Sildenafil Reverses the Neuropathological Alzheimer’s Disease Phenotype in Cholinergic-Like Neurons Carrying the Presenilin 1 E280A Mutation

Abstract

Background:

Familial Alzheimer’s disease (FAD) presenilin 1 E280A (PSEN 1 E280A) is characterized by functional impairment and the death of cholinergic neurons as a consequence of amyloid-β (Aβ) accumulation and abnormal phosphorylation of the tau protein. Currently, there are no available therapies that can cure FAD. Therefore, new therapies are urgently needed for treating this disease.

Objective:

To assess the effect of sildenafil (SIL) on cholinergic-like neurons (ChLNs) harboring the PSEN 1 E280A mutation.

Methods:

Wild-type (WT) and PSEN 1 E280A ChLNs were cultured in the presence of SIL (25μM) for 24 h. Afterward, proteinopathy, cell signaling, and apoptosis markers were evaluated via flow cytometry and fluorescence microscopy.

Results:

We found that SIL was innocuous toward WT PSEN 1 ChLNs but reduced the accumulation of intracellular Aβ fragments by 87%, decreased the non-physiological phosphorylation of the protein tau at residue Ser²⁰²/Thr²⁰⁵ by 35%, reduced the phosphorylation of the proapoptotic transcription factor c-JUN at residue Ser⁶³/Ser⁷³ by 63%, decreased oxidized DJ-1 at Cys¹⁰⁶-SO₃ by 32%, and downregulated transcription factor TP53 (tumor protein p53), BH-3-only protein PUMA (p53 upregulated modulator of apoptosis), and cleaved caspase 3 (CC3) expression by 20%, 32%, and 22%, respectively, compared with untreated mutant ChLNs. Interestingly, SIL also ameliorated the dysregulation of acetylcholine-induced calcium ion (Ca²⁺) influx in PSEN 1 E280A ChLNs.

Conclusions:

Although SIL showed no antioxidant capacity in the oxygen radical absorbance capacity and ferric ion reducing antioxidant power assays, it might function as an anti-amyloid and antiapoptotic agent and functional neuronal enhancer in PSEN 1 E280A ChLNs. Therefore, the SIL has therapeutic potential for treating FAD.

Keywords

Alzheimer’s disease amyloid-β apoptosis caspase 3 DJ-1 hydrogen peroxide sildenafil

INTRODUCTION

Familial Alzheimer’s disease (FAD) is characterized by memory loss [1] and the accumulation of extracellular amyloid-β (eAβ) plaques, i.e., eAβ_1–42, and intracellular neurofibrillary tangles composed of abnormally phosphorylated tau (p-tau) [2, 3]. Genetic analysis has shown a point mutation in exon 8 of the presenilin 1 (PSEN 1) gene (GRCh37/hg19; Ch14 : 73664808 A > C) that results in a glutamic acid-to alanine substitution (p.Glu280Ala or p.E280A) in codon 280 [4, 5]. The E280A mutation is the most common cause of familial early-onset AD and is found in a large population in Colombia [6]. Despite several attempts [7 –9], efficient therapies against PSEN 1 E280A FAD are still needed. Therefore, the search for natural or synthetic chemical compounds that retard or prevent FAD has become a research priority. In line with this view, it is proposed that drug repurposing could accelerate the identification of new treatments for individuals with Alzheimer’s disease (AD) [10 –12].

Sildenafil (SIL), also known as Viagra, is a specific and selective inhibitor of the enzyme phosphodiesterase type 5 (PDE5) [13, 14] used to treat male erectile dysfunction [15]. Indeed, PDE5 breaks down cyclic guanosine monophosphate (cGMP) into guanosine monophosphate (GMP). However, its inhibition leads to higher cGMP levels, thereby enhancing penile erection via smooth muscle relaxation and increased blood flow [16]. Given that low levels of cGMP have been associated with cognitive decline, dementia, and amyloid pathology in AD [17, 18] and that PDE5 is upregulated in AD patients [19], SIL might be a potential therapeutic agent to treat AD [20 –22] and to lower its risk [23]. SIL has been implicated in the suppression of β-site amyloid precursor protein cleaving enzyme 1 (BACE 1) expression and Aβ generation, the upregulation of antioxidant enzymes, decreased neural apoptosis, increased neurogenesis, reduced abnormal phosphorylation of the tau protein, and induced mitochondrial biogenesis, among other effects [24, 25]. However, other investigators have found no positive effects of the PDE5 inhibitor SIL on reducing the development of AD nor evidence that SIL could impact the levels of exogenous Aβ_1 - 42 fragments in in vitro assays [26]. Therefore, the molecular mechanism by which SIL protects against neuronal loss in AD remains unknown. Since there was no available information to establish whether SIL prevents the neuropathological hallmarks observed in AD, we wanted to examine whether SIL reduces the incidence of the FAD phenotype in a well-established in vitro model, wherein the intracellular accumulation of Aβ (iAβ) is critical for the mechanism of neuronal death [27]. Therefore, the depletion of iAβ would be of therapeutic value if the drug that reduces its levels is administered before the neuron is involved in the apoptotic pathway.

The aim of the present investigation was to investigate whether SIL modifies the phenotype of cholinergic-like neurons (ChLNs) bearing the PSEN 1 E280A mutation. We used ChLNs derived from wild-type (WT) and modified Wharton’s umbilical cord jelly mesenchymal cells (WJ-MSCs) harboring the PSEN 1 E280A mutation. The cells were left untreated or treated with SIL and evaluated for intracellular proteinopathy markers, oxidative stress signs, apoptosis cell signaling marker activation, and transient Ca²⁺ neuronal flux. We showed that SIL can reverse neuronal dysfunction not only by preventing the accumulation of iAβ (as detected with the antibody 1E8), oxidative stress, and p-tau (Ser²⁰²/Thr²⁰⁵) (as detected with the antibody AT8), but also by working as an antiapoptotic and restorative agent of Ca²⁺ flux. Our findings suggest that SIL has the potential to be repurposed as a drug for FAD therapy.

MATERIALS AND METHODS

Transdifferentiation of MSCs into cholinergic-like neurons (ChLNs)

ChLNs were differentiated according to previous methods [28]. Briefly, WT mesenchymal stromal cells (MSCs) PSEN 1 (Tissue Bank Code # WJMSC-19) and PSEN 1 E280A (TBC# WJMSC-24) were seeded at a density of 1–1.5×10⁴ cells/cm² on laminin-coated culture plates in regular culture medium (RCm) for 24 h. Then, the cells were incubated in cholinergic differentiation medium (Cholinergic-N-Run medium, hereafter referred to as Ch-N-Rm) at 37 °C for 7 days. The neuronal cells were termed WT PSEN 1 or PSEN 1 E280A ChLNs and further cultured in regular culture medium (RCm) for an additional 4 days post-transdifferentiation.

Assay protocol

Sildenafil (SIL)

To determine whether SIL was cytotoxic, WT ChLNs were left untreated or exposed to increasing concentrations (1, 5, 10, or 25μM) of SIL (n = 3). Given that the concentrations tested were innocuous to neurons, the highest concentration of SIL was selected as the optimal concentration for further experiments. Accordingly, four groups of ChLNs were cultured: (i) untreated WT PSEN 1, (ii) WT PSEN 1 treated with SIL (25μM), (iii) untreated PSEN 1 E280A, and (iv) PSEN 1 E280A treated with SIL (25μM).

Immunofluorescence and flow cytometry analyses

Proteinopathy, oxidative stress, and cell death signaling markers were analyzed as described previously [27]. Briefly, cells treated under different conditions were processed by standard immunofluorescence protocols, which included primary antibodies and secondary fluorescent antibodies (Table 1). Nuclei were stained with 1μM Hoechst 33342 (Life Technologies, Carlsbad, CA, USA), and images were acquired using a Zeiss Axio Vert.A1 equipped with a Zeiss AxioCam Cm1 (Carl Zeiss Microscopy, LLC, White Plains, NY, USA). Fluorescence analysis was performed using a BD LSRFortessa II flow cytometer (BD Biosciences, Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA). Cells incubated without primary antibodies were used as controls. For evaluation, 10,000 events and the quantitative data were obtained using FlowJo 7.6.2 data analysis software (TIBCO^® Data Science Palo Alto, CA, USA). Event analysis was performed by determining the cell population (forward scatter analysis, Y-axis) that exceeded the baseline fluorescence (488 nm or 594 nm, X-axis) of the no primary antibody control. Consequently, density/dot plots were created based on event analysis, and the cells located within the quadrant represented the cell population that exceeded the baseline fluorescence.

Table 1

List of antibodies used for immunocytochemistry and for flow cytometry

Antibody	Dilution	Company Cat #
Differentiation Markers
Goat anti-ChAT	1 : 200	Millipore cat# AB144P
Rabbit anti-VAChT	1 : 500	Sigma-Aldrich Cat# SAB4200559
Protein Aggregation Markers
^*Mouse anti-Amyloid βA4 clone 1E8	1 : 500	Millipore clone 1E8 cat# MABN639
Rabbit anti-total tau	1 : 500	Sigma-Aldrich cat# T6402
^**Mouse anti-phosphorylated tau	1 : 500	Thermo Fisher Scientific cat# MN1020 (AT8)
Oxidative Stress Markers
^***Rabbit anti-oxidized DJ-1-ox(Cys¹⁰⁶)DJ-1	1 : 500	Abcam cat# ab169520
Proapoptotic Markers
Rabbit anti-PUMA	1 : 500	Abcam cat# ab-9643
Mouse anti-P53	1 : 500	Millipore cat# MA5-12453
§Goat anti-phospho-c-Jun	1 : 500	Santa Cruz cat# sc-16312
Rabbit anti-caspase-3	1 : 500	Millipore cat# AB3623
Secondary Antibodies
DyLight 488 horse anti-rabbit	1 : 500	Vector laboratories DI 1088
DyLight 594 horse anti-rabbit	1 : 500	Vector laboratories DI 1094
DyLight 488 horse anti-goat	1 : 500	Vector laboratories DI 3088
DyLight 594 horse anti-goat	1 : 500	Vector laboratories DI 3094
DyLight 488 horse anti-Mouse	1 : 500	Vector laboratories DI 2488
DyLight 594 horse anti-Mouse	1 : 500	Vector laboratories DI 2594

^*This monoclonal antibody is specific for the first 2 amino acids (i.e., Asp-Ala) of the amyloid-β peptide amino terminus. ^**This antibody is specific for phospho-tau (Ser²⁰²/Thr²⁰⁵). ^***This recombinant monoclonal antibody is specific for PARK7/DJ1 – Oxidized (Cys¹⁰⁶-SO3). ^§This monoclonal antibody is specific for phospho-c-Jun (Ser⁶³/Ser⁷³).

Intracellular calcium imaging

Changes in the intracellular calcium (Ca²⁺) concentration induced by cholinergic stimulation were evaluated according to previous methods [29, 30], with minor modifications. The fluorescent dye Fluo-3 (Fluo-3 AM; Thermo Fisher Scientific, cat: F1242 168, Third Avenue, Waltham, MA, USA) was used for these measurements. The dye was dissolved in DMSO (1 mM) to form a stock solution. Prior to the experiments, the stock solution was diluted with neuronal buffer solution (NBS buffer (in mM): 137 NaCl, 5 KCl, 2.5 CaCl₂, 1 MgCl₂, pH 7.3, and 22 glucose). The working concentration of the dye was 2μM. WT and PSEN1 E280A ChLNs were incubated for 30 min at 37°C with dye-containing NBS buffer and then washed five times. Intracellular Ca²⁺ transients were evoked by acetylcholine (1 mM final concentration) 4 days after differentiation. Measurements were performed using the 20×objective of the microscope. Several regions of interest (ROIs) were defined in the camera’s visual field. One ROI was cell free, and the measured fluorescence intensity in this area was considered background fluorescence (F_bg). The time dependence of the fluorescence emission was determined, and the fluorescence intensities (thus, Ca²⁺ levels) were determined using pseudocolors. To calculate the changes in average fluorescence intensity related to Ca²⁺, the F_bg value was determined from the cell-free ROI, and the resting fluorescence intensity (F_rest) of each of the ROIs containing cells was subsequently obtained as the average of the points recorded during a consecutive 10-s period prior to acetylcholine addition. Transient fluorescence peaks were identified by averaging six consecutive points and identifying those points that yielded the highest average value (F_max). The amplitudes of Ca²⁺-related fluorescence transients were expressed relative to resting fluorescence (ΔF/F) and calculated using the following formula: ΔF/F=(F_max – F_rest)/ (F_rest – F_bg). ImageJ was used for fluorescence intensity calculations. Fluorescence intensity was used as an indirect indicator of the intracellular Ca²⁺ concentration. This evaluation was repeated three times in independent blinded experiments.

Photomicrography and image analysis

Light and fluorescence microscopy images were taken using a Zeiss Axio Vert.A1 microscope connected to an AxioCam camera (Carl Zeiss Microscopy, LLC, White Plains, NY, USA), according to previous methods [27]. The images were initially processed to remove background information using the Zen3.4 software (Zen Lite) function Background Subtraction to remove smooth backgrounds or correct uneven illumination. The implementation was adapted from the corresponding function in ImageJ and based on the rolling ball algorithm [31]. Next, fluorescence images were analyzed using ImageJ software (http://imagej.nih.gov/ij/, accessed in September, 2023). The figures were transformed into 8-bit images, and background subtraction was performed. ROIs were drawn around the nuclei (for transcription factors and apoptosis effectors) or around the cells in general (for cytoplasmic probes); subsequently, the fluorescence intensity was determined by applying the same threshold to the cells under control and treatment conditions. The mean fluorescence intensity was obtained by normalizing the total fluorescence to the number ofnuclei.

Oxygen radical absorbance capacity assay

The hydrophilic oxygen radical absorbance capacity (ORAC) assay was conducted following the protocol outlined in reference [32]. Essentially, a peroxyl radical generator, AAPH (2,2’-azobis(2-amidinopropane) dihydrochloride), a standard compound, Trolox, and a fluorescent probe, fluorescein, were employed. The ORAC values are reported as μmol Trolox equivalents (TE)/g of solution.

Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was performed as described previously [32]. Briefly, the samples and the working FRAP solution were mixed at a 1 : 25 ratio for 10 min of incubation at 37°C in the dark. The FRAP values are expressed as μmol Trolox equivalents (TE)/g of solution (μmol TE/g).

Data analysis

Data analysis was performed according to previous methods [27, 33]. Statistical significance was determined using analysis of variance (ANOVA) followed by Bonferroni’s post hoc comparison using GraphPad Prism 9 software (GraphPad Software, 225 Franklin Street, Fl. 26, Boston, MA 02110). All the data are presented as the means±SDs. Differences between groups were considered significant when the p value was <0.05 (^*), <0.01 (^**), or <0.001(^***).

RESULTS

Sildenafil is an innocuous treatment for WT ChLNs derived from Wharton’s jelly mesenchymal stromal cells

First, we confirmed that WT WJ-MSCs and PSEN 1 E280A MSCs transdifferentiated into WT ChLNs and PSEN1 E280A ChLNs (Fig. 1) to a similar extent (76–79%), as determined by flow cytometry (Fig. 1A–C) and fluorescence microscopy (FM) (Fig. 1D–G). There were no significant differences between the number of transdifferentiated WT ChLNs and the number of transdifferentiated PSEN1 E280A ChLNs (Fig. 1C, F, G), implying that this mutation did not affect the transdifferentiation of ChLNs.

Fig. 1

Wild-type (WT) presenilin 1 mesenchymal stromal cells (WT PSEN 1 MSCs) and PSEN 1 E280A MSCs transdifferentiated into cholinergic-like neurons (ChLNs). A) Representative flow cytometry dot plot of WT ChLNs in absence (black dots) or presence of cholinergic-N-Run medium (Ch-N-Run medium, blue dots). B) PSEN 1 E280A ChLNs in absence (black dots) or presence of Ch-N-Run medium (red dots) to measure the percentage of vacuolar acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) expressing the cholinergic marker alone (number in square) or simultaneously (number in circle). The plots represent 1 out of 3 independent experiments. C) Quantitative data showing the percentage of VAChT- and ChAT-positive cells. The data are expressed as the mean±standard deviation (SD); significant values were determined by one-way analysis of variance (ANOVA) test with Tukey’s post hoc test; ns = not significant. D, E) Representative fluorescence microscopy merged images of WT and PSEN 1 E280A ChLNs stained with antibodies against ChAT (D’, E’) and VAChT (D”, E”). Nuclei were stained with Hoechst (D”’, E”’). F, G) Quantitative data showing the mean fluorescence intensity (MFI) of VAChT and ChAT. The figures represent 1 out of 3 independent experiments. The data are expressed as the mean±SD; significant values were determined by one-way ANOVA with Tukey’s post hoc test. n.s., no significance.

We subsequently wanted to evaluate whether SIL affects the survival of ChLNs. WT ChLNs were exposed to increasing concentrations of SIL (0, 1, 5, 10, or 25μM) for 24 h at 37°C. We then assessed cleaved caspase 3 (CC3) expression as a marker of activated caspase 3 (CASP3), which is responsible for the terminal process of neuronal cell death by apoptosis [34]. As shown in Fig. 2B–E, SIL did not induce CC3+ neurons compared to untreated neurons (Fig. 2A), as evaluated by FM (Fig. 2G), indicating that SIL at the concentrations tested was harmless to ChLNs. As expected, the number of CC3+ PSEN 1 E280A ChLNs increased significantly, which were included as a positive control [27] (Fig. 2F, G). Based on these observations, we selected the highest concentration of SIL (25μM) for further experiments. Notably, this concentration has also been used in other in vitro experimental settings (e.g., [35, 36]).

Fig. 2

Sildenafil (SIL) is innocuous to wild-type (WT) presenilin 1 (PSEN 1) cholinergic-like neurons (ChLNs). Representative fluorescence microscopy merged imagens of (A) untreated WT ChLNs, or treated with SIL (B) 1, (C) 5, (D) 10, and (E) 25μM, or (F) untreated PSEN 1 E280A ChLNs stained with antibodies against cleaved caspase 3 (CC3, (A’-F’). Nuclei were stained with Hoechst (A”–F”). G) Quantitative data showing the mean fluorescence intensity (MFI) of CC3. The figures represent 1 out of 3 independent experiments. The data are expressed as the mean±standard deviation (SD); significant values were determined by one-way analysis of variance (ANOVA) test with Tukey’s post hoc test. Statistical significance: ^***p < 0.001.

Sildenafil decreases the accumulation of iAβ fragments and the levels of the oxidative stress marker DJ-1 and phosphorylated tau (Ser²⁰²/Thr²⁰⁵) in PSEN 1 E280A ChLNs

We next evaluated whether SIL could ameliorate the neuropathological hallmarks of AD, namely, iAβ, oxidized DJ-1 (residue Cys¹⁰⁶-SOH), and p-tau at residues Ser²⁰²/Thr²⁰⁵. To this end, WT ChLNs and ChLNs expressing PSEN 1 E280A were left untreated or treated with SIL. The flow cytometry assay data showed that SIL caused no alterations in amyloid precursor protein (APP) metabolism in WT neurons (Fig. 3A) but reduced the accumulation of iAβ by 87% in mutant ChLNs (Fig. 3B) compared to that in untreated mutant cells (Fig. 3C). Given that PSEN 1 E280A ChLNs endogenously generate reactive oxygen species, such as H₂O₂, which specifically oxidizes DJ-1 to Cys¹⁰⁶-SO₃ [37], we evaluated whether SIL prevents this chemical interaction. While SIL had no effect on the status of DJ-1 in WT neurons (Fig. 3D), it clearly diminished the level of oxidized DJ-1 Cys¹⁰⁶-SO₃ by 32% in mutant ChLNs (Fig. 3E, F). These observations were confirmed by FM analysis (Fig. 3G–L). In addition, we determined whether the decrease in the level of the oxidative stress sensor protein DJ-1 might be due to the antioxidant activity of SIL. The hydrogen atom transfer-based ORAC and single electron transfer-based FRAP assay data showed that SIL had poor antioxidant activity, yielding values of 530.96±25.85μmol Trolox equivalents (TE)/g (n = 3) and 78.58±1.68μmol TE/g (n = 3), respectively, when compared to the antioxidant minocycline (ORAC 49,297.65±3,486.75μmol TE/g (n = 3), FRAP 13,096.97±83.63μmol TE/g (n = 3)), which was used as a positive control [38].

Fig. 3

Sildenafil prevents the accumulation of iAβ and the oxidation of DJ-1 protein in PSEN 1 E280A ChLNs. After 7 days of transdifferentiation, wild-type (WT) PSEN 1 and E280A Cholinergic-like neurons (ChLNs) were left untreated or treated with SIL (25μM) in regular culture medium (RCm) for 4 days. A, B) Flow cytometry density plots showing the percentage (number in circle) of iAβ in WT PSEN 1 ChLNs and iAβ in PSEN 1 E280A ChLNs, (C) Quantification of the percentage of iAβ-positive cells, (D) Flow cytometry density plots showing percentage (number in circle) of oxidized DJ-1 in WT PSEN 1 ChLNs, (E) oxDJ-1 in PSEN 1 E280A ChLNs, (F) Quantification of the percentage of oxDJ-1-positive cells. G–J) Representative immunofluorescence merge images showing WT PSEN 1 and PSEN 1 E280A ChLNs stained with antibodies against oxidized DJ-1 (G’–J’) and iAβ (G”–J”). Nuclei were stained with Hoechst (G”’–J”’). K) Quantitative data showing the mean fluorescence intensity (MFI) of oxDJ-1. L) Quantitative data showing the MFI of iAβ. Density plots and figures represent 1 of 3 independent experiments. Data are expressed as mean±standard deviation (SD). Significant values were determined by one-way analysis of variance (ANOVA) test with Tukey’s post hoc test. Statistical significance: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Moreover, we evaluated whether SIL affects p-tau (Ser²⁰²/Thr²⁰⁵). SIL did not affect p-tau (Ser²⁰²/Thr²⁰⁵) in WT ChLNs (Fig. 4A) but SIL did reduce p-TAU (Ser²⁰²/Thr²⁰⁵) in ChLNs harboring the PSEN 1 E280A mutation (Fig. 4B) compared to that in untreated mutant cells (Fig. 4C). Similar results were obtained by FM analysis (Fig. 4D–H).

Fig. 4

Sildenafil reduces phosphorylation of tau (Ser²⁰²/Thr²⁰⁵) protein in PSEN1 E280A Cholinergic-like neurons (ChLNs). After 7 days of transdifferentiation, wild-type (WT) PSEN 1 and E280A ChLNs were left untreated or treated with SIL (25μM) in regular culture medium (RCm) for 4 days. A, B) Flow cytometry density plots showing the percentage (number in circle) of p-tau (Ser²⁰²/Thr²⁰⁵) in WT PSEN1 ChLNs and p-tau (Ser²⁰²/Thr²⁰⁵) in PSEN 1 E280A ChLNs. C) Quantification of the percentage of p-tau (Ser²⁰²/Thr²⁰⁵)-positive cells. D-G) Representative immunofluorescence merged images showing WT PSEN 1 and PSEN 1 E280A ChLNs stained with antibodies against p-tau (Ser²⁰²/Thr²⁰⁵) (D’-G’). Nuclei were stained with Hoechst (D”–G”). H) Quantitative data showing the mean fluorescence intensity (MFI) of p-tau (Ser²⁰²/Thr²⁰⁵). Density plots and figures represent 1 of 3 independent experiments. Data are expressed as mean±standard deviation (SD). Significant values were determined by one-way analysis of variance (ANOVA) test with Tukey’s post hoc test. Statistical significance: ^***p < 0.001.

Sildenafil decreases apoptosis in PSEN 1 E280A ChLNs

Next, we sought to evaluate whether SIL could decrease the activation of apoptosis-related signaling molecules in mutant ChLNs. Figure 5 shows that, compared with untreated WT ChLNs (Fig. 5C), SIL had no effect on the phosphorylation of c-JUN at Ser⁶³/Ser⁷³ in WT ChLNs (Fig. 5A) but decreased the phosphorylation of p-c-JUN at Ser⁶³/Ser⁷³ by 63% in PSEN1 E280A ChLNs (Fig. 5B, C) compared to that in untreated mutant cells. These data were confirmed by FM (Fig. 5D–H).

Fig. 5

Sildenafil reduces phosphorylation of JUN (Ser⁶³/Ser⁷³) protein in PSEN 1 E280A Cholinergic-like neurons (ChLNs). After 7 days of transdifferentiation, wild-type (WT) PSEN 1 and E280A ChLNs were left untreated or treated with SIL (25μM) in regular culture medium (RCm) for 4 days. A, B) Flow cytometry density plots showing the percentage (number in circle) of phosphorylated c-JUN (Ser⁶³/Ser⁷³) in WT PSEN 1 ChLNs and p-c-JUN (Ser⁶³/Ser⁷³) in PSEN 1 E280A ChLNs. C) Quantification of the percentage of p-c-JUN (Ser⁶³/Ser⁷³)-positive cells. D-G) Representative immunofluorescence merged images showing WT PSEN1 and PSEN 1 E280A ChLNs stained with antibodies against p-c-JUN (Ser⁶³/Ser⁷³) (D’–G’). Nuclei were stained with Hoechst (D”–G”). H) Quantitative data showing the mean fluorescence intensity (MFI) of p-c-JUN (Ser⁶³/Ser⁷³). Density plots and figures represent 1 of 3 independent experiments. Data are expressed as mean±standard deviation (SD). Significant values were determined by one-way analysis of variance (ANOVA) test with Tukey’s post hoc test. Statistical significance: ^**p < 0.01, ^***p < 0.001.

Further analysis revealed that SIL had no effect on TP53 (tumor protein p53, Fig. 6A, C) or PUMA (p53 upregulated modulator of apoptosis, Fig. 6D, F) in WT ChLNs. However, compared with untreated PSEN1 E280A ChLNs, SIL decreased the TP53 (Fig. 6B, C) and PUMA (Fig. 6E, F) levels by 20% and 32%, respectively. Similar observations were obtained by FM (Fig. 6G–L).

Fig. 6

Sildenafil reduces the cellular presence of TP53 (tumor protein p53) and PUMA (p53 upregulated modulator of apoptosis) protein in PSEN 1 E280A Cholinergic-like neurons (ChLNs). After 7 days of transdifferentiation, wild-type (WT) PSEN1 and E280A ChLNs were left untreated or treated with SIL (25μM) in regular culture medium (RCm) for 4 days. A, B) Flow cytometry density plots showing the percentage (number in circle) of TP53 (tumor protein p53) in WT PSEN1 ChLNs and TP53 in PSEN 1 E280A ChLNs. C) Quantification of the percentage of TP53-positive cells. D, E) Flow cytometry density plots show percentage (number in circle) of PUMA (p53 upregulated modulator of apoptosis) in WT PSEN 1 ChLNs and PUMA in PSEN 1 E280A ChLNs. F) Quantification of the percentage of PUMA-positive cells. G–J) Representative immunofluorescence merged images showing WT PSEN 1 and PSEN 1 E280A ChLNs stained with antibodies against PUMA (G’–J’) and TP53 (G”–J”). Nuclei were stained with Hoechst (G”’–J”’). K) Quantitative data showing the mean fluorescence intensity (MFI) of TP53.(L) Quantitative data showing the MFI of PUMA. Density plots and figures represent 1 of 3 independent experiments. Data are expressed as mean±standard deviation (SD). Significant values were determined by one-way analysis of variance (ANOVA) test with Tukey’s post hoc test. Statistical significance: ^**p < 0.01, ^***p < 0.001.

On the other hand, as expected, compared with untreated mutant ChLNs, WT ChLNs were not CC3+ after exposure to SIL (Fig. 7A, C), but the cellular expression of CC3 was reduced by 22% in mutant ChLNs (Fig. 7B, C). Similar results were obtained by FM (Fig. 7D–H).

Fig. 7

Sildenafil reduces cleaved caspase 3 in PSEN 1 E280A Cholinergic-like neurons (ChLNs). After 7 days of transdifferentiation, wild-type (WT) PSEN 1 and E280A ChLNs were left untreated or treated with SIL (25μM) in regular culture medium (RCm) for 4 days. A, B) Flow cytometry density plots showing the percentage (number in circle) of cleaved caspase 3 (CC3) in WT PSEN 1 ChLNs and CC3 in PSEN 1 E280A ChLNs. C) Quantification of the percentage of CC3-positive cells. D–G) Representative immunofluorescence merged images showing WT PSEN 1 and PSEN 1 E280A ChLNs stained with antibodies against CC3 (D’–G’). Nuclei were stained with Hoechst (D”–G”). H) Quantitative data showing the mean fluorescence intensity (MFI) of CC3. Density plots and figures represent 1 of 3 independent experiments. Data are expressed as mean±standard deviation (SD). Significant values were determined by one-way analysis of variance (ANOVA) test with Tukey’s post hoc test. Statistical significance: ^***p < 0.001.

Sildenafil attenuates dysfunctional Ca²⁺ influx in PSEN 1 E280A ChLNs

We next investigated whether SIL reverses dysfunctional Ca²⁺ influx in mutant ChLNs when they are stimulated with acetylcholine (ACh). WT and mutant ChLNs were cultured in the presence of ACh, after which cellular Ca²⁺ influx was analyzed via fluorescence microscopy [29, 30]. Figure 8 shows that ACh induced a temporary elevation in intracellular Ca²⁺ in untreated WT neurons (Fig. 8A, E) as well as in WT ChLNs treated with SIL (Fig. 8B, E) according to Ca²⁺ response imaging (Fig. 8F). Remarkably, compared with untreated mutant ChLNs, SIL significantly increased the Ca²⁺ influx response (by 2.62-fold) in ChLNs harboring the PSEN 1 E280A mutation (Fig. 8B versus 8D, 8E, and 8F).

Fig. 8

Sildenafil restores Ca²⁺ dysregulation in PSEN 1 E280A Cholinergic-like neurons (ChLNs).< /b>After 7 days of transdifferentiation, wild-type (WT) PSEN1 and E280A ChLNs were left untreated or treated with SIL (25μM) in regular culture medium (RCm) for 4 days. A–D) Time-lapse images (0, 10, 20, 30, 40, 50, and 60 s) of Ca²⁺ fluorescence in PSEN 1 WT (A, B) and E280A ChLNs (C, D) in response to ACh treatment. ACh was added to the culture at 0 s (arrow), and Ca²⁺ fluorescence of cells was monitored at the indicated times. The color contrast indicates fluorescence intensity: dark blue < light blue < green<yellow<red. E) Normalized mean fluorescence signal (ΔF/F) over time from cells indicating temporally elevated cytoplasmic Ca²⁺ in response to ACh treatment. F) Calculated area under the curve. Figures represent 1 of 3 independent experiments. Data are expressed as mean±standard deviation (SD). Significant values were determined by one-way analysis of variance (ANOVA) test with Tukey’s post hoc test. Statistical significance: ^***p < 0.001.

DISCUSSION

Previous studies have shown that PSEN 1 E280A ChLNs exhibit early intracellular accumulation of Aβ (iAβ) fragments that is concurrent with the generation of H₂O₂, oxidation of DJ-1 (at residue Cys¹⁰⁶SH) to generate DJ-1Cys¹⁰⁶SO₃, loss of ΔΨ_m, abnormal phosphorylation of the protein tau (Ser²⁰²/Thr²⁰⁵), abnormal phosphorylation of the proapoptotic transcription factor c-JUN (Ser⁶³/Ser⁷³), and activation of the proapoptotic transcription factors TP53 and PUMA, all of which are markers of proteinopathy, oxidative stress, and apoptosis [27]. Moreover, mutant ChLNs exhibit dysregulated Ca²⁺ ion flux when challenged with ACh compared to WT ChLNs [27]. Therefore, PSEN1 E280A ChLNs constitute an excellent model for screening candidate molecule(s)/drug(s) with therapeutic potential for the treatment of patients with FAD. In agreement with the view that depleting iAβ would be of therapeutic value if the drug that reduces its levels is administered before the neuron is involved in the apoptotic pathway [39], we report for the first time that the PDE5 inhibitor SIL protects PSEN 1 E280A ChLNs against iAβ-induced cytotoxic effects. Indeed, SIL prevented the accumulation of iAβ, reduced the abnormal phosphorylation of p-tau at Ser²⁰²/Thr²⁰⁵, decreased the oxidation of DJ-1Cys¹⁰⁶-SH, reduced the phosphorylation of the proapoptotic transcription factor c-JUN at Ser⁶³/Ser⁷³, decreased the expression of the proapoptotic transcription factor TP53 and its target protein PUMA, and decreased CC3. Furthermore, SIL reversed the Ca²⁺ influx dysregulation in response to ACh stimulus in mutant ChLNs. These results suggest that SIL might be a multitarget agent that functions as an anti-amyloid, antioxidant, and antiapoptotic compound. Ultimately, SIL might operate as a regulator of transient intracellular Ca²⁺ influx.

Several in vivo studies have shown that SIL decreases extracellular Aβ levels in APP/PS1 mice [40 –42]. Similarly, we found that SIL significantly decreased the accumulation of iAβ (87%) in PSEN 1 E280A ChLNs. However, the mechanism by which SIL decreases or interferes with the accumulation of iAβ has not been determined. One possible explanation is that SIL decreases BACE1 and cathepsin B levels, thereby reducing APP amyloidogenic processing in mice [43]. Another possibility is that, as a result of the inhibition of neuronal PDE5 [44], cGMP indirectly suppresses BACE1 by activating PGC-1 alpha (peroxisome proliferator-activated receptor-g coactivator 1a) [45]. We speculate that SIL might induce the degradation of iAβ fibrils or inhibit Aβ fibril and oligomer formation [46]. Given that other PDE5 inhibitors (e.g., [42]) have shown anti-amyloid activity, the anti-amyloid effect of SIL may not be unique. Therefore, we anticipate that other popular PDE5 inhibitors, such as vardenafil (brand name Levitra^®) and tadafil (Cialis^®, [47]), might also function as anti-amyloid agents when used with mutant ChLNs. However, further experiments are necessary to confirm these assumptions. Regardless of the underlying mechanism, we demonstrated the anti-amyloid activity of SIL. These observations suggest that SIL might function as an anti-amyloid agent both in vitro and in vivo.

Mounting evidence has shown that SIL exerts antioxidant activity and promotes the activity of specific enzymes involved in redox homeostasis maintenance in fibroblasts exposed to H₂O₂ [48]. However, SIL showed no antioxidant capacity in the ORAC and FRAP assays. The significant disappearance of the DJ-1Cys¹⁰⁶-SO₃-positive signal in PSEN 1 E280A ChLNs suggested that SIL might exert its antioxidant function by promoting the expression of antioxidant proteins [48] or by protecting the thiol group on Cys¹⁰⁶ of DJ-1 from H₂O₂ oxidation [37], thereby suppressing the molecular cascade triggered by DJ-1Cys¹⁰⁶-SH [49]. Alternatively, H₂O₂ might directly oxidize SIL. However, further experiments are needed to investigate this last assumption.

We report for the first time that SIL decreases the expression of p-JUN (Ser⁶³/Ser⁷³), p-tau (Ser²⁰²/Thr²⁰⁵), and TP53, three molecular targets of JNK kinase [50 –52]. This observation suggests that SIL is able to reduce the abnormal phosphorylation of tau (Ser²⁰²/Thr²⁰⁵), phosphorylated c-JUN (Ser⁶³/ Ser⁷³), and TP53 by diminishing Aβ-induced JNK activation in PSEN 1 E280A ChLNs (Fig. 9). Detection of CC3 [53] has become the gold standard marker for identifying apoptotic neural cell death [54]. In line with this, Aβ-induced apoptosis of cultured primary rat neurons was shown to depend on CASP3 activation [55 –58]. Therefore, inhibiting Aβ might restrain CASP3 activation and apoptosis. Notably, SIL reduced iAβ levels and induced a significant reduction in the CC3 signal in PSEN 1 E280A ChLNs. However, determining whether the reduction in activation of CASP3 by SIL was indirect (by diminishing iAβ) or direct (by binding to CASP3) is difficult to establish in vitro. Nonetheless, indirect and direct conjoint mechanisms might lead the ability of SIL to inhibit apoptosis in PSEN 1 E280A ChLNs. These assumptions are further supported by the observation that SIL decreases double-stranded DNA breaks in the CA1 hippocampal area, downregulates CASP3, and reduces Aβ₄₂ generation in aged mice [59].

Fig. 9

Schematic representation of the mechanism by which sildenafil reverses the neuropathological Alzheimer’s phenotype in presenilin 1 E280A (PSEN 1 E280A) cholinergic-like neurons (ChLNs). Early accumulation of iAβ inhibits the mitochondrial complex I (NADH: ubiquinone oxidoreductase), thus preventing electron transfer via flavin mononucleotide (FMN) to coenzyme Q10 (1). Consequently, interruption of the electron transport chain concomitantly generates anion superoxide (O₂^. –) and hydrogen peroxide (H₂O₂, 2). This last compound is capable of oxidizing the stress sensor protein DJ-1Cys¹⁰⁶-SH (3) into DJ-1Cys¹⁰⁶-SO₃ (4), and triggering a domino-like pro-death signaling of molecules such as JNK kinase (5), involved in the activation of the pro-apoptotic transcription factors c-JUN (i.e., p-c-JUN Ser⁶³/Ser⁷³), 6) and TP53 (7). In turn, both TP53 and p-c-JUN (Ser^63/73) increase the expression of the BH-3-only protein PUMA (8). This protein together with other proteins (e.g., BAX) affects the mitochondrial membrane potential (ΔΨ_m), leading to the release of pro-apoptogenic proteins (e.g., cytochrome C), ensuing activation of caspase-3 into cleaved caspase 3 (CC3, 9). Once active, CC3 is responsible for the dismantling of the neuronal cells, a process known as regulated cell death, apoptosis (10). Alternatively, the intracellular Aβ/H₂O₂ induces abnormal phosphorylation of protein tau at residue Ser²⁰²/Thr²⁰⁵ through JNK kinase (5), thereby contributing to its intracellular accumulation and neuronal cell death process. Additionally, eAβ42 interacts with cholinergic receptors (e.g., nicotinic (n)ACh receptors) (11), blocking the ligand-gated Ca²⁺ ions flux. Upon exposure to sildenafil (SIL, 12), ChLNs not only respond to ACh-induced transient increase of Ca²⁺ influx but also display a significant reduction in the accumulation of iAβ, DJ-1Cys¹⁰⁶-SO₃, p-tau (Ser²⁰²/Thr²⁰⁵), p-JUN (Ser⁶³/ Ser⁷³), TP53, PUMA, and CC3. All these features represent the recovery and functionality of PSEN 1 E280A ChLNs.

Currently, disrupted Ca²⁺ influx by eAβ42 through direct interaction with receptors (e.g., alpha 7 nicotinic acetylcholine receptors [60]) is known to induce synaptic deficits and memory loss in AD [61]. Interestingly, SIL reverses the dysfunction in transient Ca²⁺ influx in PSEN1 E280A ChLNs when challenged with ACh [27, 62], most likely due to its anti-amyloid property.

In conclusion, SIL can act as an anti-amyloid, antioxidant, and antiapoptotic agent by reducing the accumulation of iAβ, blocking the generation of H₂O₂, and providing a means to reduce the phosphorylation of tau (Ser²⁰²/Thr²⁰⁵) and c-JUN (Ser⁶³/Ser⁷³), TP53, PUMA, and CASP3 (Fig. 9). Our data suggest that SIL might be a promising and efficient therapeutic compound for use in the early stages of FAD. Future exposure to SIL in the population affected with the PSEN 1 E280A mutation might be highly informative for confirming or excluding the therapeutic role of SIL in FAD patients.

AUTHOR CONTRIBUTIONS

Daniela Giraldo-Berrio (Data curation; Formal analysis; Investigation; Validation; Visualization; Writing – review & editing); Marlene Jimenez-Del-Rio (Conceptualization; Funding acquisition; Methodology; Project administration; Resources; Supervision; Writing – original draft; Writing – review & editing); Carlos Velez-Pardo (Conceptualization; Funding acquisition; Methodology; Project administration; Resources; Supervision; Writing – original draft; Writing – review & editing).

Footnotes

ACKNOWLEDGMENTS

The authors have no acknowledgments to report.

FUNDING

This work was supported by the MinCiencias grants (1115-844-67062; #2020-32092) to C.V.-P.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

DATA AVAILABILITY

The datasets generated and/or analyzed during the current study are included in the manuscript.

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Sildenafil Reverses the Neuropathological Alzheimer’s Disease Phenotype in Cholinergic-Like Neurons Carrying the Presenilin 1 E280A Mutation

Abstract

Background:

Objective:

Methods:

Results:

Conclusions:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Transdifferentiation of MSCs into cholinergic-like neurons (ChLNs)

Assay protocol

Sildenafil (SIL)

Immunofluorescence and flow cytometry analyses

Intracellular calcium imaging

Photomicrography and image analysis

Oxygen radical absorbance capacity assay

Ferric reducing antioxidant power (FRAP) assay

Data analysis

RESULTS

Sildenafil is an innocuous treatment for WT ChLNs derived from Wharton’s jelly mesenchymal stromal cells

Sildenafil decreases the accumulation of iAβ fragments and the levels of the oxidative stress marker DJ-1 and phosphorylated tau (Ser202/Thr205) in PSEN 1 E280A ChLNs

Sildenafil decreases apoptosis in PSEN 1 E280A ChLNs

Sildenafil attenuates dysfunctional Ca2+ influx in PSEN 1 E280A ChLNs

DISCUSSION

AUTHOR CONTRIBUTIONS

Footnotes

ACKNOWLEDGMENTS

FUNDING

CONFLICT OF INTEREST

DATA AVAILABILITY

References

Sildenafil decreases the accumulation of iAβ fragments and the levels of the oxidative stress marker DJ-1 and phosphorylated tau (Ser²⁰²/Thr²⁰⁵) in PSEN 1 E280A ChLNs

Sildenafil attenuates dysfunctional Ca²⁺ influx in PSEN 1 E280A ChLNs