SEARCH

SEARCH BY CITATION

Keywords:

  • Akt;
  • Alzheimer disease;
  • GSK-3beta;
  • neuroglobin;
  • tau hyperphosphorylation

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

J. Neurochem. (2012) 120, 157–164.

Abstract

Neuroglobin (Ngb) is a recently identified member of hemoglobin family, distributed mainly in central and peripheral nervous systems. Recent studies suggest that Ngb can protect neural cells from β-amyloid-induced toxicity in Alzheimer disease (AD). Hyperphosphorylation of tau is another characterized pathological hallmark in the AD brains; however, it is not reported whether Ngb also affects tau phosphorylation. In this study, we found that the level of Ngb was significantly reduced in Tg2576 mice (a recognized mouse model of AD) and TgMAPt mice, and the level of Ngb was negatively correlated with tau phosphorylation. Over-expression of Ngb attenuates tau hyperphosphorylation at multiple AD-related sites induced by up-regulation of glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase. While Ngb activates Akt and thus inhibits GSK-3β, simultaneously inhibition of Akt abolishes the effects of Ngb on GSK-3β inhibition and tau hyperphosphorylation. Our data indicate that Ngb may attenuate tau hyperphosphorylation through activating Akt signaling pathway, implying a therapeutic target for AD.

Abbreviations used

β-amyloid

AD

Alzheimer disease

GFP

green fluorescence protein

GFX

GF-109203X

GSK-3β

glycogen synthase kinase-3β

PBS

phosphate-buffered saline

SDS

sodium dodecyl sulfate

TCN

Triciribine

Neuroglobin (Ngb) was identified as the third globin with its specific localization in the nervous system (Burmester et al. 2000). As a new member of globin family, Ngb participates in cellular oxygen homeostasis and acts as an endogenous neuroprotector (Kelsen et al. 2008). Expression of Ngb mRNA and protein can be induced by hypoxia in cerebral neuronal culture (Sun et al. 2001). Over-expression of Ngb in cells causes a reduced sensitivity to hypoxia or oxidative injury (Fordel et al. 2006; Li et al. 2008a; Duong et al. 2009). The mice with Ngb over-expression are resistant to ischemia in cerebral neurons and myocardial cells (Sun et al. 2003; Khan et al. 2006; Yu et al. 2009; Li et al. 2010). Additionally, Ngb over-expression protects cells from oxidative stress-induced death. These data indicate that Ngb posseses a wider neuroprotective role (Greenberg et al. 2008). Both the mRNA and protein levels of Ngb in several brain regions of rat and human are decreased with age, implying a possible relation between Ngb deficiency and age-related neurodegeneration (Sun et al. 2005; Szymanski et al. 2010).

Alzheimer’s disease (AD) is the most common neurodegenerative disorder in the elderly. Intracellular neurofibrillary tangles consisted of hyperphosphorylated tau and extracellular senile plaques deposits formed by β-amyloid (Aβ) accumulation are the most prominent pathological hallmarks of AD (Iqbal et al. 1986; Koh et al. 1990; Selkoe 1994). It is reported most recently that an age-related decline of Ngb is correlated with an increased risk of AD (Szymanski et al. 2010). Ngb Over-expression of Ngb decreases the levels of Aβ-induced reactive oxidative species and lipid peroxidation in PC12 cells (Li et al. 2008b). Ngb also reduces the toxic effects of N-methyl-d-aspartic acid (NMDA) in cortical neuron cultures, including membrane polarization, mitochondrial aggregation and cell death (Khan et al. 2007a). In AD transgenic (APPSw,Ind) mice, over-expression of Ngb decreases the levels of Aβ(1–40) and Aβ(1–42) and improves cognitive performance (Khan et al. 2007a,b). Thus, Ngb should be an effective target to arrest Aβ toxicity. To date, there has been no direct evidence showing the effects of Ngb on tau hyperphosphorylation, another characterized pathological change in the AD brains.

In the present study, we demonstrated that the level of Ngb was significant reduced in Tg2576 and TgMapt mice, well recognized AD model, and the level of Ngb was negatively correlated with the phosphorylated tau. Over-expression of Ngb not only prevents tau from hyperphosphorylation at multiple AD-related sites induced by up-regulation of glycogen synthase kinase-3β (GSK-3β), but also alleviates tau phosphorylation in the basal level. Ngb inhibits GSK-3β and attenuates tau hyperphosphorylation by activating Akt. We propose that Ngb may serve as therapeutic target for arresting AD-like tau hyperphosphorylation.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animals and treatment

C56BL/6, Tg2576 and Tg(MAPT)8cPdav/J purchased from the Jackson Laboratory, were housed with accessible food and water ad libitum. All animal experiments were performed according to the ‘Policies on the Use of Animals and Humans in Neuroscience Research’ revised and approved by the Society for Neuroscience in 1995. Mice were kept on a 12-h light/dark cycle with the light on from 7:00 am to 7:00 pm. Mice were humanely decapitated after inhalation anesthesia. The brains were rapidly removed and homogenized at 4°C using a Teflon glass homogenizer in 50 mM Tris–HCl, pH7.4, 150 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 5 mM EDTA, 2 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride. The extract was mixed with sample buffer (3 : 1, v/v) containing 200 mM Tris–HCl, pH 7.6, 8% sodium dodecyl sulfate (SDS), 40% glycerol, 40 mM dithiothreitol, boiled for 10 min and then centrifuged at 12 000 g for 10 min at 25°C. The supernatant was stored at 80°C for western blotting analysis.

Chemicals and antibodies

Wortmannin (Wort) and GF-109203X (GFX) purchased from Sigma Chemical Co. (St Louis, MO, USA). Triciribine (TCN) was from Merck Chemical, Inc. (Whitehouse Station, NJ, USA). Ngb chicken polyclonal antibody was from Biovendor Research and Diagnostic products (CTPark, Evropska, Czech Republic). Mouse monoclonal antibody (mAb) tau-5 against total tau was purchased from Lab Vision Co. (Westinghouse, CA, USA). mAb Tau-1 against unphosphorylated tau at Ser195/198/199/202 was from Chemicon International Inc (Temecula, CA, USA). Polyclonal antibodies (pAbs) pS396, pS404, pT231, and pT205 against tau phosphorylated at the corresponding sites were from Signalway antibody Co., Ltd. (Pearland, TX, USA). mAb against AT8 (1 : 1000 for Western, 1 : 200 for immunohistochemistry) from Thermo Fisher (Waltham, MA, USA). pAb anti-total GSK-3β and pAb anti-phospho-GSK-3β at Ser9, pAb anti-total p85 and pAb anti-phospho-p85 at Thr458, mAb anti-HA-tag were from Cell Signaling Technology (Beverly, MA, USA). pAb anti-total Akt, anti-phospho-Akt at Ser473 and Ser308, anti-green fluorescence protein (GFP) were from Abcam Co. (Cambridge, MA, USA). The wild type GSK-3β (wtGSK-3β) plasmid with HA-tag was a gift from DrWoodgett at Toronto University, Canada.

Cell culture

The human embryonic kidney 293 (HEK293) cells were cultured in Dulbecco’smodified eagle’s medium supplemented with 10% fetal bovine serum (Gibico BRL, Gaithersburg, MD, USA) and grown at 37°C in a humidified atmosphere containing 5% CO2. Transfection with the longest human tau (tau441) cDNA (HEK293/tau) was carried out with Lipofectamine 2000 transfection kit according to the manufacturer’s instructions. Stably tau-transfected HEK293 cells were selected, cloned by dilution, and maintained in the presence of G418 (250 μg/mL). The expression of tau was identified by western blot. Primary neuron cultures isolated from embryonic 18-day-old (E18) rats were cultured in neurobasal and 2% B27 (Invitrogen, Carlsbad, CA, USA) as previously describe (Zhu et al. 2007, 2010).

For plasmids transfection, HEK293 was seeded in six-well plates and grown to 60–70% confluence, and then cultured in serum- and antibiotic-free OPTI-MEM for 4 hours. Plasmids were transfected by using Lipofectamine 2000 according to the manufacturer’s instruction. Briefly, Lipofectamine (10 μL) and DNA plasmid (Ngb), or its vector pEGFP-N1 (4 μg) were diluted in 250 μL of OPTI-MEM followed by equilibration at 15–25°C for 10 min after mixing. The Lipofectamine-DNA complex was added to HEK293 and the cells was incubated at 37°C for 6–8 h. Cells transfected with GFP constructs were visualized at 48 h after the transfection by an Olympus IX70 microscope with a 209LCPlanF1 lens. For double-transfection, the Ngb and GSK-3β plasmids were mixed with the ratio of 1 : 1. To induce tau hyperphosphorylation, 293/tau cell was incubated with Wort (1 μΜ) and GFX (1 μΜ) for 1 h.

Western blotting

Western blotting was carried out according to the well-established method in our laboratory. Briefly, after rinsing twice by ice-cold phosphate-buffered saline (PBS) (pH 7.5), the cells were lysed with RIPA buffer (50 mM Tris–Cl, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 0.02% NaN3, 100 μg/mL phenylmethanesulfonyl fluoride, and 10 μg/mL each of the protease inhibitors) and subjected to sonication for 5 s on ice. Then, an extracting buffer (200 mM Tris–Cl, pH 7.6, 8% SDS, 40% glycerol) was added to the homogenate with a ratio of 1 : 3. The protein concentration in the extracts was measured by BCA kit (Pierce, Rockford, IL, USA). The protein was separated by 10% SDS–polyacrylamide gel electrophoresis gel, and then transferred to NC membrane. After blocking in 5% non-fat milk for 1 h at 15–25°C, the membranes were then incubated with primary antibodies at 4°C overnight. Finally, the blots were incubated with anti-rabbit or anti-mouse IgG conjugated to IRDye™ (800CW) for 1 h at 15–25°C and visualized using the Odyssey Infrared Imaging System (Licor biosciences, Lincoln, NE, USA).

Immunofluorescence and confocal microscopy

For brain section immunofluorescence studies, mice were killed by overdose chloral hydrate (1 g/kg) and perfused through aorta with 100 mL 0.9% NaCl followed by 400 mL phosphate buffer containing 4% paraformaldehyde. Brains were removed and post-fixed in perfusate overnight and then cut into sections (20 μm) with a vibratome (Leica, Nussloch, Germany; S100, TPI). The sections of mice brain were collected consecutively in PBS for immunofluorescence staining. Free-floating sections were incubated bovine serum albumin to block non-specific sites for 30 min at 15–25°C. Sections were then incubated overnight at 4°C with primary antibodies chicken polyclonal Ngb antibody for 48 h. After washing with PBS, sections were subsequently incubated with pT231 for 48 h. After washed with PBS for 30 min, sections were subsequently incubated secondary antibodies Alexa Fluor 488 (donkey anti chicken) or Cy5 (goat anti chicken) and Alexa Fluor 546 (goat anti rabbit) for 1 h at 37°C. The images were captured with a laser confocal microscope (LSM710 Carl Zeiss).

Cells or primary neurons 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% bovine serum albumin in PBS for 1 h and incubated with primary antibody at 4°C overnight. The cell was probed using Alexa Fluor 488 (donkey anti chicken) and Alexa Fluor 546 (goat anti rabbit or mouse) secondary antibodies (Invitrogen) for 2 h at 15–25°C. All the procedures were performed according to the established method (Zhu et al. 2007). The images were visualized with a laser confocal microscope (LSM710 Carl Zeiss).

Fluorescent intensity analysis

The quantitative data of the immunofluorescence intensity measured from about 40 to 90 cells in three independent experiments. The quantitative data of the immunofluorescence intensity measures in an individual cell body or neurite as indicated. The quantitative analysis was carried out by using the ‘measure IOD’ function of Image Pro plus according to the manufacture’s instruction (see page 1–66 of the IPP startUP manual) as reportedly previously (Shi et al. 2008).

Statistical analysis

All the data was analyzed by using SPSS 10.0 statistical software (SPSS Inc., Chicago, IL, USA). The different means among groups was determined by one-way anova procedure followed by LSD’s post hoc test.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Negative correlation of Ngb with the phosphorylated tau levels in mice

To explore the possible involvement of Ngb in tau hyperphosphorylation, we first examined the protein level in Ngb in Tg2576 mice. We found that with the dramatically elevated phosphorylation level of tau in 13 month Tg2576 mice, the level of Ngb display a significant decrement compared with the normal littermates (Fig. 1a and b, Figure S1a and b). We then measured the co-localization of the phosphorylated tau with Ngb in the brain of Tg2576 (well-known AD mice models) or TgMapt (human tau over-expression) and control mice (C57BL/6). We found that the immunoreaction of Ngb was significantly decreased with an enhanced phosphorylation level of tau in both Tg2576 mice and TgMapt mice (Fig. 1a, Figure S1). By employing a well-known analyzing method for the fluoresenct intensity, we also found a negative correlation of Ngb with the hyperphosphorylated tau at both pT231 (Fig. 1c and d) and pS396 (Fig. 1e and f) sites but not total tau (Tau5, Fig. 1g and h) in the neurons of Tg2576 mice. Similar data were also got from the TgMapt mice (Figure S1c). These data suggest that Ngb may arrest tau hyperphosphorylation.

image

Figure 1.  Reduction of Ngb in the brain of Tg2576 mice and a negative correlation of Ngb with the phosphorylated Tau. (a, b) Brain lysis from 13-month-old Tg2576 and C57BL/6. Tau phosphorylation was analyzed by western blotting using a panel of phosphorylation sites-specific antibodies as labeled. **p < 0.01 vs C57BL/6. (c, e, g) Brain sections prepared from 13-month-old Tg2576 were respectively co-stained with pT231, pS396, Tau5 (red) and Ngb (green); the neurons with higher Ngb expression show weaker staining of p-Tau. (arrows), and the neurons with lower Ngb expression show stronger staining of p-Tau. (arrowheads). Scale bar = 20 μm. (d, f, h) Correlative analysis of the fluorescent intensity of p-Tau or total tau and Ngb in Tg2576 mice. The brain regions include parietal lobe, temporal lobe, thalamus, hypothalamus, amygdaloid nucleus for analysis. Each neuron includes cell body, neurite for intensity analysis (about 90 cells were analyzed by Image Pro Plus 4.5). p < 0.01, R 2 = 0.689(pT231), R2 = 0.672 (pS396), p > 0.05, R 2 = 0.007 (Tau5).

Download figure to PowerPoint

Over-expression of Ngb attenuates tau hyperphosphorylation induced by up-regulation of GSK-3β

To verify the role of Ngb in tau phosphorylation, we measured whether over-expression of Ngb could attenuate tau hyperphosphorylation induced by Wort and GFX (activation of GSK-3β) in HEK293/tau cells. As reported previously, treatment of the cells with Wort and GFX for 1 h induced tau hyperphosphorylation at Ser396 (pS396), Ser404 (pS404), Thr231 (pT231) and Ser195/198/199/202 (Tau-1) epitopes (Liu et al. 2003; Shi et al. 2008), all of which are GSK-3β dominated sites (Wang et al. 2007) (Fig. 2a) We found that over-expression of Ngb effectively attenuated tau hyperphosphorylation at multiple sites (Fig. 2a and c). We also observed that Ngb could inhibit endogenous tau phosphorylation at pS214, pT231 pS396 and pS404 in the absence Wort/GFX (Fig. 2a and b). No obvious change was detected in total tau level probed by Tau5 after normalization to GFP (Fig. 2a). By double-immunofluorescent imaging, we observed that the fluorescent intensity of tau at pT231, pS404 and pS396 (Fig. 3a and b, red) was much weaker and Tau1 much stronger in the cells with Ngb over-expression (Fig. 3a and b, green), indicating that Ngb over-expression reduces tau phosphorylation at Thr231, Ser404, Ser396 and Ser195/198/199/202 sites. A negative correlation of Ngb and p-Tau was also detected in primary hippocampal neuron cultures (Fig. 3c). Concomitantly, Ngb over-expression can also reduce the endogenous tau phosphorylation both in the HEK293/tau (Figure S2a and b) and primary cultures (Figure S2c). These results suggest that Ngb can attenuate GSK-3-induced tau hyperphosphorylation at multiple AD-related sites.

image

Figure 2.  Over-expression of Ngb attenuates tau hyperphosphorylation induced by Wort+GFX in HEK293 cells. (a) HEK 293/tau cells were transfected with pEGFP-N1 (Con) or EGFP-Ngb (Ngb) for 48 h, and then treated with Wort+GFX (1 μM each) or vehicle for 1 h. Tau phosphorylation was analyzed by western blotting using a panel of phosphorylation sites-specific antibodies as labeled. (b, c) Quantitative analyses of the blots without (b) or with (c) Wort + GFX treatment. The phosphorylation level of tau was normalized against total tau probed by Tau-5, and the total level of tau was normalized against GFP. *p < 0.05, **p < 0.01 versus pEGFP-N1.

Download figure to PowerPoint

image

Figure 3.  Over-expression of Ngb attenuates tau hyperphosphorylation with Wort+GFX in cell culture. (a, b) Co-immunofluorescent staining of tau and Ngb in HEK 293/tau cells and the quantitative analyses of the cells (including cell body, neurite for intensity analysis in each neuron, n = ∼30) with (arrow) or without (arrowhead) Ngb over-expression. **p < 0.01 versus pEGFP-N1. (c) The neurons were transfected with pEGFP-N1 or EGFP-Ngb at 7 days in vitro (div) and then fixed at 9 div after treated with Wort + GFX (1 μM each) for 1 h for staining Tau (red) and Ngb (green). pT231, pS214, and pS396 react with the phosphorylated tau and Tau-1 reacts with the unphosphorylated tau.

Download figure to PowerPoint

To confirm the role of Ngb in attenuating GSK-3β-induced tau hyperphosphorylation, we co-transfected Ngb and GSK-3β (wtGSK-3β, HA-tagged) or control plasmid into HEK293/tau cells and measured the phosphorylation level of tau. We found that co-expression of Ngb and GSK-3β attenuated GSK-3β-induced tau hyperphosphorylation at pS195/198/199/202, pT205, pS396, pS214, pS404, and pT231 epitopes without affecting the total tau level (Fig. 4a and c). Simultaneously, co-transfection with Ngb and the vector of GSK-3β (pcDNA) also show decreasing tau phosphorylation level compared with the cells with pcDNA transfected alone (Fig. 4a and b). The expression of exogenous GSK-3β and Ngb were verified respectively by western blotting with anti-HA-tag and anti-GFP (Fig. 4a). These data further confirm that Ngb can attenuate GSK-3β-induced tau hyperphosphorylation.

image

Figure 4.  Over-expression of Ngb attenuates tau hyperphosphorylation induced by over-expression of GSK-3β. (a) HEK293/tau cells were co-transfected with pcDNA and pEGFP-N1, or pcDNA and EGFP-Ngb for 48 h as control. And HEK293/tau cells were co-transfected with wild-type GSK-3β (wtGSK-3β) and pEGFP-N1, or wtGSK-3β and EGFP-Ngb for 48 h. Then the level of the phosphorylated tau was measured by western blotting. (b) Quantitative analysis of tau phosphorylation with or without Ngb over-expression by transfection of pcDNA. (c) Quantitative analysis of tau phosphorylation with or without Ngb over-expression by transfection of GSK-3β.*p < 0.05, **p < 0.01 versus pEGFP-N1.

Download figure to PowerPoint

Ngb attenuates tau hyperphosphorylation by activating Akt signaling

To explore the regulation of Ngb on GSK-3β, we first measured alteration of GSK-3β activity by detecting the inhibitory phosphorylation at Ser9. We found that the level of pS9-GSK-3β in HEK293/tau cells decreased remarkably after Wort/GFX treatment, and expression of Ngb increased the level of pS9-GSK-3β in the presence and absence of Wort/GFX (Fig. 5a and b), suggesting that Ngb can inhibit GSK-3β by enhancing its phosphorylation at Ser9. It is well known that phosphoinositide 3-kinases (PI3K)/Akt is the upstream regulator of GSK-3β for its phosphorylation modification in mammalian cells (Cross et al. 1995). Therefore, we detected the activity of PI3K/Akt by measuring their phosphorylation levels. We found that over-expression of Ngb increased the phosphorylation level of Akt at Ser473 and Ser308 without affecting the phosphorylation level of p85-PI3K at Thr458 (Fig. 5c and d), indicating that Ngb can activate Akt but with no effect on p85-PI3K.

image

Figure 5.  Ngb inhibits GSK-3β, activates Akt with no effects on PI3K activity. (a, b) HEK293 cells were treated as described in Fig. 2. The levels of total GSK-3β and GSK-3β phosphorylated at serine-9 (pS9-GSK3β) were measured by western blotting and quantitative analysis (b). (c, d) HEK293 cells were transfected with pEGFP-N1 or EGFP-Ngb for 48 h. Then the levels of total Akt (t-Akt), Ser473-phosphorylated Akt (pS473-Akt), Ser308-phosphorylated Akt (pS308-Akt), p85-PI3K(p85), and Thr458-phosphorylated p85-PI3K(pT458-p85) were measured by western blotting (c) and quantitative analysis (d). *p < 0.05, **p < 0.01 versus pEGFP-N1.

Download figure to PowerPoint

To further verify the role of Akt in mediating the protection of Ngb on tau phosphorylation, we used TCN, a cell-permeable and reversible tricyclic nucleoside that selectively inhibits the cellular phosphorylation/activation of Akt1/2/3 without any inhibition to the known upstream activators of Akt, that is, PI3K or phosphoinositide-dependent kinase (PDK) (Yang et al. 2004; Karst et al. 2006). By examining the phosphorylation of Akt at Ser473, we tested the concentration gradients of TCN in inhibiting Akt. We observed that treatment of the cells with 2 μΜ TCN for 2 h could inhibit Akt to 30% of the control level and could also increase GSK-3β by decreasing its phosphorylation at Ser9 (Fig. 6a–c). With inhibition of Akt by TCN, the Ngb-induced tau dephosphorylation at Ser396, Ser404, Thr231, and Ser/195198/199/202 sites was diminished (Fig. 6d and e). Furthermore, TCN can also antagonist the effect of Ngb on alleviating endogenous tau phosphorylation (Fig. 6f and g). These data suggest that Ngb protects tau from GSK-3β-induced hyperphosphorylation through activating Akt.

image

Figure 6.  Ngb attenuates GSK-3β-induced tau phosphorylation by activating Akt signaling. (a) HEK293/tau cells were treated by TCN with different concentrations and the phosphorylation level of Akt and GSK-3β was detected by western blotting and quantitative analysis. (b, c) HEK293/tau cells were transfected with GSK-3β alone or co-transfected with GSK-3β and EGFP-Ngb for 48 h, then TCN (2 μM) was added for 2 h. The levels of pS473-Akt, t-Akt, p-Tau at Ser396, Ser404 and Thr231, and unP-Tau at Tau-1 epitopes, and total tau (Tau-5) were detected by western blotting (d) and quantitative analyses (e) *p < 0.05, **p < 0.01 versus co-transfected with GSK-3β and EGFP-Ngb. HEK293/tau cells were transfected with EGFP-Ngb for 48 h, then TCN (2 μM) was added for 2 h. The levels of pS473-Akt, t-Akt, p-Tau at Ser396, Ser404 and Thr231, and unP-Tau at Tau-1 epitopes, and total tau (Tau-5) were detected by western blotting (f) and quantitative analyses (g). #p < 0.05, ##p < 0.01 versus untransfected without TCN. *p < 0.05, **p < 0.01 versus untransfected with TCN.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In the present study, we observed that Ngb level was negatively correlated with tau phosphorylation in Tg2576 and TgMapt mice. By employing a well-established cell model with pharmacological or genetical up-regulation of GSK-3β (Shi et al. 2008), a critical tau kinase that also mediates Aβ production in AD models (Sun et al. 2002; Ryder et al. 2003; Wang et al. 2007), we found that Ngb over-expression could effectively rescue tau hyperphosphorylation at multiple AD-related sites by activating Akt. It is well known that tau hyperphosphorylation is crucial for the AD synaptic disorder and the number of neurofibrillary tangles (consists of hyperphosphorylated tau) is highly correlated with the degree of dementia. Given that Ngb could also attenuate Aβ toxicity, alleviate oxidative stress, eliminate reactive oxidative species (Li et al. 2008a, 2010, 2011), prevent lipid peroxidation, maintain mitochondrial function and resist to apoptosis (Brittain et al. 2010a,b; Raychaudhuri et al. 2010), all of which also participate in the AD pathogenesis, we propose that Ngb could be a promising therapeutic target for AD.

We also observed that over-expression of Ngb inhibits GSK-3β, demonstrated by an increased phosphorylation at Ser9. It is well known that Akt, a member of serine/threonine protein kinases, is an important upstream regulator for the inhibitory phosphorylation of GSK-3β at Ser9. Moreover, Akt-dependent signaling is linked to age-related structural and functional changes in human (Wu et al. 2010). Akt also plays crucial roles in cell survival, cell growth, gene expression, apoptosis, protein synthesis, energy metabolism and oncogenesis (Scheid and Woodgett 2001; Vivanco and Sawyers 2002). Therefore, we studied the involvement of Akt in Ngb-mediated GSK-3β inhibition and attenuation of tau phosphorylation. We found that over-expression of Ngb could activate Akt by enhancing its phosphorylation at Ser473 and Ser308, and simultaneous inhibition of Akt by TCN abolished Ngb-induced GSK-3β inhibition and tau hyperphosphorylation.

To explore whether Ngb affects PI3K, an immediate upstream kinase of Akt, we measured the activity-dependent phosphorylation of p85-PI3K at Thr458 (active form of PI3K). Unexpectedly, we observed that over-expression of Ngb did not change the phosphorylation level of p85-PI3K, suggesting that the increased Akt phosphorylation induced by Ngb over-expression was independent of p85-PI3K activation. A most recent study showed that Ngb could protect the H2O2 insults in SH-SY5Y cell via PI3K/Akt signaling (Antao et al. 2010). Given that HEK293 cells do not express endogenous Ngb, whereas the SH-SY5Y cells used by Witting, Paul K group express robustedly the endogenous Ngb. The discrepancy could be caused by the application of different cell lines. Moreover, the methods used for the detection of Akt/PI3K were also different (Antao et al. 2010). The detailed mechanism underlying the increased Akt phosphorylation/activation by Ngb deserves further investigation.

Taken together, we have found in the present study that over-expression of Ngb could protect GSK-3β-induced tau hyperphosphorylation at multiple AD-related sites, and the mechanism involves activation of Akt. Our findings suggest that Ngb could be a promising therapeutic target for AD-like tau hyperphosphorylation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported in part by grants from the Natural Science Foundation of China (30800342 and 30971478).

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Figure S1. Reduction of Ngb in the cortex of Tg2576 and TgMAPt mice.

Figure S2. Over-expression of Ngb attenuates endogenous tau phosphorylation in cell culture.

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
JNC_7275_sm_FigS1-2.pdf980KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.