The neuroblastoma SK-N-MC cell lines were obtained by Korean Cell Line Bank (Seoul, South Korea). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, MD, USA). The antibodies of p-Akt (Thr 308, sc-16646-R), p-Akt (Ser 473, sc-7985-R), Akt (sc-8312), eukaryotic translation initiation factor 4E (eIF4E; sc-9976), cyclin D₁ (sc-753), cyclin E (sc-481), cyclin-dependent kinase 4 (CDK4; sc-749), CDK2 (sc-748), tau (sc-390476), p-tau (Thr 212, sc-135643), p-tau (Ser 396, sc-101815), rapamycin-insensitive companion of mTOR (RICTOR; sc-271081), α-tubulin (sc-32293) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated IgG was obtained from Jackson Immunoresearch (West Groove, PA, USA). The antibodies of cleaved caspase 3(#9661), mammalian target of rapamycin (mTOR; #2983), p-mTOR (Ser 2448, #5536), p-mTOR (Ser 2481, #2974), p-regulatory-associated protein of mTOR (RAPTOR; Ser 792, #2083), p70 S6 kinase 1 (S6K1, #2708), p-p70S6K1 (Thr 389, #9234), eIF4E binding protein (4EBP1; #9644), p-4EBP1 (Thr 37/46, #9459) and p35/25 (#2680) were acquired from Cell Signaling Technology (Beverly, MA, USA). The antibodies of hypoxia inducible factor (HIF1α; NB100-105), p70S6K1 (NB600-1049), LC3 (NB100-2220) and p62 (NBP1-48320) were obtained from Novus Biologicals (Littleton, CO, USA) and the HRP-conjugated goat anti-rabbit IgG was purchased from Santa Cruz Biotechnology. NeuN (ab77315), RAPTOR (ab26264) antibodies were purchased from Abcam (Cambridge, England). Small interfering RNA (siRNA) for HIF1A was purchased from GenePharma (Sanghai, China). CDK2, CDK4 and non-targeting (NT) siRNAs were purchased from Dharmacon (Lafayette, CO, USA). EGTA, BAPTA-AM, ionomycin, nifedipine, N-acetyl-L-cysteine (NAC), PF4708671, cycloheximide, LY294002, rapamycin, trehalose, flavopiridol, paclitaxel were purchased from Sigma Aldrich (St. Louis, MO, USA). Akt inhibitor was purchased from Calbiochem (La Jolla, CA, USA). All chemicals used in this study were of the highest quality commercially available forms.

The SK-N-MC cells were cultured with Dulbecco Modified Eagle Medium (DMEM, Thermo Fisher, Waltham, MA, USA). Cells were grown in 10% FBS with a 1% antibiotics mixture. Cells were plated on 35, 60, or 100 mm diameter culture dishes, or in 6- or 12-well plates in an incubator kept at 37°C with 5% CO₂. The medium was changed to serum-free medium. After 24 h of incubation in serum-free medium, cells were washed twice with phosphate buffered solution (PBS), and placed in medium that included supplements.

Hippocampal primary neurons were isolated from prenatal ICR mice (18–19 days) to confirm the effect of Aβ in neurons. Isolation was performed as described (Seibenhener and Wooten, 2012). Hippocampus was isolated from the prenatal mice (18–19 days), and gently minced by using sterile scalpel. Minced hippocampus was treated with trypsin (0.025%) and dissociated by trituration. 2.5 × 10⁶ cells were plated at poly-D-lysine-coated 35 mm dish in neurobasal plating media (neurobasal Media containing B27 Supplement [1 ml/50 ml], 0.5 mM glutamine, 25 μM glutamate, 1% antibiotics, 1 mM HEPES, 10% heat inactivated donor horse serum) and placed in an incubator kept at 37°C with 5% CO_2. After 24 h, growth media is changed to neurobasal feeding media (Neurobasal media containing B27 supplement [1 ml/50 ml], 0.5 mM glutamine, 1% antibiotics, 1 mM HEPES. To avoid glial cell contamination, cytosine arabinoside (AraC) was treated early time points in the culture. NeuN antibody was used for neuronal nucleus staining. The protocol for mouse hippocampal neuron primary culture was approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-151116-1). All procedures involving mice were performed following the National Institutes of Health Guidelines for Humane Treatment of Animals.

The β-Amyloid [1-42] (Human) peptide was purchased from Invitrogen Corporation (Camarillo, CA, USA). Peptide was dissolved at a concentration of 1 mg/ml in 100% HFIP (Sigma Aldrich), and incubated at room temperature for 1 h with occasional vortexing. The sonicated for 10 min in a water bath sonciator. After sonication, it was freeze-dried for 3 h. When the film is formed, it dissolved in DMSO. Scrambled β-Amyloid [1-42] (scrambled Aβ) was purchased from ANASPEC (San Jose, CA, USA). Protein sequence of scrambled Aβ is as follows. AIAEGDSHVLKEGAYMEIFDVQGHVFGGKIFRVV DLGSHNVA.

The total RNA of cells was extracted using MiniBEST Universal RNA Extraction Kit (TaKaRa, Otsu, Shinga, Japan). Reverse transcription (RT) was carried out using a Maxime™ RT-PCR PreMix kit (Intron Biotechnology, Seongnam, Korea) with the oligo(dT18) primers. RT was performed at 45°C for 60 min to cDNA synthesis and 95°C for 5 min RTase inactivation step. RT products were amplified using QuantiSpeed SYBR Kits (Life technologies, Gaithersburg, MD, USA). Real-time quantification of RNA targets was performed in a Rotor-Gene 6000 real-time thermal cycling system (Corbett Research, NSW, Australia). The primers used are described in the Supplementary Table S1. The reaction mixture (10 μl) contained 50 ng of total RNA, 0.5 mM of each primer, and appropriate amounts of enzymes and fluorescent dyes as recommended by the supplier. The Real-time PCR were performed as follows: 15 min at 95°C for DNA polymerase activation; 15 s at 95°C for denaturing; and 40 cycles of 15 s at 94°C, 30 s at 54°C and 30 s at 72°C. Data was collected during the extension step (30 s at 72°C) and analysis was performed using the software provided. Following real-time PCR, melting curve analysis was performed to verify the specificity and identity of the PCR products.

Harvested cells were washed once with cold PBS prior to incubation in lysis buffer (1 mM EDTA, 1 mM EGTA, 20 mM Tris (pH 7.5), 1% Triton X-100, 1 mg/ml aprotinin, and 1 mM phenylmethylsulfonylfluoride (PMSF)) for 30 min on ice. The lysates were then cleared by centrifugation (16,000 g at 4°C for 30 min). The BCA assay with lysates was performed to determine protein concentration. Samples containing 10 μg of protein were prepared for 10% sodium dodecyl sulfate polyacryl-amide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. Protein-containing membranes were washed Tris-buffered saline containing 0.1% Tween-20 (TBST) solution (10 mM Tris-HCl (pH 7.6), 150 mM NaCl and 0.1% Tween-20) blocked with 5% bovine serum albumin (BSA) for 30 min. Blocked membranes were washed with TBST for three times every 10 min, and incubated with primary antibody overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated secondary antibody at room temperature for 2 h. The western blotting bands were visualized by using chemiluminescence (Bio-Rad, Hercules, CA, USA). Densitometric analysis was carried out by using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA¹).

Prior to Aβ treatment, siRNAs specific for HIF1A, CDK2, CDK4, or NT were transfected to cell for 24 h with Turbofect™ (Thermo Fisher) according to the manufacturer’s instructions. The concentration of each transfected siRNA was 25 nM. The NT siRNA was used as the negative control. The siRNA sequences are described in Supplementary Table S2.

Cells were incubated with a 0.05% Trypsin and 0.5 mM EDTA solution. Detached cells were treated with soybean trypsin inhibitor (0.05 mg/ml) to quench trypsin. To exclude dead cells, 0.4% trypan blue was added to the cell suspension. Subsequently, unstained and stained cells were counted by using a Petroff-Hausser counting chamber (Hausser Scientific, Horsham, PA, USA). The equation used to determine cell viability was Cell viability = [{1 − (number of trypan blue-stained cells/number of total cells)}] × 100.

The cells were cultured in a 12-well plate. Prior to harvesting, each well was treated with 100 pg/ml of MTT reagent (Sigma Aldrich) for 4 h. Harvested cells were washed with PBS and incubated with 150 μl of DMSO (Sigma Aldrich) for 20 min. 1 × 10⁵ cells were collected, and resuspended in 150 μl of PBS. Absorbance at 540 nm was measured by using a micro plate reader (Bio-Rad).

Cells were fixed with 4% paraformaldehyde (Sigma Aldrich) for 10 min, followed by permeabilization with 0.1% Triton X-100 (Sigma Aldrich) in PBS followed by washing with PBS. Cells were incubated with 1% normal goat serum to block non-specific binding of antibody, and then incubated with 1:100 dilution of primary antibody for overnight in 4°C. Next, the cells were incubated for 2 h at room temperature with Alexa Fluor^®-conjugated secondary antibodies (1:100 dilution) and propidium iodide (PI, Invitrogen, Carlsbad, CA, USA) in PBS. Images were obtained by using a FluoView™ 300 fluorescence microscope (Olympus, Tokyo, Japan). Three independent experiments were performed and 60 cells (20 cells per experiment) were used for statistical analysis.

The changes in [Ca²⁺]_i were observed using Fluo 3-AM dissolved in DMSO. The cells were washed once with a PBS, incubated in PBS containing 3 μM of Fluo 3-AM with 5% CO₂ at 37°C for 40 min, and washed once with the PBS and scanned every second using confocal microscopy (FluoView™ 300, Olympus, Tokyo, Japan). The fluorescence was excited at 488 nm and the emitted light was read at 515 nm. In order to verify the assay, ionomycin was applied to the cells as a positive control. All of the analysis of [Ca²⁺]_i were processed at a single cell level are expressed as the relative fluorescence intensity (RFI).

The cells were plated on 12-well dish and confocal dishes. The cells were then incubated in the dark with 10 μM CM-H₂DCFDA for 1 h at 37°C. The cells were visualized by using laser confocal microscopy (FluoView™ 300, Olympus, Tokyo, Japan) with an excitation wavelength of 448 nm and an emission wavelength of 515 nm. To quantify the ROS production, cells were incubated with 10 μM of CM-H₂DCFDA, and washed twice with PBS. The 1 × 10⁵ cells were loaded into a 96-well black plate and assessed by using a luminometer (Victor3, Perkin-Elmer, Waltham, MA, USA).

Cells were lysed with the co-immunoprecipitation buffer (1% Triton X-100 in 50 mM Tris-HCl [pH 7.4] containing 150 mM NaCl, 5 mM EDTA, 2 mM NA₃VO₄, 2.5 mM Na₄PO₇, 100 mM NaF, 200 nM microcystin lysin-arginine, and protease inhibitor). Cell lysates (300 μg) were incubated with 10 μg of anti-tau antibody. The samples were incubated for 4 h, and Protein A/G PLUS-agarose immunoprecipitation reagent was added. The samples were incubated for an additional 12 h. The beads were washed three times with the co-immunoprecipitation buffer. Beads were added to sample buffer, and eluted by boiling. Samples were analyzed by western blotting.

Apoptosis and necrosis of cells were measured with an Annexin V and PI staining kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Briefly, the cells were detached with 0.05% trypsin/EDTA and 1 × 10⁵ cells were suspended with Annexin V binding buffer. The cells were stained with Annexin V and PI and incubated for 20 min at room temperature in the dark. The sample was read by flow cytometry and analyzed using CXP software (Beckman Coulter, Brea, CA, USA). Samples were gated to exclude debris (forward light scatter [FSC] area vs. side scatter area), and then any cell doublets were excluded using FSC area vs. FSC width analysis.

Results are expressed as mean value ± standard error of mean (SE). All experiments were analyzed by analysis of variance (ANOVA), and some experiments which needed to compare with two groups were examined by comparing the treatment means to the control using a Student’s test. A result with a p value of < 0.05 was considered statistically significant.

Aβ significantly induced the cytotoxicity of SK-N-MC cells from 1 μM to 20 μM for 48 h (Figure 1A). For instance, cell viability decreased by 36% at 5 μM of Aβ compared to a control sample. The results of a trypan blue exclusion assay also revealed that cell viability decreased by 34% when the cells were treated with 5 μM of Aβ (Figure 1B). In addition, we found that Aβ has the ability to induce caspase 3 cleavage, suggesting that neuronal cell death as induced by Aβ is related to apoptotic cell death (Figure 1C). It was also found that Aβ increased ROS production by as much as 87% in 3 h, with the rate strongly maintained for up to 48 h (Figure 1D). The stimulatory effect of Aβ on ROS production was further visualized with a florescent dye, CM-H₂DCFDA (Supplementary Figure S1A). Importantly, the cell viability decreased by the Aβ treatment was significantly recovered by a treatment with the ROS scavenger NAC (Figure 1E). Consistent with this, we found through flow cytometric analyses that Aβ significantly induced the necrotic cell death (a 1.3 ± 0.8-fold increase compared to a control) as well as apoptosis (a 3.0 ± 0.2-fold increase compared to a control; Figure 1F). In addition, we performed CM-H₂DCFDA staining to confirm the effect of NAC in ROS production. We observed that ROS level of Aβ-treated SK-N-MC was increased to 143%. And, those of NAC-pretreated SK-N-MC with either vehicle or Aβ were decreased to 85% and 72%, respectively. (Supplementary Figure S1B). Aβ has greater stimulatory potency on apoptotic cell death than necrotic cell death, confirming that Aβ is essential for triggering apoptotic cell death. However, this increase in apoptotic cell death was significantly blocked by a treatment with NAC. Next, we determine whether ROS production induced by Aβ causes alterations in the Ca²⁺ influx [Ca²⁺]_i level. As shown in Figure 1G, Aβ caused an increased level of [Ca²⁺]_i, which was enhanced by the Ca²⁺ ionophore ionomycin as a positive control. However, ROS production as induced by Aβ was significantly blocked by a pretreatment with the extracellular calcium chelator EGTA but not with the intracellular calcium chelator BAPTA-AM in SK-N-MC cells, indicating that Aβ-induced ROS is dependent on extracellular [Ca²⁺]_i (Figure 1H). Interestingly, the [Ca²⁺]_i induced by Aβ was blocked by a treatment with an L-type calcium channel blocker, Nifedipine (Figure 1I and Supplementary Figure S2). These results suggest that [Ca²⁺]_i is highly dependent on extracellular calcium influx.

As shown in the Figure 2A and Supplementary Figure S3, Aβ significantly induced the phosphorylation of Akt at Thr 308 and Ser 473, which was blocked by NAC pretreatment in SK-N-MC and mouse hippocampal neuron. Aβ also significantly induced the phosphorylation of mTOR at Ser 2448, but not at Ser 2481 (Figure 2B). mTOR was more likely to bind RAPTOR, while decreasing the binding of RICTOR when the cells were treated with Aβ for 24 h, suggesting that Aβ uniquely regulates the phosphorylation of mTOR Complex 1 (mTORC1) rather than mTORC2 (Figure 2C). Importantly, the inhibition of PI3K with LY294002 and Akt with an Akt inhibitor significantly blocked the phosphorylation of mTORC1 and RAPTOR, suggesting that Aβ triggers mTORC1 activation through the PI3K/Akt pathway (Figure 2D). The mTOR phosphorylation at Ser 2448 residue in Aβ-treated SK-N-MC cells and hippocampal primary neurons were increased to 238% and 208%, respectively (Figures 2E,F). Given that Aβ specifically induces mTOR phosphorylation, we further assessed whether mTOR activation is involved in the regulation of neuronal apoptosis. The cytotoxicity (Figure 3A) and reduced number of cells (Figure 3B) induced by Aβ were significantly inhibited by a treatment with the mTOR inhibitor, rapamycin. The result in flow cytometric analyses also revealed that the rapamycin significantly blocked the apoptosis induced by Aβ (Figure 3C). Moreover, the levels of caspase-3 cleavage (Figure 3D) and cytotoxicity (Figure 3E) induced by Aβ in primary neurons were significantly blocked by rapamycin. These results indicate that Aβ stimulates the PI3K/Akt pathway required for mTORC1 activation to stimulate neuronal apoptosis.

Despite the fact that Aβ is a strong regulator of ROS production, it was previously reported that Aβ reduces HIF1α expression via a ROS-independent mechanism in astrocytes (Schubert et al., 2009). However, our results showed that the protein level of HIF1α is increased by Aβ for 48 h in a time-dependent manner (Figure 4A). The increased HIF1α level was significantly inhibited by the ROS scavenger NAC (Figure 4B). To determine how ROS induced by Aβ regulates the level of the HIF1α protein, we focused on 4EBP1 and S6K1, which are involved in the mRNA translation of HIF1α. As shown in Figure 4C, Aβ induced the phosphorylation of 4EBP1 and p70S6K1, which was blocked by rapamycin, suggesting that mTOR activation is mediated by the activation of 4EBP1 and p70S6K1 in Aβ-treated SK-N-MC cells. In addition, Aβ-mediated 4EBP1 phosphorylation was closely related to a decreased level of 4EBP1 interaction with its binding protein eIF4E, in which a rapamycin treatment significantly recovered this interaction to the control level (Figure 4D). Importantly, the increased protein level of HIF1α induced by Aβ was markedly inhibited by a treatment with rapamycin (Figure 4E), the p70S6K1 inhibitor PF4708671 (Figure 4F), or the translation inhibitor cycloheximide (Figure 4G). These results indicate the novel role of Aβ in the promotion of HIF1α synthesis though the mTORC1 pathway, which is required for the phosphorylation of 4EBP1 and p70S6K1. Another major role of mTOR is known to be the inhibition of autophagy. As shown in Figure 4H, we found that Aβ reduces the expression of LC3-II and the degradation of p62, and that the inhibition of autophagy induced by Aβ significantly blocked by a treatment with rapamycin. We confirmed using confocal microscopy that Aβ inhibits the formation of autophagic vesicles via mTOR (Figure 4I). Interestingly, the cytotoxicity induced by Aβ was recovered by a treatment with the autophagy inducer trehalose (Figures 4J,K). Together, these results suggest that Aβ has the ability to induce two pathogenic pathways related to HIF1α synthesis and autophagy inhibition through the regulation of mTORC1 activation to promote neuronal apoptosis. Moreover, we pretreated inhibitors used in this study to demonstrate the sequential activation of Aβ-induced ROS/Akt/mTOR/HIF1α pathway. As shown in the Supplementary Figure S4, mTOR phosphorylation (Ser 2448) and HIF1α expression were suppressed by NAC or Akt inhibitor or rapamycin. However, Akt phosphorylations at Thr 308 and Ser 473 residues were inhibited by NAC or Akt inhibitor, but not rapamycin. These findings indicate the ROS-induced Akt is an upstream regulator of Akt/mTOR/HIF1α pathway.

To determine how two pathogenic pathways triggered by Aβ influence the neuronal cell death process, we subsequently investigated the role of Aβ at the level of cell cycle regulatory proteins including cyclin 2, cyclin 4, CDK2, CDK4 and CDK5, which are known as critical regulators of neuronal cell death in AD patients (Frade and Ovejero-Benito, 2015; Liu et al., 2016). Interestingly, Aβ did not have an effect on the mRNA expression of CDK5 or its regulators, p35 and p39, which are well-known candidate molecules responsible for the hyper-phosphorylation of tau protein in AD patients (Figure 5A). Moreover, our result showed that Aβ did not affect p35/p25 expression in SK-N-MCs (Supplementary Figure S5). Instead, we found that Aβ significantly induced the expression of cyclin D₁/CDK4 and cyclin E/CDK2 (Figures 5B,C). However, the level of cell cycle regulatory proteins was inhibited by the knockdown of HIF1A (Figure 5B) and by a treatment with the trehalose (Figure 5C), respectively. In addition, the expression of cell cycle regulatory proteins induced by Aβ is markedly inhibited by a treatment with the rapamycin (Figure 5D), the p70S6K1 inhibitor PF4708671 (Figure 5E), and the translation inhibitor cycloheximide (Figure 5F). Importantly, our results with Aβ-treated primary hippocampal neurons revealed that the increase in the level of cell cycle regulatory proteins was blocked by a treatment with HIF1A siRNA (Figure 5G) and trehalose (Figure 5H). These results indicate that Aβ-induced mTOR signaling pathway stimulates the expression of cell cycle regulatory proteins.

Aβ significantly induced the phosphorylation of tau at Thr 212 and Ser 396 (Figure 6A). Interestingly, the Aβ-induced phosphorylations of tau (Thr 212, Ser 396 and Ser 262) were significantly blocked by the knockdown of CDK2 in SK-N-MC and mouse hippocampal neuron (Figure 6B and Supplementary Figures S6A,B), suggesting that tau phosphorylation induced by Aβ is CDK2-dependent. Furthermore, pretreatment with the CDK inhibitor flavopiridol also prevented the tau phosphorylation induced by Aβ in SK-N-MC cells (Supplementary Figure S7A) and primary neurons (Supplementary Figure S7B). It was noted that tau is co-immunoprecipitated with CDK2, and importantly, that these interactions were enhanced by the Aβ treatment (Figure 6C). Aβ also decreased tubulin interaction with tau, which was blocked by the knockdown of CDK2 (Figure 6C). Consistent with this, pretreatment with the CDK inhibitor flavopiridol also prevented microtubule destabilization as induced by Aβ (Supplementary Figure S7C). Finally, the silencing of CDK2 attenuated the level of cleavage of caspase-3 (Figure 6D), cytotoxicity (Figure 6E) and apoptotic cell death (Figure 6F) as stimulated by an Aβ treatment. In addition, we screened the effect of scrambled Aβ (1-42) peptide to check the nonspecific effect of Aβ. Our results showed that scrambled Aβ treatment did not affect the phosphorylations of Akt (Thr 308 and Ser 473), mTOR (Ser 2448), and tau (Thr 212 and Ser 396) and expressions of HIF1α and CDK2 (Supplementary Figure S8). These findings suggest that CDK2 plays a critical role in tau activation and in the detachment of tau from tubulins in promoting neuronal apoptosis. Interestingly, treatment with a microtubule stabilizer, paclitaxel, significantly blocked Aβ-induced neuronal cell death, although paclitaxel reduced cell viability by 20% (Figure 6G). As tau proteins aggregate after detachment from microtubules, we investigated whether autophagy is associated in any way with aggregated tau. Interestingly, the induction of autophagy with trehalose significantly inhibited the tau phosphorylation induced by Aβ but not the total level of the tau protein, suggesting that autophagy negatively regulates the accumulation of cell cycle regulatory proteins, but not tau itself (Supplementary Figure S9A). Moreover, tau proteins aggregated by Aβ did not co-localize with LC3 puncta despite the fact that autophagy was induced by trehalose (Supplementary Figure S9B). These results suggest that the accumulation of cell cycle regulatory proteins induced by Aβ is necessary for tauopathy, which leads to neuronal apoptosis.