Beta-amyloid oligomers induce phosphorylation of tau and inactivation of insulin receptor substrate via c-Jun N-terminal kinase signaling: suppression by omega-3 fatty acids and curcumin

Abstract

Both insulin resistance (type II diabetes) and beta-amyloid (Abeta) oligomers are implicated in Alzheimer's disease (AD). Here, we investigate the role of Abeta oligomer-induced c-Jun N-terminal kinase (JNK) activation leading to phosphorylation and degradation of the adaptor protein insulin receptor substrate-1 (IRS-1). IRS-1 couples insulin and other trophic factor receptors to downstream kinases and neuroprotective signaling. Increased phospho-IRS-1 is found in AD brain and insulin-resistant tissues from diabetics. Here, we report Abeta oligomers significantly increased active JNK and phosphorylation of IRS-1 (Ser616) and tau (Ser422) in cultured hippocampal neurons, whereas JNK inhibition blocked these responses. The omega-3 fatty acid docosahexaenoic acid (DHA) similarly inhibited JNK and the phosphorylation of IRS-1 and tau in cultured hippocampal neurons. Feeding 3xTg-AD transgenic mice a diet high in saturated and omega-6 fat increased active JNK and phosphorylated IRS-1 and tau. Treatment of the 3xTg-AD mice on high-fat diet with fish oil or curcumin or a combination of both for 4 months reduced phosphorylated JNK, IRS-1, and tau and prevented the degradation of total IRS-1. This was accompanied by improvement in Y-maze performance. Mice fed with fish oil and curcumin for 1 month had more significant effects on Y-maze, and the combination showed more significant inhibition of JNK, IRS-1, and tau phosphorylation. These data indicate JNK mediates Abeta oligomer inactivation of IRS-1 and phospho-tau pathology and that dietary treatment with fish oil/DHA, curcumin, or a combination of both has the potential to improve insulin/trophic signaling and cognitive deficits in AD.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1.

Redistribution of phosphorylated IRS-1 in AD and in 3xTg-AD mice and its enhancement by high-fat bad diet in 3xTg-AD mice. Using an antibody to the pS312 phosphoepitope of IRS-1, confocal immunocytochemistry illustrated that neurons from control human hippocampus (hippo) expressed high levels in the nucleus (A), whereas in AD patients, it was redistributed in the cytosol, sometimes in a granulovacuolar pattern (B). Around plaques, increased IRS-1 staining colocalized with dystrophic neurites (C). Antibodies to phosphorylated IRS-1 Ser616 typically stained tangles (D) and dystrophic neurites (E). IRS-1 pS312 colocalized with PHF-1 (F). Compared with 3-month-old control mice, which showed hippocampal CA1 nuclear staining of IRS-1 pS312 (G), 3xTg-AD mice had elevated IRS-1 pS312 in cytosol (H), which increased at 7–8 months of age (I). By 18 months of age, there was extensive colocalization of IRS-1 ps312 with PHF-1 (J). This transgene effect was also seen in animals using another IRS-1 antibody (pS616) in an experiment in which 5-month-old 3xTg-AD mice were fed with a HFBD (see text) for 4 months, and the phosphorylation of IRS-1 Ser616 was examined. Compared with Tg− control mice (K), 3xAD-Tg+ mice showed more cytosolic pS616 IRS-1 labeling and minimal nuclear labeling at 9 months of age (L). Scale bars, 20 μm.

Figure 2.

Immunostaining of phosphorylated IRS-1 in AD brain was variable and partially colocalized with the GVD. A, Immunostaining of phospho-IRS-1 Ser312 showed increased IRS-1 staining (green) typically in tangle-bearing neurons (AT8; red), and this colocalized with AT8. B, C, pIRS-1 Ser312 labeled apparent GVD. pIRS-1 was occasionally colocalized with phosphorylated tau in GVD (arrowheads indicate neurons containing granules). D, No phospho-IRS-1 and tau staining were observed in the absence of primary antibodies. Scale bar, 20 μm.

Figure 3.

Total IRS-1 was reduced in membrane extract of hippocampus in 3xTg-AD compared with Tg− control mice. Detergent lysis buffer extracts of hippocampal membrane pellets from 3xTg-AD (n = 8 for standard diet; n = 9 for high-fat bad diet) and Tg− controls (n = 8 for each group) were evaluated by Western for total IRS-1 (tIRS-1). Total IRS-1 was significantly reduced in transgene-positive mice (4.324 ± 0.248 for standard diet group; 5.015 ± 0.483 for high-fat bad diet group) compared with transgene-negative controls (6.232 ± 0.537 for standard group; 6.816 ± 0.576 for high-fat bad diet group) (p < 0.01, p < 0.05, respectively). Shown are mean ± SE. The Western immunoblotting was repeated three times. No change was observed in IRS-1 in TBS fractions (data not shown).

Figure 4.

Aβ42 oligomers induced aberrant inactivation of IRS-1 Ser616 in cultured hippocampal neurons. Western immunoblot analysis of pIRS-1 induced by Aβ42 oligomers in primary hippocampal neurons. Hippocampal neurons cultured for 9 DIV were treated with 100 nm Aβ oligomers. Harvested cells were then sonicated in lysis buffer and Western blotted with anti-pIRS-1Ser616 antibody. pIRS-1 levels were significantly increased in the membrane fraction after 5 h of Aβ42 oligomer-treatment (17.218 ± 0.922) when compared with controls (7.170 ± 0.674) (p = 0.013). β-Actin was used to normalize protein loading. Shown are mean ± SE. The experiment was repeated three times.

Figure 5.

Aβ42 oligomers induced aberrant activation of JNK-sensitive tau Ser422 in cultured hippocampal neurons. Western immunoblot analysis of ptau induced by Aβ42 oligomers in primary hippocampal neurons. Lysis fractions from 9 DIV hippocampal neurons treated with or without 100 nm Aβ oligomers were Western blotted with an anti-ptau-1Ser422 antibody. ptau levels were significantly increased in the cytosol fraction after 5 h in Aβ42 oligomer-treated neurons (21.161 ± 2.407) when compared with controls (5.488 ± 1.000) (p = 0.004). β-Actin was used for normalization. Shown are mean ± SE. The experiment was repeated three times.

Figure 6.

JNK inhibitor SP600125 blocked Aβ oligomer-induced aberrant inactivation of IRS-1 Ser616 and JNK-sensitive tau Ser422 phosphorylation in cultured hippocampal neurons. Hippocampal neurons were grown for 9 DIV and then pretreated with 10 μm JNK inhibitor, SP600125, for 30 min, followed by 100 nm Aβ42 oligomers for 5 h. After harvesting cells, pIRS-1 and ptau were evaluated by Western blot. A, B, Western immunoblot analysis of IRS-1 phosphorylation revealed induction by Aβ42 oligomers, which was antagonized by JNK inhibitor SP600125. pIRS-1 protein levels in membrane fractions were significantly increased by Aβ (B) (**p < 0.01), whereas JNK inhibitor SP600125 blocked the phosphorylated IRS-1 when compared with control (CTRL) (B) (**p < 0.01). A, C, Western immunoblot showed ptau was elevated by Aβ42 oligomers and JNK inhibitor SP600125 blocked this. ptau protein levels were significantly increased in cytosol (C) (***p = 0.001), whereas JNK inhibitor SP600125 significantly blocked the elevated ptau when compared with CTRL (C) (***p = 0.001). β-Actin was used for normalization. Error bars indicate SEM.

Figure 7.

DHA suppressed Aβ oligomer-induced aberrant inactivation of IRS-1 Ser616 and JNK-sensitive tau Ser422 phosphorylation in cultured hippocampal neurons. Hippocampal neurons were cultured for 9 DIV and then pretreated either with 2.5 μm DHA for 48 h or with the JNK inhibitor SP600125 (10 μm) for 30 min, followed by 100 nm Aβ42 oligomers for 5 h, after which pIRS-1 and ptau were evaluated by Western blot. A, B, Western immunoblot analysis of pIRS-1 showed induction by Aβ42 oligomers, a response antagonized by either DHA or JNK inhibitor (SP600125). pIRS-1 protein levels were significantly increased in membrane fractions (B) (***p < 0.001) by Aβ42 oligomers, but not if DHA or JNK inhibitor SP600125 was also present (B). A, C, Western immunoblot analysis shows that ptau was induced by Aβ42 oligomers, a response that was blocked by JNK inhibitor SP600125. ptau protein levels were significantly increased in cytosol (C) (***p < 0.001), but not if DHA or JNK inhibitor SP600125 was also present. β-Actin was used for normalization for protein loading. Error bars indicate SEM.

Figure 8.

Effect of HFBD, fish oil, curcumin, and the combination on phosphorylation of JNK, IRS-1, and tau in 3xTg-AD mice. A–C, Five-month-old 3xTg-AD transgenic-positive mice were fed standard mouse chow or with a HFBD (see text) for 4 months, and the phosphorylation of JNK, IRS-1 Ser616, and tau Ser422 were evaluated from detergent lysis buffer-extracted hippocampal membrane pellet fractions by Western blot. The levels of pJNK, pIRS-1 Ser616, and ptau Ser422 were significantly increased from HBFD 3xTg-AD mice when compared with control standard diet mice (A; pJNK, *p = 0.022) (B; pIRS-1, *p = 0.053) (C; ptau, **p = 0.004). For treatments, 5-month-old 3xTg-AD mice were fed with HFBD or HBFD plus 2.4% fish oil (FO), or HBFD plus 500 ppm curcumin or HBFD plus both FO and curcumin for 4 months, and the phosphorylation of JNK, IRS-1 Ser616, and tau Ser422 were evaluated from detergent lysis buffer-extracted hippocampal membrane pellet fractions by Western blot. The levels of pJNK were significantly reduced from fish oil-treated HFBD 3xTg-AD mice (A; *p = 0.025), the serine phosphorylated (inactivated) IRS-1 (B; p = 0.073), and phospho-tau (C; p = 0.078) showed a trend toward reduction when compared with HFBD alone control mice. The activated JNK (A; p = 0.059) showed a trend to be reduced by curcumin treatment in 3xTg-AD, whereas IRS-1 Ser616 (B; *p = 0.05) and ptau Ser422 (C; *p = 0.027) were significantly reduced when compared with 3xAD-Tg mice on HFBD alone. The combination of fish oil and curcumin significantly suppressed all three endpoints, pJNK (A; **p = 0.007), pIRS-1 (B; *p = 0.04), and ptau (C; *p = 0.016) in 3xTg-AD mice when compared with 3xAD-Tg mice on HFBD alone. β-Actin was used for normalization. Error bars indicate SEM.

Figure 9.

Effect of HFBD, fish oil, curcumin, and the combination on total IRS-1 in 3xTg-AD mice. Five-month-old 3xTg-AD mice were fed with HFBD or HBFD plus fish oil or curcumin alone or in combination for 4 months and total IRS-1 (tIRS-1) was evaluated by Western blot. The levels of IRS-1 were significantly increased in membrane fractions from all treatment groups; fish oil increased levels by 17.3% (**p < 0.01), curcumin by 11.2% (*p < 0.05), and the combination of them by 11.2% (*p < 0.05) when compared with HFBD alone in 3xTg-AD mice. Error bars indicate SEM.

Figure 10.

Curcumin and/or fish oil prevent cognitive deficits. Spontaneous alternation tests of 3xTg-AD mice compared with wild-type (WT) controls of the same age and background strain using Y-maze were performed after 1 or 2 months of treatment. WT mice had no decline with age on the HFBD. In contrast, the 3xTg-AD mice exhibited a significant behavior deficit developing from 5 to 7 months of age, which was more evident in the HFBD (significant from WT after 1 month on diet, **p < 0.01) (6 months of age). Only fish oil plus curcumin (Cur) added to HFBD base diet corrected the deficit at 1 month (**p < 0.01), but all three treatments (fish oil, curcumin, fish oil plus curcumin) prevented additional cognitive decline after 2 months of treatment (**p < 0.01 for fish oil and curcumin, *p < 0.05 for fish oil plus curcumin). Error bars indicate SEM.