A Curcumin Analog Exhibits Multiple Biologic Effects on the Pathogenesis of Alzheimer's Disease and Improves Behavior, Inflammation, and β-Amyloid Accumulation in a Mouse Model

Abstract

Drugs for the treatment of Alzheimer's disease (AD) are in urgent demand due to the unmet need and the social burden associated with the disease. Curcumin has been historically considered as a beneficial product for anti-aging and AD. However, many efforts to develop curcumin for clinical use are hindered mainly due to its poor bioavailability. Recent development in drug delivery and structural design has resolved these issues. In this study, we identified a small molecule, TML-6, as a potential drug candidate for AD through screening a panel of curcumin derivatives using six biomarker platforms related to aging biology and AD pathogenesis. The structural modification of TML-6 is designed to improve the stability and metabolism of curcumin. Cell biological studies demonstrated that TML-6 could inhibit the synthesis of the β-amyloid precursor protein and β-amyloid (Aβ), upregulate Apo E, suppress NF-κB and mTOR, and increase the activity of the anti-oxidative Nrf2 gene. In the 3x-Tg AD animal model, TML-6 treatment resulted in significant improvement in learning, suppression of the microglial activation marker Iba-1, and reduction in Aβ in the brain. Although TML-6 exhibited a greater improvement in bioavailability as compared to curcumin, formulation optimization and toxicological studies are under development to assure its druggability. Taken together, TML-6 meets the current strategy to develop therapeutics for AD, targeting the combination of the Aβ cascade and aging-related biology processes.

Keywords: Alzheimer’s disease therapy; TML-6; aging; bioavailability; curcumin analog.

Figure 1

Comparison of traditional curcumin and TML-6 through its chemical structure. (A) The medical use of traditional curcumin is limited by its water insolubility (hydrophobic), low intestinal absorption, rapid metabolism, which leads to poor bioavailability. (B) TML-6 modified from traditional curcumin conserves the bioactivity of curcumin, becomes more stable, and slows down in metabolism. However, preliminary studies revealed that both curcumin and TML-6 had poor water solubility and need to be optimized by formulation.

Figure 2

The cytotoxicity studies, inhibitory concentration (IC)50, and biologic studies of TML-6. TML-6 could transcriptionally activate the Nrf2 promoter, suppress the expression of the amyloid precursor protein (APP) and phospho-NF-κB, and in contrast, increases the ApoE and phospho-mTOR expression in Huh-7 cell line. (A) The cytotoxicity (IC50 assay) of traditional curcumin and TML-6 were examined by CCK-8 in Huh-7 cells for 24 h. The working concentration of traditional curcumin and TML-6 was expressed in μM and μg/mL separately for comparison. The IC50 of curcumin and TML-6, 60 and 8 μM, respectively, is expressed at the right side of Figure 2A. In the following studies, the dose concentration was expressed in μg/mL only, as indicated in Figure 2B–D. (B) The luciferase plasmid contains the Nrf2 promoter co-transfected with pRL-TK (Renilla) into Huh-7 cells by Lipofectamine 2000. Transfected cells were treated with curcumin and TML-6 for 24 h, and the Nrf2 promoter activity was analyzed by the Dual-Luciferase Report assay system. TML-6 could exhibit transcriptional activation of the Nrf2 gene in a dose-dependent manner, with the highest activity at a concentration of 1.32 µg/mL, which was 12.1- and 6.6-fold stronger than the traditional curcumin at a dose of 10 and 20 µg/mL, respectively. Data represent the mean with a standard deviation (SD) error bar and p value < 0.05 was considered significant (* p < 0.05, ** p < 0.01). The Nrf2 promoter plasmid revealed a higher cytotoxicity beyond 5.24 μg/mL. (C) Huh-7 cells were treated with different doses of traditional curcumin and TML-6 for 24 h. Western blotting was performed to examine Alzheimer’s-related protein molecules such as APP, Apo E, inflammatory marker-phosphorylated NF-κB (C), and phosphorylated mTOR (D). The quantification of protein expression levels in APP, ApoE, p-NFkB and p-mTOR were normalized by β-Actin and are shown as fold induction in comparison to the untreated control (CTRL) under immunoblotting data in each panel. These AD biomarker proteins revealed a dose-dependent manner in response to TML-6 treatment.

Figure 3

TML-6 suppresses Aβ production in the N2a/APPswe stable cell line. (A) The N2a cell line showed stable over-expression of APP with wild-type full-length (N2a/APP) and Swedish mutant (N2a/APPswe) cells by Western blotting. The N2a/APPswe stable cell line was used to examine Aβ 40 and Aβ42 levels in culture medium in response to traditional curcumin and TML-6 by ELISA test. N2a/APPswe stable cells were treated with different doses of traditional curcumin (B) and TML-6 (C) as indicated in the figures. The cell morphological change or viability in each treatment was captured and represented for comparison at the lower panel, as indicated. After 24 h incubation, the culture medium was examined for levels of Aβ40 and Aβ42 by LEGEND MAX™ β-Amyloid X-40 and X-42 ELISA Kit. The significant differences between the treated and control cells are represented as the mean with a standard deviation (SD) error bar, and p value < 0.05 was considered significant (* p < 0.05, ** p < 0.01, *** p < 0.001). The cell density, viability, and morphological changes by micrographs were similar between curcumin and TML-6, probably due to cytotoxicity at the highest doses of curcumin (20 μg/mL) and TML-6 (4.19 μg/mL) in N2a/APPswe stable cells.

Figure 4

TML-6 improved the behavior test and reduced the brain levels of Aβ and the inflammatory biomarker Iba1 in the 3x-Tg AD mouse model. (A) Six-month old 3x-Tg AD mice were fed with normal diets (vehicle), or diets containing traditional curcumin and TML-6 for four months at a dosage of 150 mg/kg (each group contained five mice, n = 5). After four months of treatment on the experimental diet, animals were subjected to a behavior test in the form of the Morris water maze. The figure shows data from vehicle, curcumin and TML-6 fed different chow diets, with triplex-Tg AD mice and non-transgenic mice (Non-Tg) serving as the control. (B) The Aβ levels in mouse brains in 3x-Tg AD mice were examined at the end of behavior test after four-month feeding with the treatment of normal diets, traditional curcumin and TML-6. Mouse brain sections were examined by immunofluorescence staining. The immunofluorescence staining of Aβ was quantified and is represented in the panel. Scale bar: 200 μm. (C) Mouse brain sections were also examined for the inflammatory biomarker Iba1 by immunofluorescence staining. The images of Iba-1 levels were represented and quantified as indicated in the panel. The significance of the behavior test, Aβ, and Iba-1 levels between the mice treated with normal diets (vehicle) and either curcumin or TML-6 was determined by paired t-test. Asterisks denote data representing the mean with a standard deviation (SD) error bar, and p value < 0.05 was considered significant (* p < 0.05 and ** p < 0.01).

Figure 5

TML-6 exhibited significantly improved bioavailability as compared to traditional curcumin. (A) Summary of the absorption data on traditional curcumin and TML-6 in male Sprague–Dawley rats after oral administration of curcumin and TML-6 of 150 mg/kg. Values represented the mean ± SD of three rats in each treatment group. Abbreviations: ND, not detectable. (B) Bioavailability (serum concentration) of traditional curcumin and TML-6 was plotted against the time after oral administration to rats. The pK of curcumin was almost non-detectable (ND), consistent with the previous reports. Cmax indicates the observed maximum blood concentration and the time to reach the maximum blood concentration, while Tmax was determined directly from the experimental values. AUC (0-last) indicates the area under the concentration–time curve from time 0 to the last measurable concentration and MRT indicates the mean residence time.

Figure 6

A hypothetical scheme on the potential action mechanism of TML-6 in the therapy of Alzheimer’s disease. Curcumin derivative-TML-6 treatments can induce (1) transcriptional activation of the Nrf2 promoter, which can reduce free radicals, the hallmark of the aging process; (2) following the aging process, the dysfunction of autophagy machinery and activation of mTOR will reduce the autophagolysosomal function to remove the misfolded proteins and lead to the accumulation of Aβ. TML-6 can effectively inhibit the phosphorylation of mTOR and maintain the normal function of autophagy to assure Aβ clearance; (3) TML6 can inhibit the production of Aβ from APP and (4) suppress the synthesis and secretion of Aβ from neurons; (5) TML6 can transcriptionally activate APOE and enhance the clearance of Aβ; (6) TML6 can inhibit the activation of microglial cells and reduce inflammatory injuries in the brain. The future design of therapeutics should combine targeting APP/the Aβ cascade and improving aging-related biological processes.