The development of SH-SY5Y cells stably expressing the APP wild-type (APP WT), APP Swedish (APP Swe), and APP Swedish/Indiana (APP Swe/Ind) has been described in our previous study (Pahrudin Arrozi et al., 2017). Briefly, SH-SY5Y cells were transfected with three different plasmids carrying the wild-type (WT), swedish (Swe), or swedish/Indiana (Swe/Ind) form of APP gene using Lipofectamine 3,000 (Life Technologies, Carlsbad, CA, United States), following the protocol from the manufacturer. After 72 h of transfection, the positive green fluorescent protein (GFP)-expressing cells were selected using a selection medium containing 400 μg/ml of geneticin (G418) (Life Technologies, United States). The level of Aβ-40 and 42 in each cell group was also examined, which showed that the ratio of Aβ42/40 was increased in the following order: SH-SY5Y APP WT < SH-SY5Y APP Swe < SH-SY5Y APP Swe/Ind (Pahrudin Arrozi et al., 2017).

The non-transfected SH-SY5Y cells were cultured in a complete culture medium (CCM) containing a 1:1 ratio of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12 medium (Gibco, Life Technologies, United States) supplemented with 1% penicillin/streptomycin (Gibco, Life Technologies, United States) and 10% fetal bovine serum (FBS) (HyClone, Logan, UT, United States). The stably transfected SH-SY5Y cells were cultured in CCM without penicillin/streptomycin and with 400 μg/ml of geneticin (Gibco, Life Technologies, United States). All cell types were cultured in a humidified atmosphere of 5% CO2 at 37°C.

The tocopherol isomers were obtained from ChromaDex, Los Angeles, CA, United States. The stock solution of 0.5 M for each isomer was freshly prepared with 100% undenatured ethanol and stored at −20°C for approximately 1 month. The undenatured ethanol was used as it has no additives and denaturants. Before treatment, 15 μl of tocopherol isomer from the 0.5 M stock was pre-incubated with 20 μl FBS overnight. The incubation with serum is necessary as the protein constituents must deliver this water-insoluble compound to cells (Sontag and Parker, 2007). Then, 18 μl of CCM and 21 μl of 100% undenatured ethanol were added to the activated isomer to make a 0.1 M stock solution. Next, further dilution was carried out to 0.05 M with 72 μl of a mixture of CCM and undenatured ethanol in a 1:1 ratio. After that, a stock solution of 0.025 M was prepared with 146 μl of CCM. Next, 60 μl from 0.025 M stock was taken out and added with 240 μl of CCM to make a 0.05 M stock solution. Finally, the desired working concentration of tocopherol isomer was prepared for 1, 3, 5, 10, 20, 40, 60, 80, and 100 μM. The final concentration of undenatured ethanol and FBS was kept constant (below 0.1%) (calculated from the overnight incubation with 20 μl of FBS). The treatment was carried out for 24 h.

The concentration of β- (BTF) and δ- tocopherol (DTF) from 1 to 100 μM were screened by cell viability assay using CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, United States). The kit supplemented 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulphonyl)-2H-tetrazolium (MTS) and the electron coupling agent phenazine methosulfate (PMS). The dehydrogenase enzyme reduced the MTS compound into a formazan product soluble in the medium in active cells. This colored formazan product is proportional to the number of viable cells. Briefly, the MTS solution was premixed with media at the ratio of 2:3 before adding 50 μl of the solution to each well of the cells and incubated in a humidified incubator at 37°C in 5% CO₂ for 2–4 h. The quantity of formazan product was determined by measuring the absorbance at 490 nm with a microplate reader (Enspire, Perkin Elmer, Waltham, MA, United States).

According to the manufacturer’s protocol, RNA extraction was carried out with TRI Reagent^® (Molecular Research Centre, Cincinnati, OH, United States). qRT-PCR was performed using the KAPA SYBR FAST One-Step qPCR kit (Kapa Biosystems, Boston, MA, United States). Each sample was tested in triplicate. The expression level of each gene was normalized to that of GAPDH. The relative transcription was analyzed by the 2^–ΔΔCT method. The fold change was calculated relative to control non-transfected when comparing the group of cells before treatment. The fold change was calculated relative to untreated control when comparing different isomers in each group of cells. The primers used in this study are listed in Table 1.

Whole-cell extracts were prepared from SH-SY5Y cell cultures. After 24 h treatment with tocopherol isomers, cells were washed in ice-cold phosphate buffered saline (PBS) and removed from the flask’s surface by scraping the cells in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fischer Scientific, Waltham, MA, United States). Cell extracts were sonicated, collected, and stored at −80°C until protein concentration was measured by Bradford assay (Bradford, 1976). According to the manufacturer’s instructions, electrophoresis and blotting were performed using the Mini-PROTEAN^® Tetra Cell system (Bio-rad, Hercules, CA, United States). Denatured samples with an equal amount of protein per lane (20 μg) were separated on gradient gels (10% SDS-PAGE gel) electrotransferred on nitrocellulose membrane (GE Healthcare, Marlborough, MA, United States). They blocked in 5% skimmed milk for 1 h at room temperature. The membranes were then incubated overnight with primary antibodies at 4°C (rabbit anti-APP, 1:1,000, Abcam, Cambridge, United Kingdom; mouse anti-β-actin, 1:5,000 (Santa Cruz, Dallas, TX, United States). Before incubation with primary antibody, the membranes were washed in triphosphate buffered saline (TPBS) several times, and then incubated with secondary antibody (anti-rabbit IgG, 1:2,000, Abcam, United Kingdom; anti-mouse, 1:2,000, Abcam, United Kingdom)) for 1 h at room temperature. Peroxidase enzyme activity was detected using WesternBright™ Sirius Western blotting detection kit (Advansta, Menlo Park, CA, United States) and visualized on Gel-Doc FluorChem FC2 (Alpha Innotech, San Leandro, CA, United States).

Conditioned media was collected from cells. Protein inhibitors were added to the media to prevent the degradation of Aβ protein. According to the manufacturer’s instructions, the concentration of Aβ42/40 was determined by using human Aβ42 or Aβ40 ELISA kit (Elabscience, Wuhan, China).

Data obtained were expressed as mean ± SD, and statistical analysis was carried out using GraphPad PRISM v8 software (GraphPad Software, La Jolla, CA, United States). Statistical comparisons between groups were performed using one-way ANOVA followed by the post-hoc test. The statistical significance of all tests was set at p < 0.05.

To determine the cytotoxicity effect of the different tocopherol isomers, a range of doses from 1 to 100 μM were screened by cell viability assay in non-transfected and stably transfected SH-SY5Y with three different types of APP gene. In our previous data, the incubation with ATF and GTF for 24 h did not show any significant cytotoxic effect in all groups of cells. In addition, ATF at 5 and 100 μM and GTF at 5 and 80 μM increased the cell viability in SH-SY5Y stably transfected with APP WT, APP Swe, and APP Swe/Ind (Pahrudin Arrozi et al., 2020). The present study also showed similar findings. There was no cytotoxicity effect with BTF and DTF treatment from 1 to 100 μM in all cells. Furthermore, BTF treatment at a lower dose (3 μM) significantly increased the cell viability in APP WT and APP Swe/Ind-expressing cells (p < 0.01 and p < 0.05 respectively) (Figures 1B,D). Still, DTF treatment at the same dose significantly increased the cell viability only in APP Swe-expressing cells compared to untreated control (p < 0.01) (Figure 1C). Moreover, the treatment with BTF at 80 μM and DTF at 100 μM demonstrated a significant increase in cell viability in non-transfected (Figure 1A), APP WT (Figure 1B), APP Swe (Figure 1C), and APP Swe/Ind-expressing cells (Figure 1D) compared to untreated control (p < 0.0001, p < 0.01, and p < 0.05 respectively) in respective groups of cells. Based on these findings, ATF at 5 and 100 μM, BTF at 3 and 80 μM, DTF at 3 and 100 μM, and GTF at 5 and 80 μM were chosen for further screening for their effects on APP expression at gene and protein levels, and also on the level of Aβ-42.

Previously, we showed that APP gene and protein level increased significantly in SH-SY5Y stably transfected with APP WT or mutant (Swe or Swe/Ind) compared to non-transfected control (Pahrudin Arrozi et al., 2017). On the other hand, the ratio of Aβ42/Aβ40 was shown in the following order: non-transfected SH-SY5Y < SH-SY5Y APP WT < SH-SY5Y APP Swe < SH-SY5Y APP Swe/Ind (Pahrudin Arrozi et al., 2017). As the ratio of Aβ42/Aβ40 in SH-SY5Y expressing APP Swe represent the intermediate level between cells expressing APP WT and APP Swe/Ind, further screening was done to investigate the effects of selected doses of different tocopherol isomers on the level of APP expression and Aβ-42 only in APP Swe-expressing cells. The non-transfected SH-SY5Y act as a control group without APP overexpression.

The treatment with ATF at 5 and 100 μM significantly decreased the level of the APP mRNA expression (p < 0.01 and p < 0.001) (Figure 2A), but there were no changes in the level of the APP protein expression (Figure 2B) in non-transfected cells compared to untreated control. Similarly, the treatment with ATF at 100 μM and GTF at 80 μM significantly decreased the level of the APP mRNA expression (p < 0.05 and p < 0.001, respectively). In contrast, at 5 μM, both treatments only showed a decreasing trend without statistical significance compared to untreated control in APP Swe-expressing cells (Figure 2C). Although reduced significantly at the gene level, treatment with ATF or GTF did not show statistical significance and only showed a slight reduction on the level of the APP protein expression compared to untreated control (Figure 2D). Whereas the treatment with BTF and DTF at selected doses showed no significant changes in the APP gene and protein expression level compared to untreated control in both groups of cells (Figure 2).

We further evaluate the effect of the tocopherol isomers on the level of Aβ-42. Before treatment, all types of APP-expressing cells showed a significantly higher level of Aβ42 compared to non-transfected and transfected cells delivered the following order: SH-SY5Y APP WT < SH-SY5Y APP Swe < SH-SY5Y APP Swe/Ind (Figure 3A). There was no significant difference with all tocopherol isomer treatment on the level of Aβ-42 compared to untreated control in non-transfected cells (Figure 3B). Interestingly, the treatment with ATF (5 and 100 μM; p < 0.05 and p < 0.0001, respectively) and GTF (5 and 80 μM; p < 0.0001) significantly decreased the level of Aβ-42, whereas BTF and DTF showed no significant difference compared to untreated control in APP Swe-expressing cells (Figure 3C). Based on these findings, ATF and GTF reduced the level of Aβ42 without affecting APP protein expression. Therefore, investigating their role in the APP processing pathway at the transcriptional level was conducted to elucidate their mechanism of action in SH-SY5Y, further stably expressing the APP WT, single-mutant (Swe), and double-mutant (Swe/Ind).

The expression of several genes involved in the regulation of the APP processing pathway was measured by qPCR. The genes are ADAM10, which encodes the major α-secretase in the brain; BACE1, which encodes a transmembrane aspartyl protease responsible for β-secretase processing; APH1B, which encodes a multi-pass transmembrane protein that is a functional component of the gamma-secretase complex; NCSTN, which encodes a type I transmembrane glycoprotein that is an integral component of the multimeric γ-secretase complex; and SIRT1, which encodes sirtuin 1, NAD⁺ dependent histone deacetylase.

Before treatment, the baseline level in each cells group was measured. There was no significant difference between the groups in the expression level of ADAM10 (Figure 4A). Whereas the expression level of BACE1 was significantly upregulated in APP Swe and APP Swe/Ind-expressing cells compared to non-transfected cells (p < 0.01 and p < 0.001, respectively; Figure 4B). The expression level of BACE1 in APP Swe/Ind was also significantly upregulated compared to APP WT (p < 0.05; Figure 4B). Meanwhile, the expression level of APH1B was significantly upregulated in all APP type expressing cells compared to non-transfected cells (p < 0.0001 for all groups; Figure 4C). Furthermore, the expression level of NCSTN was significantly upregulated in APP WT- and APP Swe/Ind-expressing cells compared to non-transfected cells (p < 0.05 and p < 0.001, respectively). In addition, the level of upregulation in APP Swe/Ind was significant when compared to APP WT and APP Swe-expressing cells (p < 0.05 and p < 0.01, respectively) (Figure 4D). In contrast, the expression level of SIRT1 was significantly downregulated in all APP types expressing cells compared to non-transfected cells (p < 0.001 for APP WT and Swe, p < 0.0001 for APP Swe/Ind), which is the level of downregulation in APP Swe/Ind that was significant when compared to APP WT-expressing cells (p < 0.05; Figure 4E). These results suggested that the genes regulating the APP processing pathway were altered in SH-SY5Y cells with overexpressed. A more substantial alteration effect was seen in cells with overexpressed mutant APP.

In APP WT-expressing cells, the treatment with ATF at 5 and 100 μM significantly downregulated the expression of APH1B without affecting the expression of other genes compared to untreated control (p < 0.05; Figure 5A). On the other hand, treatment with GTF did not alter the expression of all candidate genes in the same group of cells (Figure 5A). Meanwhile, in APP Swe-expressing cells, the treatment with ATF and GTF, especially at a higher dose, significantly downregulated the expression of BACE1 (p < 0.05 and p < 0.01, respectively) and APH1B (p < 0.01 for both) compared to untreated control (Figure 5B). The expression of the other genes, such as ADAM10, NCSTN, and SIRT1, was unchanged with ATF or GTF treatments (Figure 5B). However, in APP Swe/Ind-expressing cells, ATF and GTF affected several genes. For example, ATF and GTF significantly downregulated the expression of BACE1 regardless of the concentration compared to untreated control (p < 0.0001 and p < 0.01, respectively; Figure 5C). Similarly, the treatment with ATF at both lower and higher doses and GTF at a higher concentration significantly downregulated the expression of APH1B compared to untreated control (p < 0.0001 and p < 0.01, respectively; Figure 5C). Furthermore, the treatment with ATF at a higher concentration also significantly downregulated (p < 0.001) the expression of NCSTN. Meanwhile, the treatment with GTF showed different effects at different concentrations, such as significantly downregulated (p < 0.05) and upregulated (p < 0.01) with lower and higher concentration, respectively, compared to untreated control (Figure 5C). In addition, the treatment with ATF and GTF at a higher concentration significantly upregulated the expression of SIRT1 compared to untreated control (p < 0.001 and p < 0.01, respectively; Figure 5C). There was no significant difference in the expression of ADAM10 with ATF and GTF treatment in all APP types expressing cells compared to untreated control (Figures 5A–C).

According to these results, the treatment with ATF and GTF for 24 h differently modulated the expression of genes involved in regulating the APP processing pathway in SH-SY5Y with different types of overexpressed APP. Specifically, ATF and GTF downregulated the expression of genes involved in mediating the APP processing toward the amyloidogenic pathway, such as BACE1, APH1B and NCSTN, and upregulated the expression of a gene involved in mediating the amyloidogenic pathway, such as SIRT1.