Novel Hybrid Acetylcholinesterase Inhibitors Induce Differentiation and Neuritogenesis in Neuronal Cells in vitro Through Activation of the AKT Pathway

Abstract

Background:

Alzheimer’s disease (AD) is characterized by a progressive loss of episodic memory associated with amyloid-β peptide aggregation and the abnormal phosphorylation of the tau protein, leading to the loss of cholinergic function. Acetylcholinesterase (AChE) inhibitors are the main class of drugs used in AD therapy.

Objective:

The aim of the current study was to evaluate the potential of two tacrine-donepezil hybrid molecules (TA8Amino and TAHB3), which are AChE inhibitors, to induce neurodifferentiation and neuritogenesis in SH-SY5Y cells.

Methods:

The experiments were carried out to characterize neurodifferentiation, cellular changes related to responses to oxidative stress and pathways of cell survival in response to drug treatments.

Results:

The results indicated that the compounds did not present cytotoxic effects in SH-SY5Y or HepG2 cells. TA8Amino and TAHB3 induced neurodifferentiation and neuritogenesis in SH-SY5Y cells. These cells showed increased levels of intracellular and mitochondrial reactive oxygen species; the induction of oxidative stress was also demonstrated by an increase in SOD1 expression in TA8Amino and TAHB3-treated cells. Cells treated with the compounds showed an increase in PTEN_{(Ser380/Thr382/383)} and AKT_(Ser473) expression, suggesting the involvement of the AKT pathway.

Conclusion:

Our results demonstrated that TA8Amino and TAHB3 present advantages as potential drugs for AD therapy and that they are capable of inducing neurodifferentiation and neuritogenesis.

Keywords

Acetylcholinesterase inhibitors AKT pathway Alzheimer’s disease neuronal differentiation oxidative stress SH-SY5Y cells

INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by neuronal loss, neurofibrillary tangles, and amyloid-β (Aβ) deposition in the brain [1]. Aβ peptides are derived from the abnormal proteolytic processing of amyloid-β protein precursor (AβPP) by β- and γ-secretases, resulting in the accumulation and subsequent aggregation of Aβ_1–40 and Aβ_1–42 amyloid proteins [2, 3]. The abnormal hyperphosphorylation of the tau protein causes a specific type of slow and progressive neurodegeneration, leading to neurofibrillary degeneration, which can be observed as neurofibrillary tangles, neuropilar wires, and dystrophic neurites [4]. There is evidence that these changes are directly associated with the clinical characteristics of AD, which in turn cause the depletion of cerebral acetylcholine. This depletion is one of the causes of cognitive decline in older adults and patients with AD, since the cholinergic system plays an important role in regulating the processes of learning and memory [5, 6].

The main therapeutic strategy applied in AD is based on the inhibition of acetylcholinesterase (AChE), aiming to reduce the symptoms of the disease [3], leading to an increase in acetylcholine (ACh) levels in the brain [7]. AChE inhibitors, such as donepezil, galantamine, rivastigmine, and tacrine (the latter is no longer prescribed due to its hepatotoxicity), have been indicated as pharmacological interventions for patients with mild or moderate AD [5, 8]. Additionally, drugs targeting N-methyl-D-aspartate (NMDA) receptors, such as memantine, are also prescribed for AD patients [9]. In general, these drugs are well tolerated in clinical practice, but some patients may suffer from several side effects, such as nausea, vomiting, diarrhea, and tremors [5, 10]. In fact, the use of AChE inhibitors (AChEI) in patients with mild, moderate, or severe AD provides modest improvements in cognition, function, and behavior [11]. Thus, there is currently a large worldwide demand for new therapies that are effective in slowing the progression of AD and are also able to reverse the symptoms of the disease [12].

In the last few years, several different therapy strategies were developed for AD, such as those based on the inhibition of AChE, or butyl cholinesterase (BChE), Aβ, β-secretase-1 (BACE), antioxidant properties of metals, and capacity of free radical scavenging [13]. Among these strategies, a new series of hybrid molecules of donepezil and tacrine were synthesized as new dual AChE inhibitors, with the ability to simultaneously bind to the peripheral and catalytic sites of the enzyme [7]. Several of these hybrid molecules incorporate the main substructures derived from various chemotypes, such as acridine, quinoline, carbamates, and other heterocyclic analogs that have the desired pharmacological activity in a single hybrid molecule [13]. However, it is known that tacrine bearing an acridine ring promotes a high rate of serum aminotransferase elevations during therapy, thus causing acute liver injury [14].

Taking into account the inhibitory activity of these hybrids, in addition to the requirement of reducing the hepatotoxicity potential due to the tacrine moiety, Chierrito et al. [14] designed an alternative approach to prevent these undesirable effects using a simplified scaffold of tacrine structure related to the quinoline core that may display two favorable properties: 1) maintenance of the main interactions at AChE active site and 2) decrease in the toxicity attributed to the acridine nucleus of tacrine. Thus, the authors synthesized novel hybrid donepezil-tacrine molecules, which are potent allosteric modulators of AChE, and capable of achieving cholinergic functions by fixing a specific AChE conformation, confirmed by STD-NMR (Saturation Transfer Difference Nuclear Magnetic Resonance) and molecular modelling studies. In addition, biological assays performed in SH-SY5Y cells indicated that the two new hybrid compounds are not cytotoxic when tested at concentrations within the levels of the IC₅₀ (50% inhibition of AChE activity); furthermore, the hybrids were capable of inducing neurodifferentiation [14].

According to the literature, the neuroblastoma cell line SH-SY5Y is commonly used as a model of neuronal differentiation and maturation [15 –17]. The neuronal differentiation of these cells is typically blocked at an early stage, and cells exhibit low levels of neuronal markers [15, 18]. Despite their tumor origin, SH-SY5Y cells present a functional and morphological neuronal phenotype when treated with several agents, such as retinoic acid (RA) and its active metabolite [19]. When mature neurons derived from SH-SY5Y cells were treated with RA for 7 to 10 days, it was found that they express catecholaminergic/neuronal markers, such as tyrosine hydroxylase (TH), enolase-2, and β-III-tubulin [18, 20]. Furthermore, previous studies have shown that RA-treated neurodifferentiated SH-SY5Y cells showed an increase in acetylcholinesterase enzyme activity [17, 21]. Thus, this model is applicable to different approaches, including neuronal differentiation, disease modelling, and drug screening.

In this context, a promising emerging therapeutic strategy for patients with neurodegenerative diseases is based on targeting the regulation of niches residing in neural stem cells (NSCs) in the adult brain. The stimulation of neurogenesis in the adult hippocampus can be used as a strategy to compensate the neuronal death by using molecules that are capable to modulate the differentiation of NSCs and, consequently, the rate of neurogenesis [12, 22]. Studies have shown that the drug donepezil, which has AChE inhibitory function, also promotes neurogenesis in the hippocampus of adult rats [23, 24]. Therefore, the discovery of new compounds that can target aspects of the differentiating function of NSCs is promising. Thus, the treatment of patients with compounds that act in adult neurogenesis may be considered a viable therapeutic approach, and this strategy has also the potential to delay the development of neurodegenerative diseases [25].

The major intracellular signaling pathways that control neurite outgrowth and neurogenesis include those of growth factors, such as the protein kinase B (AKT), phosphatidylinositol 3-kinase (PI3K), glycogen synthase kinase 3β (GSK-3β), and protein kinase C (PKC) proteins [26]. Hence, the activation of the PI3K/AKT signaling pathway has been studied for its involvement in the stimulation of neurogenesis and neuronal survival. The PI3K/AKT pathway plays an essential role in brain development, promoting neuritogenesis, the extension of neurites (dendrites and axons), and the regulation of neuronal synaptic plasticity, especially in the hippocampus. It has been demonstrated that the activation of this pathway in rat embryos can inhibit autophagy, protecting hippocampal neurons [27]. Furthermore, the activation of PI3K/AKT has been found to be correlated with increased cell survival, partially due to the phosphorylation and inhibition of death-inducing proteins, including GSK-3, Bcl-xL/Bcl-2-associated death promoter (BAD), and caspase-9. The misregulation of cell death mechanisms has been reported in AD brains [28]. Still according to Jiang et al. [29], AChE inhibitors, such as donepezil and galantamine, may exert a neuroprotective effect through the activation of the PI3K/AKT pathway, thus promoting a pro-survival response in neurons exposed to various apoptosis-inducing stimuli.

Based on the above information, the current study was performed to test the hypothesis that the activity of AChE inhibitors (novel synthetic donepezil-tacrine hybrids) relies on the induction of neurodifferentiation and neuritogenesis in neuronal cells derived from SH-SY5Y cells. Thus, the purpose was to evaluate the potential of two tacrine-donepezil hybrid molecules (TA8Amino and TAHB3) to induce neuronal differentiation and neuritogenesis in SH-SY5Y cells, as well as to characterize cellular alterations related to oxidative stress responses and their association with cell survival pathways in response to treatment.

MATERIALS AND METHODS

Chemicals

The AChE inhibitor compounds were kindly supplied by Prof. Dr. Ivone Carvalho (Laboratory of Medicinal Chemistry of the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo). The compounds, TA8Amino and TAHB3, were donepezil-tacrine hybrids that were obtained by molecular hybridization [14]. The compounds were prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), and the stock solutions were diluted in phosphate buffered saline (PBS). The concentrations used were based on the IC₅₀ of AChE activity (Fig. 1) [14]. DMSO was used as a solvent at a concentration of 0.1% and doxorubicin (0.17 μM) was used as a positive control.

Fig.1

Compounds with Human hAChE inhibitory activity. IC₅₀, 50% of maximal inhibitory concentration [14].

Cell lines and treatment conditions

Human SH-SY5Y neuroblastoma and HepG2 cell lines were purchased from Rio de Janeiro Cell Bank (CBRJ, Rio de Janeiro, SP, Brazil), and the American Type Culture Collection (ATCC), respectively; they were kept frozen in N₂ (liquid nitrogen). After thawing, the cells were maintained in a mixture of HAM F10 and DMEM (1:1) (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, New York, USA) and penicillin (100 U/mL)/streptomycin (100 mg/mL) (Sigma-Aldrich) and incubated at 37°C in 5% CO₂ and a 95% humidified atmosphere. All experiments were conducted in cell cultures that reached 70–80% confluence. The HepG2 hepatic cell line was used for the evaluation of AChE inhibitors as a model of cellular metabolic systems, especially cytochrome P450 (CYP) [30]. To investigate the neurotoxic effects of AChE inhibitors, the cells were treated with different concentrations of TA8Amino (0.0035–0.112 μM) and TAHB3 (0.088–2.84 μM) for 24 h. Donepezil (0.0057 μM) and tacrine (0.23 μM) were also tested in parallel as single-treatment controls. Based on the cell viability results, the IC₅₀ values of AChE activity were determined for each hybrid compound, and they were determined to be 0.014 μM for TA8Amino, and 0.71 μM for TAHB3; these values were chosen based on previous results generated in neuronal differentiation assays. Each experiment was repeated at least three times.

Cytotoxicity assay

Cytotoxicity assays were performed using an XTT kit (Cell Proliferation Kit II; XTT, Roche Molecular Biochemicals) according to the manufacturer’s instructions. After 24 h of treatment, the medium was removed and replaced with fresh medium containing 10% FBS, and the cell cultures were incubated at 37°C for different times: 48, 72, or 120 h. SH-SY5Y cells were seeded in 24-well plates; 8×10⁴ cells/well were plated for the 48 h experiment, and 4×10⁴ cells/well for plated for the 72 h and 120 h experiments. HepG2 cells were seeded in 96-well plates; 10⁴ cells/well were plated for the 48 h experiment, and 2×10³ cells/well were plated for the 72 h and 120 h experiments. The absorbance was measured at 492 and 690 nm (Epoch Microplate Spectrophotometer, BioTek Instruments, Winooski, VT, USA). The results are expressed as percentages, and for each experiment, the value obtained for the negative control was considered 100%.

Neuronal differentiation assay

Neuronal differentiation was studied in the SH-SY5Y cell line. The cells were grown in 6-well plates (1×10⁶ cells/well) for 24 h. Neuronal differentiation assays were performed in HAM F10 and DMEM (1:1) containing 1% FBS, and the cell cultures were treated with the following compounds: TA8Amino (0.014 μM), TAHB3 (0.71 μM), donepezil (0.0057 μM), and tacrine (0.23 μM). Retinoic acid (RA, 10 μM) (Sigma-Aldrich) is an important inducer of neuronal differentiation in the SH-SY5Y cell line [18], being adequate to be used as a positive control for the neuronal differentiation process. The culture medium was replaced every two days to maintain the cell treatments for up to 8 days (Fig. 2) [18].

Fig.2

Neurodifferentiation protocol applied to SH-SY5Y cells. At day – 1, cells were seeded and cultured in 10% FBS medium; after 24 h (day 0), the medium was removed and replaced by treatment medium (TM) containing 1% FBS, following drug treatments: DMSO, 0.1% (negative control); Retinoic acid, 10 μM (RA, positive control); TA8Amino (0.014 mM); TAHB3 (0.71 μM); donepezil (0.0057 μM); tacrine (0.23 μM). For all cell cultures, the medium was replaced every two days.

Immunofluorescence staining

SH-SY5Y cells were grown on glass coverslip (1×10⁵ cells) and let to neurodifferentiate along 8 days. After this period, cells were fixed with methanol/acetate acid (1:1) for 10 min, and permeabilized with a 0.2% solution of TritonX-100 (Sigma-Aldrich) for 15 min. A blocking solution containing 2% of bovine serum albumin (BSA - Sigma-Aldrich) was added, and after 1 h, cells were incubated with the primary antibody anti-β-III-tubulin (5 μg/ml) for 2 h at room temperature in a solution containing 2% BSA and 0.1% Tween20. Then, cells were washed with 0.1% Tween20 solution and the secondary antibody (Alexa Fluor 488 chicken, Invitrogen, diluted 1:200 in solution containing 2% BSA and 0.1% Tween20) was added for 40 min, and kept in the dark. Cells were washed with 0.1% Tween20 solution, following staining with DAPI (0.15 μg/mL) for 10 min. The coverslips were assembled with Vectashield (Vector Labs) solution. The images were analyzed in inverted-phase microscope, 40× objective (Zeiss).

Quantification of neuronal differentiation and neurite outgrowth

Cells were grown in 6-well plates (1×10⁶ cells/well) and let to achieve neurodifferentiation along 8 days. After this period, neuronal differentiation was analyzed in images captured by using light microscopy. Twenty microscopic fields were randomly selected, and photographs were taken under an inverted-phase microscope using a 20× objective. The cells were analyzed by using the ImageJ software with the Fiji image processing package. Initially, a micrometer set scale (μm) was defined. Then, the images were processed using the Simple Neurite Tracer plugin and 2D tracing view. This plugin provides the tracking path that will be considered to determine the neurite length (μm unit). Approximately 100 cells per sample were analyzed to determine the rates of neurite-bearing cells [31].

Western blot analysis

Cells were lysed in 100 μL of RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with protease inhibitor cocktails (Sigma-Aldrich) on ice and shaken for 10 min. After centrifugation at 14000×g for 15 min at 4°C, the supernatant was collected, and protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. Proteins (40 μg) were separated by electrophoresis on a NuPAGE 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and blotted onto a PVDF membrane (Invitrogen). The membrane was incubated in blocking buffer and kept at 4°C for 16 h in the presence of antibodies: beta-III-tubulin (1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), phospho-AKT_(Ser473) (1:500 dilution; Cell Signaling Technologies, Danvers, MA, USA), AKT (1:500 dilution; Cell Signaling Technologies), PTEN (1:500 dilution; Cell Signaling Technologies), phospho-PTEN_{(Ser380/Thr382/383)} (1:500 dilution; Cell Signaling Technologies), COX2 (1:500 dilution; Cell Signaling Technologies), SOD1 (1:500 dilution; Cell Signaling Technologies), and β-actin (1:1000 dilution; Cell Signaling Technologies). The membrane was incubated with secondary antibody (CA Breeze Chemiluminescent Kit, Invitrogen). The band intensity was quantified using Image Studio Lite Software, Version 5.0. The proteins were marked separately and, when necessary, the membrane was submitted to stripping and re-labelling.

Analysis of cell cycle

The cell cycle was analyzed with the use of propidium iodide, a fluorescent nuclear marker. After 8 days of cell differentiation, the samples were collected, processed and stored at –20°C in 70% cold ethanol for 16 h. The samples were then resuspended in 200 μL of the propidium iodide (5 μg/mL) solution and maintained for 30 minutes at 37°C. The cells were analyzed with a Guava EasyCyte Mini System flow cytometer (Merck Millipore) using Guava CytoSoft software, version 4.2.1 (Merck Millipore).

Cell proliferation assay

The cell proliferation assay was carried out using flow cytometry with a Guava ViaCount Kit (Merck Millipore, Billerica, MA, USA). Cells were collected at days 0, 2, 6, and 8 and analyzed using Guava CytoSoft Software 4.2.1 (Guava Technologies). The cell number (proliferation index) was calculated as the sum of the numbers of cells that were observed for all generations (including parental) divided by the number of cells at the beginning of the experiment [32].

Superoxide production

The generation of intracellular and mitochondrial superoxide was measured using flow cytometry using dihydroethidium (DHE) (Molecular Probes, Thermo Fisher Scientific Inc.) and MitoSOX Red (Molecular Probes, Thermo Fisher Scientific Inc.), respectively. After 8 days of neurodifferentiation, cells were incubated with DHE (1.5 μM) or MitoSOX Red (1.5 μM) diluted in PBS containing 5 mM glucose and kept at 37°C in the dark for 30 min. The cells were analyzed with a Guava EasyCyte Mini System flow cytometer (Merck Millipore) using Guava CytoSoft software, version 4.2.1 (Merck Millipore).

Measurement of the mitochondrial membrane potential and mitochondrial mass

The mitochondrial membrane potential (ΔΨ) was measured with non-quenching tetramethylrhodamine methyl ester (TMRM) (Molecular Probes, Thermo Fisher Scientific Inc.), and mitochondrial mass was measured with MitoTracker Green (Molecular Probes, Thermo Fisher Scientific Inc.). After 8 days of neurodifferentiation, cells were incubated with TMRM (40 nM) or MitoTracker Green (50 nM) in PBS containing 5 mM glucose and kept at 37°C in the dark for 30 min. The cells were analyzed using a Guava EasyCyte Mini System flow cytometer (Merck Millipore) and Guava CytoSoft software, version 4.2.1 (Merck Millipore).

Statistical analysis

For each assay, the results obtained from at least three independent experiments are expressed as the mean±SEM. Statistical analysis was performed using one-way ANOVA and Newman-Keuls post-test or Kruskal-Wallis test using GraphPad Prism software, v. 5.0. p < 0.05 was considered the limit for a statistically significant difference between the results.

RESULTS

AChE inhibitors do not induce cytotoxicity in SH-SY5Y and HepG2 cells

Cell viability assays were performed in SH-SY5Y cells treated with TA8Amino (0.0035 to 0.112 μM) and TAHB3 (0.088 to 2.84 μM), which, compared with the negative control (DMSO) did not induce significant cytotoxic effects on cell viability at 48, 72, or 120 h (Fig. 3a, b); however, 0.17 μM doxorubicin (positive control) induced a significant decrease (p < 0.001) in cell viability. Similarly, HepG2 cells treated with TA8Amino and TAHB3, compared to those treated with the control (DMSO), did not show statistically significant (p > 0.05) changes in cell viability at the time points studied (Fig. 3c, d), whereas doxorubicin (positive control) induced a significant decrease (p < 0.001) in cell viability at 48 h; however, the cells were partially recovered at 72 and 120 h. Donepezil and tacrine were not cytotoxic in all tested times. These results indicate a low cytotoxic effect of the hybrid compounds, as well as donepezil and tacrine. The subsequent assays were performed at concentrations based on the IC₅₀ of AChE activity, specifically 0.014 μM TA8Amino, 0.71 μM TAHB3, 0.0057 μM donepezil, and 0.23 μM tacrine.

Fig.3

Cytotoxic effects of AChEI treatments. Cell viability in cells treated with TA8Amino (a) and TAHB3 (b) in SH-SY5Y cell line (N = 3). Cell viability in cells treated with TA8Amino (c) and TAHB3 (d) in HepG2 cell line (N = 3). Cell viability was determined by the XTT assay. The results are expressed as mean±SEM. Three independent experiments were performed. Statistical analysis: One-Way ANOVA and Newman-Keuls post-test. ^***p < 0.001; ^*p < 0.05 indicate statistically significant differences compared to the negative control (0.1% DMSO). DO, Doxorubicin (positive control, 0.17 μM); donepezil (0.0057 μM); tacrine (0.23 μM).

AChE inhibitors induce morphological differentiation by expression of neuronal marker β-III-tubulin

Morphological analysis determined by immunofluorescence images in neurodifferentiated cells treated with the hybrid compounds demonstrated that TA8Amino and TAHB3 were capable of inducing morphological alterations, including an increase in neurite projections and decreases in cytoplasm mass and the number of cells per field, as analyzed by microscopy, mainly in the differentiated group induced by TA8Amino. For RA-treated cells, such morphological changes were observed in practically all cells. Likewise, we observed similar morphological alterations in cells treated with the hybrid compounds, as well as with donepezil and tacrine (tested separately) although to a lesser extent (Fig. 4a).

Fig.4

Neurodifferentiation assay in SH-SY5Y cell line. a) Representative immunofluorescence images showing the cytoskeleton labelled with anti-β-III-tubulin (5 μg/mL) and nuclear DAPI (0.15 μg/mL) staining in neurodifferentiated cells. Morphological changes observed under 20× magnification, using an inverted microscope (Zeiss). b) Representative image of the neurite tracing (pink line) of the neurite length quantification experiments using the ImageJ software (Fiji) for analysis. c) The length of neurites was evaluated by the ImageJ software (Fiji) in a total of 100 cells (N = 3). Statistical analysis: One-Way ANOVA and Newman-Keuls post-test. d) Expression of β-III-Tubulin (∼55 kDa) and β-actin (∼45 kDa) proteins analyzed by western blotting following neurodifferentiation. The images were obtained in the same gel/blot. e) Levels of β-III-Tubulin expression after neurodifferentiation (N = 4). The values were normalized with the endogenous β-actin protein, using the software Image Studio Lite Ver 5.0 (Lite Software). DMSO 0.1% (negative control); Retinoic acid 10 μM (RA, positive control); TA8Amino compound (0.014 mM); TAHB3 (0.71 μM); donepezil (0.0057 μM); tacrine (0.23 μM). Statistical analysis was performed by the One-Way ANOVA and Newman-Keuls post-test, for the flow cytometry data and Kruskal-Wallis test for the protein expression (western blot). Three or four independent experiments were performed. Data are expressed as mean±SEM. ^***p < 0.001; ^**p < 0.01; ^*p < 0.05 indicate statistically significant differences when the results were compared to the negative control and between groups.

AChE inhibitors (TA8Amino, TAHB3, donepezil, and tacrine) administered during the period of neurodifferentiation increased the neurite length. While the neurite length observed for untreated cells (negative control) was 21.9 μm, RA-treated cells showed a significant increase (73.4 μm; p < 0.001). The cells treated with TA8Amino and TAHB3 also showed a significant (p < 0.01) increase, with average length of 37.4 and 32.7 μm, respectively, while the cells treated with donepezil and tacrine also showed a significant (p < 0.01) increase, with length of 31.5 and 30.6 μm, respectively (Fig. 4c). These results indicate that the all compounds efficiently increased neurite outgrowth.

Neuronal differentiation was confirmed by the quantification of β-III-tubulin expression, which was evaluated in cells collected after 8 days (neurodifferentiation assay). TA8Amino induced a significant increase (p < 0.05) in the expression of β-III-tubulin compared to that induced by the negative control, but cells treated with TAHB3 and RA showed only a slight increase in β-III-tubulin expression; donepezil and tacrine did not induce a significant (p > 0.05) increase in the expression of β-III-tubulin (Fig. 4d, e).

Alterations of cell cycle and cell proliferation index in AChE inhibitor-treated neurodifferentiated cells

The cell cycle analysis, TA8Amino and TAHB3-treated cells did not show significant differences in the cell cycle progression compared with the negative control (DMSO) that showed a regular proportion of cells (53.3%; 23.3%; 25.0%) at the G0/G1, S, and G2/M phases, respectively. However, donepezil and tacrine significantly reduced (p < 0.01 and p < 0.001, respectively) the proportion of cells (18.3%; 17.5%) at the G2/M phase; tacrine also induced a significant increase (p < 0.001) in sub-G1 fraction (17.6%). The positive control (RA) induced a significant increase (p < 0.001) in the proportion of cells at G0/G1 phase (75.3%), with a consequent reduction (p < 0.001) of cells at S (10.3%) and G2/M (14.2%) phases of the cell cycle (Fig. 5a, b). In addition, the cell proliferation capacity was analyzed during the neurodifferentiation of cells treated with the hybrid compounds by counting the total number of viable cells and calculating the doubling time following 2, 6, and 8 days of treatment. As expected, the cells treated with the positive control (RA) showed a significant decrease (day 6, p < 0.05; day 8, p < 0.01) in doubling times after differentiation into mature neurons. Only TA8Amino induced a significant increase (p < 0.05) in the number of cell duplications on day two, but following 8 days of neurodifferentiation, the results were similar to those observed with the negative control (DMSO). The other treatments did not show significant differences in comparison with the negative control (Fig. 5c).

Fig.5

Cell cycle kinetics and Cell proliferation index during 8 days of neurodifferentiation in drug-treated SH-SY5Y cells. a) The cell cycle analysis of differentiated cells was performed using PI staining and the proportions of cells were determined for each cell cycle phase. b) Distribution of cells at different cell cycle and Sub-G1 phases following 8 days of neurodifferentiation in drug-treated SH-SY5Y cells (N = 3). c) The cell proliferation was measured using the ViaCount-kit (Merck Millipore), which determines the number of viable cells by flow cytometry (N = 4). DMSO 0.1% (negative control); Retinoic acid 10 μM (RA, positive control); TA8Amino compound (0.014 mM); TAHB3 (0.71 μM); donepezil (0.0057 μM); tacrine (0.23 μM). Values are expressed as mean±SEM. Three or four independent experiments were performed. Statistical analysis: One-Way ANOVA and Newman-Keuls post-test. ^**p < 0.01; ^*p < 0.05 indicate statistically significant differences when compared to the negative control.

AChE inhibitors induced superoxide production and SOD1 protein expression in neurodifferentiated cells

The evaluation of intracellular reactive oxygen species (ROS; superoxide radicals-DHE dye), RA treatment induced a 62.74% increase in the production of intracellular superoxide (p < 0.01) compared to that induced by the negative control, while treatment with TA8Amino (but not TAHB3) induced a significant (p < 0.05) increase (48.27%) in the production of intracellular superoxide radicals in the neurodifferentiation assay, compared to that induced by the negative control (Fig. 6a, b). Regarding mitochondrial ROS production (superoxide radicals-MitoSox dye) indicated that TA8Amino induced a significant increase (p < 0.05) in ROS production (67.60%) compared to that induced by the negative control (0.1% DMSO). On the other hand, in the generation of mitochondrial superoxide radicals, significant (p < 0.05) reductions of 56.44, 56.95, and 67.15% were observed in response to treatments with RA, donepezil, or tacrine, respectively. In contrast, compared with the negative control, TAHB3 did not induce changes (Fig. 6c, d). Additionally, SOD1 protein expression was slightly increased (p > 0.05) following 8 days of neuronal differentiation in the presence of RA, TA8Amino, and TAHB3 (Fig. 6e, f).

Fig.6

Effects of AChEI on superoxide production and SOD1 protein expression in neurodifferentiated cells. a) Intracellular ROS levels were measured by flow cytometry with DHE dye in undifferentiated cells with DMSO in comparison with differentiated cells AChEI-treatments. b) Quantification of Intracellular ROS production by DHE dye measurement (N = 3). c) Mitochondrial ROS levels were measured by flow cytometry with MitoSox red dye in undifferentiated cells with DMSO in comparison with differentiated cells AChEI-treatments. d) Quantification of mitochondrial ROS production MitoSox red dye measurement (N = 3). e) Expression of SOD1 (∼18 kDa) and β-actin (∼45 kDa) analyzed by western blotting after neurodifferentiation. The images were obtained in the same gel/blot and image contrast adjustment was performed when needed for better viewing. f) Levels of expression of SOD1 protein after neurodifferentiation (N = 3). The values were normalized with the endogenous β-actin protein, using the Image Studio Lite Ver 5.0 software (Lite Software). Three independent experiments were performed. DMSO, 0.1% (negative control); Retinoic acid, 10 μM (RA, positive control); TA8Amino compound (0.014 μM); TAHB3 (0.71 μM); donepezil (0.0057 μM); tacrine (0.23 μM). Values are expressed as mean±SEM. Three independent experiments were performed. Statistical analysis was performed by the One-Way ANOVA and Newman-Keuls post-test, for the flow cytometry data and Kruskal-Wallis test for the protein expression (western blot). ^***p < 0.001; ^**p < 0.01; ^*p < 0.05 indicate statistically significant differences when compared to the negative control and between groups.

Effects of AChE inhibitors on the ΔΨ and mitochondrial mass in neurodifferentiated cells

TA8Amino and TAHB3 did not induce significant changes in the ΔΨ, and similar results were observed for donepezil and tacrine, and only RA promoted a 60.21% reduction in the ΔΨ compared to that induced by the negative control (p < 0.05) (Fig. 7a, b). Regarding mitochondrial mass, we found that TA8Amino, TAHB3, donepezil, and tacrine did not promote changes in mitochondrial mass, and only RA was capable of inducing a significant increase in mitochondrial mass (150.43%) compared to the negative control (p < 0.05) (Fig. 7c, d).

Fig.7

Effects of AChEI on ΔΨ and Mitochondrial Mass in neurodifferentiated cells. a) ΔΨ was measured by flow cytometry using TMRM dye in undifferentiated cells with DMSO in comparison with differentiated cells AChEI-treatments. b) Quantification of ΔΨ by TMRM dye measurement (N = 3). c) Mitochondrial mass was measured by flow cytometry with MitoTracker green dye in undifferentiated cells with DMSO in comparison with differentiated cells AChEI-treatments. d) Quantification of mitochondrial mass by MitoTracker green dye measurement (N = 3). DMSO, 0.1% (negative control); Retinoic acid, 10 μM (RA, positive control); TA8Amino compound (0.014 μM); TAHB3 (0.71 μM); donepezil (0.0057 μM); tacrine (0.23 μM). Values are expressed as mean±SEM. Three independent experiments were performed. Statistical analysis: One-Way ANOVA and Newman-Keuls post-test. ^***p < 0.001; ^**p < 0.01; ^*p < 0.05 indicate statistically significant differences when compared to the negative control and between groups.

AKT pathway activation by AChE inhibitors

The expression of proteins involved in the AKT signaling pathway was evaluated in SH-SY5Y cells after 8 days of neuronal differentiation in the presence of AChE inhibitors (Fig. 8a). A significant increase (p < 0.05) was observed in the relative expression of phospho-PTEN_{(Ser380/Thr382/383)} upon treatment with the hybrid compound TA8Amino, and only a slight increase (P = 0.06) was observed in cells treated with TAHB3; single treatments with donepezil and tacrine did not cause significant changes compared to the negative control (Fig. 8b). A slight increase was also observed in the relative expression for phospho-AKT_(Ser473) in cells treated with all the compounds, but AKT expression levels were not significantly different between treatments (Fig. 8c). Regarding COX2 relative expression, a significant (p < 0.05) higher level was observed for TA8Amino treatment compared to the negative control. RA, TAHB3, donepezil, and tacrine did not induce a significant (p > 0.05) alteration in the expression levels (Fig. 8d).

Fig.8

Protein expression after neurodifferentiation with AChEI-treatment in SH-SY5Y cells. a) Expression of PTEN (∼54 kDa), phospho-PTEN_{(Ser380/Thr382/383)} (∼54 kDa), phospho-AKT_(Ser473) (∼60 kDa), AKT (∼60 kDa) COX2 (∼69 KDa), and β-actin (∼45 kDa) analyzed by western blotting after neurodifferentiation. The images were obtained in the same gel/blot and image contrast adjustment was performed when needed for better viewing. b) Protein expression of phospho-PTEN_{(Ser380/Thr382/383)} (N = 4); c) phospho-AKT_(Ser473) (N = 4); d) COX2 (N = 3). The values were normalized with the endogenous β-actin protein, using the software Image Studio Lite Ver 5.0 (Lite Software). Three independent experiments were performed. DMSO, 0.1% (negative control); Retinoic acid, 10 μM (RA, positive control); TA8Amino compound (0.014 μM); TAHB3 (0.71 μM); donepezil (0.0057 μM); tacrine (0.23 μM). Values are expressed as mean±SEM. Three or four independent experiments were performed. Statistical analysis: Kruskal-Wallis test. ^*p < 0.05 indicate statistically significant differences when compared to the negative control.

DISCUSSION

AChE inhibitors are considered one of the main therapeutic strategies for AD but offer symptomatic relief [33]. Tacrine-donepezil hybrid compounds (TA8Amino and TAHB3) were synthesized based on the three-dimensional structure of the AChE enzyme, and these hybrid compounds were described as potent allosteric modulators of the enzyme [14]. Based on a previous result, the present study confirmed the advantageous effects of TA8Amino and TAHB3 regarding the induction of neuronal differentiation in SH-SY5Y cells in vitro. In addition, the cell viability assay performed in SH-SY5Y and HepG2 cells did not show cytotoxic effects for any of the concentrations or time points analyzed. The therapeutic doses of donepezil approved by the Food and Drug Administration (FDA), within the range of 5 to 10 mg, are capable of inhibiting cortical AChE activity by 20 to 40% in vivo in patients with AD [34]. Therefore, the IC₅₀ for AChE inhibitory activity of the compounds was chosen for the neuronal differentiation assays.

The limited potential for neuronal regeneration is a major challenge in the prevention and treatment of AD and other neurodegenerative disorders. Within this context, when performing assays to characterize the biological activities of new compounds with potential neuroprotective effects, it becomes relevant to study mechanisms related to neuronal development, particularly neuritogenesis and neuronal migration, as well as synaptic plasticity in mature neurons [35]. Neurogenesis, a process of neuronal differentiation in postmitotic cells, is characterized by the elongation of cellular extensions, called neurites, which in turn may differentiate into axons or a dendritic tree complex [36]. In our study, we evaluated the ability of TA8Amino and TAHB3 to induce neurodifferentiation and neuritogenesis in SH-SY5Y cells. These compounds were capable of inducing neurodifferentiation and neurite outgrowth in SH-SY5Y cells, making them morphologically similar to primary neurons; especially in TA8Amino-treated cells, we observed mature neurons with neurite extensions following 8 days of neurodifferentiation. These effects are vital for neural networks and may represent a great contribution to neuronal regeneration, which could be helpful for patients with neurodegenerative disorders since abnormalities in neuritogenesis are considered a hallmark of these diseases [36].

Although endogenous nerve growth factor (NGF) has been proposed for the treatment of AD due to its ability to promote neuritogenesis, its application has been hampered by the fact that NGF does not cross the blood-brain barrier and elicits multiple side effects, such as cognitive disturbances, neuropathic pain, and exacerbated inflammation [37]. Thus, there is still a need for the development of new therapeutic compounds that promote neurogenesis and neuritogenesis, which are important mechanisms related to neuroprotection.

The analysis of β-III tubulin expression was performed in cells treated with the hybrid compounds, based on the observation that mature neurons derived from RA-treated SH-SY5Y cells express adult neuronal markers [18]. In fact, cells treated with the hybrid compounds TA8Amino and TAHB3 presented an increase in the expression of β-III-tubulin protein. Donepezil and tacrine did not cause any increase, indicating that they did not induce neurodifferentiation, at least at the concentrations tested.

The cell proliferation rate and cell cycle progression were analyzed in neurodifferentiated drug-treated cells. TA8Amino induced a significant increase in cell proliferation on the second day; however, after 8 days of neurodifferentiation, none of the AChE inhibitors (TA8Amino, TAHB3, donepezil, and tacrine) altered the cell proliferation capacity. Although a significant reduction occurred in the proportion of cells at the G2/M phase in donepezil and tacrine-treated cells, the cell proliferation rates did not suffer significant changes. In contrast, RA induced a significant reduction in the cell proliferation rate, thus corroborating the blockade observed at the G0/G1, with a concurrent decrease in S- and G2/M phases. This result was already expected for the positive control (RA), since G1-arrest and reduction in proliferation rates have been previously reported [38, 39]. It should be emphasized that the hybrid compounds (TA8Amino and TAHB3) induced an increase of neurites and β-III-tubulin protein expression in neurodifferentiated cells, but maintaining the cell growth for 8 days without causing cell toxicity and alterations in the cell cycle progression.

Experiments were also carried out to evaluate oxidative changes in SH-SY5Y cells treated with hybrid compounds after 8 days of neurodifferentiation. The results demonstrated that TA8Amino was able to induce the production of intracellular and mitochondrial ROS, thus promoting oxidative stress. TAHB3, donepezil, and tacrine did not promote differences in intracellular and mitochondrial ROS production, whereas the positive control (RA) induced intracellular ROS but decreased the production of mitochondrial ROS. Similar effects of RA were previously observed; mitochondrial superoxide radical production increased after RA treatment for 48 h and decreased after 72 h, in parallel with a significant increase in hydrogen peroxide (H₂O₂) and SOD activity in SK-N-SH cells [40]. Similar to RA, TA8Amino caused oxidative stress while promoting neuronal differentiation.

To evaluate whether hybrid molecules affect the antioxidant defense system in differentiated cells, we evaluated the expression of SOD1 (CuZnSOD), an intracellular antioxidant enzyme. The results demonstrated a tendency of increase in SOD1 expression in cells treated with RA, besides hybrid compounds TA8Amino and TAHB3. Most likely, the decrease in the production of mitochondrial ROS observed after 8 days of neurodifferentiation in RA-treated cells was associated with the increase in SOD1 expression and led to the conversion of the superoxide radicals into hydrogen peroxide as an antioxidant defense mechanism. The oxidative stress induced by TA8Amino might activate the antioxidant defense system through the induction of SOD1 expression in neurodifferentiated cells.

It is well known that oxidative stress can lead to the accumulation of mitochondrial damage and dysfunction and thus to the formation of aberrant mitochondria and changes in the membrane potential [41]. In this context, we evaluated mitochondrial mass and the membrane potential in differentiated cells following 8 days of treatment with the hybrid compounds. TA8Amino and TAHB3 did not promote changes in mitochondrial mass and membrane potential at the concentrations tested and conditions used in the present study; only the positive control (RA) significantly decreased the membrane potential and increased the mitochondrial mass. The results obtained for TA8Amino and TAHB3 demonstrated that they did not cause mitochondrial changes in differentiated cells and were able to induce neurodifferentiation without promoting mitochondrial damage in SH-SY5Y cells. The alterations induced by RA were previously reported and are probably due to its ability to promote the permeabilization of the mitochondrial membrane to protons [42]. In other studies, scanning electron microscopy demonstrated that RA triples the number of mitochondria in differentiated cells over 7 days [43].

To study the molecular pathways involved in the neurodifferentiation activity of AChE inhibitors, we evaluated the protein expression of phospho-PTEN_{(Ser380/Thr382/383)} (inactive form), phospho-AKT_(Ser473) (active form) and COX2 in cells collected after 8 days of neuronal differentiation induced by AChE inhibitors. The results indicated that TA8Amino, TAHB3, and donepezil induced an increase in the phosphorylation of PTEN at Ser380/Thr382/383 and the phosphorylation of AKT at Ser473. In addition, TA8Amino also induced a significant increase in COX2 protein expression. In general, the hybrid compounds tested in SH-SY5Y cells, especially TA8Amino, led to PTEN inactivation and, consequently, the activation of the AKT pathway, thus promoting neuronal differentiation, neurite outgrowth, and cell survival (Fig. 9).

Fig.9

Model for the induction of the AKT signaling pathway by AChEI. TA8Amino and TAHB3 could be promoting positive regulation AKT pathway. The compounds reduce PTEN signaling and consequently activate the AKT pathway. TA8Amino may induce oxidative stress with consequent production of ROS. Consequently, all these alterations could be promoting the activation of genes, such as COX2 and PGE2, leading to neuronal differentiation.

It has been widely reported that AKT pathway activation is involved in neuronal differentiation [44]. The activated PI3K/AKT signaling cascade is an important cell survival pathway that can also regulate differentiation processes, and the overexpression of constitutively active PI3K induces neurite outgrowth and the expression of neuronal markers [45]. Furthermore, according to researchers, the PI3K inhibitor LY294002 blocks RA-induced neurite growth and the expression of neuronal markers, suggesting that the activation of PI3K signaling is involved in the regulation of neuronal differentiation. Zhang et al. [46] observed that formyl peptide receptors regulate the differentiation of NSCs by ROS and the PI3K-AKT signaling pathway, promoting neurogenesis. Likewise, especially for TA8Amino, we can assume that AChE inhibitors-stimulated neural differentiation was mediated by ROS and involved PI3K/AKT induction, thereby promoting neurogenesis in SH-SY5Y cells.

AD is characterized by a progressive neurodegenerative process, and the disease is mainly a condition of neuronal and synaptic loss [12]. Such characteristics of AD can provide the basis for new therapies for patients with AD. An important approach to be considered is that, while neurogenesis implies the development of new neurons, neuritogenesis allows sensory and dendritic formation through the neurite outgrowth [47]. Considering that the loss of neuritis is an early event of the neurodegenerative process, the regeneration of the neuritis network appears as a promising therapeutic strategy to be studied for neurodegenerative disorders [48]. Furthermore, it is known that neurotrophic factors play a critical role in neuronal regeneration, but their clinical use is limited as to the possibility of crossing the blood-brain barrier [48]. Since single-target drugs that have reached clinical trials have not shown effectiveness in the treatment of AD, there is currently a search for an emerging strategy for the development of multifunctional drugs, although there is still a limited number of studies focusing on this approach [49]. In the present study, we demonstrated that two new hybrid compounds, TA8Amino and TAHB3, promoted the induction of neurite outgrowth, possibly involving the AKT signaling pathway, and this information indicates the potential of these compounds to act as multiple targets in AD therapy, and may provide some light towards a possibility to improve clinical benefits for AD patients.

In conclusion, the hybrid compounds TA8Amino and TAHB3 (AChE inhibitors derived from donepezil and tacrine) were able to induce neuronal differentiation and neuritogenesis in SH-SY5Y cells (used as an in vitro model); TA8Amino induced a significant increase in β-III-tubulin expression, a neurodifferentiation marker, which confirms the formation of mature neurons after 8 days. It is important to note that the hybrid compounds did not exhibit cytotoxicity in the SH-SY5Y and HepG2 cells at any of the tested concentrations or at any of the different time-points evaluated, and this characteristic is relevant in clinical practice. In addition, although TA8Amino induced intracellular and mitochondrial ROS, mitochondrial dysfunction was not observed in differentiated cells. In addition, TA8Amino led to changes in PTEN and AKT phosphorylation and in the expression of the COX2, demonstrating that the AKT pathway is probably involved in the neuronal differentiation mechanism. Thus, these results provide important information about the characterization of the biological properties of two new hybrid compounds (donepezil-tacrine), with multi-target activities that can not only promote an increase in cholinergic activity by inhibiting AChE, but can also act in the modulation of adult neurogenesis, therefore being considered as potential therapeutic candidates for patients with AD.

Footnotes

ACKNOWLEDGMENTS

The authors are grateful to Ana P.L. Montaldi for suggestions and technical advice, and Luiz A. Costa Junior for technical assistance. The financial support provided by the São Paulo Research Foundation (FAPESP), National Council for Scientific and Technological Development (CNPq, Brazil) and Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) is also acknowledged.

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Proc. 2017/15123-1, and 2018/21709-1), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Proc. 309854/2017-2), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Authors’ disclosures available online ().

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