Materials

Phosphate-buffered saline (PBS) was obtained from Biomed Lublin (Lublin, Poland). Neurobasal medium and B27 were purchased from Gibco (Grand Island, NY, USA). Culture plates for cell cultures were obtained from Techno Plastic Products AG (Trasadingen, Switzerland), Corning (Corning, NY, USA) and Ibidi (Gräfelfing, Germany). Fetal bovine serum (FBS), L-glutamine, dimethyl sulfoxide (DMSO), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate hydrate (CHAPS), ammonium persulfate, N,N,N′,N′-tetramethylethane-1,2-diamine (TEMED), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Trizma base), sodium deoxycholate, DL-dithiothreitol, poly-L-ornithine, (S)-5-(4-hydroxy-3,5-dimethyl-phenyl)-indian-1-ol (PaPE-1), Tween 20, radioimmunoprecipitation assay buffer (RIPA) and protease inhibitor cocktail, SP600125, and GenElute™ Mammalian Genomic DNA Miniprep Kits were purchased from Sigma‒Aldrich (St. Louis, MO, USA). Amyloid-β was obtained from rPeptide (Watkinsville, GA, USA). The RNeasy Mini Kit, EpiTect MethyLight PCR Kit, and EpiTect Bisulfite Kit were obtained from Qiagen (Hilden, Germany). High-Capacity cDNA-Reverse Transcription Kit, Neurite Outgrowth Staining Kit, TaqMan Gene Expression Master Mix, and TaqMan probes for specific genes encoding Hprt, Actb, Gapdh, Fas, Fasl, Bax, Bcl2, Gsk3b, Rbfox, Ache, Apoe, Chat, Bace1, Bace2, Mapt, App, Rcan1, Ide, and Ngrn were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Sodium dodecyl sulfate (SDS), Bradford reagent, Laemmli sample buffer, and 0.5 M Tris–HCL buffer 4–15% Mini-PROTEAN TGX Precast Protein Gels were purchased from Bio-Rad Laboratories (Munich, Germany). The ROS-Glo™ H2O2 assay was obtained from Promega (Madison, WI, USA). JC-10 Mitochondrial Membrane Potential Assay Kit, Z-IETD-FMK, Z-LEHD-FMK, TDZD 8, and SB203580 were purchased from Abcam (Cambridge, UK). 2-Mercaptoethanol was obtained from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Immobilon-P membranes were purchased from Millipore (Bedford, MA, USA). Fluoro-Jade C was obtained from Biosensis Pty Ltd. (Thebarton, Australia). Antibodies used for western blot and immunofluorescence staining were as follows: anti-GAPDH (MAB374), anti-BCL-2 (SAB5700155), and anti-MAP2 (M9942)-obtained from Sigma‒Aldrich (St. Louis, MO, USA); anti-amyloid β (bs-0107R)-purchased from Thermo Fisher Scientific (Waltham, MA, USA); and anti-BAX (SC-7480), anti-GSK3β (sc-9166), anti-FAS (sc-74540), anti-FASL (sc-19681), and anti-MAP2 (sc-20172)-purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Primary Neuronal Cell Culture

Primary neuronal cell cultures were established using Swiss CD1 mice obtained from Charles River Laboratory (Germany) as described previously [23, 24]. Cortices acquired from embryos were fragmented and incubated with 0.1% trypsin at 37 °C for 15 min. Next, the cells were centrifuged for 5 min at 1500 × g in medium with 10% FBS. The obtained neuronal cells were then seeded on multiwell plates coated with poly-L-ornithine (0.1 mg/ml) at a density of ~2.0 × 105 and further cultured in Neurobasal medium with the addition of L-glutamine, B27, and an antibiotic cocktail containing penicillin and streptomycin. Cells were cultured for 7 days at 37 °C with humidified air with a CO2 concentration of 5%. For the first 2 days, the cells were cultured with FBS added to the culture medium. All animals used in the research were maintained according to the principles of the Three Rs in compliance with European Union Legislation (Directive 2010/63/EU, amended by Regulation (EU) 2019/1010).

Treatment

Preparation of Aβ1-42 was conducted as previously described [22]. Non-specific aggregation of Aβ1-42 was negated using HFIP (hexafluoroisopropanol). Next, HFIP was removed under N2 flux, and Aβ1-42 was dissolved using DMSO to prepare a stock solution, which was further dissolved in culture medium. The obtained solution of Aβ1-42 was then incubated overnight to induce specific aggregation and then used to treat the cell cultures. After 24-h treatment with preaggregated Aβ1-42 (10 µM), PaPE-1 (at concentrations of 5 and 10 µM) was applied for the following 6 h. To determine the contribution of apoptotic signaling, we applied Z-IETD-FMK (caspase-8 inhibitor; 40 µM), Z-LEHD-FMK (caspase-9 inhibitor; 40 µM), TDZD 8 (GSK3β inhibitor; 1 µM), SP600125 (JNK inhibitor; 1 µM), and SB 203580 (p38 MAPK inhibitor; 1 µM) to cells treated with Aβ. DMSO was used as a solvent for all compounds at concentrations not exceeding 0.1% in the culture medium.

Assessment of Caspase Activity

In this research, we assessed the activity of caspase-3, caspase-8, and caspase-9. The assessment procedure is identical for each of the enzymes, with the only difference being the substrate used and, consequently, the product of the reaction.

For caspase-3, the colorimetric substrate was Ac-DEVD-pNA (N-acetyl-asp-glu-val-asp-p-nitroanilide, Sigma‒Aldrich, St. Louis, MO, USA), and the product was p-nitroanilide. For caspase-8, the substrate was Ac-VETD-AMC (Ac-val-glu-thr-asp-AMC), and the product was 7-amino-4-methylcoumarin. For caspase-9, the substrate was Ac-DL-Leu-DL-Glu-DL-His-DL-Asp-pNA, and the product is p-nitroanilide.

The activity of caspases was measured as described earlier [25]. Samples were first incubated with CAB (Caspase Assay Buffer) for 15 min at 4 °C and then with substrate specific for each caspase for 60 min at 37 °C. The levels of caspase reaction products were measured with an Infinite M200PRO microplate reader (Tecan, Switzerland) at excitation = 400 nm and emission = 530 nm for caspase-8 activity measurement and absorbance = 405 nm for caspase-3 and caspase-9 activity measurements. The obtained data were analyzed with i-control software and normalized to the blank, and the final results are presented as a percentage of the control ± SEM.

Identification of Living Cells

In this experiment, living cells were identified via calcein AM staining as previously described by Kajta et al. [26]. First, the cells grown on glass cover slips were washed with 10 mM PBS and then incubated with 2 mM calcein AM/PBS solution at room temperature for 10 min. As described above, qualitative analysis was conducted using a Leica DM IL LED Inverted Microscope (Leica Microsystems, Wetzlar, Germany) coupled with a CoolSnap camera (Vision Systems GmbH, Puchheim, Germany) with MetaMorph software (MetaMorph® Microscopy Automation & Image Analysis Software, Molecular Devices LLC, California, United States). Cells presenting bright green cytoplasm were considered living cells. In this case, the intensity of fluorescence was measured from entire photos using ImageJ software. The  final results are presented as a percentage of the control ± SEM.

Identification of Apoptotic Cells

Detection of apoptotic cells was conducted with Hoechst-33342 staining after the initial experiment as described [27]. Cortical primary cells cultured on glass cover slips were washed with 10 mM phosphate-buffered saline (PBS) and incubated with Hoechst-33342 at a concentration of 0.6 mg/ml at room temperature for 5 min. Qualitative analysis was conducted using a Leica DM IL LED Inverted Microscope (Leica Microsystems, Wetzlar Germany) coupled with a CoolSnap camera (Vision Systems GmbH, Puchheim, Germany) with MetaMorph software (MetaMorph® Microscopy Automation & Image Analysis Software, Molecular Devices LLC, California, United States). Bright blue stained nuclei with condensed chromatin are widely recognized as a symptom of apoptosis. Fluorescence intensity was measured based on singular nuclei using ImageJ software. The  final results are presented as a percentage of the control ± SEM.

mRNA Analysis Using qPCR

Total RNA from primary cell cultures was obtained using reagents from an RNeasy Mini Kit (Qiagen, USA) in accordance with the manufacturer’s protocol, as previously described by Wnuk et al. [28, 29]. The RNA was eluted in 40 µl of RNAse-free water. Then, the amount of RNA was assessed using a NanoDrop spectrophotometer at 260 nm, and the 260/280 ratio was obtained (ND/1000UV/VIS; Thermo Fisher Scientific, Waltham, MA, USA). A A260/A280 ratio of ~ 2.0 is considered to be an honest indicator of pure RNA. Subsequently, after isolation, the RNA extract was reverse transcribed to avoid freeze‒thaw cycles. Reverse transcription was conducted with a High-Capacity cDNA Reverse Transcription Kit in accordance with the manufacturer’s protocol using a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). The collected cDNA was stored at − 20 °C overnight and subsequently subjected to quantitative polymerase chain reaction (qPCR). Amplification of the cDNA was conducted using FastStart Universal Probe Master containing TaqMan Gene Expression Assays specific for Bax, Bcl2, Gsk3b, Fas, Fasl, Rbfox, Ache, Apoe, Chat, Bace1, Bace2, Mapt, App, Rcan1, Ide, Ngrn, Hprt1, Gapdh, and Actb. In amplification, the following substances were used: a mixture containing 10 µl of FastStart Universal Probe Master, 1 µl of cDNA as template, 1 µl of the TaqMan Gene Expression Assay mix, and 8 µl of RNAse-free water in a total volume of 20 µl. qPCR was conducted using a CFX96 Real-Time system (Bio-Rad, USA), and the procedure consisted of intervals of varying temperatures as follows: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The obtained data were analyzed with the delta delta Ct method. The reference gene was chosen with the use of the following algorithms: geNorm, NormFinder, BestKeeper, and delta Ct; the three algorithms recommended hypoxanthine–guanine phosphoribosyltransferase (Hprt1).

Western Blot Analysis

Once the experiment was concluded, neocortical cells underwent lysis in RIPA buffer with the addition of protease inhibitor. After lysis, the cells were sonicated to obtain homogenous solution, which was subsequently centrifuged at 15,000 × g for 20 min at 4 °C. To assess the protein concentration, a Bradford assay was conducted using Bradford reagent and bovine serum albumin as standards. Samples were then reconstituted and denatured in Laemmli sample buffer with β-mercaptoethanol. Subsequently, the samples underwent electrophoresis in 15-well [4–15%] SDS polyacrylamide gels (Bio-Rad, USA), and then, proteins were electrotransferred to PVDF membranes with the Bio-Rad Mini Trans-Blot apparatus as previously described [30, 31]. To block the non-specific binding sites, the PVDF membranes were incubated for 2 h with a solution of dried milk (5%) and Tween-20 (0.2%) in 0.02 M Tris-buffered saline (TBS). Next, the membranes were incubated overnight with primary antibodies at 4 °C. Primary antibodies were diluted using solution of Tween-20 and TBS in proportions as follows: anti-GAPDH mouse monoclonal antibody (diluted 1:3500), anti-BCL-2 rabbit polyclonal antibody (diluted 1:100), anti-BAX mouse monoclonal antibody (diluted 1:100), anti-GSK3β rabbit polyclonal antibody (diluted 1:700), anti-FAS mouse monoclonal antibody (diluted 1:80), and anti-FASL mouse monoclonal antibody (diluted 1:80). Next, the membranes were washed with Tween-20/TBS solution and incubated for 1 h with secondary antibodies coupled with horseradish peroxidase diluted in Tween-20/TBS solution 1:100 and 1:3500. Detection of the chemiluminescent signal was conducted employing BM (Chemiluminescence Blotting Substrate) and visualization using a Luminescence Image Analyzer Fuji-Las 4000 (Fuji, Japan). The intensity of the obtained bands was quantified with the MultiGauge V3.0 program (ScienceLab).

Bax and Bcl2 Gene-Specific Methylation

Specific methylation of Bax and Bcl2 genes was measured as described previously [32, 33]. Genomic DNA was obtained with GenElute™ Mammalian Genomic DNA Miniprep Kits, and the quantity of obtained DNA was assessed spectrophotometrically at wavelengths of 260 nm and 260/280 nm with a NanoDrop ND-1000 UV‒Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, denaturation followed by bisulfite conversion of GC-rich DNA was conducted with an EpiTect Bisulfite Kit obtained from Qiagen (Hilden, Germany). Samples were then eluted in a 10 µl volume and underwent qPCR (MethyLight) with an EpiTect MethyLight PCR Kit. The methylation regions in the Bax and Bcl2 genes were verified in CpG hot spots in the 5′ flanking sequence (2000 bp). Methyl Primer Express Software 1.0 was employed to design primers for methylated and unmethylated target sequences. Fully methylated and fully unmethylated TaqMan probes were designed for the Bax and Bcl2 promoters, and the internal reference set for the Hprt1 gene was designed to control the input of DNA. For the EpiTect MethyLight assays, the specific TaqMan probes contained FAM™ as the reporter dye. The degree of methylation of each sample was calculated by taking the threshold cycles determined as percentage of methylation [%]: Cmeth = 100/[1 + 2(∆Ctmeth − ∆Ctunmeth)].

Immunofluorescence Staining

To visualize the cellular localization of the studied proteins and to confirm the neuronal character of cultured cells, immunofluorescence labeling followed by confocal microscopy was employed as previously described [34]. The cortical cells were cultured on glass coverslips and subjected to immunofluorescence labeling. Cells were fixed with a 4% paraformaldehyde solution in PBS for 15 min at room temperature and further incubated with blocking buffer, i.e., 5% normal donkey serum and 0.3% Triton X-100 in 0.01 PBS for 1 h. Subsequently, the neurons were incubated with the following primary antibodies for 24 h at 4 °C: anti-Aβ rabbit (diluted 1:50), anti-MAP2 mouse (1:100), anti-BAX mouse (1:50), anti-BCL2 rabbit (1:50), anti-FAS mouse (1:50), anti-FASL mouse (1:50), and anti-GSK3β rabbit (1:50). Next, the neocortical cells were incubated with secondary antibodies: Alexa Fluor Plus 488-conjugated goat anti-mouse IgG (1:200 and 1:600) and Alexa Fluor Plus 647-conjugated goat anti-rabbit IgG (1:200). Finally, the microscopic preparations were washed with PBS, mounted, and cover-slipped. For viewing the preparations, a Leica TCS SP8 WLL confocal laser scanning microscope (DMi8-CS, Leica Microsystem, Wetzlar, Germany) was employed.

Fluoro-Jade C Staining

Fluoro-Jade C staining was used to assess the level of degenerating neurons as previously described [33, 35]. To conduct the staining, a stock solution with a concentration of 0.01% was prepared by diluting the Fluoro-Jade C in distilled water. The stock solution was then further diluted into a 0.005% working solution in Neurobasal medium. Then, the culture medium was removed from the 96-well plates, and the working solution was added at 100 µl per well. The plates were incubated for 45 min at room temperature. Then, the level of fluorescence was measured, with excitation = 490 nm and emission = 525 nm, using an Infinite M200 PRO microplate reader (Tecan Mannedorf, Switzerland). The results were analyzed by i-control software and presented as the percentage of the control ± SEM. Fluoro-Jade C is a green fluorescence dye. Microscopic images were obtained with a Leica DM IL LED Inverted Microscope (Leica Microsystems, Wetzlar, Germany). In this study, we used ImageJ to change the color to magenta.

Neurite Outgrowth Staining

To assess neurite outgrowth, the Neurite Outgrowth Staining Kit was employed. First, the cells were washed with PBS, and then, the appropriate amount of 1X working Fix/Stain Solution was added to each well. Then, the cells were incubated for 15 min at room temperature. Next, the staining was visualized using a Leica TCS SP8 WLL confocal laser scanning microscope (DMi8-CS, Leica Microsystem, Wetzlar, Germany). The obtained images were analyzed with ImageJ, and the fluorescence intensity from entire images was measured. The data were normalized to the signal intensity of vehicle-treated cells and expressed as a percentage of the control ± SEM.

ROS Activity Measurement

The ROS-Glo™ H2O2 Assay was used to assess the level of reactive oxygen species (ROS) in neocortical cells exposed to Aβ and PaPE-1. The assay was performed according to the manufacturer’s protocol. The bioluminescence was measured using a GloMax® Navigator Microplate Luminometer (Promega, Madison, WI, USA). The detected signal was proportional to the amount of H2O2 in cultured cells. The data were normalized to the signal intensity of vehicle-treated cells and expressed as a percentage of the control ± SEM.

Assessment of Mitochondrial Membrane Potential

The JC-10 dye is a commonly employed fluorescent marker that forms aggregates (emitting red fluorescence) within mitochondria when the mitochondrial membrane potential is high. When mitochondrial membrane potential is decreased, JC-10 stays in the cytoplasm in monomeric form (green fluorescence). The assay was performed following the manufacturer’s protocol and fluorescence measurements were taken at Ex/Em = 490/525 nm and Ex/Em = 540/590 nm using an Infinite M200 PRO microplate reader (Tecan Mannedorf, Switzerland). The fluorescence intensity data were used to calculate the ratio, and the results were expressed as a percentage relative to the control, with the standard error of the mean (SEM). The data were normalized to the fluorescence intensity of control cells.

Data Analysis

Statistical analysis was performed on raw data. The obtained results are presented as the mean absorbance, fluorescence or luminescence units per well containing 50,000 cells for the caspase activity assessment, JC-10, ROS activity and Fluoro-Jade C, mean fluorescence of the whole picture or single nuclei for calcein AM, Hoechst 33342 and neurite outgrowth staining, the fluorescence units per 1.5 million cells for qPCR and specific gene methylation, and the mean optical density per 10 µg of protein for western blot assays. To determine overall significance, an analysis of variance (ANOVA) was used. The differences between the control and experimental groups were defined with a post hoc Newman‒Keuls test that was preceded by Levene’s test for homogeneity.

Differences of statistical significance were indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001 (versus control cultures) and ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 (versus cultures exposed to Aβ). The results are expressed as the mean ± SEM.