Cholinergic-like neurons carrying PSEN1 E280A mutation from familial Alzheimer’s disease reveal intraneuronal sAPPβ fragments accumulation, hyperphosphorylation of TAU, oxidative stress, apoptosis and Ca2+ dysregulation: Therapeutic implications

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and cognitive disturbance as a consequence of the loss of cholinergic neurons in the brain, neuritic plaques and hyperphosphorylation of TAU protein. Although the underlying mechanisms leading to these events are unclear, mutations in presenilin 1 (PSEN1), e.g., E280A (PSEN1 E280A), are causative factors for autosomal dominant early-onset familial AD (FAD). Despite advances in the understanding of the physiopathology of AD, there are no efficient therapies to date. Limitations in culturing brain-derived live neurons might explain the limited effectiveness of AD research. Here, we show that mesenchymal stromal (stem) cells (MSCs) can be used to model FAD, providing novel opportunities to study cellular mechanisms and to establish therapeutic strategies. Indeed, we cultured MSCs with the FAD mutation PSEN1 E280A and wild-type (WT) PSEN1 from umbilical cords and characterized the transdifferentiation of these cells into cholinergic-like neurons (ChLNs). PSEN1 E280A ChLNs but not WT PSEN1 ChLNs exhibited increased intracellular soluble amyloid precursor protein (sAPPf) fragments and extracellular Aβ42 peptide and TAU phosphorylation (at residues Ser202/Thr205), recapitulating the molecular pathogenesis of FAD caused by mutant PSEN1. Furthermore, PSEN1 E280A ChLNs presented oxidative stress (OS) as evidenced by the oxidation of DJ-1Cys106-SH into DJ-1Cys106-SO3 and the detection of DCF-positive cells and apoptosis markers such as activated pro-apoptosis proteins p53, c-JUN, PUMA and CASPASE-3 and the concomitant loss of the mitochondrial membrane potential and DNA fragmentation. Additionally, mutant ChLNs displayed Ca2+ flux dysregulation and deficient acetylcholinesterase (AChE) activity compared to control ChLNs. Interestingly, the inhibitor JNK SP600125 almost completely blocked TAU phosphorylation. Our findings demonstrate that FAD MSC-derived cholinergic neurons with the PSEN1 E280A mutation provide important clues for the identification of targetable pathological molecules.

Despite advances in the understanding of the physiopathology of AD [21], there are no efficient therapies to date. Although limitations in culturing brain-derived live neurons might slow AD research, the rapid advances in cellular genetic reprogramming, in particular the induction of somatic cells (e.g., fibroblast) into stem cells (e.g., human induced pluripotent stem cells, hiPSCs), has led to the modeling of FAD PSEN1 mutations in vitro [22][23][24][25]. Obtaining iPSCs from patients bearing PSEN1 mutations is appealing; however, the isolation and purification procedures are technically challenging, expensive, time consuming and labor intensive. Alternatively, the human mesenchymal stromal (stem) cells derived from Wharton's jelly tissue (WJ-MSCs) are multipotent cells that can differentiate and/or transdifferentiate into mesodermal and ectodermal lineage cells [26][27][28][29]. Because MSCs might be equivalent to human embryonic stem cells (hESCs) and hiPSCs [30,31]; these cells have become an interesting and promising tool for modeling FAD PSEN1 E280A in vitro.
The aim of the present study was to establish an in vitro cellular model that reveals the major pathologic features of the FAD PSEN1 E280A mutation, thereby enabling investigation of the pathomechanisms of early onset FAD. Therefore, Aβ42 accumulation, Aβ42 production, TAU phosphorylation, oxidative stress (OS), cell death, and neuronal dysfunction were investigated in cholinergic-like neurons (ChLNs) derived from wild-type (control) and PSEN1 E280A MSCs. We demonstrate for the first time that FAD PSEN1 E280A pathology can be recapitulated in MSC-derived ChLNs. These findings in ChLNs show great promise for modeling human FAD in vitro and identifying therapeutic targets for AD treatment.

Materials and methods
The collection and use of umbilical cords from newborns was approved by Ethics Committee of the Hospital San Vicente Fundacion Research act # 13-2015 Colombia, and was provided following natural childbirth with written consent. Donors had a familial background of AD. The mother's medical history was negative for human pathogens, such as human immunodeficiency virus 1/2, hepatitis B and C virus, and syphilis. The cord (~7 cm long) was immersed in low-glucose DMEM (Sigma) supplemented with 100 U Penicillin/streptomycin (Sigma) and 5 μg/ml Plasmocin (Invivogen) and immediately transported to the laboratory.

Isolation and expansion of hWJ-MSCs
The human umbilical cords were obtained from ten healthy, natural childbirths (Tissue Bank Code (TBC) # WJMSC- 11, -12, -13, -14, -15, -16, -17, -18, -19, -20) and aseptically stored at 4 C PBS containing 1% penicillin and streptomycin. The cords were rinsed several times to drain blood from vessels, cut into 2-3-cm-long segments and rinsed again. Umbilical arteries and veins were removed, and the remaining tissue was transferred to a sterile container and chopped into small fragments in PBS. The explants were digested with an enzyme mixture containing 0.25% trypsin, 0.1% Dispase and 0.5% collagenase II for 2 h at 37˚C under constant agitation. Then, the digestion products were centrifuged at 447 x g for 40 min, and the pellet was cultured in T75 cell culture flasks (Corning) in hWJ-MSC regular culture medium (lowglucose DMEM supplemented with 20% fetal bovine serum (FBS, Sigma), 100 U penicillin/ streptomycin and 5 μg/ml Plasmocin). Once confluence had been reached, adherent cells (passage 0) were detached with 0.25% trypsin and passaged at 13,000 cells/ cm 2 in a T75 flask. Cells from passages 2 or 4 were harvested during the first expansion period for further characterization and cryopreservation.
APOE genotyping analysis. Genotyping of the APOE polymorphism (n = 10) was performed using polymerase chain reaction amplification of a 244-bp fragment followed by digestion with HhaI as described by [33].
Karyotyping. Karyotype analysis was performed by the Medical Genetics Unit of the Faculty of Medicine-UdeA, using standard cytogenetic protocols. At 60-70% confluence, WJMSC-11 WT and WJMSC-12 PSEN1 E280A cells were incubated with 0.1 mg/ml Colcemid (Sigma) for 90 min at 37˚C. Then, the cells were detached with 0.25% trypsin and centrifuged at 591 x g for 20 min. The medium was removed, and the hypotonic solution (0.075 M KCl, 0.017 M Na-citrate) was added and incubated for 20 min at 37˚C. After a new centrifugation, cells were fixed with freshly prepared Carnoy's solution. Metaphase spreads were analyzed after staining with quinacrine (Sigma) for karyotyping. Analysis was performed on three different primary cultures counting 20 metaphases for each sample.

Colony-forming units assay
The colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony [34]. WJMSC-11 WT and WJMSC-12 PSEN1 E280A cells were seeded at a density of 200 cells/ well on 6-well plates followed by the addition of 3 mL of regular culture medium. The cultures were left to grow in a humidified atmosphere with 5% CO 2 at 37˚C for 15 days. The culture medium was changed twice a week. After 15 days of cultivation, both WT and mutant PSEN-1 cells were stained with 0.5% crystal violet and counted using the cell counter plugin from ImageJ program. The experiment was conducted three times.

Immuno-phenotypic characterization
Standard flow cytometry techniques were used to determine the cell surface epitope profile (CD9, CD73, CD90, CD34 and CD45) of both WT and mutant PSEN1 MSCs. Briefly, hWJ-MSCs were incubated with saturating concentrations (1:500) of mouse monoclonal antibodies conjugated to human CD9-peridinin chlorophyll protein (PerCP)-cy5.5, CD73-phycoerythrin (PE), CD90 PE-cy5.5, CD34 PE, and CD45-fluorescein isothiocyanate (FITC). All antibodies were purchased from BD Biosciences (San Diego, CA). Cells were incubated for 1 h at 4˚C. Prior to antibody labeling, the cells were preincubated with 5% fetal bovine sera (FBS) for 10 min to block nonspecific binding. Cell suspensions were washed and resuspended in PBS for analysis on an LSRFortessa (BD Biosciences). Ten thousand events were acquired, and the acquisition analysis was performed using FlowJo 7.6.2. Positive staining was defined as the fluorescence emission that exceeded levels obtained by more than 99% of cells from the population stained with the corresponding negative controls. The isotype (negative) control used in this study was IgG1 PE-Cy5.5, IgG1-PE and IgG1-FITC (BD Biosciences).
Osteogenic differentiation. Osteogenic differentiation was performed according to [35] with minor modifications. Briefly, WT and mutant cells at passages 4-7 were plated at a density of 10,000 cells/ cm 2 in 12-well plates in regular culture medium. After 72 h, the culture medium was replaced by osteogenic differentiation medium containing high-glucose DMEM (Sigma), 10% FBS, 1 μM dexamethasone (Alfa Aesar, cat # A17590), 250 μM sodium ascorbate (Sigma, cat # A4034), and 10 mM β-glycerophosphate (Alfa Aesar, cat # L03425). The medium was changed every 3-4 days. Control cells were kept in regular culture medium. After 28 days of induction, cells were fixed in 4% FA and stained with standard Von Kossa Staining.
Chondrogenic differentiation. Chondrogenic differentiation was performed according to ref. [36] with minor modifications. Briefly, 2.5×10 5 WT and mutant cells were left aggregated in microwell plates and then provided with chondrogenic medium containing high-glucose DMEM, 10% FBS, 10 μg/ L TGF-β3, 0.1 μmol/ L dexamethasone, 50 μmol/ L vitamin C, and 6.25 mg/L insulin. The medium was changed every 3-4 days. Control cells were kept in regular culture medium. After 28 days of induction, cells were fixed in 4% formaldehyde, stained with toluidine blue for 2 min at room temperature and viewed by light microscopy.

Western blot analysis
Cells were incubated as described above, detached with 0.25% trypsin and lysed in 50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), and 0.1% sodium dodecyl sulfate and a protease inhibitor cocktail (Sigma-Aldrich). All lysates were quantified using the bicinchoninic acid assay (Thermo Scientific cat # 23225). Extracted samples (30 μg of proteins) were heated at 95˚C for 5 min in 2 x SDS and 20x reducing agent (except for protein oxDJ-1) and loaded into 12% Bis/Tris gels at 120 V for 90 min, and the bands were transferred onto nitrocellulose membranes (Hybond-ECL, Amersham Biosciences) at 270 mA for 90 min using an electrophoretic transfer system (BIO-RAD) according to ref. [37] with minor modifications. The membranes were incubated overnight at 4˚C with anti-GFAP, MAP2, β-tubulin III, ChAT, amyloid β 1-42, total TAU, phospho-TAU, ox(Cys 106 ) DJ1, PUMA, p53, p-c-Jun, and caspase-3 primary antibodies (1:5000). The anti-actin antibody (1:1000, cat #MAB1501, Millipore) was used as an expression control. Secondary infrared antibodies (goat anti-rabbit IRDye 1 680RD, cat #926-68071; donkey anti-goat IRDye 1 680RD, cat # 926-68074; and goat anti mouse IRDye 1 800CW, cat #926-32270; LI-CORBiosciences) at 1:1000 were used for western blotting analysis, and data were acquired using Odyssey software. To directly control the conformation-dependent differences among Aβ assemblies, we prepared a homogenous synthetic unaggregated (i.e., monomers) and large oligomeric Aβ 42 assemblies according to ref. [38]. Briefly, after solubilization of the peptide (Sigma Cat #A9810) in DMSO, the "unaggregated" peptide was obtained by dissolving the DMSO-solubilized peptide in water and used immediately (0 days). To obtain the "large oligomers", 10 mM Tris was added to DMSO-solubilized peptide solution and incubated it for 15 days at 4˚C. The determination of the aggregation state of Aβ 42 was performed by Western analysis of SDS-PAGE as described above. The assessment was repeated three times in independent experiments.

Mass spectrometry analysis
Gel region between 60 and 125 kDa was excised from the experimental lane, cut into 1 x 1 mm cubes, incubated consequently with 10 mM dithiothreitol and 55 mM iodacetamid in 100 mM ammonium bicarbonate, and dehydrated with acetonitrile. Then, the proteins were in-gel digested overnight at 37˚C with LysC protease (Promega, Mannheim). The resulting peptide mixture was extracted twice with exchange of 5% formic acid and 100% acetonitrile, the extracts pulled together and dried down. The peptides were re-suspended in 25 μl of 5% formic acid and 5 μl were taken for LC-MS/MS analysis. The analysis was performed on a nano-UPLC Ultimate 3000 interfaced on-line to a Q-Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer (both Thermo Fischer Scientific, Bremen). The UPLC system was equipped with Acclam PepMap™ 100 75 μm x 2 cm trapping column and 75 μm x 15 cm separating column packed with 3 μm diameter C18 particles (Thermo Fischer Scientific, Bremen). Peptides were separated using 180 min linear gradient (solvent A-0.1% formic acid in water, solvent B-0.1% formic acid in neat acetonitrile). Spectra were acquired in DDA mode using top 20 method. Spectra were then converted into (.mgf) format and searched human sequences in the UniProt database (January 2020) using MASCOT software (version 2.2.04) and against human Amyloid-beta precursor protein (ACC No P05067) without enzyme specificity. Mass tolerance was set on 5 ppm and 0.025 Da for precursor and fragment ions respectively; variable modification-Cysteine carbamidomethyl and propionamide, methionine oxidation, protein N-terminal acetyl. The result of the database search was evaluated by Scaffold software (v.4.7.5, Proteome Software, Portland) and the spectra matched Amyloid-beta precursor protein also manually inspected. To investigate the presence of Aβ 42 , the spectra were acquitted in DDA targeted mode using masses of C-and N-terminal peptides which are unique for 671-713 fragment, and then processed by SkyLine software.

Analysis of cells Evaluation of intracellular hydrogen peroxide (H 2 O 2 ) by fluorescence microscopy.
To determine the levels of intracellular H 2 O 2 , we used 2 0 ,7 0 -dichlorofluorescein diacetate (5 μM, DCFH2-DA; Invitrogen). hWJ-MSCs or ChLNs were left in RCm for 0, 2 and 4 days. Then, the cells (5 x 10 3 ) were incubated with the DCFH 2 -DA reagent for 30 min at 37˚C in the dark. Cells were then washed, and DCF fluorescence intensity was determined by analysis of fluorescence microscopy images. The assessment was repeated three times in independent experiments. The nuclei were stained with 0.5 μM Hoechst 33342 (2.5 μM) staining compound. The assessment was repeated three times in independent experiments blind to experimenter.

Evaluation of intracellular hydrogen peroxide (H 2 O 2 ) by flow cytometry. H 2 O 2 was
determined with 2',7'-dichlorofluorescein diacetate (1 μM, DCFH 2 -DA). ChLNs were left in regular medium (RCm) for 0, 2 and 4 days. Then, the cells (1×10 5 ) were incubated with DCFH 2 -DA reagent for 30 min at 37˚C in the dark. Cells were then washed, and DCF fluorescence was determined using an LSRFortessa (BD Biosciences). The assessment was repeated 3 times in independent experiments. Quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software. The assessment was repeated three times in independent experiments blind to experimenter and flow cytometer analyst.
Analysis of mitochondrial membrane potential (ΔCm) by fluorescence microscopy. The hWJ-MSCs or ChLNs were left in regular culture medium (RCm) for 0, 2 and 4 days. Then, the cells (5 x 10 3 ) were incubated with the passively diffusing and active mitochondriaaccumulating dye deep-red MitoTracker compound (20 nM, final concentration) for 20 min at RT in the dark (Invitrogen, cat # M22426). Cells were then washed twice with PBS. Mito-Tracker fluorescence intensity was determined by analysis of fluorescence microscopy images. The assessment was repeated three times in independent experiments. The nuclei were stained with 0.5 μM Hoechst 33342 (2.5 μM) staining compound. The assessment was repeated three times in independent experiments blind to experimenter and flow cytometer analyst.
Analysis of mitochondrial membrane potential (ΔCm) by flow cytometry. ChLNs were left in regular medium for 0, 2 and 4 days. Then, the cells (1×10 5 ) were incubated for 30 min at RT in the dark with MitoTracker (20 nM, final concentration). The cells were analyzed using an LSRFortessa (BD Biosciences). The experiment was performed three times in independent experiments, and 10,000 events were acquired for analysis. Quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software. The assessment was repeated three times in independent experiments blind to experimenter and flow cytometer analyst.
Determination of DNA fragmentation by flow cytometry. DNA fragmentation was determined using a hypotonic solution of PI. Cells entering the cellular cycle phase sub-G 0 were identified as those undergoing apoptosis. ChLNs were left in regular medium for 0, 2 and 4 days. Then, the cells (1x10 5 ) were detached, washed twice with PBS (pH 7.2) and stored in 95% ethanol overnight at -20˚C. The cells were washed and incubated in 400 μL solution containing propidium iodide (PI; 50 μg/ml), RNase A (100 μg/ mL), EDTA (50 mM), and Triton X-100 (0.2%) for 60 min at 37˚C. The cell suspension was analyzed for PI fluorescence using an Epics XL flow cytometer (Beckman Coulter). DNA fragmentation was assessed 3 times in independent experiments. Quantitative data and figures from the sub-G 0 /G 1 population were obtained using FlowJo 7.6.2 Data Analysis Software. The assessment was repeated three times in independent experiments blind to experimenter and flow cytometer analyst.
Acetylcholinesterase activity measurement. We determined the acetylcholinesterase (AChE) activity using the AChE Assay Kit (Abcam, Cat# ab138871) according to the manufacturer's protocol. Briefly, ChLNs at days 0, 2 and 4 of post differentiation were detached with 0.25% trypsin and mechanically lysed by freezing/sonication. Lysates were centrifuged at 18,894 x g for 15 min, and supernatants were used for protein quantification by the BCA method (see above) and the detection of AChE activity. AChE degrades the neurotransmitter acetylcholine (ACh) into choline and acetic acid. We used the DTNB (5,5 0 -dithiobis(2-nitrobenzoic acid)) reagent to quantify the thiocholine produced from the hydrolysis of acetylthiocholine by AChE. The absorption intensity of the DTNB adduct was used to measure the amount of thiocholine formed, which was proportional to AChE activity. We read the absorbance in a microplate reader at~410 nm. The data obtained were compared to the standard curve values, and the AChE amounts (mU) were normalized to protein values (mU/ mg protein). The assessment was repeated three times in independent experiments blind to experimenter.
Measurement of Aβ 1-42 peptide in culture medium. The level of Aβ 1-42 peptide was measured according to a previous report [39] with minor modifications. Briefly, WT and PSEN1 E280A ChLNs were left in regular medium for 0, 2 and 4 days. Then, 100 μl of conditioned medium was collected, and the levels of secreted Aβ 1-42 peptides were determined by a solid-phase sandwich ELISA (Invitrogen, Cat# KHB3544) following the manufacturer's instructions. The assessment was repeated three times in independent experiments blind to experimenter.
Intracellular calcium imaging. Intracellular calcium (Ca 2+ ) concentration changes evoked by cholinergic stimulation were assessed according to refs. [40,41] with minor modifications. For the measurement, the fluorescent dye Fluo-3 (Fluo-3 AM; Thermo Fisher Scientific, cat: F1242) was employed. The dye was dissolved in DMSO (1 mM) to form a stock solution. Before the experiments, the stock solution was diluted in neuronal buffer solution (NBS buffer: 137 mM NaCl, 5 mM KCl, 2.5 mM CaCl 2 , 1 mM MgCl 2 , pH 7.3, and 22 mM glucose). The working concentration of the dye was 2 μM. The WT and PSEN1 E280A ChLNs were incubated for 30 min at 37˚C with the dye-containing NBS and then washed five times. Intracellular Ca 2+ transients were evoked by acetylcholine (1 mM final concentration) at 0, 2 and 4 days post differentiation. The measurements were carried out using the 100x objective of the microscope. Several regions of interest (ROIs) were defined in the visual field of the camera. One of the ROIs was cell-free, and the fluorescence intensity measured here was considered background fluorescence (F bg ). The time dependence of the fluorescence emission was acquired, and the fluorescence intensities (hence the Ca 2+ levels) were represented by pseudocolors. To calculate the changes of the average Ca 2+ -related fluorescence intensities, the F bg value was determined from the cell-free ROI, and then the resting fluorescence intensities (F rest ) of the cell-containing ROIs were obtained as the average of the points recorded during a period of 10 s prior to the addition of acetylcholine. The peaks of the fluorescence transients were found by calculating the average of four consecutive points and identifying those points that gave the highest average value (F max ). The amplitudes of the Ca 2+ -related fluorescence transients were expressed relative to the resting fluorescence (ΔF/F) and were calculated by the following formula: ΔF/F = (F max -F rest )/(F rest -F bg ). For the calculation of the fluorescence intensities, ImageJ was used. The terms fluorescence intensity was used as an indirect indicator of intracellular Ca 2+ concentration. The assessment was repeated three times in independent experiments blind to experimenter.
JNK inhibition experiment. The ChLNs were left in regular medium for 0, 2 and 4 days alone or co-incubated with the anthrapyrazolone JNK inhibitor SP600125 (1 μM final concentration). This compound competes with ATP to inhibit the phosphorylation of c-JUN. After this time, cells were evaluated for p-TAU and t-TAU protein expression by immunofluorescence, as described above. The assessment was repeated three times in independent experiments blind to experimenter.

Photomicrography and image analysis
Light microscopy photographs were taken using a Zeiss Axiostart 50 Fluorescence Microscope equipped with a Canon PowerShot G5 digital camera (Zeiss Wöhlk-Contact-Linsen, Gmb Schcönkirchen, Germany), and fluorescence microscopy photographs were taken using a Zeiss Axiostart 50 Fluorescence Microscope equipped with a Zeiss AxioCam Cm1 and (Zeiss Wöhlk-Contact-Linsfluoreen, Gmb Schcönkirchen, Germany) and Floyd Cells Imaging Station microscope. Fluorescence images were analyzed by ImageJ software (http://imagej.nih. gov/ij/). The figures were transformed into 8-bit images, and the background was subtracted. The cellular measurement regions of interest (ROIs) were drawn around the nucleus (for the case of transcription factors and apoptosis effectors) or over all cells (for cytoplasmic probes), and the fluorescence intensity was subsequently determined by applying the same threshold for cells in the control and treatment conditions. Mean fluorescence intensity (MFI) was obtained by normalizing total fluorescence to the number of nuclei.

Data analysis
In this experimental design, a vial of MSCs was thawed, cultured and the cell suspension was pipetted at a standardized cellular density of 2.6 x 10 4 cells/cm 2 into different wells of a 24-well plate. Cells (i.e., the biological and observational unit [42]) were randomized to wells by simple randomization (sampling without replacement method), and then wells (i.e., the experimental units) were randomized to treatments by similar method. Experiments were conducted in triplicate wells. The data from individual replicate wells were averaged to yield a value of n = 1 for that experiment and this was repeated on three occasions blind to experimenter and/ or flow cytometer analyst for a final value of n = 3 [42]. Based on the assumptions that the experimental unit (i.e. the well) data comply with independence of observations, the dependent variable is normally distributed in each treatment group (Shapiro-Wilk test), and there is homogeneity of variances (Levene's test), the statistical significance was determined by a One-way analysis of variance (ANOVA) followed by Tukey's post hoc comparison calculated with GraphPad Prism 5.0 software. Differences between groups were only deemed significant when a p-value of < 0.05 ( � ), < 0.001 ( �� ) and < 0.001 ( ��� ). All data are illustrated as the mean ± S.D.
Additionally, MSCs were transdifferentiated into cholinergic-like neurons (ChLNs) from WJ-MSCs using a new method [29]. As shown in Fig 2, WT PSEN1 and PSEN1 E280A WJ-MSCs cultured in minimal culture medium (MCm) for 7 days expressed basal levels of protein MAP2 (Fig 2A and 2B) and β Tub III (Fig 2A and 2C) and undetectable levels of   (Fig 2A and 2D) and ChAT (Fig 2A and 2E). As expected, when the cells were exposed to cholinergic-N-Run medium (Ch-N-Rm) for 7 days [29], the levels of protein MAP2 (Fig 2A  and 2B), β Tub III (Fig 2A and 2C) and ChAT (Fig 2A and 2E) were significantly higher than those in cells exposed to MCm. Noticeably, ChLNs remained negative for the specific glial cell lineage marker GFAP (Fig 2A and 2D). These observations were confirmed by immunofluorescence (Fig 2G-2J). Because the enzyme AChE catalyzes the breakdown of the neurotransmitter acetylcholine (ACh, [44]), we evaluated whether ChLNs expressed a catalytically functional AChE enzyme. As shown in Fig 2F, the AChE enzyme presented basal activity in WJ-MSCs under MCm culture conditions (~8 ± 1.2 mU/mg protein), while the AChE enzyme activity was significantly higher (at least a 3-fold increase) in both control and mutant ChLNs (~26 ± 1.6 mU/mg protein). Remarkably, there was no significant difference in AChE enzymatic activity (p< 0.05) between WT and PSEN1 E280A ChLNs (at day 0 of post differentiation).

PSEN1 E280A Cholinergic-Like Neurons (ChLNs) show high levels of intracellular sAPPβf, oxidized DJ-1, reactive oxygen species (ROS), loss of mitochondrial membrane potential (ΔC m ) and DNA fragmentation, but none of those markers are detected in WT PSEN1 ChLNs
The above observations prompted us to evaluate the same cell parameters in ChLNs. We initially verify that the antibody clone 6E10 recognizes Aβ monomers as well as large aggregates of Aβ 42 and APP. Effectively, the antibody recognized the "unaggregated" (monomers), and large aggregates forms of synthetic Aβ 42 peptide (Fig 3A). The WT PSEN1 and PSEN1 E280A ChLNs (obtained after 7 days in Ch-N-Rm) were left in regular culture medium (RCm) for 0, 2 and 4 additional days post transdifferentiation (hereafter labelled as 0, 2, or 4). Western blot also revealed that WT PSEN1 ChLNs displayed undetectable levels of intracellular APP/ Aβ 42 aggregates (Fig 3A and 3B) and oxidized DJ-1 (Fig 3A and 3C), whereas flow cytometry showed no loss of ΔC m (Fig 4A-4C) and no ROS generation (Fig 4D-4F) at any time tested. However, PSEN1 E280A ChLNs exhibited significantly higher levels of intracellular APP/ Aβ 42 aggregates (Fig 3A and 3B) and oxidized DJ-1 (Fig 3A and 3C) and lower ΔC m than WT PSEN1 ChLNs at days 2 and 4 (Fig 4B and 4C). These observations were confirmed by tubulin III (green; G"' and I"') and GFAP (red; G" and I") or MAP2 (green; H"' and J"') and ChAT (red; H" and J"). The nuclei were stained with Hoechst 33342 (blue; G'-J'). Data are expressed as the mean ± SD; � p<0.05; �� p<0.01; ��� p<0.001. The blots and figures represent 1 out of 3 independent experiments. Image magnification, 200x.

PSEN1 E280A ChLNs display activation of p53, PUMA, c-JUN and CASPASE-3
Next, we wanted to investigate whether the intracellular sAPPβf induced cell death signaling in PSEN1 ChLNs. Therefore, we used the transcription factors p53 and c-JUN, pro-apoptotic BH3-only protein PUMA, and protease CASPASE-3 as cell death markers to examine the prodeath activity of sAPPβf in ChLNs over time. As shown in Fig 5, while WT PSEN1 ChLNs showed no detectable levels of apoptogenic proteins at any time tested (Fig 5A-5E), the PSEN1 E280A ChLNs displayed significant detection of c-JUN (at day 0, 2 and 4, Fig 5A and 5B), p53 (at day 2 and 4, Fig 5A and 5C), PUMA (at day 4, Fig 5A and 5D) and CASP-3 (at day 4,  Fig 5A and 5E). These observations were confirmed by immunofluorescence analysis at day 4 (Fig 5F-5M).
The above findings compelled us to evaluate whether ChLNs expressed a catalytically functional acetylcholinesterase (AChE) enzyme at similar times post of transdifferentiation. As shown in Fig 6K, the AChE enzyme showed similar catalytic activity at days 2 and 4 in both WT PSEN1 and PSEN1 E280A ChLNs. However, the AChE enzyme activity in PSEN1 E280A ChLNs was significantly lower (at least 5-fold) than that in WT PSEN1 ChLNs at day 4.

Discussion
Currently, the neuropathology of AD includes extracellular deposits of Aβ in plaques, intracellular neurofibrillary tangles comprising hyperphosphorylated TAU, synaptic dysfunction, and neuronal death. In an effort to explain such observations, several theories have been proposed [48]; however, the amyloid cascade hypothesis has prevailed for more than 25 years [49]. The Aβ hypothesis postulates that an imbalance in the production of extracellular Aβ42 plaques by mutations in at least three genes (e.g., APP, PSEN1; PSEN2) is an early initiating factor in AD.  However, experimental therapies targeting Aβ have thus far been unsuccessful [50]. Several factors have probably contributed to the failures in AD drug development, including unsuitable preclinical research models that do not fully recapitulate the human disease; consequently, druggable targets remain missing [51]. Furthermore, the mechanism(s) by which hAPP/ Aβ42 might induce toxicity is not yet fully established. Because UC-MSCs possess plasticity properties enabling them to transdifferentiate into non-mesenchymal lineages, they provide a unique opportunity to study the effect of PSEN1 mutations in neuronal cells. Most importantly, MSCderived neurons are natural, non-genetically modified cells of the FAD PSEN1 mutation. Here, we used UC-MSCs bearing the mutation PSEN1 E280A for the first time and found that the mutation alters none of the typical MSC characteristics, such as colony-forming capacity, fibroblast-like morphology, immunophenotype and/or ability to differentiate or transdifferentiate into ChLNs. Furthermore, we did not detect any APP/ Aβ 42 produced by the cells or any other cellular alterations (e.g., ROS production, mitochondria depolarization, and oxidation of stress sensor DJ-1) in mutant MSCs. These observations suggest that the PSEN1 E280A mutation might not affect the physiology of the multipotent MS cells at this stage of development.
In this study, we report for the first time that ChLNs derived from hMSCs carrying the PSEN1 E280A mutation recapitulate typical pathologic features of AD at 4 days post transdifferentiation, i.e., eleven days of culture (7 days in Ch-N-Rm and 4 days in RCm) including increased secretion of Aβ 42 , intracellular accumulation of sAPPβf, and TAU phosphorylation. Furthermore, PSEN1 E280A ChLNs not only show OS and apoptosis markers but also display Ca 2+ flux dysregulation and altered acetylcholinesterase activity. Our data provide evidence that intracellular sAPPβf specifically induces neurotoxicity through a temporal and sequential order of molecular events in PSEN1 E280A ChLNs. In agreement with commercial laboratory Western blot data (https://www.biolegend.com/en-us/products/anti-beta-amyloid-1-16-antibody-10998) , the antibody 6E10 recognized monomers (4 kDa), and aggregated (~100 kDa) synthetic Aβ 42 , and high molecular weight APP/ Aβ 42 (~100 kDa) in PSEN1 E280A ChLNs. Interestingly, no Aβ 42 monomers were detected in mutant ChLNs. These observations suggested aggregation of APP. In agreement with this assumption, mass spectrometry (MS) analysis identified several APP fragments (e.g., APP714, APP733, APP751, APP752, collectively named sAPPβf) but no Aβ 42 fragment was present at the gel region of HMW proteins (60-120 kDa). Outstandingly, the sAPP fragments can be generated not only by the β-secretase but also by the δ-secretase, η-secretase or Meprin β [4]. Taken together these observations suggest that sAPP fragments rather than Aβ 42 are accumulating early in the PSEN1 E280A cholinergic neurons. Accordingly, we found intracellular sAPPβf accumulation in ChLNs after 7 days of transdifferentiation (day 0), at which time fully developed ChLNs were obtained [29]. Whether intracellular APP fragments build up because full length APP processing is altered and consequently some APP fragments remain intracellular leading to cell death require further investigation. However, recent data support this view. Indeed, it has been shown that hAPP overexpression causes Aβ-independent neuronal death in olfactory sensory neurons via intrinsic apoptosis pathway [5,6]. In contrast, others have found that the PSEN1 L166P and G384A mutations cause relocalization of γ-secretase, which significantly promotes the generation of intracellular long Aβ 42 [52]. Although it is not yet known whether the PSEN1 E280A mutation induces relocalization of γ-secretase, similar to PSEN1 mutations, our data suggest that the PSEN1 E280A mutation strongly enhances intraneuronal sAPPβf aggregates in ChLNs. These results support the view that intracellular accumulation of sAPPβf is the earliest event in the development of the neuropathological changes of AD [53]. Since the PSEN1 E280A mutation might represent a typical trans-dominant negative mutation on γ-secretase [15,16], we do not discard the possibility that APP-derived intracellular Aβ 42 aggregates might be a late event in the process of neurodegeneration [7,54,55]. Interestingly, we simultaneously found a significant increase in DCF fluorescent-positive cells, DJ-1 Cys 106 -sulfonate (DJ-1 Cys 106 SO 3 ), which is the most sensitive thiol group towards H 2 O 2 reactivity [56], and activation of transcription factor c-JUN in PSEN1 E280A ChLNs. These observations suggest that sAPPβf (aggregates) > H 2 O 2 > oxDJ-1Cys 106 -SO 3 and c-JUN are the earliest events detectable in PSEN1 E280A ChLNs. These findings imply that hAPP and/or sAPPβf (high molecular weight aggregates) can trigger events related to oxidative stress and cell death [5,6]. How then does intracellular sAPPβf generate H 2 O 2 ? Several lines of evidence for the intracellular toxicity of APP have been suggested, including inhibition of mitochondrial import channels (e.g., TOM40 and TIM23), impairment of mitochondrial transport (e.g., Cytochrome c oxidase), and disruption the electron transfer [57]. Whatever the mechanism, we demonstrated for the first time that sAPPβf endogenously produces H 2 O 2 in PSEN1 E280A ChLNs. Because H 2 O 2 can function as a second messenger [58], it might also activate other redox proteins, such as apoptosis-signal regulating kinase 1 (ASK-1, [59], which in turn directly or indirectly activate other signaling pathways, e.g., JNK/c-JUN [60]. We found that sAPPβf induced phosphorylation of c-JUN [61], p53 and PUMA in PSEN1 E280A ChLNs. Notably, c-JUN-and p53-dependent apoptosis is triggered by transactivation of the pro-apoptotic gene PUMA [62][63][64]. Remarkably, JNK can also stabilize and activate p53 [65]. Taken together, these results suggest that sAPPβf > H 2 O 2 activates a cascade of events leading to the JNK> c-JUN, p53 > PUMA pathway. Although PUMA has been shown to cooperate with direct activator proteins (e.g., BAX) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis [66], the exact mechanism by which MOMP occurs is not fully understood [67]. Interestingly, PSEN1 E280A ChLNs showed loss of ΔC m concomitant with overexpression of CASPASE-3 and fragmentation of nuclei at day 4 post transdifferentiation. These observations suggest that mitochondria play an important role in intracellular sAPPβf-induced apoptosis in mutant ChLNs. However, whether ΔC m dysfunction is a consequence of a direct effect of intracellular sAPPβf on the organelle or whether the damage is the result of the impact of PUMA on mitochondria is an unresolved issue. Our findings suggest that both sAPPβf / PUMA might separately or jointly damage ΔC m . Taken together, these data indicate that sAPPβf induce a cascade of events in PSEN1 E280A ChLNs through the H 2 O 2 signaling pathway involving CASP-3, as an end executer protein, and DNA fragmentation of nuclei, all indicative of apoptosis. Taken together, these data comply with the idea that intraneuronal accumulation of the APP fragments (sAPPβf) are the first step of a lethal cascade in FAD neurons.
Neuronal calcium (Ca 2+ ) dyshomeostasis has been proposed to play a crucial role in AD disease progression [68]. However, the mechanisms of Ca 2+ dysregulation are not clear. In contrast to Demuro and Parker [69], who found that intracellular Aβ42 oligomers disrupted cellular Ca 2+ regulation, we observed no Ca 2+ dysregulation in PSEN1 E280A ChLNs evaluated at 0, 2, and 4 days post transdifferentiation. This discrepancy can be explained by differences in experimental methodology. Whereas those authors elucidated the actions of intracellular Aβ42 by imaging Ca 2+ responses to injections of Aβ42 oligomers into Xenopus oocytes, we directly imaged Ca 2+ responses to endogenously generated intracellular APP in mutant ChLNs. Under the present experimental conditions, we concluded that intracellular sAPPβf did not affect Ca 2+ flux in PSEN1 E280A ChLNs. However, increasing evidence has shown that extracellular Aβ specifically interacts with nAChRs, resulting in Ca 2+ dysregulation [70]. We found that the PSEN1 E280A ChLN response to ACh was significantly reduced by day 4 post transdifferentiation. Notably, Aβ has been shown to directly affect α7 nicotinic ACh receptor (α7 nAChR) function by acting as an agonist (~100 nM) and a negative modulator (at high concentrations) [71]. Consistent with this view, we confirmed that PSEN1 E280A ChLNs secreted aberrant amounts of Aβ42 (e.g.,~2500-f.i.) compared to WT PSEN1 ChLNs. These observations confirm that overproduction of extracellular Aβ42 is a paramount feature of the majority of PSEN1 mutations in vitro and in vivo [9], including the E280A mutation [25]. Despite these observations, further investigation is required to determine whether α7 nAChRs specifically are affected by Aβ 42 in PSEN1 E280A ChLNs. In contrast to others (e.g., [72]), our observations do not support the common view that extracellular Aβ is capable of increasing neuronal Ca 2+ flux through Aβ42-forming pores. However, we do not discard the possibility that given a longer incubation time, Aβ42 could affect Ca 2+ flux via Aβ42-forming pores in PSEN1 E280A ChLNs. Taken together, and our data suggest that extracellular Aβ42 might bind to nAChRs in PSEN1 E280A ChLNs, affecting neuronal Ca 2+ flux.
The accumulation of hyperphosphorylated TAU in neurons leads to neurofibrillary degeneration in AD [73]. Mounting evidence suggests that TAU pathogenesis is promoted by Aβ42 [74,75]. We found that PSEN1 E280A ChLNs showed hyperphosphorylation of TAU protein.
In fact, PSEN1 E280A ChLNs exhibited a significantly higher p-TAU/ t-TAU ratio than WT PSEN1 ChLNs at 4 days post transdifferentiation. Because absence of Aβ42, these observations imply that sAPPβf rather than Aβ42 signaling precedes TAU phosphorylation [75,76] in PSEN1 E280A ChLNs. These findings suggest that sAPPβf accumulation might affect TAU pathology. However, the molecular link between sAPPβf and TAU is still not yet completely defined. In agreement with others [77], our data suggest that JNK is a strong candidate TAU kinase involved in the hyperphosphorylation of TAU in PSEN1 E280A ChLNs. This assumption is supported by two observations. First, JNK phosphorylates TAU at Ser 202 / Thr 205 [47], two phosphorylation epitopes identified in the present study. Second, PSEN1 E280A ChLNs exposed to the JNK inhibitor SP600125 significantly reduced TAU phosphorylation. Given that JNK plays a pivotal role in both OS-induced apoptosis and TAU phosphorylation, these findings identify JNK as a potential therapeutic target [78]. Although we do not discard the possibility that other kinases might be implicated in TAU pathology (e.g., LRRK2, GSK-3, Cdk5), our findings suggest that JNK plays a key role in TAU hyperphosphorylation in PSEN1 E280A ChLNs. Our results suggest that PSEN1 E280A-induced neural alterations may precede Aβ42 deposition and that those alterations represent longstanding effects of intracellular sAPPβf toxicity and possibly even developmental changes. The molecular alterations might start when neurons develop into neuron-specific cholinergic-type cells or may even exist at birth. These findings may explain why functional and structural brain changes manifest in children (9-17 years old) and young individuals (18-26 years old) who are carriers of the PSEN1 E280A mutation [79,80]. Furthermore, these observations suggest that intracellular sAPPβf toxicity is an early and slowly progressive process that might damage neuronal cells in a TAU-dependent and independent fashion (OS, ΔC m shutdown, apoptosis and intraneuronal Ca 2+ dysregulation) more than two decades before the stage of dementia [10,81].

Conclusion
We demonstrate that intracellular accumulation of sAPPβf, generation of H 2 O 2 , oxidation of the DJ-1 protein (DJ-1 Cys 106 -SO 3 ), and activation of the pro-apoptosis protein c-JUN were the earliest cellular changes in PSEN1 E280A ChLNs (obtained after 7 days in Ch-N-Rm (i.e., day 0 in RCm), Fig 8 step 1, s2, s3, s4/s5). These changes were followed by the activation of pro-apoptosis proteins p53 (s6) and PUMA (s7), loss of mitochondria membrane potential (ΔC m , s8), activation of CASP-3 (s9), fragmentation of nuclei (s10), and complete expression of markers of apoptosis (at day 4 post transdifferentiation in RCm). These biochemical abnormalities were found concomitant with irregular secretion of Aβ42 (day 4, s11), Ca 2+ flux dysregulation (s12), diminished secretion of AChE (s13), and hyperphosphorylation of TAU protein (day 4, s14). Therefore, our data support the view that FAD PSEN1 E280A cholinergic neuronal pathology is instigated by early intracellular accumulation of sAPPβf [55,82]. These findings raise the question of whether strategies to remove extracellular Aβ 42 such as immunotherapy [83] should be complemented with treatments to remove intracellular sAPPβf [84] and OS (i.e., H 2 O 2 ) to avoid apoptosis and TAU pathology and treatment with nAChR agonists to increase neuronal functionality. The present findings naturally (i.e., not genetically manipulated) recapitulated for the first time the neuropathological features of FAD PSEN1 E280A. We anticipate that the present in vitro model will inspire new and innovative therapies for early onset PSEN1 E280A patients. Despite the fact that the mutation PSEN1 E280A is 100% penetrant in those individuals bearing such mutation, and given that our findings are based on ChLNs obtained from a single PSEN1 E280A MSCs, the conclusions from such analyses require verification through additional studies with larger sample size.