Aluminum chloride and D-galactose induced a zebrafish model of Alzheimer's disease with cognitive deficits and aging

Abstract

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Transgenic and pharmacological AD models are extensively studied to understand AD mechanisms and drug discovery. However, they are time-consuming and relatively costly, which hinders the discovery of potential anti-AD therapeutics. Here, we established a new model of AD in larval zebrafish by co-treatment with aluminum chloride (AlCl₃) and D-galactose (D-gal) for 72 h. In particular, exposure to 150 μM AlCl₃ + 40 mg/mL D-gal, 200 μM AlCl₃ + 30 mg/mL D-gal, or 200 μM AlCl₃ + 40 mg/mL D-gal successfully induced AD-like symptoms and aging features. Co-treatment with AlCl₃ and D-gal caused significant learning and memory deficits, as well as impaired response ability and locomotor capacity in the plus-maze and light/dark test. Moreover, increased acetylcholinesterase and β-galactosidase activities, β-amyloid 1-42 deposition, reduced telomerase activity, elevated interleukin 1 beta mRNA expression, and enhanced reactive oxygen species production were also observed. In conclusion, our zebrafish model is simple, rapid, effective and affordable, incorporating key features of AD and aging, thus may become a unique and powerful tool for high-throughput screening of anti-AD compounds in vivo.

Keywords: Aluminum chloride; Alzheimer's disease; D-galactose; High-throughput screening; Zebrafish.

Graphical abstract

Fig. 1

Assessment of color-associated learning and memory in the plus-maze test. A: Schematic diagram of plus-maze and representative swimming trajectories in the plus-maze test. B: Quantitative analysis of the ratio of distance swam in the blue area to the total distance swam in 2 dpf zebrafish after exposure to four different combinations of AlCl₃ and D-gal for 72 h. Data are presented as the mean ± SEM (n = 30 zebrafish larvae per group), and the distance of five zebrafish represents one data point. Statistical significance of treatment effects was assessed using one-way ANOVA with Dunnett’s multiple comparison test and illustrated as follows: * * p < 0.01 (versus the control group).

Fig. 2

Assessment of the response ability and locomotor behavior in the light-dark test. A: Locomotor patterns and average speed during dark-light stimulations of zebrafish after exposure to four different combinations of AlCl₃ and D-gal from 2 to 5 dpf. B: Quantitative analysis of the average speed change during light-dark stimulations. C: Representative traces of total locomotor activity in different groups during all light-dark stimulations. D: Quantitative analysis of total distance swam during all light-dark stimulations. In B and D, data are presented as the mean ± SEM (n = 10 zebrafish larvae per group). Statistical significance of treatment effects was assessed using one-way ANOVA with Dunnett’s multiple comparison test and illustrated as follows: * * p < 0.01 (versus the control group).

Fig. 3

Determination of AChE activity and Aβ_1–42 content. A, B: Quantitative analysis of AChE fluorescence intensity (A) and Aβ_1–42 plaques (B) after exposure of 2 dpf zebrafish to four combinations of AlCl₃ and D-gal for 72 h. Data are presented as the mean ± SEM (n = 3). Statistical significance of treatment effects was assessed using one-way ANOVA with Dunnett’s multiple comparison test and illustrated as follows: * p < 0.05, * * p < 0.01 (versus control group).

Fig. 4

Determination of β-galactosidase and telomerase levels. A: Representative images of staining for β-galactosidase. B, C: Quantitative analysis of β-galactosidase (B) and telomerase (C) activities after exposure of 2 dpf zebrafish to four different combinations of AlCl₃ and D-gal for 72 h. Data are presented as the mean ± SEM (n = 10 in B and n = 3 in C). In B and C, statistical significance of treatment effects was assessed using one-way ANOVA with Dunnett’s multiple comparison test and illustrated as follows: * * p < 0.01 (versus control group).

Fig. 5

Determination of il1b expression and ROS levels. A: Quantitative analysis of the relative il1b mRNA expression normalized by actb1 mRNA level in 2 dpf zebrafish exposed to four combinations of AlCl₃ and D-gal for 72 h. B: Quantitative analysis of the fluorescence intensity of ROS levels after exposure of 2 dpf zebrafish to four different combinations of AlCl₃ and D-gal for 72 h. Data are presented as the mean ± SEM (n = 3 in A and n = 10 in B). Statistical significance of treatment effects was assessed using one-way ANOVA with Dunnett’s multiple comparison test.