Article Figures & Data
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.
Tables
- Table 1.
Cases of autopsy-confirmed AD and healthy control subjects used in the immunofluorescent staining
Postmortem cases No. of cases Age (range) (years) Postmortem interval (h) Senile plaques Braack stage Other pathologic changes Healthy controls 6 50∼90 11∼19 None I One case has a small aneurysm and shows only mild atherosclerotic change AD 8 64∼93 3.5∼15 Moderate to abundant plaques are mainly in dense core or diffuse III∼IV None Formula (g/kg) High-fat bad diet 0.6% DHA diet Curcumin diet 0.6% DHA plus curcumin diet Casein, ″vitamin-free″ test 200 200 200 200 dl-Methionine 3 3 3 3 Sucrose 329.1887 329.1887 329.1887 329.1887 Maltodextrin 50 50 50 50 Corn starch 100 100 100 100 Coconut oil 160 136 160 136 Safflower oil, linoleic 50 50 50 50 Fish oil (25% DHA) 0 24 0 24 Curcumin 0 0 0.5 0.5 Cholesterol 1.5 1.5 1.5 1.5 Cellulose 50 50 50 50 Mineral mix, AIN-93G-MX (94046) 42 42 42 42 Calcium phosphate, dibasic 4 4 4 4 Vitamin mix, without choline, A, D, E (83171) 6 6 6 6 Vitamin A palmitate (500,000 IU/g) 0.048 0.048 0.048 0.048 Vitamin D3, cholecalciferol (500,000 IU/g) 0.0053 0.0053 0.0053 0.0053 Vitamin E, dl-α -tocopheryl acetate (500 IU/g) 0.054 0.054 0.054 0.054 Choline dihydrogen citrate 4.2 4.2 4.2 4.2 TBHQ, antioxidant 0.004 0.004 0.004 0.004 -
Note: Prolab RMH 2500 5P14 was used for normal control standard diet, which supplied for 4.5% fat and 101 ppm of cholesterol. TBHQ, tert-Butylhydroquinone.
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