Reagents

Trypsin, penicillin, streptomycin, resazurin sodium salt, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA), caspase-3 substrate (Ac-DEVD-pNA), hydroxyethyl piperazineethanesulfonic acid (HEPES), sodium chloride, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), ethylenediaminetetraacetic acid (EDTA), glycerol, dithiothreitol (DTT), protease inhibitor cocktail, Bradford reagent, bovine serum albumin (BSA), Tween-20, alcohol dehydrogenase, epinephrine, ammonium metavanadate, hydrogen peroxide (H2O2), methanol, all-trans retinoic acid (RA), and acrylamide/bisacrylamide were purchased from MERCK (Darmstadt, Germany). The DMEM/F12 and phosphate buffer saline (PBS) were purchased from Corning (New York, USA). The fetal bovine serum heat inactivated (FBS HI), radioimmunoprecipitation assay (RIPA) buffer, Universal RNA Purification Kit, Perfect Tricolor Protein Ladder and Fast Probe qPCR Master Mix (2x), and plus ROX Solution were purchased from EURx (Gdańsk, Poland). The anti-GAPDH (sc-47724), anti-ac-α-tubulin (sc-23950) mouse primary antibodies and AGK2 (Sirtuin 2 inhibitor) were purchased from SantaCruz Biotechnology (Santa Cruz, USA). The goat anti-rabbit (#31460) and goat anti-mouse (#31430) secondary antibodies HRP-conjugated, TaqMan probes and starters complementary to genes encoding GAPDH (Hs02758991_g1), TUBB3 (Hs00801390_s1), SIRT2 (Hs01560289_m1), P53 (Hs01034249_m1), CAT (Hs00156308_m1), and SOD2 (Hs00167309_m1), and the High-Capacity cDNA Reverse Transcription Kit were purchased from Thermo Fisher Scientific (Waltham, USA). The Laemmli Sample Buffer and β-mercaptoethanol (BME) were purchased from Bio-Rad (Hercules, USA). The rabbit anti-SIRT2 primary antibodies (A3967) were purchased from ABClonal (Woburn, USA). The mouse anti-α-tubulin primary antibodies (66031–1-Ig) were purchased from Proteintech (Düsseldorf, Germany).

Cell Culture and Differentiation Protocol

The human neuroblastoma cell line (SH-SY5Y, ATCC® CRL-2266) was purchased from the American Type Culture Collection (ATCC). The cells were cultured in DMEM/F12 with 10% of FBS HI, supplemented with 0.1% of penicillin/streptomycin, in a humidified atmosphere (37°C, 5% CO2). After reaching confluency, the cells were seeded on a 96-well plate at the density of 4.5 × 103 cells/well, on a 12-well plate at the density of 9 × 104 cells/well, on a 6-well plate at the density of 12 × 105 cell/well, or on a ⌀35 mm culture dish at the density of 1 × 105 cells/well 24 h before the experiment. After this time, the differentiation was initiated as in Zhang et al. with the use of a differentiation medium containing DMEM/F12, 1% of FBS HI, and 10 µM of all-trans retinoic acid (RA) [21]. The medium was subsequently replaced every 3 culture days up to the 14th day to obtain mature neuron model. The effectiveness of the differentiation was assessed based on TUBB3 mRNA expression (a well-established marker of neurons) and visualized using fluorescence staining. Next, on the 14th day of differentiation (mature neurons), the differentiation medium was removed and replaced with medium containing 10 µM of AGK2 and 10 nM of VGVAPG. The concentrations of the tested compounds were chosen based on a previous study [9] and literature data [22]. Additionally, to determine the effect of the tested peptide on Sirtuin 2, the cells were first pretreated with 10 µM of AGK2 (for 0.5 h); afterwards, the cells were treated with 10 nM of VGVAPG for 24h or 48h,. The differentiated neuronal SH-SY5Y cells were used as a model of mature neurons in all the experiments.

Resazurin Reduction Assay

The resazurin reduction assay was performed as in Szychowski et al. [9]. Briefly, on the 14th day of differentiation, the medium was removed and replaced with medium containing 10 µM of AGK2 or 10 nM of VGVAPG. Additionally, pretreatment with 10 µM of AGK2 was performed for 0.5h before the treatment of the SH-SY5Y cells with 10 nM of VGVAPG. After 24h or 48h, the medium was removed and replaced with serum-free medium containing 1% resazurin sodium salt. The measurement of fluorescence was performed at λex. = 530 nm and λem. = 590 nm using a microplate reader (FilterMax F5). The results were expressed as a percentage (%) of the control (DMSO-treated cells).

Intracellular Reactive Oxygen Species (ROS) Level

The 2',7'–dichlorofluorescin diacetate (H2DCF-DA) assay was performed as proposed by Skóra et al. [23]. Briefly, on the 14th day of differentiation, the medium was replaced with serum-free medium containing 5 µM of H2DCF-DA and incubated for 30 min at 37°C with 5% CO2. After this time, the cells were washed once with warm PBS to remove probe residues, and fresh medium containing 10 µM of AGK2 or 10 nM of VGVAPG was added. Additionally, pretreatment with 10 µM of the AGK2 was performed for 0.5h before the treatment of the neuronal cells with 10 nM of VGVAPG. After 24h and 48h, the measurement of fluorescence was performed at λex. = 485 nm and λem. = 535 nm using a microplate reader (FilterMax F5). The results were expressed as a percentage (%) of the control (DMSO-treated cells).

Caspase-3 Activity Assay

The caspase-3 activity assay was performed as in Nicholson et al. with minor modifications [24]. Briefly, after 24h or 48h of treatment of the cells with the tested compounds, the cells were frozen at -80°C for 24h. Afterwards, the cells were lysed using CAB buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, 10 mM DTT) for 10 min at 4°C. Subsequently, the caspase-3-specific substrate (Ac-DEVD-pNA) was added for 30 min. Next, absorbance was measured using a microplate reader (FilerMax F5) at λ = 405 nm. The results were expressed as a percentage (%) of the control (DMSO-treated cells).

Confocal Fluorescence Microscopy

Fluorescence confocal microscopy was used in this study to determine the impact of the tested compounds on degeneration of axons, which is a well-described indicator of the phenotype of AD-like neurons. This method was used to confirm the effectiveness of the neuron differentiation procedure. Briefly, on the 14th day of differentiation, the cells were treated with the tested compounds for 72h as described above. Next, the medium was removed and the cells were washed 3 times with warm PBS and stained with serum-free medium containing 10 µM of Hoechst 33342 (cell nucleus dye) and 10 µM of Calcein-AM (cell cytoplasm dye) for 5 min (37°C, 5% CO2). Next, the cells were visualized by confocal microscopy with a laser scanner module at × 100 magnification (ZEISS LSM700). The mean axon length was quantified using the ImageJ and AutoNeuriteJ module as proposed by Boulan et al. [25]. The results were expressed as a percentage (%) of the control (DMSO-treated cells).

Superoxide Dismutase and Catalase Activity

Superoxide dismutase (SOD) and catalase (CAT) activities were used in this study as markers of oxidative stress in the cells. After treatment with the tested compounds on the 14th day of differentiation, the cells were collected using 50 mM TRIS (pH = 7.2) and frozen at -80°C. Subsequently, the SOD and CAT activity measurement was performed as described by Piechowiak et al. with modifications introduced by Szychowski et al. [23, 26]. The absorbance was measured at λ = 490 nm and λ = 374 nm for SOD and CAT activity, respectively, using a microplate reader (EPOCH). The results were expressed as a percentage (%) of the control (DMSO-treated cells).

Real-Time Polymerase Chain Reaction (RT-PCR)

The RT-PCR assay was performed as in Szychowski et al. with minor modifications [27]. Briefly, after 6h or 24h (in the case of SIRT2) of treatment of the neurons with the tested compounds, the total mRNA was obtained using the Universal RNA Purification Kit according to the manufacturer’s manual (EURx). Subsequently, the reverse transcription reaction was performed using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher). Next, the obtained cDNA template was used in the RT-PCR reaction mixture (total volume 20 µL) containing Fast Probe qPCR Master Mix (2x), primers, and TaqMan probes specific for genes encoding GAPDH, TUBB3, SIRT2, PPARγ, CAT, and SOD2. The RT-PCR method used in this study consisted of the following steps: 2 min at 50 °C and 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C and 1 min at 60 °C. The threshold value (Ct) for each sample was calculated during the exponential phase and ΔΔCt was used to determine the Average Fold (Avg. Fold) of the expression of certain genes. GAPDH was used as a reference gene.

Western Blot Analysis

The protein expression was performed as in Szychowski et al. with minor modifications [28]. Briefly, after 24h and 48h of treatment, the differentiated SH-SY5Y cells were lysed using RIPA buffer containing protease inhibitor cocktail and sonicated (amp. 10%, 8s.) on ice. Subsequently, the total protein content in each sample was measured using the Bradford method (BSA as a standard), and the concentration of protein in each sample was standardized. Next, 40 µg of each sample was mixed with 5 × Laemmli buffer and the samples were separated in 7.5% SDS–polyacrylamide gel electrophoresis (Bio-Rad). Afterwards, the proteins were transferred onto the PVDF membrane and left overnight at 4°C with 35V. Subsequently, non-specific side blocking was performed using 1% BSA in TBST buffer for 1 h, followed by overnight incubation of the membranes with anti-GAPDH (1:1500), anti-SIRT2 (1:2000), and anti-Ac-α-Tubulin (1:400) primary antibodies at 4°C. After this time, the membranes were washed 3 times with TBST and horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:10 000 for anti-Sirtuin 2 and Ac.-α-tubulin, 1:20 000 for anti-GAPDH) were added for 1h at RT. Next, the membranes were washed 3 times with TBST buffer and once with 1 × TBS for 10 min. The protein amount was measured using the chemiluminescence method induced by luminol reagent (Li-Cor). The results were expressed as a relative protein level normalized to GAPDH as a reference protein in each sample and compared to the control (DMSO-treated cells).

Statistical Analysis

In this study, the data are presented as mean values ± standard deviations (SD). Each experiment was performed at least 3 times (n ≥ 3). The results were subsequently subjected to statistical analysis performed using the GraphPad Prism 8.0 Statistical Analysis Mode. The analysis of the statistical differences between the control and treated cells was performed using one-way ANOVA with Tukey’s post-hoc test. The statistical difference between certain groups was measured using the t-test. The results of the statistical analyses (p values) are presented under the bars in each graph.