overexpression-induced tau phos- phorylation, the cells were treated with SB 2.5 or 5 μΜ for 24 h after transfection of Bip plasmids. To study the effect of SIL1 on Bip overexpression inducing tau hyperphosphorylation, plas- mids of SIL1 and Bip were co-transfected into HEK293/tau cells.

Sample Preparation, Immunoprecipitation, and Western Blotting

To prepare the protein extract of brain sample, the different brain regions from decapitated animals were rapidly removed and homogenized on ice using a Teflon glass homogenizer in 50 mM Tris-HCl, pH 7.4; 1 % NP-40; 0.25 % Nadeoxycholate; 150 mM NaCl; 1 mM Na3VO4; 1 mM EDTA; 1 mM PMSF; 1 mM NaF; and a mixture of aprotinin, leupeptin, and pepstatin A (10 μg/ml each), and then centri- fuged at 14,000×g for 15 min at 4 °C. To prepare the protein extract from cultured cells, 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 NaCl; 1 % NP-40; 0.5 % Na-deoxycholate, 0.1 % sodium dodecyl sulfate (SDS); 0.02 % NaN3; 100 μg/ml PMSF; and 10 μg/ml prote- ase 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). Protein concentration was estimated by a BCA kit.

Immunoprecipitation was performed as described [19]. Briefly, the hippocampal extracts containing 500 μg protein were incubated overnight with 2 μg Bip antibody, and then with protein G for 2 h at 4 °C with gentle rotation. After centrifugation at 14,000×g for 1 min at 4 °C, the supernatant was removed and the protein G agarose beads were collected and washed with PBS. Then, the bound proteins were disso- ciated by boiling for 5 min the beads in 2× Laemmli sample buffer and analyzed by Western blotting. Samples were boiled for 10 min before electrophoresis.

For Western blotting, the proteins were separated by 10 % SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5 % nonfat milk dissolved in TBS (50 mM Tris-HCl, pH 7.6; 150 mM NaCl) for 1 h and probed with primary antibody (see Table 1 for detail) at 4 °C for overnight. The blots were probed by using IRDye 800CW-conjugated secondary antibody (1:10,000) and visualized by infrared fluorescence imaging.

The intensity of the protein bands was quantified by Odyssey system (Li-Cor Bioscience, Lincoln, NE).

Immunohistochemistry and Immunofluorescence Staining

The mice were killed by overdose of chloral hydrate (1000 mg/kg) and perfused through the aorta with 100 ml 0.9 % NaCl followed by 400 ml phosphate buffer containing 4 % paraformaldehyde. Coronal sections of brain were cut at 20 μm using a vibratome (Leica, Nussloch, Germany; S100, TPI). For immunohistochemistry, the sections were incubated for 48 h at 4 °C with primary antibodies, and the slices were developed with Histostain-SP kits (Zymed, South San Francisco, CA) and visualized with diaminobenzidine (DAB) (Sigma, St. Louis, USA). The images were observed by using a microscope (Olympus BX60, Tokyo, Japan).

For immunofluorescence staining, cells were cultured on coverslips and fixed with 4 % paraformaldehyde for 1.5 h at 4 °C and then incubated overnight at 4 °C with primary antibody followed by 1 h at ~25 °C with rhodamine red-X- or Cy5-conjugated secondary antibodies. The rest of the pro- cesses were the same as described above [22]. The images were observed by using a laser confocal microscope (Zeiss LSM710, Germany).

Statistical Analysis

Data were expressed as means±SD and analyzed using SPSS 12.0 statistical software (SPSS Inc., Chicago, IL, USA). The one-way analysis of variance (ANOVA) procedure followed by LSD’s post hoc tests was used to analyze the differences among groups.

Results

Overexpressed Bip Induced Tau Hyperphosphorylation

Increased expression of Bip in the cortex and hippocampus of AD patients has been demonstrated [7, 8, 22]. By contrast, reduced Bip has been illustrated in the cortex of aging brain [23]. In this research, we detected the expres- sion of Bip in the cortex and hippocampus of 4-, 8-, and 12-month-old Tg2576 mice (Tg) or age-matched C57/BL6 mice (C57) by Western blotting. We observed significantly increased Bip in both cortex and hippocampus of Tg2576 mice compared with age-matched control (Fig. 1a–d), and younger C57/BL6 mice had lower Bip level in both cortex and hippocampus (Fig. 1a–d). Previously, we found ER stress inducers, TG and TM, could induce increased Bip and tau phosphorylation both in vivo and in vitro [18, 19] and promote the binding of GSK-3β to tau [19]. To further confirm the role of Bip in ER stress-induced tau hyperphosphorylation, we transfected the plasmid of Bip- EGFP into HEK293/tau and N2a cells and found Bip- transfected cells showed significantly increased tau phos- phorylation at Ser396, Ser262, and Thr231, and no alter- ation in the total level of tau (Fig. 1e–h). All these data suggested aging-related increased Bip participated in tau hyperphosphorylation of AD.

Overexpression of Bip Resulted in GSK-3β Activation

GSK-3β and PP2A are respectively the most implicated pro- tein kinase and phosphatase in tau hyperphosphorylation [24, 25]. Therefore, we tested activity-dependent phosphorylation of GSK-3β after transfecting Bip-EGFP in HEK293/tau and N2a cells. We found that the phosphorylation of GSK-3β at Try216 (Y216-GSK-3β, activation) was significantly elevat- ed and phosphorylation of GSK-3β at Ser9 (S9-GSK-3β, inactivation) was markedly reduced in Bip-transfected HEK293/tau and N2a cells (Fig. 2a–d). The level of total GSK-3β was unaffected (Fig. 2a–d). Immunofluorescence staining also showed overexpressed Bip co-localized with the active form of GSK-3β (Y216-GSK-3β) and the hyperphosphorylated tau (pS396) (Fig. 2e, white arrowhead). And phosphorylation of tau (pS396) and Y216-GSK-3β was increased in HEK293/tau cells transfected by Bip-EGFP plas- mids compared with untransfected cells (Fig. 2e. pS396, red; Y216-GSK-3β, blue). Whereas, the activity-dependent phos- phorylation of the PP2A catalytic subunit (PP2Ac) and its total level was unchanged after Bip transfection in HEK293/ tau cells (data not shown). Then, we used 0, 2.5, and 5 μM SB216763 (SB), a specific GSK-3β inhibitor, to treat the HEK293/tau cells at 24 h after Bip transfection. Twenty-four hours later, we found 5 μM SB-treated Bip-transfected HEK293/tau cells showed obviously decreased tau phosphor- ylation levels at Ser396 and Thr231 epitopes compared with 0 and 2.5 μM SB-treated cells (Fig. 3a, b). The data together suggested that overexpression of Bip induce tau phosphory- lation by activating GSK-3β.

To explore the possible mechanisms of Bip overexpression-induced GSK-3β activity-related phosphory- lation, we detected upstream factors of GSK-3β. A member of Src tyrosine kinase family, Fyn [22, 26], and Akt/PKB [27, 28] could respectively phosphorylate GSK-3β at Tyr216 and Ser9. We found phosphorylated Fyn (Y416-Fyn), total Fyn (t- Fyn), phosphorylated Akt (T308-Akt, S473-Akt), and total Akt (t-Akt) had no marked alteration (Fig. 3c), suggesting Bip-induced GSK-3β phosphorylation did not depend on Akt- and Fyn-related pathways. Protein tyrosine phosphatase 1B (PTP1B), a tyrosine phosphatase, is located in the ER that may dephosphorylate GSK-3β at Tyr216 [24]. Phosphorylat- ed protein kinase C (PKC) phosphorylates GSK-3β on the Ser9 position inactivating GSK-3β [29, 30]. We tested PTP1B and phosphorylation of PKC in Bip-EGFP-transfected HEK293/tau cells and found the level of PTP1B and the phosphorylation level of PKC at the Ser738 site were reduced (Fig. 3d, e). So we presumed that overexpression of Bip- induced GSK-3β activation is via reduction of PTP1B and PKC activities.

Fig. 1 Increased Bip in brains of Tg2576 mice and old C57/BL6 mice and Bip overexpression induced tau hyperphosphorylation in HEK293/tau cells and N2a cells. Bip in the cortex (a) of 4-month- old (4m), 8-month-old (8m), and 12-month-old (12m) Tg2576 mice (Tg) and the age-matched C57/BL6 mice (C57), and its levels in the hippocampus (c) of 4-month-old (4m) and 12-month- old (12m) Tg2576 mice (Tg) and the age-matched C57/BL6 mice (C57) were tested by Western blotting (a, c) and quantitative analysis (b, d). Data are presented as means±SD (n=6); *p<0.05,**p<0.01, ***p<0.001, vs age- matched C57 mice. △p<0.05, △△p<0.01 vs 4-month-old (4m) C57 mice. ▲▲▲p<0.001 vs 4- month-old (4m) Tg2576 mice. The plasmids of Bip-EGFP and its vector as control were transfected into HEK293/tau cells (e, f) and N2a cells (g, h); 48 h later, the levels of Bip, tau phosphorylation at Ser 396 (pS396), Ser262 (pS262), Thr23 (pT231), and total tau (Tau5) in cell lysate were tested by Western blotting and quantitative analysis. Data are presented as means±SD (n=6). #p<0.05, ##p<0.01 vs vector transfected cells SIL1 Was Decreased in Brains of Tg2576 Mice and ER Stress SIL1, one important co-chaperon of Bip, regulates the ATPase activity of Bip [11] and plays an important role in the neurodegeneration of Marinesco-Sjögren syndrome disease [31–34]. Here, we detected SIL1 level in the brains of Tg2576 mice and C57/BL6 mice. With the increased Bip (Fig. 1a–d), we found that levels of SIL1 were reduced in the cortex and hippocampus of Tg2576 mice compared with age-matched C57/BL6 mice by Western blotting (Fig. 4a–d) and immunohistochemistry (Fig. 4e, f). To further confirm the alteration of SIL1 in ER stress, we treated HEK293/tau cells with 2 μM TG and observed decreased SIL1 with the in- creased Bip at 3 h after TG treatment (Fig. 4g, h). In 4- month SD rats, 10 μl of 50 μM TM treatment via lateral ventricle injection induced decreased SIL1 in the hippocam- pus and cortex (Fig. 4i, j), while Bip was increased [31, 33] at 48 h after TM injection. All these data indicated the deficiency of SIL1 in ER stress compared with increased Bip. Fig. 2 Overexpression of Bip induces activation of GSK-3β. The plasmids of Bip-EGFP and its vector as control were transfected into HEK293/tau cells and N2a cells. Forty-eight hours later, phosphorylation levels of GSK-3β at Tyr216 (Y216-GSK-3β, activation of GSK-3β) and Ser9 (S9-GSK-3β, inactivation of GSK-3β) and its total level were tested by Western blotting (a, HEK293/tau cells; c, N2a cells) and quantitative analysis (b, d). Data are presented as means±SD (n=4). *p <0.05, ***p < 0.001 vs vector. Immunofluorescence staining also showed overexpressed Bip (green) co-localized with Ser396 phosphorylated tau (red) and Tyr216 phosphorylated GSK-3β (blue) in cultured HEK293/tau cells. Scale bar=20 μm. SIL1 Rescued Tau Hyperphosphorylation Induced by Overexpressed Bip and TG To further confirm the role of SIL1 in ER stress or Bip overexpression-induced tau hyperphosphorylation, the cul- tured HEK293/tau cells were co-transfected with Bip and SIL1 plasmids. In SIL1- and Bip-co-transfected cells, tau phosphorylation levels at Ser396, Ser262, and Thr231 were lower than those in Bip-solo-transfected cells. SIL1 and Bip co-transfection did not affect tau phosphorylation and its total level (Fig. 5a, b). SIL1- and Bip-co-transfected cells showed higher phosphorylation of GSK-3β at Ser9 (S9-GSK-3β) and less phosphorylation at Tyr216 (Y216-GSK-3β) (Fig. 5c, d). The levels of PTP1B and phosphorylated PKC were much higher in SIL1- and Bip-co-transfected cells than those in Bip- solo-transfected cells (Fig. 5e, f). In addition to phosphorylation regulation, protein complex formation also affects the activity of GSK-3β. Previously, we detected in ER stress an increasing binding of Bip with GSK- 3β and tau proteins (but not β-catenin) accompanying with an increased dissociation of Bip with protein kinase-like ER kinase (PERK), inositol-requiring enzyme-1 (IRE-1), and ac- tivating transcription factor-6 (ATF-6) both in vitro and in vivo [19]. By immunoprecipitation, we found in SIL1/Bip-co- transfected cells and SIL1-solo-transfected cells that the level of Bip-bound tau and GSK-3β decreased, while Bip-bound SIL1 increased (Fig. 6a, b). To further investigate whether SIL1 could rescue ER stress-induced tau hyperphosphorylation, the plasmid of SIL1 was transfected into HEK293/tau cells, and 48 h the cells received a further 3-h treatment of 2 μM TG. We found that SIL1-transfected cells showed lower tau phosphor- ylation levels at Ser396 and Thr231 sites compared to the vector-transfected cells (Fig. 6c, d). All these data indicated the protective role of SIL1 in ER stress-induced tau hyperphosphorylation. Fig. 3 SB216763 (SB) prevents Bip overexpression-induced tau hyperphosphorylation and PTP1B and PKC involved in Bip overexpression-induced GSK-3β activation. Twenty-four hours after Bip transfection into HEK293/tau cells, 0, 2.5, and 5 μM SB216763 (SB), a specific GSK-3β inhibitor, were used to treat the cells. Then, levels of Bip, phosphorylation of tau at Ser396, Ser262, and Thr231 sites, and the total tau (Tau5) were tested by Western blotting (a) and quantitative analysis (b). To explore the possible mechanisms of Bip overexpression-induced GSK-3β activation, phosphorylation of some related kinases including Fyn, Akt, and PKC and their total levels, and the level of an important protein phosphatase, tyrosine phosphatase 1B (PTP1B), were tested by Western blotting (c, b) and quantitative analysis (e). Data are presented as means±SD (n= 5). *p<0.05 vs 0 μM SB-treated cells in b and vector-transfected cells in e Discussion Bip is an ER chaperon and normally associated with the ER membranous proteins, such as PERK, IRE-1, and ATF-6 (Fig. 7). In ER stress, Bip is increased and dissociated with PERK, IRE-1, and ATF-6, which will be phosphorylated after disso- ciation from Bip [35, 36]. ER stress is a defense system for dealing with the accumulation of unfolded proteins in the ER lumen. Damages including oxidative stress, disturbed protein glycosylation and calcium homeostasis, and deprivation of glucose and oxygen can induce ER stress by expressing chaperon proteins and trigger many rescuer responses, includ- ing unfolded protein response and ER-associated degradation [36]. ER stress is involved in the pathogenesis of AD, which is characterized by the accumulation and aggregation of misfolded proteins [7]. ER stress markers, including elevated levels of Bip [14, 15] and increased phosphorylation of PERK [17–19], were observed in the brains of AD individuals. Here, we observed significantly elevated Bip in both cortex and hippocampus of Tg2576 mice compared with age-matched C57 mice. ER stress features are prominent in the brain of AD patients but not in prion diseases [15], suggesting a specific role of Bip in the pathophysiological process of AD. Hyperphosphorylated microtubule-associated protein tau is the major component of NFTs and mediates amyloid-β (Aβ), a key component of plaques, and induces impairment of long- term potentiation [3, 5]. Therefore, proper modulation of tau phosphorylation may be a step in arresting AD [7]. However, the upstream effectors leading to tau hyperphosphorylation are still not fully understood. Although elevated levels of Bip were found in the temporal cortex and hippocampus of AD patien t s , th e e videnc e l in king Bip w ith tau hyperphosphorylation and the underlying mechanism has been missing. Previously, we found that ER stress inducers, TG and TM, could induce increased Bip and tau phosphory- lation both in vivo and in vitro; downregulating Bip by transfecting its siRNA plasmid significantly rescued tau hyperphosphorylation in TG-treated HEK293/tau cells [18, 19]. Here, we directly transfected the plasmid of Bip into HEK293/tau and N2a cells and found Bip-transfected cells showed obviously increased tau phosphorylation at Ser396, Ser262, and Thr231 sites, which are important sites in AD-like phosphorylation of tau. Our data have provided the in vitro evidences showing that increasing of Bip could induce AD- like tau hyperphosphorylation. Fig. 4 Decreased SIL1 was found in ER stress and in the brains of Tg2576 mice. The levels of SIL1 in the cortex and hippocampus of 4- month-old (4m), 8-month-old (8m), and 12-month-old (12m) Tg2576 mice (Tg) and the age-matched C57/BL6 mice (C57) were tested by Western blotting (a, c) and quantitative analysis (b, d). Data are presented as means±SD (n=6). *p<0.05, **p<0.01 vs age-matched C57 mice. SIL1 in CA3 of the hippocampus (e) and cortex (f) of 2- month-old (2m), 5-month-old (5m), and 8-month-old (8m) Tg2576 mice (Tg) and the age-matched C57/BL6 mice (C57) was shown by immunohistochemistry staining (scale bar =20 μm). In 2 μM thapsigargin (TG)-treated (3 h) HEK293/tau cells (g, h) and brains of 50 μM tunicamycin (TM, 10 μl, 48 h) lateral ventricle-injected 4-month SD rats (same volume of solvent injection in rats as control, i and j), Bip and SIL1 were tested by Western blotting (g, i) and quantitative analysis (h, j). Data are presented as means±SD (n=6). *p<0.05, **p<0.01 vs control. The SIL1 antibodies used in Western blotting (a, c, g) were gifts from Dr. Linda M. Hendershot at the University of Tennessee Health Science Center (Memphis, TN). Tau hyperphosphorylation is the consequence of an imbalanced protein kinases and phosphatases. Among var- ious enzymes, GSK-3β and PP2A are respectively among the most implicated kinase and phosphatase in AD-like tau hyperphosphorylation [18, 19, 33, 34]. ER stress will trig- ger GSK-3β activation [37]. We previously found that TM and TG could induce tau hyperphosphorylation with si- multaneous activation of GSK-3β, whereas inhibiting GSK-3β by a specific inhibitor or expression of a domi- nant negative GSK-3β plasmid efficiently prevented tau from hyperphosphorylation [38]. By immunoprecipitation, we also found that the binding levels of Bip to tau and GSK-3β were significantly increased with the elevation of Bip in brains of TM-treated rats [19]. In this research, HEK293/tau and N2a cells overexpressing Bip showed increased phosphorylation of GSK-3β at Tyr 216 and decreased phosphorylation at Ser9, indicating the activa- tion of GSK-3β. The treatment of SB, an inhibitor of GSK- 3 β , r escued overexpressed B ip-induced tau hyperphosphorylation. These data showed that increased Bip could induce AD-like tau hyperphosphorylation by activating GSK-3β. The location of NFTs displays a region-specific pattern [5, 6]. In the human brain, the earliest NFTs are observed in the transentorhinal and entorhinal cortex (stages I and II). A more extensive involvement of the entorhinal cortex and the forma- tion of NFTs in sector CA1 of the hippocampus correspond to stages III and IV. In stages V and VI, abundant NFTs are presented in neocortical association areas. Subjects with stages I and II are cognitively unimpaired, whereas the stage III and IV ones may present with mild cognitive impairment, and stage Vand VI ones are severely demented. Till now, there is no evidence showing NFTs involve the cerebellum. Recent- ly, in 4-month-old SD rats, we observed increasing of Bip; increased phosphorylation of PERK, IRE-1, and ATF-6; acti- vation of GSK-3β; and increased phosphorylation of tau in the temporal cortex, frontal cortex, and hippocampus, but not the cerebellum [39]. Interestingly, the cerebellum keeps the highest Bip level in these four regions, nearly 2.2 times of that in the temporal cortex. The levels of PERK and IRE-1 and their phosphorylation have no difference in the cerebellum, temporal cortex, frontal cortex, and hippocampus. However, the level of ATF-6 and its phosphorylation are much lower in the cerebellum. And in the cerebellum, the phosphorylation of GSK-3β at Tyr216 is almost half and its phosphorylation at Ser9 is about twice as much as those in the temporal cortex, indicating the inactivation of GSK-3β in the cerebellum. These data indicate that in the cerebellum, ER stress has a higher stimulation threshold, which might be determined by the ratio of Bip and PERK, IRE-1, and/or ATF-6. And GSK- 3β activation is triggered by increasing of Bip, but not the level of Bip. Fig. 5 SIL1 rescued Bip overexpression-induced tau hyperphosphorylation and GSK- 3β activation. HEK293/tau cells were transfected with plasmids of vector, Bip, SIL1, and Bip together with SIL1, respectively. Forty-eight hours later, the phosphorylation levels of tau at Ser262 (pS262), Ser396 (pS396), Thr231 (pT231) sites; GSK-3β at Ser9 (S9-GSK-3β) and Tyr216 (Y216-GSK-3β) sites and PKC; and the total levels of tau, GSK- 3β, PKC, and PTP1B were tested by Western blotting (a, c, e) and quantitative analysis (b, d, f). Fig. 6 SIL1 rescued ER stress-induced tau hyperphosphorylation by reducing the association between Bip with tau and GSK-3β. HEK293/ tau cells were transfected with plasmids of vector, Bip, SIL1, and Bip together with SIL1, respectively. Forty-eight hours later, the cell lysate of different groups containing 500 μg protein was incubated overnight with 2 μg Bip antibody, respectively. Immunoprecipitation was performed as described in the “Materials and Methods,” and then the protein was tested by Western blotting (a) and quantitative analysis (b). To further investigate whether SIL1 could rescue ER stress-induced tau hyperphosphorylation, the plasmid of SIL1 and its vector were transfected into HEK293/tau cells, and 48 h later, cells were treated with 2 μM TG for 3 h. Then, the phosphorylation levels of tau at Ser396 (pS396) and Thr231 (pT231) sites and the total levels of tau were tested by Western blotting (c) and quantitative analysis (d). Data are presented as means±SD (n=4). *p<0.05, **p<0.01, ***p<0.001 vs vector-transfected cells; #p<0.05, ###p<0.001 vs Bip-solo-transfected cells. The activity of GSK-3β is regulated by the activity- dependent phosphorylation at Ser9 (inhibitory) and Tyr216 (activating). Fyn [22, 26] and Akt/PKB [27, 28] could phos- phorylate GSK-3β at Tyr216 and Ser9, respectively. In this research, overexpressing Bip did not affect the activities of these two enzymes. Inhibition of PKC [40] and PTP1B [24] directly increased GSK-3β activity. PTP1B, a ubiquitous tyrosine phosphatase, locates in the ER. In this research, PTP1B was decreased in Bip-overexpressed cells and is spec- ulated as the main reason of GSK-3β dephosphorylation at Tyr216. PKC phosphorylates GSK-3β at Ser9, and in Bip- overexpressed cells, the phosphorylation of PKC at Ser738 was decreased, indicating its inactivation. Besides the phos- phorylation regulation, protein complex formation also affects the activity of GSK-3β. Previously, we detected in ER stress an increasing association of Bip with GSK-3β and tau pro- teins (but not β-catenin) accompanied with an increased dis- sociation of Bip with PERK, IRE-1, and ATF-6 both in vitro and in vivo [19]. Here, in SIL1-transfected cells, Bip mainly combined with GSK-3β and SIL1, but not tau protein, which may be another reason to explain why SIL1-transfected cells showed less tau phosphorylation. In this research, SIL1 treat- ment rescued Bip overexpression-induced inhibition of PTP1B and PKC, and the activity of GSK-3β in Bip overex- pression was downregulated after SIL1 treatment. The under- lying mechanism needs further investigations. Fig. 7 The biochemical pathway regulating tau phosphorylation in this study. Bip is normally associated with the endoplasmic reticulum (ER) membranous proteins, such as protein kinase-like ER kinase (PERK) and inositol-requiring enzyme-1 (IRE-1). In ER stress, with the decreasing of SIL1, Bip is increased and dissociated with PERK and IRE-1, which will be phosphorylated after dissociation from Bip. Increased Bip activates GSK-3β, an important tau phosphorylation-related kinase in AD, by inhibiting PTP1B and PKC. Increased Bip also promotes its combination with GSK-3β and tau. Overexpressing SIL1 rescues increased Bip-induced tau hyperphosphorylation by decreasing the activation of GSK-3β and the binding of Bip with GSK-3β and tau. SIL1, a co-chaperon of Bip, stimulates the ATPase activity of Bip by promoting the release of ADP from Bip [11, 12]. Mutations in SIL1 cause Marinesco-Sjögren syndrome, a cerebellar ataxia [32]. Disruption of SIL1 contributes to pro- tein accumulation and neurodegeneration in the woozy mutant mouse [13]. In this research, SIL1 levels both in the cortex and hippocampus of Tg2576 mice were much lower than those in age-matched C57 mice, while Bip levels were higher. In TM- treated cell, SIL1 is decreased while Bip is increasing. These data showed the importance of SIL1 in ER stress-induced tau hyperphosphorylation. In summary, our data showed higher Bip and lower SIL1 in ER stress and in the brains of Tg2576 mice. Overexpression of Bip induced tau hyperphosphorylation by activating GSK-3β and promoting its combination with GSK-3β and tau. Increased SIL1 in cells made Bip combine with less tau and rescued overexpressed Bip- induced GSK-3β activation, which leads to downregula- tion of tau hyperphosphorylation. All these data indicate important roles of Bip and its co-chaperon SIL1 in ER stress-induced tau hyperphosphorylation.