Multi-Target Effects of the Cannabinoid CP55940 on Familial Alzheimer's Disease PSEN1 E280A Cholinergic-Like Neurons: Role of CB1 Receptor.

Abstract

Background:

Alzheimer’s disease (AD) is characterized by structural damage, death, and functional disruption of cholinergic neurons (ChNs) as a result of intracellular amyloid-β (Aβ) aggregation, extracellular neuritic plaques, and hyperphosphorylation of protein tau (p-Tau) overtime.

Objective:

To evaluate the effect of the synthetic cannabinoid CP55940 (CP) on PSEN1 E280A cholinergic-like nerve cells (PSEN1 ChLNs)—a natural model of familial AD.

Methods:

Wild type (WT) and PSEN1 ChLNs were exposed to CP (1μM) only or in the presence of the CB₁ and CB₂ receptors (CB₁Rs, CB₂Rs) inverse agonist SR141716 (1μM) and SR144528 (1μM) respectively, for 24 h. Untreated or treated neurons were assessed for biochemical and functional analysis.

Results:

CP in the presence of both inverse agonists (hereafter SR) almost completely inhibits the aggregation of intracellular sAβPPβf and p-Tau, increases ΔΨ_m, decreases oxidation of DJ-1Cys¹⁰⁶-SH residue, and blocks the activation of c-Jun, p53, PUMA, and caspase-3 independently of CB₁Rs signaling in mutant ChLNs. CP also inhibits the generation of reactive oxygen species partially dependent on CB₁Rs. Although CP reduced extracellular Aβ₄₂, it was unable to reverse the Ca²⁺ influx dysregulation as a response to acetylcholine stimuli in mutant ChLNs. Exposure to anti-Aβ antibody 6E10 (1:300) in the absence or presence of SR plus CP completely recovered transient [Ca²⁺]_i signal as a response to acetylcholine in mutant ChLNs.

Conclusion:

Taken together our findings suggest that the combination of cannabinoids, CB₁Rs inverse agonists, and anti-Aβ antibodies might be a promising therapeutic approach for the treatment of familial AD.

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Keywords: Apoptosis, cannabinoids, cholinergic neurons, CP55940, E280A mutation, familial Alzheimer disease, neuronal dysfunction, oxidative stress, PSEN1, sAβPPβf, tau

Article notes

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Accepted 2020 Oct 12; Prepublished 2020 Nov 23; Collection date 2021.

Author notes

INTRODUCTION

Alzheimer’s disease (AD) is a multifactorial neu-rodegenerative condition [1] characterized by severe loss of cholinergic neurons from the nucleus basalis magnocellularis of Meynert and cholinergic projections to the cortex and hippocampus [2]. The neuronal destruction is associated with the extracellular accumulation of insoluble forms of amyloidβ (Aβ) in plaques, mostly fragment Aβ_1–42 (hereafter Aβ₄₂), intracellular aggregation of the microtubule protein tau in neurofibrillary tangles, neuronal cell death, and synaptic dysfunction [3]. The Aβ₄₂ peptide is derived from the amyloid-β protein precursor (AβPP) via sequential cleavage by the beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and by the gamma-site aspartyl protease γ-secretase [4]. This last protein is composed of four subunits, namely presenilins (PSEN1 and PSEN2), presenilin enhancer 2 (PEN2), anterior pharynx-defective 1 (APH-1), and nicastrin. While PEN2, APH-1, and nicastrin stabilize the complex, PSEN1 and PSEN2 function as the catalytic core of the protease [5]. Over two hundred autosomal dominant mutations in the presenilin 1 (PSEN 1) gene result in the overproduction of extracellular (e)Aβ₄₂ [6] and occur most frequently in familial AD (FAD; https://www.alzforum.org/mutations/psen-1). Among PSEN1 mutations, the Glu280Ala (p. E280A, c.839A>C, exon 8) in PSEN1 causes FAD with complete penetrance in a large kindred localized in Antioquia, Colombia [7–10]. As the majority of dominant-negative PSEN1 mutations [11], PSEN1 E280A produces increased Aβ₄₂ deposition [12], hippocampal neuron loss [13], and Aβ/tau accumulation in young adults [14, 15]. Recently, we have recapitulated the molecular pathogenesis of FAD caused by PSEN1 E280A in umbilical cord mesenchymal stem cells-derived cholinergic-like neurons (ChLNs [16]). It has demonstrated that PSEN1 E280A ChLNs effectively exhibited not only an increased eAβ₄₂ but also an early increased of intracellular sAβPPβ fragments (but not Aβ₄₂ peptide) and tau phosphorylation. Moreover, PSEN1 E280A ChLNs exhibited oxidized DJ-1 (at Cys¹⁰⁶SO₃) indicative of oxidative stress (OS), and concomitant loss of the mitochondrial membrane potential (ΔΨ_m), activation of apoptogenic proteins, and DNA fragmentation, markers of apoptosis—a type of regulated cell death. Additionally, mutant ChLNs displayed Ca²⁺ flux dysregulation when challenged to acetylcholine (ACh) and deficient acetylcholinesterase (AChE) activity compared to wild type (WT) ChLNs most probably due to eAβ₄₂ peptide. Taken together these observations suggest that PSEN1 E280A ChLNs display the typical neuropathological markers of AD; therefore, mutant ChLNs provide a unique FAD model for screening of candidate molecule(s)/drug(s).

Due to the lack of effective therapies to date for AD, the need for new drugs has become increasingly urgent. Phytocannabinoids, a group of biologically active compounds isolated from the plant Cannabis sativa [17], have been postulated to possess therapeutic potential for clinical purposes in AD [18]. Cannabis contains at least 120 kinds of cannabinoids mainly classified as Δ⁹-trans-tetrahydrocannabidiol (THC), cannabidiol (CBD), CBG (Cannabigerol), and CBN (Cannabinol), among others [19]. Biologically, cannabinoids, endocannabinoids (e.g., anandamide, 2-arachidonoylglycerol), and synthetic cannabimimetic compounds (e.g., CP55940) bind cannabinoid type 1 and 2 receptors (CB₁R/ CB₂R), two G-protein-coupled receptors (GPCR) [20, 21]. Interestingly, it has been shown in vitro that 9 out of 11 cannabinoids studied, including THC, were able to protect cells (e.g., MC65—a human neuron-like cell line, HT22 mouse hippocampal nerve cell line, primary cortical neurons from 18-day-old rat embryos, BV2—a microglial cell line) in four distinct phenotypic neurodegeneration screening assays including proteotoxicity, loss of trophic support, OS, energy loss, and inflammation [22]. Outstandingly, cannabinoids were able to remove intraneuronal Aβ, reduce oxidative damage, and protect from the loss of energy or trophic support in neurons in which CB₁ and CB₂ receptors were lacking (e.g., MC65 and HT22 cells). Furthermore, CB₁ agonists (e.g., CP55940) were also effective neuroprotective agents against Aβ toxicity in MC65 neurons [22]. These data suggest that cannabinoids might be antioxidant and anti-amylogenic compounds through receptor-independent mechanisms in a cell- and species-specific manner [22–28]. Despite these observations, no data are available to establish whether cannabinoids might reverse the neuropathological markers, i.e., intracellular sAβPPβf aggregation, hyperphosphorylation of tau protein, and DJ-1 oxidation in PSEN1 E280A ChLNs. Furthermore, it is not yet known whether cannabinoids might be able to block neuronal apoptosis, eAβ₄₂, and/or prevent Ca²⁺ dysregulation in those mutant cholinergic neurons.

To get insight into these issues, we have selected the commercially available CP55940 ((-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol, hereafter CP), a CB₁ and CB₂ potent bicyclic analog of THC [29], to evaluate its effect on wild-type and PSEN1 E280A ChLNs concerning the intracellular sAβPPβf accumulation, tau phosphorylation, OS, cell death, and Ca²⁺ neuronal dysfunction in ChLNs. We demonstrate for the first time that CP can protect FAD PSEN1 E280A ChLNs against neuronal structural damage and OS-induced cell death signaling and that it can reverse the cholinergic dysfunction in the presence of eAβ₄₂ blocking agents (e.g., anti-Aβ₄₂ antibody 6E10). These findings might favor the use of cannabinoids as potential lead compound candidates as a combined therapy for FAD.

MATERIALS AND METHODS

Cholinergic-like neuron (ChLN) differentiation

ChLN differentiation was performed according to [30]. The WT (TBC# WJMSC-11) and PSEN1 E280A (TBC# WJMSC-12) [16] MSCs were seeded at 1–1.5×10⁴ cells/cm² in laminin-treated culture plates for 24 h in regular culture medium (RCm). Then, the medium was removed, and cells were incubated in cholinergic differentiation medium (Cholinergic-N-Run medium, hereafter Ch-N-Rm) containing DMEM/F-12 media 1:1 Nutrient Mixture (Gibco cat# 10565018), 10 ng/ mL basic fibroblast growth factor (bFGF) recombinant human protein (Gibco Cat# 13256029), 50μg/ mL sodium heparin (Hep, Sigma-Aldrich cat# H3393), 0.5μM all-trans retinoic acid, 50 ng/ml sonic hedgehog peptide (SHH, Sigma cat# SRP3156) and 1% FBS at 37°C for 7 days. After this process of transdifferentiation, the cells were labeled as WT PSEN1 or PSEN1 E280A ChLNs. Since Ch-N-Rm contains several factors that might interfere with the experiment interpretation and measurements, WT PSEN1 and PSEN1 E280A ChLNs (obtained after 7 days in Ch-N-Rm) were left in regular culture medium (RCm) for 4 additional days of post transdifferentiation.

Immunofluorescence analysis

For the analysis of markers of AD, oxidative stress, and cell death, the cells treated under different conditions were fixed with 4% paraformaldehyde for 20 min, followed by Triton X-100 (0.1%) permeabilization and 10% bovine serum albumin (BSA) blockage. Cells were incubated overnight with primary antibodies against APP₇₅₁ and/or protein amyloid β_1 - 42 (1:500; clone 6E10 cat# 803014, Biolegend), total tau (1:500; t-Tau; cat# T6402, Sigma), and phospho-tau (p-Tau, 1:500, Ser202/Thr205, cat# MN1020 (AT8), Thermo Fisher Scientific); and primary antibodies against oxidized DJ-1 (1:500; ox(Cys106)DJ-1; spanning residue C¹⁰⁶ of human PARK7/DJ1; oxidized to produce cysteine sulfonic (SO₃) acid; cat # ab169520, Abcam). To assess cell death, we used primary antibodies against p53-upregulated modulator of apoptosis (1:500; PUMA, cat# ab-9643, Abcam), p53 (1:500; cat# MA5-12453, Millipore), phospho-c-Jun (1:250; c-Jun (S63/73) cat# sc-16312, Santa Cruz), and caspase-3 (1:250; cat # AB3623, Millipore). After exhaustive rinsing, we incubated the cells with secondary fluorescent antibodies (DyLight 488 and 594 horse anti-rabbit, -goat and -mouse, cat DI 1094, DI 3088, and DI 2488, respectively) at 1:500. The nuclei were stained with 1μM Hoechst 33342 (Life Technologies), and images were acquired on a Floyd Cells Imaging Station microscope.

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 (30μg of proteins) were heated at 95°C for 5 min in 2×SDS and 20× reducing agent (except for protein oxDJ-1) and loaded on to 12% 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 Bio-Rad protocol (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6376). The membra-nes were incubated overnight at 4°C with anti-APP₇₅₁, total tau, phospho-Tau, ox(Cys¹⁰⁶) 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® 680 RD, cat #926-68071; donkey anti-goat IRDye ® 680 RD, cat # 926-68074; and goat anti-mouse IRDye ® 800 CW, cat #926-32270; LI-CORBiosciences) at 1:1000 were used for western blotting analysis, and data were acquired using Odyssey software. The determination of the aggregation state of APP₇₅₁ (i.e., sAβPPβf) was performed by western analysis of SDS-PAGE as described above. The assessment was repeated three times in independent experiments.

Analysis of cells

Assay protocol

The methodology for both WT and PSEN1 E280A ChLNs cell culture assays was the same. Initial CP55940 screening was performed at least twice in triplicate between 10 nM and 1μM. Subsequently, CP55940 (1μM) was established as an optimal concentration for further experiments. ChLNs were divided in four groups: 1) untreated; 2) treated with SR141716 (CB₁ receptor inverse agonist) and SR144528 (CB₂ receptor inverse agonist) at 1μM final concentration each (hereafter SR); 3) CP55940 (or CP); 4) RS + CP55940 (also named as SR + CP cocktail). To block eAβ₄₂ [31], WT and mutant ChLNs were incubated four days with anti-Aβ₄₂ antibody 6E10 (1:300 in RCm) after differentiation in the presence or absence of CP or CP + RS.

Evaluation of intracellular hydrogen peroxide (H₂O₂) by fluorescence microscopy

To determine the levels of intracellular H₂O₂, we used 2^′,7^′-dichlorofluorescein diacetate (5μM, DCFH2-DA; Invitrogen). ChLNs were left in RCm for 4 days. Then, the cells (5×10³) were incubated with the DCFH₂-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 0.5μM Hoechst 33342 staining compound. The assessment was repeated three times in independent experiments blind to the experimenter.

Analysis of mitochondrial membrane potential (ΔΨm) by fluorescence microscopy

The ChLNs were left in regular culture medium (RCm) for 4 days. Then, the cells (5×10³) were incubated with the passively diffusing and active mitochondria-accumulating dye deep red MitoTracker compound (20 nM, final concentration) for 20 min at room temperature in the dark (Invitrogen, cat # M22426). Cells were then washed twice with PBS. MitoTracker fluorescence intensity was determined by analysis of fluorescence microscopy images. The assessment was repeated three times in independent experiments. The nuclei were stained with 0.5μM Hoechst 33342 staining compound. The assessment was repeated three times in independent experiments blind to the experimenter and flow cytometer analyst.

Measurement of Aβ₄₂ peptide in culture medium

The level of Aβ_1–42 peptide was measured according to a previous report [32] with minor modifications. Briefly, WT and PSEN1 E280A ChLNs were left in RCm for 4 days. Then, 100μl of conditioned medium was collected, and the levels of secreted Aβ₄₂ peptides were determined by a solid-phase sandwich ELISA (Invitrogen, Cat# KHB3544) following the manufacturer’s instructions. The ass-essment was repeated 4 times in independent experiments blind to the experimenter.

Intracellular calcium imaging

Intracellular calcium (Ca²⁺) concentration chan-ges evoked by cholinergic stimulation were assessed according to [33, 34] 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 CaCl₂, 1 mM MgCl₂, pH 7.3, and 22 mM glucose). The working concentration of the dye was 2μM. 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 Ca²⁺ transients were evoked by acetylcholine (1 mM final concentration) at 4 days post differentiation. The measurements were carried out using the 20× 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 Ca²⁺ levels) were represented by pseudo colors. To calculate the changes of the average Ca²⁺ -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 consecutive period of 10 s before the addition of acetylcholine. The peaks of the fluorescence transients were found by calculating the average of six consecutive points and identifying those points that gave the highest average value (F_max). The amplitudes of the Ca²⁺ -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 Ca²⁺ concentration. The assessment was repeated three times in independent experiments blind to the 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 ROIs were drawn around the nucleus (for the case of transcription factors and apoptosis effectors) or overall cells (for cytoplasmic probes), and the fluorescence intensity was subsequently determined by applying the same the 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 2.6×10⁴ cells/ cm² into different wells of a 24-well plate. Cells (i.e., the biological and observational unit [35]) were randomized to wells by simple randomization (sampling without replacement method), and then wells (i.e., the experimental units) were randomized to treatments by a 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 the experimenter and/ or flow cytometer analyst for a final value of n = 3 [35]. Based on the assumption that the experimental unit (i.e., the well) data comply with the independence of observations, the dependent variable is normally distributed in each treatment group (Shapiro-Wilk test), and variances are homogeneous (Levene’s test), the statistical significance was determined by 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.

RESULTS

CP55940 restores the mitochondrial membrane potential (ΔΨ_m) and blunts generation of ROS in PSEN1 E280A ChLNs in an independent concentration fashion

Previously, it has been shown that PSEN 1 E280A ChLNs present aggregation of sAβPPβf, endogenously generated OS, and loss of ΔΨ_m as the earliest neuropathological markers in mutant ChLNs [16]. Therefore, we initially determined whether CP could recover ΔΨ_m and scavenge ROS in those cells. To this aim, cholinergic cells were exposed to increasing concentrations of CP. As shown in Fig. 1A’-E’, CP did not affect ΔΨ_m in wild type ChLNs (Fig. 1A–E, K). However, it significantly increased the ΔΨ_m in PSEN1 E280A ChLNs in a concentration-independent fashion (Fig. 1F’-J’). Accordingly, we found 500 nM –1000 nM (= 1μM), but not low concentrations (10, 100 nM), as the maximal optimal concentrations to raise ΔΨ_m in mutant cells (Fig. 1F-J, K). Figure 1 shows mutant ChLNs (Fig. 1F”) but no WT ChLNs (Fig. 1A”) endogenously produce ROS. CP blunted ROS yield in mutant ChLNs (Fig. 1G”-J”) compared to WT ChLNs (Fig. 1B”-E”) in an independent concentration fashion. Since CP (1μM) recovered ΔΨ_m to control levels and completely diminished ROS in mutant ChLNs (Fig. 1K, L), we selected this concentration for further experiments.

CP55940 restores the mitochondrial membrane potential (ΔΨm) and reduces the levels of intracellular reactive oxygen species independent of its concentration in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with CP at increasing concentrations (0, 10, 100, 500, and 1000 nM) in regular culture medium (RCm) for 4 days. Representative MitoTracker (A’-J’), DCF (A”-J”), Hoechst (A”’-J”’), and merge (A-J) pictures of WT PSEN1 and PSEN1 E280A ChLNs treated as described. K) Quantification of MitoTracker fluorescence intensity. L) Quantification of DCF fluorescence intensity. Data are expressed as the mean±SD; *p < 0.05; **p < 0.01; ***p < 0.001. The histograms and figures represent 1 out of 3 independent experiments. Image magnification, 200×. Inset magnification, 800×. — Fig. 1CP55940 restores the mitochondrial membrane potential (ΔΨ_m) and reduces the levels of intracellular reactive oxygen species independent of its concentration in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with CP at increasing concentrations (0, 10, 100, 500, and 1000 nM) in regular culture medium (RCm) for 4 days. Representative MitoTracker (A’-J’), DCF (A”-J”), Hoechst (A”’-J”’), and merge (A-J) pictures of WT PSEN1 and PSEN1 E280A ChLNs treated as described. K) Quantification of MitoTracker fluorescence intensity. L) Quantification of DCF fluorescence intensity. Data are expressed as the mean±SD; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. The histograms and figures represent 1 out of 3 independent experiments. Image magnification, 200×. Inset magnification, 800×.

CP55940 restores ΔΨ_m in a CB₁ receptor-independent manner but diminishes ROS production partially dependent on CB₁ receptors in PSEN1 E280A ChLNs

We wanted to establish whether the neuroprotective effect on mitochondria and antioxidant activity of CP was due to interaction with CB₁Rs. Although these receptors are expressed in cholinergic neurons [36] and CP is a CB₁ and CB₂ receptor agonist, we used both potent and highly specific CB₁ and CB₂ receptor inverse agonist SR141716 and SR144528 (hereafter SR at 1μM each) only or with CP agonist (i.e., SR + CP cocktail) to avoid ambiguous observations. Figure 2 shows that while SR only neither affects ΔΨ_m in wild type ChLNs (i.e., high ΔΨ_m Fig. 2C) nor in mutant ChLNs (i.e., low ΔΨ_m Fig. 2D) when compared to untreated neurons (Fig. 2A, B), CP (Fig. 2F) or CP + SR (Fig. 2H) significantly increased the ΔΨ_m in mutant ChLNs (Fig. 2I). Neither CP nor the cocktail altered ΔΨ_m in WT ChLNs (Fig. 2E, G, I). However, analysis of ROS disappearance in ChLNs shows that while CP only almost completely scavenged ROS in mutant ChLNs (Fig. 2J), the SR + CP cocktail diminished ROS by almost half of the value of untreated or treated with SR only in PSEN1 E280A ChLNs (Fig. 2J). WT ChLNs produced no detectable ROS in any experimental condition (Fig. 2J). Of note, SR only treatment showed no effect in ΔΨ_m and ROS conditions in both WT (Fig. 2I) and mutant ChLNs (Fig. 2J).

CP55940 augments the Ψm independent of CB1R but reduced the levels of intracellular reactive oxygen species partially dependent on those receptors in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with SR, CP, or SR + CP in RCm for 4 days. A-H) Representative MitoTracker/DCF/Hoechst merge pictures of WT PSEN1 and PSEN1 E280A ChLNs treated as described. I) Quantification of MitoTracker fluorescence intensity. J) Quantification of DCF fluorescence intensity. Data are expressed as the mean±SD; *p < 0.05; **p < 0.01; ***p < 0.001. The histograms and figures represent 1 out of 3 independent experiments. Image magnification, 400×. — Fig. 2CP55940 augments the Ψ_m independent of CB₁R but reduced the levels of intracellular reactive oxygen species partially dependent on those receptors in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with SR, CP, or SR + CP in RCm for 4 days. A-H) Representative MitoTracker/DCF/Hoechst merge pictures of WT PSEN1 and PSEN1 E280A ChLNs treated as described. I) Quantification of MitoTracker fluorescence intensity. J) Quantification of DCF fluorescence intensity. Data are expressed as the mean±SD; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. The histograms and figures represent 1 out of 3 independent experiments. Image magnification, 400×.

CP55940 reduces intracellular sAβPPβf aggregation in a CB₁Rs-independent fashion but it reduces oxidized DJ-1 partially dependent of CB₁Rs in PSEN1 E280A ChLNs

Next, we assessed whether CP could inhibit intracellular aggregation of sAβPPβf and avoid oxidation of the stress sensor protein DJ-1. To this aim, WT and mutant ChLNs were left untreated or exposed to CP in the absence or presence of SR. Western blot analysis revealed that CP only or with SR reduced the aggregation of sAβPPβf in a CB₁Rs-independent manner (Fig. 3A, B) but blunted the oxidation of DJ-1 partially dependent of CB₁Rs (Fig. 3A, C). As expected, WT ChLNs remained unaltered to CP and to the cocktail exposure. Neither sAβPPβf nor oxidized DJ-1 were detected in those neurons (Fig. 3A–C). These results were confirmed by fluorescent microscopy (Fig. 3D–M).

CP55940 reduced intracellular sAβPPβf and oxidized DJ-1 independent of CB1Rs in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with SR, CP, or SR + CP in RCm for 4 days. Further, the proteins in the extracts were blotted with primary antibodies against Aβ42, oxDJ-1Cys106, and actin proteins. The intensities of the western blot bands shown in (A) were measured (B, C) by an infrared imaging system (Odyssey, LI-COR), and the intensity was normalized to that of actin. Additionally, cells were double-stained as indicated in the figure (D-K) with primary antibodies against oxDJ-1Cys106 (red; D’-K’) and APP751/ Aβ42 (green; D”- K”). The nuclei were stained with Hoechst 33342 (blue; D”’- K”’). L) Quantification of Aβ42 fluorescence intensity. M) Quantification of oxDJ-1Cys106 fluorescence intensity. 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, 200×. — Fig. 3CP55940 reduced intracellular sAβPPβf and oxidized DJ-1 independent of CB₁Rs in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with SR, CP, or SR + CP in RCm for 4 days. Further, the proteins in the extracts were blotted with primary antibodies against Aβ₄₂, oxDJ-1Cys¹⁰⁶, and actin proteins. The intensities of the western blot bands shown in (A) were measured (B, C) by an infrared imaging system (Odyssey, LI-COR), and the intensity was normalized to that of actin. Additionally, cells were double-stained as indicated in the figure (D-K) with primary antibodies against oxDJ-1Cys¹⁰⁶ (*red*; D’-K’) and APP₇₅₁/ Aβ₄₂ (*green*; D”- K”). The nuclei were stained with Hoechst 33342 (*blue*; D”’- K”’). L) Quantification of Aβ₄₂ fluorescence intensity. M) Quantification of oxDJ-1Cys¹⁰⁶ fluorescence intensity. 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, 200×.

CP55940 blocks apoptosis in a CB₁Rs-independent manner in PSEN1 E280A ChLNs

Cell death by apoptosis is a prominent feature in mutant ChLNs [16]. We used, therefore, the activation of the transcription factors p53 and c-Jun, pro-apoptotic BH3-only protein PUMA, and protease caspase-3 as apoptosis markers to examine the effect of CP and/or SR plus CP on those neuronal cells. Effectively, we confirmed that ChLNs displayed high levels of protein c-Jun (Fig. 4A, B), p53 (Fig. 4A, C), PUMA (Fig. 4A, D), and caspase-3 (Fig. 4A, E) in mutant but not in WT ChLNs (Fig. 4A–E). No significant difference was found between untreated and SR treatment for all the apoptosis markers in both types of cells. However, CP only or CP + SR significantly reduced c-Jun (Fig. 4A, B), p53 (Fig. 4A, C), PUMA (Fig. 4A, D), and caspase-3 (Fig. 4A, E) in PSEN1 E280A neurons when compared to untreated cells or WT ChLNs. Interestingly, mutant neurons exposed to CP + SR displayed almost basal levels of all proteins comparable to WT ChLNs. These observations were confirmed by fluorescent microscopy (Fig. 4F–Y).

CP55940 reduced the activation of p53, PUMA, c-Jun, and caspase-3 independent of CB1Rs signaling in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left Untreated or treated with SR, CP, or SR + CP in regular culture medium for 4 days. After this time, the proteins in the extracts were blotted with primary antibodies against phosphorylated c-Jun (p-c-JUN)/total c-Jun, p53, PUMA, caspase-3 (CASP-3) and actin proteins. The intensities of the western blot bands shown in (A) were measured (B-E) by an infrared imaging system (Odyssey, LI-COR), and the intensity was normalized to that of actin. Additionally, cells were double-stained as indicated in the figure (F-U) with primary antibodies against p53 (green; F’-M’), PUMA (red; F”-M”), c-JUN (green; N’-U’), and CASP-3 (red; N”-U”). The nuclei were stained with Hoechst 33342 (blue; F”’-U”’). V-X) Quantification of c-JUN (V), p53 (W), PUMA (X), and CASP-3 (Y) fluorescence intensity. 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, 200×. — Fig. 4CP55940 reduced the activation of p53, PUMA, c-Jun, and caspase-3 independent of CB₁Rs signaling in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left Untreated or treated with SR, CP, or SR + CP in regular culture medium for 4 days. After this time, the proteins in the extracts were blotted with primary antibodies against phosphorylated c-Jun (p-c-JUN)/total c-Jun, p53, PUMA, caspase-3 (CASP-3) and actin proteins. The intensities of the western blot bands shown in (A) were measured (B-E) by an infrared imaging system (Odyssey, LI-COR), and the intensity was normalized to that of actin. Additionally, cells were double-stained as indicated in the figure (F-U) with primary antibodies against p53 (*green*; F’-M’), PUMA (*red*; F”-M”), c-JUN (*green*; N’-U’), and CASP-3 (*red*; N”-U”). The nuclei were stained with Hoechst 33342 (*blue*; F”’-U”’). V-X) Quantification of c-JUN (V), p53 (W), PUMA (X), and CASP-3 (Y) fluorescence intensity. 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, 200×.

CP55940 inhibits tau phosphorylation in a CB₁Rs-independent fashion PSEN1 E280A ChLNs

It has been established that intracellular sAβPPβf induce phosphorylation of tau [16]. We then investigated whether CP could inhibit tau phosphorylation in PSEN1 E280A ChLNs. As shown in Fig. 5, while the level of tau phosphorylation was detected to a similar extend in WT ChLNs under any experimental conditions (Fig. 5A, B), CP or CP + SR cocktail dramatically reduced phosphorylation of protein tau when compared to untreated or SR only treatment in mutant ChLNs (Fig. 5A, B). No statistically differences were observed between CP and CP + SR treatments in mutant ChLNs (Fig. 5B). These observations were confirmed by fluorescent microscopy (Fig. 5C–K).

CP55940 reduced the phosphorylation of tau protein independent of CB1Rs in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with SR, CP, or SR + CP in regular culture medium for 4 days. After this time, the proteins in the extracts were blotted with primary antibodies against phosphorylated tau (p-Tau), total tau (t-Tau), and actin proteins. The intensities of the western blot bands shown in (A) were measured (B) by an infrared imaging system (Odyssey, LI-COR), and the p-Tau/t-Tau ratio was normalized to that of actin. Additionally, cells were double-stained as indicated in the figure (C-J) with primary antibodies against p-Tau (green; C’-J’) and t-Tau (red; C”-J”). The nuclei were stained with Hoechst 33342 (blue; C”’-J”’). K) Quantification of the p-Tau/t-Tau fluorescence ratio. 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, 200×. — Fig. 5CP55940 reduced the phosphorylation of tau protein independent of CB₁Rs in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with SR, CP, or SR + CP in regular culture medium for 4 days. After this time, the proteins in the extracts were blotted with primary antibodies against phosphorylated tau (p-Tau), total tau (t-Tau), and actin proteins. The intensities of the western blot bands shown in (A) were measured (B) by an infrared imaging system (Odyssey, LI-COR), and the p-Tau/t-Tau ratio was normalized to that of actin. Additionally, cells were double-stained as indicated in the figure (C-J) with primary antibodies against p-Tau (*green*; C’-J’) and t-Tau (*red*; C”-J”). The nuclei were stained with Hoechst 33342 (*blue*; C”’-J”’). K) Quantification of the p-Tau/t-Tau fluorescence ratio. 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, 200×.

CP55940 reduces the levels of eAβ₄₂ protein fragment independent of CB₁Rs in PSEN 1 E280A ChLNs

Based on the observation that CP inhibited the intracellular aggregation of sAβPPβf (see Fig. 3), we assessed whether CP could have the same effect on eAβ₄₂ [16]. Effectively, CP and CP + SR but not SR only significantly diminished the amounts of eAβ₄₂ compared to untreated neurons in mutant ChLNs according to the ELISA technique (Fig. 6). No statistically differences were observed between CP and CP + SR treatments in mutant ChLNs (Fig. 6). The amount of eAβ₄₂ was not affected by any of the treatments in WT ChLNs (Fig. 6).

CP55940 reduced the levels of extracellular Aβ42 peptide in PSEN1 E280A ChLNs by a CB1R-independent mechanism. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with SR, CP, or SR + CP in RCm for 4 days. ELISA quantification of extracellular Aβ42 peptide in supernatants. Data are presented as means±SD. *p < 0.05; **p < 0.01; ***p < 0.001. The histograms and figures represent 1 out of 4 independent experiments. — Fig. 6CP55940 reduced the levels of extracellular Aβ₄₂ peptide in PSEN1 E280A ChLNs by a CB₁R-independent mechanism. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with SR, CP, or SR + CP in RCm for 4 days. ELISA quantification of extracellular Aβ₄₂ peptide in supernatants. Data are presented as means±SD. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. The histograms and figures represent 1 out of 4 independent experiments.

CP55940 does not recover Ca²⁺ dysregulation in PSEN1 E280A ChLNs

The above observations prompted us to test whether CP could ameliorate the physiological response of mutant ChLNs to ACh neurotransmitter stimuli. As expected, ACh induced a transient elevation of intracellular Ca²⁺ in WT ChLNs (control, the average maximum fluorescence change (ΔF/F) was 3.95±0.19 at 10 s (n = 20 ChLN cells imaged, N = 3 dishes were used as a standard condition in this and following experiments) according to cytoplasmic Ca²⁺ responses to Fluo-3-mediated imaging (Fig. 7A, E). We found no statistical differences between SR (Fig. 7B, E, ΔF/F = 5.05±0.35), SR + CP (Fig. 7D, E; ΔF/F = 4.31±0.26) and untreated conditions (Fig. 7E). In contrast, CP only and ACh significantly depressed intracellular Ca²⁺ (by –68%) in WT ChLNs (Fig. 7C, E, ΔF/F = 2.35±0.12). When mutant neurons were exposed to ACh only (Fig. 7F, J, ΔF/F = 0.02±0.00) and CP (Fig. 7H, J, ΔF/F = 0.06±0.00), mutant cells were irresponsive to both stimuli (Fig. 7J). Interestingly, while SR only slightly increased intracellular Ca²⁺ (Fig. 7G, J, ΔF/F = 0.63±0.07), the CP + SR cocktail and ACh increased inflow Ca²⁺ (Fig. 7H, J, ΔF/F = 3.32±0.23) in PSEN 1 E280A ChLNs to similar extend as [Ca²⁺]i in WT ChLNs (Fig. 7E, J).

CP55940 does not recover Ca2+ dysregulation in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with SR, CP, or SR + CP in regular culture medium for 4 days. A-D, F-I) Time-lapse images (0, 10, 20, 30, 40, 50, and 60 s) of Ca2+ fluorescence in WT PSEN1 and PSEN1 E280A ChLNs on day 4 in response to ACh treatment. ACh was puffed into the culture at 0 s (arrow). Then, the Ca2+ fluorescence of the cells was monitored at the indicated times. Color contrast indicates fluorescence intensity: dark blue < light blue < green<yellow<red. E, J) Normalized mean fluorescence signal (ΔF/F) over time from the cells indicating temporal cytoplasmic Ca2+ elevation in response to ACh treatment. Data are presented as the mean±SD. *p < 0.05; **p < 0.01; ***p < 0.001 compared to UNT. The histograms and figures represent 1 out of 3 independent experiments. Image magnification, 200×. — Fig. 7CP55940 does not recover Ca²⁺ dysregulation in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were left untreated or treated with SR, CP, or SR + CP in regular culture medium for 4 days. A-D, F-I) Time-lapse images (0, 10, 20, 30, 40, 50, and 60 s) of Ca²⁺ fluorescence in WT PSEN1 and PSEN1 E280A ChLNs on day 4 in response to ACh treatment. ACh was puffed into the culture at 0 s (*arrow*). Then, the Ca²⁺ fluorescence of the cells was monitored at the indicated times. Color contrast indicates fluorescence intensity: dark blue < light blue < green<yellow<red. E, J) Normalized mean fluorescence signal (ΔF/F) over time from the cells indicating temporal cytoplasmic Ca²⁺ elevation in response to ACh treatment. Data are presented as the mean±SD. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 compared to UNT. The histograms and figures represent 1 out of 3 independent experiments. Image magnification, 200×.

Anti-Aβ₄₂ antibodies completely recuperate Ca²⁺ influx in PSEN1 E280A ChLNs

Since PSEN1 E280A ChLNs response to ACh decline in a time-dependent fashion [16], and since SR alone treatment was innocuous for WT and E280A ChLNs, we evaluated whether early inhibition of eAβ₄₂ by anti-Aβ antibodies could ameliorate the mutant ChLNs response to ACh under CP and CP + SR treatments. As shown in Fig. 8, incubation of WT with anti-Aβ antibody 6E10 only (Fig. 8A, D; ΔF/F = 4.27±0.21) or in the presence of CP (Fig. 8B, D; ΔF/F = 3.11±0.25) or CP + SR (Fig. 8C, D; ΔF/F = 4.01±0.20) showed similar transient intracellular Ca²⁺. We found no statistical differences between those treatments (Fig. 8D). Remarkably, ACh and the anti-Aβ antibody 6E10 completely recovered Ca²⁺ influx in mutant ChLNs (Fig. 8E, H; ΔF/F = 3.37±0.20). The anti-Aβ antibody 6E10 and CP (Fig. 8F, H; ΔF/F = 5.78±0.87) and antibody + CP + SR (Fig. 8G, H; ΔF/F = 5.93±0.29) dramatically induced a transient elevation of intracellular Ca²⁺ in mutant ChLNs (Fig. 8H).

CP55940 plus SR and anti-Aβ42 antibody 6E10 completely recover Ca2+ influx in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were incubated with 6E10 antibody as described in Materials and Methods section. Simultaneously cells were left untreated or treated with CP or SR + CP in RCm for 4 days. A-C, E-G) Time-lapse images (0, 10, 20, 30, 40, 50, and 60 s) of Ca2+ fluorescence in WT PSEN1 and PSEN1 E280A ChLNs at day4 in the presence of 6E10 antibody as a response to ACh treatment. ACh was puffed into the culture at 0 s (arrow). Then, the Ca2+ fluorescence of the cells was monitored at the indicated times. Color contrast indicates fluorescence intensity: dark blue < light blue < green<yellow<red. D, H) Normalized mean fluorescence signal (ΔF/F) over time from the cells indicating temporal cytoplasmic Ca2+ elevation in response to ACh treatment. Data are presented as means±SD. *p < 0.05; **p < 0.01; ***p < 0.001 compared to UNT. The histograms and figures represent 1 out of 3 independent experiments. Image magnification, 200×. — Fig. 8CP55940 plus SR and anti-Aβ₄₂ antibody 6E10 completely recover Ca²⁺ influx in PSEN1 E280A ChLNs. After 7 days of transdifferentiation, WT PSEN1 and PSEN1 E280A ChLNs were incubated with 6E10 antibody as described in Materials and Methods section. Simultaneously cells were left untreated or treated with CP or SR + CP in RCm for 4 days. A-C, E-G) Time-lapse images (0, 10, 20, 30, 40, 50, and 60 s) of Ca²⁺ fluorescence in WT PSEN1 and PSEN1 E280A ChLNs at day4 in the presence of 6E10 antibody as a response to ACh treatment. ACh was puffed into the culture at 0 s (arrow). Then, the Ca²⁺ fluorescence of the cells was monitored at the indicated times. Color contrast indicates fluorescence intensity: dark blue < light blue < green<yellow<red. D, H) Normalized mean fluorescence signal (ΔF/F) over time from the cells indicating temporal cytoplasmic Ca²⁺ elevation in response to ACh treatment. Data are presented as means±SD. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 compared to UNT. The histograms and figures represent 1 out of 3 independent experiments. Image magnification, 200×.

DISCUSSION

In the present investigation, we demonstrate that CP inhibits the aggregation of (i)sAβPPβf, and p-Tau, increases ΔΨ_m, inhibits the generation of ROS and oxidation of DJ-1Cys¹⁰⁶-SH to DJ-1Cys¹⁰⁶-SO₃, blocks the activation of the transcription factor p53 and c-Jun, pro-apoptotic protein PUMA, and protease caspase-3—all markers of cell death by apoptosis, and reduce eAβ₄₂ in a CB₁R-dependent and-independent fashion in PSEN1 E280A ChLNs, used as a neuronal cell model of FAD [16]. Although CP was unable to reverse the Ca²⁺ influx dysregulation as a response to ACh stimuli, it is demonstrated that CP affects several cellular molecular targets. Several observations support these assumptions. First, in agreement with previous work [27], we found that CP reduced OS by directly scavenging ROS (e.g., H₂O₂) [22–28] and partially diminished ROS through CB₁Rs signaling. These observations may indicate that CP operates in a dual-way: as an antioxidant and as a CB₁Rs ligand. Second, CP dramatically increases the ΔΨ_m in PSEN1 E280A ChLNs. Several data indicate that CP and other cannabinoids (e.g., THC, arachidonyl-2-chloroethylamide) can protect mitochondria against OS stimuli by inhibition of mitochondria permeability transition pore (mPTP) opening [37] or through activation of mitochondrial (mt)CB₁Rs [38, 39]. Even though, mtCB₁Rs activation have been linked to an intra-mitochondrial signaling cascade involving lessening of the activity of the electron transport system, oxygen consumption, and ATP production [40], our data favor the view that inhibition of mPTP opening by CP might be responsible for neuroprotective action on mutant ChLNs [37]. Indeed, it has been shown that the CB₂ agonist JWH-015 attenuated mitochondrial damage against OS stimuli by scavenging anion superoxide radical (O₂^. -) and H₂O₂, maintaining ΔΨ_m and avoiding Ca²⁺-induced mitochondrial swelling [37]. Additionally, CP may indirectly protect mitochondria by blocking the interaction of sAβPPβf and mitochondrial respiratory complexes [41, 42] or by inhibition of mitochondria-targeted pro-apoptotic proteins (e.g., PUMA, this work). Taken together our findings suggest that CP reverses the loss of ΔΨ_m and mitochondria damage independently of plasma membrane CB₁ receptors or (mt)CB₁Rs. Third, it is known that cannabinoids (e.g., THC) stimulate the removal of intracellular Aβ through a dependent [43] or independent CB₁Rs mechanism [22]. Here, we report for the first time that CP inhibits the aggregation of (i)sAβPPβf in PSEN1 E280A ChLNs independently of CB₁ receptor signaling. Since Aβ₄₂ is still detected extracellularly in mutant neurons treated with CP, it might imply that this cannabinoid does not interfere with the AβPP metabolism. Finally, CP significantly diminished oxidation of protein DJ-1, as evidence of OS. Because DJ-1Cys¹⁰⁶SH residue is specifically sensible to H₂O₂ oxidation [44], it is likely that CP might directly interact with ROS or induces its suppression via CB₁Rs activation. Taken together our results comply with the view that early aggregation of (i)sAβPPβf and generation of H₂O₂ can be blocked by the direct action of cannabinoid CP and by partial activation of CB₁Rs, respectively.

Recent data have shown that (i)sAβPPβf (/H₂O₂) induce activation of JNK/c-Jun and p53, which in turn activate PUMA and caspase-3 [16]. Interestingly, we found that CP blocks activation of transcription factors c-Jun, p53, PUMA, and caspase-3 independently of CB₁Rs stimulation. These observations suggest that CP exerted a direct role in protecting PSEN1 E280A ChLNs against endogenously OS-induced cell death signaling. Since downregulation of c-Jun and p53 may reduce transcription of PUMA [45–47], CP may restrain mitochondria from leaking pro-apoptogenic proteins such as caspase-9, and ensuing activation of caspase-3. These actions avoid neuronal dismantling and increase the survival of mutant ChLNs.

Previously, it has been shown that (i)sAβPPβf induces p-Tau through JNK kinase in PSEN 1 E280A ChLNs [16]. In this work, we demonstrate that CP almost completely inhibits the phosphorylation of protein tau independently of CB₁Rs. One possible explanation is that CP can suppress H₂O₂ generation, which serves as a messenger molecule that triggers activation of JNK [48, 49], and phosphorylation of tau [50]. It is therefore not surprising that p-Tau was untraceable in PSEN1 E280A ChLNs. However, we do not discard the possibility that CP blocks p-Tau by alternative CB₁Rs-independent mechanisms in ChLNs [51].

Mounting evidence has demonstrated that eAβ is neurotoxic before the appearance of plaques [16, 52, 53]. Interestingly, CP reduced the amounts of eAβ₄₂ in a CB₁Rs-independent manner. This observation suggests that CP directly modulates intracellular and extracellular AβPP-derived protein fragments. However, the action of CP on eAβ₄₂ did not impact on the response of mutant ChLNs to ACh stimuli. Here, we confirm that ACh receptors (AChRs) are physiologically altered in PSEN 1 E280A ChLNs [16] most probably through Aβ-induced inhibition of nicotinic (n)AChRs [54, 55], which normally revealed a rapid elevation of intracellular Ca²⁺ [56–58] as shown in WT ChLNs. Indeed, AChRs were irresponsive to ACh stimuli, thereby nullifying the transient rise of intracellular Ca²⁺ in mutant ChLNs (i.e., –99% reduction in [Ca² ⁺]_i) compared to wild type ChLNs. On the other hand, CP decreased [Ca² ⁺]_i by –68% in WT ChLNs. These observations suggest that CP either directly inhibited the function of AChRs [59, 60] and/ or inhibited the inflow of Ca²⁺ via activation of CB₁Rs on calcium channels (e.g., T, N- and P/Q-type [20]). Since the response of PSEN 1 E280A ChLNs to ACh is already compromised, CP was not able to modify the influx of Ca²⁺ when challenged with the neurotransmitter. Interestingly, SR only or with CP increased [Ca² ⁺]_i in PSEN 1 E280A ChLNs compared to untreated mutant ChLNs. This positive improvement of Ca²⁺ inflow was comparable to WT ChLNs (see CP + SR effect on neurons). These observations can be explained by the fact that SR can either stimulate Ca²⁺ influx through Ca²⁺ channels (e.g., Ca_v2: N-, P/Q channels [61]) or block the negative interaction of CP with nAChRs in WT ChLNs when exposed to ACh (this work). Our findings suggest that CB₁Rs are marginally active in PSEN 1 E280A ChLNs. Interestingly; anti-Aβ₄₂ antibody completely recovered transient intracellular Ca²⁺ signal in mutant ChLNs. The Ca²⁺ signal was further increased when neurons were co-cultured with CP only or SR plus CP. These last observations reinforce the view that Aβ₄₂ and SR interfere with CP and AChRs. Similar to PSEN 1 E280A ChLNs, antibody anti-Aβ₄₂ increased transient intracellular Ca²⁺ signals in WT ChLNs. These observations imply that eAβ₄₂ secreted in low amounts by WT ChLNs or abnormally secreted in high amounts by mutant ChLNs is an AChRs regulator in ChLNs. Taken together these observations suggest that a combination of CP with anti-Aβ₄₂ would be therapeutically beneficial to FAD patients.

Conclusion

In an attempt to modify the neuropathological expression of ChLNs bearing the mutation PSEN1 E280A (e.g., intracellular aggregation of sAβPPβf, tau phosphorylation, OS, apoptosis, and Ca²⁺ influx dysfunction) (Fig. 9A), we found that cannabinoid CP is a multi-target molecule that promotes the recovery FAD cholinergic neurons by increasing ΔΨ_m, impedes phosphorylation of protein tau, and blocks OS-induced apoptosis signaling (Fig. 9B). However, CP was unable to modify the neuronal functionality as evidenced by an inability of ACh to increase Ca²⁺ influx in PSEN1 E280A ChLNs. Since the nature of the neuronal cholinergic dysfunctionality stems from the blocking of nAChRs by Aβ₄₂ [54, 55], drugs that interfere with the interaction of Aβ and the AChRs might ameliorate the ligand-gated ion Ca²⁺ channel interactions (e.g., [62]). In this respect, we show that anti-Aβ₄₂ antibody 6E10 plus CP or anti-Aβ₄₂ and SR + CP change mutant ChLNs response to ACh by increasing Ca²⁺ influx to similar [Ca²⁺]_i in WT ChLNs (Fig. 9C, D). It is therefore proposed that combinations of cannabinoids, anti-Aβ₄₂ antibodies (e.g., crenezumab [63, 64]), and CB₁ inverse agonists might be a promising multi-target drugs for therapy [65] in the early treatment of FAD PSEN 1 E280A ChLNs neurodegeneration.

Schematic representation of the protective effect of CP55940 on PSEN 1 E280A ChLNs. A) Intracellularly accumulation of sAβPPβf (step 1) generates H2O2 (s2), which in turn oxidized the OS sensor protein DJ-1 at Cys-106-SH residue into DJ-1Cys106-SO3 (s3) and activates a domino-like pro-death signaling mechanism by triggering kinases (s4, e.g., ASK1) and JNK kinase (s5). This last kinase phosphorylates protein tau (s6) and activates the transcription factors c-Jun (s7) and p53 (s8). Both transcription factors transcribe the BH-3-only protein PUMA (s9), which together with other pro-apoptosis proteins (e.g., BAX), it induces depolarization of mitochondria, opening of the mitochondrial transition pore, and loss of mitochondrial membrane potential (ΔΨm, s10). Damage of mitochondria potential provokes the release of pro-apoptogenic protein cytochrome C, ensuing activation of caspase-3 (s11). In turn, this protease activates the endonuclease DFF40 by cleaving the nuclease’s inhibitor DFF45. Finally, DFF40 causes the nuclear chromatin fragmentation, typical of apoptosis (s12). Additionally, ChLNs do not respond to ACh stimuli, i.e., intracellular transient Ca2+ increase is missing due to extracellular interaction between Aβ42 (eAβ42) and nicotinic (n)ACh receptors (s13). The sAβPPβf-induced process (s1-s13) leads ChL neurons to structural alterations (e.g., aggregation/ hyperphosphorylation of protein tau), cell death (apoptosis) and intracellular Ca2+ dysfunction. B) Upon exposure to CP55940 (CP), ChLNs show normal features such as no oxidized protein DJ-1 (s3), unaltered ΔΨm (s10), and intact nuclei morphology (s12). Moreover, CP inhibits aggregation of sAβPPβf (s1), and blocks generation of H2O2 (s2) most probably by quenchinganion superoxide radicals (O2.―). As a consequence, no further activation of kinases (s4, s5), phosphorylation of protein tau (s6), and c-Jun(s7), and activation of p53 (s8), PUMA (s9), and caspase-3 (s11) occurs in PSEN1 E280A ChLNs just leaving mitochondria and nuclei unchanged from sAβPPβf-induced damage. C) Since nAChRs are blocked by eAβ42 (s13), CP was unable to modify the response of mutant ChLNs to ACh (inset graph i). Interestingly, the CB1 and CB2 receptor inverse agonists SR141716 plus SR144528 ( = SR) only or in the presence of CP induce a significant increase in intracellular Ca2+ (s14) most probably through activation of Ca2+ channels (s15 and inset graph i). D) When mutant ChLNs were exposed to CP, SR, and anti-Aβ42 antibody 6E10 (s16), the mixture induced a dramatic increase in the transient intracellular Ca2+ flow (s13, s17 and inset graph ii). — Fig. 9Schematic representation of the protective effect of CP55940 on PSEN 1 E280A ChLNs. A) Intracellularly accumulation of sAβPPβf (step 1) generates H₂O₂ (s2), which in turn oxidized the OS sensor protein DJ-1 at Cys-¹⁰⁶-SH residue into DJ-1Cys¹⁰⁶-SO₃ (s3) and activates a domino-like pro-death signaling mechanism by triggering kinases (s4, e.g., ASK1) and JNK kinase (s5). This last kinase phosphorylates protein tau (s6) and activates the transcription factors c-Jun (s7) and p53 (s8). Both transcription factors transcribe the BH-3-only protein PUMA (s9), which together with other pro-apoptosis proteins (e.g., BAX), it induces depolarization of mitochondria, opening of the mitochondrial transition pore, and loss of mitochondrial membrane potential (ΔΨ_m, s10). Damage of mitochondria potential provokes the release of pro-apoptogenic protein cytochrome C, ensuing activation of caspase-3 (s11). In turn, this protease activates the endonuclease DFF40 by cleaving the nuclease’s inhibitor DFF45. Finally, DFF40 causes the nuclear chromatin fragmentation, typical of apoptosis (s12). Additionally, ChLNs do not respond to ACh stimuli, i.e., intracellular transient Ca²⁺ increase is missing due to extracellular interaction between Aβ₄₂ (eAβ₄₂) and nicotinic (n)ACh receptors (s13). The sAβPPβf-induced process (s1-s13) leads ChL neurons to structural alterations (e.g., aggregation/ hyperphosphorylation of protein tau), cell death (apoptosis) and intracellular Ca²⁺ dysfunction. B) Upon exposure to CP55940 (CP), ChLNs show normal features such as no oxidized protein DJ-1 (s3), unaltered ΔΨ_m (s10), and intact nuclei morphology (s12). Moreover, CP inhibits aggregation of sAβPPβf (s1), and blocks generation of H₂O₂ (s2) most probably by quenchinganion superoxide radicals (O².―). As a consequence, no further activation of kinases (s4, s5), phosphorylation of protein tau (s6), and c-Jun(s7), and activation of p53 (s8), PUMA (s9), and caspase-3 (s11) occurs in PSEN1 E280A ChLNs just leaving mitochondria and nuclei unchanged from sAβPPβf-induced damage. C) Since nAChRs are blocked by eAβ42 (s13), CP was unable to modify the response of mutant ChLNs to ACh (inset graph i). Interestingly, the CB₁ and CB₂ receptor inverse agonists SR141716 plus SR144528 ( = SR) only or in the presence of CP induce a significant increase in intracellular Ca²⁺ (s14) most probably through activation of Ca²⁺ channels (s15 and inset graph i). D) When mutant ChLNs were exposed to CP, SR, and anti-Aβ42 antibody 6E10 (s16), the mixture induced a dramatic increase in the transient intracellular Ca²⁺ flow (s13, s17 and inset graph ii).

ACKNOWLEDGMENTS

This study was funded by MinCiencias grant # 1115-844-67062, contract #830-2019 to CV-P, MJ-Del-Rio, and MM-P. VS-M is a doctoral student of the Basic Biomedical Sciences Academic Corporation program at the Universidad de Antioquia (UdeA) funded by the “2019 Bicentennial Doctoral Excellence Scholarship”, MinCiencias-Colombia.

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/20-1045r2).

REFERENCES

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Studies

Conditions

Cannabinoids and active compounds

Related research

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Untitled section

Article notes

Untitled section

Author notes

INTRODUCTION

MATERIALS AND METHODS

Cholinergic-like neuron (ChLN) differentiation

Immunofluorescence analysis

Western blot analysis

Analysis of cells

Assay protocol

Evaluation of intracellular hydrogen peroxide (H2O2) by fluorescence microscopy

Analysis of mitochondrial membrane potential (ΔΨm) by fluorescence microscopy

Measurement of Aβ42 peptide in culture medium

Intracellular calcium imaging

Photomicrography and image analysis

Data analysis

RESULTS

CP55940 restores the mitochondrial membrane potential (ΔΨm) and blunts generation of ROS in PSEN1 E280A ChLNs in an independent concentration fashion

CP55940 restores ΔΨm in a CB1 receptor-independent manner but diminishes ROS production partially dependent on CB1 receptors in PSEN1 E280A ChLNs

CP55940 reduces intracellular sAβPPβf aggregation in a CB1Rs-independent fashion but it reduces oxidized DJ-1 partially dependent of CB1Rs in PSEN1 E280A ChLNs

CP55940 blocks apoptosis in a CB1Rs-independent manner in PSEN1 E280A ChLNs

CP55940 inhibits tau phosphorylation in a CB1Rs-independent fashion PSEN1 E280A ChLNs

CP55940 reduces the levels of eAβ42 protein fragment independent of CB1Rs in PSEN 1 E280A ChLNs

CP55940 does not recover Ca2+ dysregulation in PSEN1 E280A ChLNs

Anti-Aβ42 antibodies completely recuperate Ca2+ influx in PSEN1 E280A ChLNs

DISCUSSION

Conclusion

ACKNOWLEDGMENTS

REFERENCES

Untitled section

References

Additional materials

Evaluation of intracellular hydrogen peroxide (H₂O₂) by fluorescence microscopy

Measurement of Aβ₄₂ peptide in culture medium

CP55940 restores the mitochondrial membrane potential (ΔΨ_m) and blunts generation of ROS in PSEN1 E280A ChLNs in an independent concentration fashion

CP55940 restores ΔΨ_m in a CB₁ receptor-independent manner but diminishes ROS production partially dependent on CB₁ receptors in PSEN1 E280A ChLNs

CP55940 reduces intracellular sAβPPβf aggregation in a CB₁Rs-independent fashion but it reduces oxidized DJ-1 partially dependent of CB₁Rs in PSEN1 E280A ChLNs

CP55940 blocks apoptosis in a CB₁Rs-independent manner in PSEN1 E280A ChLNs

CP55940 inhibits tau phosphorylation in a CB₁Rs-independent fashion PSEN1 E280A ChLNs

CP55940 reduces the levels of eAβ₄₂ protein fragment independent of CB₁Rs in PSEN 1 E280A ChLNs

CP55940 does not recover Ca²⁺ dysregulation in PSEN1 E280A ChLNs

Anti-Aβ₄₂ antibodies completely recuperate Ca²⁺ influx in PSEN1 E280A ChLNs