Glucose deprivation increases tau phosphorylation via P38 mitogen-activated protein kinase

Abstract

Alterations of glucose metabolism have been observed in Alzheimer's disease (AD) brain. Previous studies showed that glucose deprivation increases amyloidogenesis via a BACE-1-dependent mechanism. However, no data are available on the effect that this condition may have on tau phosphorylation. In this study, we exposed neuronal cells to a glucose-free medium and investigated the effect on tau phosphorylation. Compared with controls, cells incubated in the absence of glucose had a significant increase in tau phosphorylation at epitopes Ser202/Thr205 and Ser404, which was associated with a selective activation of the P38 mitogen-activated protein kinase. Pharmacological inhibition of this kinase prevented the increase in tau phosphorylation, while fluorescence studies revealed its co-localization with phosphorylated tau. The activation of P38 was secondary to the action of the apoptosis signal-regulating kinase 1, as its down-regulation prevented it. Finally, glucose deprivation induced cell apoptosis, which was associated with a significant increase in both caspase 3 and caspase 12 active forms. Taken together, our studies reveal a new mechanism whereby glucose deprivation can modulate AD pathogenesis by influencing tau phosphorylation and suggest that this pathway may be a new therapeutic target for AD.

Keywords: Alzheimer's disease; amyloid beta; glucose deprivation; mitogen-activated protein kinase; neuronal cells; tau phosphorylation.

Figure 1

Glucose deprivation modulates tau phosphorylation in neuronal cells. (A) Representative Western blot analyses for total tau (Tau‐1) and phosphorylated tau at residues S202/T205 (AT8), T231/S235 (AT180), T181 (AT270), S396/S404 (PHF‐1), and S396 (PHF‐13) in lysates from neuronal cells incubated 24 h in either normal DMEM (CTR) or DMEM without glucose (‐GLU). (B) Densitometric analyses of the immunoreactivities shown in the previous panel (n = 6; *P < 0.05; **P < 0.001). (C) Representative Western blot analyses for (SAPK)/JNK and (pSAPK)/JNK, GSK‐3α, GSK‐3β, pGSK‐3α, pGSK3β, CDK5, P35 P25, PP‐2A, P38, and pP38 in lysates from neuronal cells incubated 24 h in either normal DMEM (CTR) or DMEM without glucose (‐GLU). (D) Densitometric analyses of the immunoreactivities shown in the previous panel (n = 6; *P < 0.05; **P < 0.001). Results are mean ± SEM.

Figure 2

P38 mitogen‐activated protein kinase mediates glucose deprivation‐induced tau phosphorylation. (A) Representative Western blot analyses of P38, pP38, phosphorylated tau at residues S202/T205 (AT8), and S396/S404 (PHF‐1), in lysates from cells treated with vehicle or SB20358 (20 μm) (SB), in either normal DMEM (CTR) or DMEM without glucose (‐GLU). (B) Densitometric analyses of the immunoreactivities to the antibodies presented in the previous panel (n = 4; *P = 0.05). Results are mean ± SEM. (C) Representative images of immunofluorescence analysis for pP38 and AT8 in neuronal cells treated with vehicle or SB20358 (20 μm) (SB), in either normal DMEM (CTR) or DMEM without glucose (‐GLU) (scale bar: 20 μm). (D) Quantitative analysis of the immune co‐localization signal for pP38 and AT8 as observed in panel C.

Figure 3

Involvement of ASK1 in the glucose deprivation‐induced P38 activation and tau phosphorylation. (A) Representative Western blot analysis for ASK1, total tau (Tau‐1), and tau phosphorylated at S202/T205 as recognized by the antibodies AT8, P38, and pP38 in lysates from neuronal cells preincubated with siRNA ASK1 (0.1 μmol L^‐1) and then challenged with glucose‐free media (‐GLU) or regular medium (CTR). (B) Densitometric analyses of the immunoreactivities shown in the previous panel (n = 4; *P < 0.05; **P < 0.005). Results are mean ± SEM.

Figure 4

Glucose deprivation‐dependent neuronal apoptosis is mediated by caspases 12 and 3. (A) TUNEL assay. N2A cells treated with vehicle or SB20358 (20 μm) (SB), in either normal DMEM (CTR) or DMEM without glucose (‐GLU), were subjected to TUNEL assay and counterstained with propidium iodide (PI). Positive controls were prepared using DNase I (scale bar: 50 lm). (B) Representative Western blot analysis for procaspase‐12, procaspase‐3, procaspase‐7, caspase‐12, caspase‐3, and caspase‐7 in lysates from neuronal cells treated with vehicle or SB20358 (20 μm) (SB) in normal DMEM (CTR) or glucose‐free DMEM (‐GLU). (C) Densitometric analyses of the immunoreactivities are shown in the previous panel (n = 4; *P < 0.05; **P < 0.001). Results are mean ± SEM.