Walnut Oil Reduces Aβ Levels and Increases Neurite Length in a Cellular Model of Early Alzheimer Disease

Abstract

(1) Background: Mitochondria are the cells' main source of energy. Mitochondrial dysfunction represents a key hallmark of aging and is linked to the development of Alzheimer's disease (AD). Maintaining mitochondrial function might contribute to healthy aging and the prevention of AD. The Mediterranean diet, including walnuts, seems to prevent age-related neurodegeneration. Walnuts are a rich source of α-linolenic acid (ALA), an essential n3-fatty acid and the precursor for n3-long-chain polyunsaturated fatty acids (n3-PUFA), which might potentially improve mitochondrial function. (2) Methods: We tested whether a lipophilic walnut extract (WE) affects mitochondrial function and other parameters in human SH-SY5Y cells transfected with the neuronal amyloid precursor protein (APP695). Walnut lipids were extracted using a Soxhlet Extraction System and analyzed using GC/MS and HPLC/FD. Adenosine triphosphate (ATP) concentrations were quantified under basal conditions in cell culture, as well as after rotenone-induced stress. Neurite outgrowth was investigated, as well as membrane integrity, cellular reactive oxygen species, cellular peroxidase activity, and citrate synthase activity. Beta-amyloid (Aβ) was quantified using homogenous time-resolved fluorescence. (3) Results: The main constituents of WE are linoleic acid, oleic acid, α-linolenic acid, and γ- and δ-tocopherol. Basal ATP levels following rotenone treatment, as well as citrate synthase activity, were increased after WE treatment. WE significantly increased cellular reactive oxygen species but lowered peroxidase activity. Membrane integrity was not affected. Furthermore, WE treatment reduced Aβ_1-40 and stimulated neurite growth. (4) Conclusions: WE might increase ATP production after induction of mitochondrial biogenesis. Decreased Aβ_1-40 formation and enhanced ATP levels might enhance neurite growth, making WE a potential agent to enhance neuronal function and to prevent the development of AD. In this sense, WE could be a promising agent for the prevention of AD.

Keywords: PUFA; aging; mitochondria; neurodegeneration; poly-unsaturated fatty acids; vitamin E; walnut.

Figure 1

Effects of 10 µg/mL walnut fatty acid extract in SY5Y-APP695 cells. EtOH-12%BSA mixture served as control. (A) Cellular concentrations of reactive oxygen species (ROS) in SY5Y-APP695 cells; n = 8. (B) Peroxidase activity in SY5Y-APP695 cells; n = 8. (C) Relative mRNA expression of Keap1; n = 10. (D) Relative mRNA expression of NFE2L2; n = 10. PGK1, GAPDH, and ACTβ were used as reference genes according to the MIQE guidelines [32]. Data are displayed as mean ± SEM. Significant differences (student’s t-test) are indicated as follows: * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 2

Effects of 10 µg/mL walnut lipid extract in SY5Y-APP695 cells. EtOH-12%BSA mixture served as control. (A) Mitochondrial membrane integrity reflected as the capacity to retain Calcein-AM (CAM) fluorescence dye. Displayed CAM fluorescence is adjusted to protein levels of the samples and reflects CAM captured in mitochondria only. Cytosolic CAM is quenched by CoCl₂. Positive control ionomycin led to permeabilization of mitochondrial membrane and quenching of the majority of CAM. Cyclosporin A reduced the probability of opening of the mitochondrial permeability transition pore resulting in less CAM to be channeled from mitochondria to cytosol; n = 11. (B) Basal ATP concentrations; n = 8. (C) ATP concentrations following rotenone-induced (25 µM) complex I inhibition. White column represents medium control treated with rotenone; n = 10. (D) MMP determined as fluorescence of dye R123 taken up by the mitochondria; n = 7. (E) Citrate synthase activity adjusted to the protein content of the sample; n = 10. (F) Content of Aβ_1–40 in SY5Y-APP695 cells were determined via the HTRF method and adjusted to protein levels of the samples; n = 9. Displayed are means ± SEM. Significant differences (student’s t-test) are displayed as: ^ns p > 0.05, * p < 0.05, ** p < 0.01.

Figure 3

Effects of 10 µg/mL walnut lipid extract on gene expression of markers of mitochondrial biogenesis. EtOH-12%BSA mixture served as control. (A) Gene expression of PGC1α; n = 10. (B) Gene expression of NRF1; n = 10. (C) Gene expression of TFAM; n = 10. To evaluate statistical significance, a student’s t-test was performed in (A,B), while a Mann-Whitney test was performed in (B) due to data not fulfilling normality according to a Shapiro–Wilks test. Significant differences are displayed as: ^ns p ≥ 0.05, * p < 0.05. Results displayed as means ± SEM.

Figure 4

Effects of 10 µg/mL walnut extract (WE) in SY5Y-APP695 cells on neurite growth. EtOH-12%BSA mixture served as control. (A) Microscopic view of SY5Y-APP695 cells differentiated with retinoic acid (10 µM) and 10 µg/mL WE. (B) Microscopic view of cells treated with retinoic acid (10 µM) and EtOH-BSA control (Ctrl). (C) Quantification of neurite length of differentiated SY5Y-APP695 cells treated with 10 µg/mL WE. Data displayed as means ± SEM; n = 6. For each n, at least 10 neurites were measured in 3 separate images. Significance (student’s t-test) is displayed as *** p < 0.001.