The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse

Abstract

Inflammation in Alzheimer's disease (AD) patients is characterized by increased cytokines and activated microglia. Epidemiological studies suggest reduced AD risk associates with long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs). Whereas chronic ibuprofen suppressed inflammation and plaque-related pathology in an Alzheimer transgenic APPSw mouse model (Tg2576), excessive use of NSAIDs targeting cyclooxygenase I can cause gastrointestinal, liver, and renal toxicity. One alternative NSAID is curcumin, derived from the curry spice turmeric. Curcumin has an extensive history as a food additive and herbal medicine in India and is also a potent polyphenolic antioxidant. To evaluate whether it could affect Alzheimer-like pathology in the APPSw mice, we tested a low (160 ppm) and a high dose of dietary curcumin (5000 ppm) on inflammation, oxidative damage, and plaque pathology. Low and high doses of curcumin significantly lowered oxidized proteins and interleukin-1beta, a proinflammatory cytokine elevated in the brains of these mice. With low-dose but not high-dose curcumin treatment, the astrocytic marker GFAP was reduced, and insoluble beta-amyloid (Abeta), soluble Abeta, and plaque burden were significantly decreased by 43-50%. However, levels of amyloid precursor (APP) in the membrane fraction were not reduced. Microgliosis was also suppressed in neuronal layers but not adjacent to plaques. In view of its efficacy and apparent low toxicity, this Indian spice component shows promise for the prevention of Alzheimer's disease.

Fig. 1.

Effect of curcumin on IL-1β on APPSw brains.A, Effect of low-dose curcumin on IL-1β. Protein levels were measured in TBS-extracted supernatant from Tg+ mice fed low-dose curcumin diet and untreated Tg+ mice. Levels of IL-1β were significantly decreased by 61.8%. in curcumin-treated animals. *p < 0.05. B, Effect of high-dose curcumin on IL-1β. Protein levels were measured in TBS-extracted supernatant from Tg+ mice fed a high-dose curcumin diet and untreated Tg+ mice. Two-way ANOVA revealed a 57% reduction in IL-1β levels in Tg+ mice receiving a high dose of curcumin compared with untreated animals. ***p < 0.001.

Fig. 2.

Reductions in percentage of stained microglia in response to dietary curcumin (160 ppm) were observed in neuronal layers in every region examined except the hilus of the hippocampus (A). Layers 1 of the cortex and the stratum oriens of the hippocampus were minimally affected, whereas the most robust reductions were observed in layer 2 of the cortex (40% reduction) and the outer molecular layer (OML) of the dentate gyrus (53% reduction). An example of the PT staining quantified in the OML is shown in B. D, Percentage of PT staining was quantified within plaques (ring 1) and associated within 1 plaque radius (ring 2). Whether curcumin altered the association of microglia with plaques was analyzed by analysis of the staining within these rings, using a 2 × 2 ANOVA (treatment diet × ring) of microglial staining (percentage area). A ring effect signifies that microglial staining is dependent on proximity to the plaque. The treatment × ring interaction signifies that curcumin treatment effects depend on the ring analyzed. As shown in C, microglial PT staining was not reduced within plaques (ring 1) but was even increased in ring 2 around plaques in curcumin-treated animals.IML, Inner molecular layer.

Fig. 3.

Measurement of oxidized proteins in APPSw mice.A, Representative example of Oxyblot using 10 μg of protein from Tg− untreated and Tg+ untreated entorhinal cortex samples. B, Transgene effect on oxidized proteins, as measured by Oxyblot, of SDS-extracted supernatant from hippocampus and entorhinal cortex of Tg− untreated (n = 9) and Tg+ untreated (n = 6). Two-way ANOVA showed a highly significant transgene effect, in which the levels of oxidized proteins were 12-fold higher in Tg+ animals compared with Tg− animals. ***p < 0.001. C, High-dose curcumin effect. Levels of oxidized proteins in oxyblots of SDS-extracted supernatant from hippocampal, entorhinal, and piriform cortices of Tg+ untreated (n = 6) and Tg+ high-dose curcumin (n = 8). Two-way ANOVA showed that levels of oxidized proteins were 46% lower in mice fed a diet containing a high-dose of curcumin (HD curcumin). *p < 0.05. D, Low-dose curcumin effect. Amounts of oxidized proteins in residual cortex and piriform cortex of Tg+ untreated and Tg+ curcumin-treated animals. Two-way ANOVA revealed a significant treatment effect. *p < 0.05. OD, Optical density.

Fig. 4.

Effect of low-dose curcumin on SDS-insoluble Aβ and plaque pathology. A, Formic acid extracted (SDS-insoluble) Aβ as measured by sandwich ELISA. Aβ was measured in the three regions of the brain: hippocampus, entorhinal cortices, and residual cortex. A two-way ANOVA (treatment × region) showed significant treatment effects in insoluble Aβ levels (***p < 0.001). Homogeneity of variance was obtained using a natural log transformation of square root transformed values. B, Plaque burden (percentage of hippocampal and cortical area stained with amyloid) in Tg+ untreated and Tg+ low-dose curcumin mice. Image analysis was performed on amyloid-positive structures (DAE-positive) in hemibrain cryostat sections. Two-way ANOVA revealed a significant 43% reduction in plaque burden in curcumin-treated animals (*p = 0.03).C, Soluble Aβ in Tg+ untreated and Tg+ low-dose curcumin mice as measured by sandwich ELISA. Aβ levels were measured in hippocampus, entorhinal cortex, piriform cortex, and residual cortex regions. Two-way ANOVA (treatment - region) showed significant treatment effects in decreasing the levels of soluble Aβ (*p < 0.05).

Fig. 5.

Curcumin blocks AD pathogenesis at multiple sites. Curcumin can act as a scavenger of ROS, including NO and peroxynitrite generated by reactive glia and hydroxyl radicals generated by neurons as a result of direct Aβ toxicity. Ibuprofen (NSAID action at site 1) can inhibit microglial activation and cytokine production but was not sufficient to reduce oxidative damage. Antioxidants that can block ROS at multiple sites may be required. Curcumin can also limit damage by inhibiting NFκB-induced iNOS, cyclooxygenase 2, and inflammatory cytokine production by reactive glia. By blocking NFkB and reducing IL-1β, IL-6, and ApoE, curcurmin should reduce proamyloidogenic factors (APOE, α1ACT). Finally, curcumin can lower plasma and tissue cholesterol, potentially lowering Aβ production. LOX, Lipoxygenase; Cox-2, cyclooxygenase 2; SCR, Scavenger receptors;Fc, Fc Ig receptors.