Neuroprotective Effect of Combined Treatment with Epigallocatechin 3-Gallate and Melatonin on Familial Alzheimer’s Disease PSEN1 E280A Cerebral Spheroids Derived from Menstrual Mesenchymal Stromal Cells

Isolation and characterization of MenSCs derived from human MensB

The MensB samples were collected from one healthy female and one female carrier of the mutation PSEN1 E280A aged 30 years (Tissue Bank Code, TBC # 69308) and 25 years (TBC # 04335), respectively. Donors signed an informed consent accepted by the Ethics Committee of the Sede de Investigación Universitaria -SIU-, University of Antioquia, Medellín, Colombia. The specimens (MensB) were collected, purified, and maintained as previously described in [20]. The isolated MenSCs were used for the generation of cerebral spheroids [20].

Ethical Approval Menstrual specimen donors provided a signed informed consent approved by the ethics committee of the Sede de Investigación Universitaria (SIU), University of Antioquia, Medellín, Colombia (Act 2020-10854).

Generation of cerebral spheroids derived from MensB-MenSCs and induced to cholinergic-like neuronal cell lineage

CSs were obtained by differentiation of WT or PSEN1 E280A MenSCs as previously described in [20]. Briefly, WT and mutant MenSCs were cultured in a brand-new medium called Fast-N-Spheres V2 composed of serum-free culture medium containing reagents as described in [20] plus Matrigel^® and 1% fetal bovine serum. Cultures were constantly shacked for 4 and 11 days. The MenSCs-derived CSs were examined for the expression of neuronal and astrocyte markers according to [20].

Immunofluorescence and fluorescence microscopy analysis

The WT or PSEN1 E280A CSs cultured with Fast-N-Spheres V2 medium for 4 and 11 days without or with EGCG (25μM) and/or melatonin (50μM) were stained with primary antibodies against the glial fibrillary acidic protein (GFAP, cat. no. sc6170, Santa Cruz), β-tubulin III (β-TUB III cat. no. G712A, Promega), choline acetyltransferase (ChAT, cat. no. AB144P, Millipore) primary antibodies (1:500); primary antibodies against APP751 and/or protein amyloid β1–42 (1:500; clone 6E10 cat# 803014, Biolegend), oxidized DJ-1 (1:500; ox(Cys¹⁰⁶)DJ-1; spanning residue Cys¹⁰⁶ of human PARK7/DJ1; oxidized to produce cysteine sulfonic (SO₃) acid; cat # ab169520, Abcam), and total tau (1:500; t-Tau; cat# T6402, Sigma), and phospho-tau (p-tau, 1:500, Ser²⁰²/Thr²⁰⁵, cat# MN1020 (AT8), Thermo Fisher Scientific) antibodies. Incubation with secondary fluorescent antibodies and nuclei staining were performed according to [20]. Fluorescent Thioflavin-T derivative BTA-1 staining was performed by incubating CSs slides with a BTA-1 (Sigma) solution (500 nM) for 30 min. Nuclei were identified by incubating CSs with propidium iodide (1μM). Postmortem temporal cortical slices from an Alzheimer’s disease patient (case #3396) and a healthy individual (case #3648) were used as a positive and negative control, respectively [31]. Imaging processing data was performed according to an online guidance program (https://imagej.nih.gov/ij/).

Flow cytometry analysis

Flow cytometry analysis was performed according to [32] with minor modifications. Briefly, the WT or PSEN1 E280A CSs cultured with Fast-N-Spheres V2 medium for 11 days without or with EGCG (25μM) and/or melatonin (50μM) were detached with Accutase Cell Detachment Solution (Beckton Dickinson) and washed in PBS. Cells (10×10⁵) were resuspended in PBS and fixated with cold ethanol overnight and used for further flow cytometry analysis. Cell markers were analyzed by incubating cells with primary antibodies against caspase-3 (1:250; cat # AB3623, Millipore), APP751 and/or protein amyloid-β1-42 (1:500; clone 6E10 cat# 803014, Biolegend), oxidized DJ-1 (1:500; ox(Cys¹⁰⁶)DJ-1; spanning residue Cys¹⁰⁶ of human PARK7/DJ1; oxidized to produce cysteine sulfonic (SO₃) acid; cat # ab169520, Abcam), and total tau (1:500; t-Tau; cat# T6402, Sigma), and phospho-tau (p-tau, 1:500, Ser²⁰²/Thr²⁰⁵, cat# MN1020 (AT8), Thermo Fisher Scientific) antibodies, followed incubation with secondary fluorescent antibodies according to [23]. Resuspended cells were analyzed using BD LSR Fortessa II flow cytometer (BD Biosciences). Ten thousand events were acquired, and the acquisition analysis was performed using FlowJo 7.6.2 Data Analysis Software.

Cytochemical detection of DNA fragmentation

DNA fragmentation was determined in fixed untreated or treated CSs to polylysine coated slides by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique according to the manufacturer’s protocol (Promega catalog #G7130). Briefly, CSs samples were treated for 10 min with 25μg/ml protease K and fixed in 4% methanol in PBS for 5 min. CSs slides were pre-incubated with equilibration buffer (EB) (50μl, 200 mM potassium cacodylate, pH 6.5; 25 mM Tris-HCl pH 6.6; 0.2 mM DTT; 0.25 mg/ml BSA; 2.5 mM cobalt chloride) for 15 min. Then, slides were incubated at 37°C for 60 min with 25μl TdT incubation buffer (23μl EB; 2μl nucleotide mix; 0.5μl TdT enzyme). After the appropriate incubation time, tailing reaction was terminated by immersing the slides in 2X SSC for 15 min and rinsing twice with PBS. Then after, slides were incubated with streptavidin HRP (diluted 1:500 in PBS) for 30 min at room temperature. Finally, slides were incubated with DAB solution (prepared immediately prior to use by adding 100μl DAB 10X Chromogen to 900μl DAB Substrate 1X Buffer). Slides were stained with safranine for 10 s at room temperature and washed twice in deionized water. Immediately, slides were coverslipped using aqueous mounting media.

Oxygen radical absorbance capacity (ORAC) assay

The hydrophilic ORAC assay was performed as described in [33]. The AAPH (2,2’-Azobis(2-amidinopropane) dihydrochloride) was used as a peroxyl radical generator, Trolox was used as a standard, and fluorescein was used as the fluorescent probe. Briefly, fluorescein, AAPH, and the samples were prepared in a phosphate buffer (75 mM, pH 7.4). First, 25μL of sample solution or Trolox standard was mixed with 150μL of fluorescein (1 mM) and pre-incubated at 37°C for 30 min before the addition of 25μL of AAPH solution (200 mM). The fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 520 nm was measured every 2 min for 120 min using a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Inc.; Winooski, USA). The relative ORAC values were calculated using the differences of areas under the decay curves and were expressed as μmol Trolox equivalents (TE)/g of solution.

Intracellular calcium imaging

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was measured according to Pap et al. [34]. Fluorescence microscopy photographs were taken using a Zeiss Axiostart 50 Fluorescence Microscope equipped with a Zeiss AxioCam Cm1 (Zeiss Wöhlk-Contact-Linsfluoreen, Gmb Schcönkirchen, Germany), and imaging processing data was performed according to online guidance program (http://imagej.nih.gov/ij/) [35].

Data analysis

This experimental design was performed as previously reported in [20] and based on the statistics considerations described in [36]. Provided that the experimental unit (i.e., the well) data fulfill 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 Student’s t-test, one-way, or two-way ANOVA followed by Bonferroni’s or 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.

PSEN1 E280A CSs spontaneously express cholinergic and astrocytic markers

Previous work has shown that WT CSs derived from MensB spontaneously express neuronal markers such as β-TUB III and ChAT, as well as the astrocyte marker GFAP, when cultured in Fast-N-Spheres medium for 3 days [20]. Likewise, WT and PSEN1 E280A CSs showed similar expression of neuronal (β-TUB III, Fig. 1A–D), cholinergic (ChAT, Fig. 1E–H), and astrocytic (GFAP, Fig. 1I–L) lineage cell markers when grown in Fast-N-Spheres V2 medium for 4 (Fig. 1M) or 11 days (Fig. 1N).

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Fig. 1

MenSCs-derived CSs show cholinergic and astrocytic marker. After 4 and 11 days, WT PSEN1 and PSEN1 E280A CSs were triple-stained as indicated in the figure with primary antibodies against β-TUB III (blue; A–D), ChAT (green; E–H), and GFAP (red; I–L). M) Mean fluorescence intensity (MFI) quantification of images obtained by immunofluorescence analysis on day 4. N) MFI quantification of images obtained by immunofluorescence analysis on day 11. Images represent one out of three independent experiments. Statistical significance was determined by one-way ANOVA with a Tukey post hoc test; ^**p < 0.01. Image magnification 10x.

PSEN1 E280A CSs show an early increase in iAβPPf and OS

Next, we wanted to determine whether CSs express the typical neuropathological features of AD, such as Aβ and OS. Figure 2 shows that WT NS displayed neither iAβPPf accumulation (Fig. 2A, C) nor OS markers (Fig. 2E, G), as reflected by the absence of iAβPPf (Fig. 2M, N), oxidized protein DJ-1 (Fig. 2M, N) and nuclear-positive cells (Fig. 2I–L). In contrast, PSEN1 E280A CSs displayed enhanced iAβPPf (Fig. 2B, D, M, N) and oxidation of protein DJ-1 (Fig. 2F, H, M, N) as early as 4 days in culture (Fig. 2M, N).

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Fig. 2

PSEN 1 E280A cerebral spheroids (CSs) accumulate intracellular AβPP fragments (iAβPPf) and oxidized (Cys¹⁰⁶) DJ-1 protein (Ox-DJ-1). After 4 and 11 days, WT PSEN1 and PSEN1 E280A CSs were double-stained as indicated in the figure with primary antibodies against (i)AβPPf (red; A–D) and Ox-DJ-1 (green; E–H). Nuclei were stained with Hoechst (blue; I–L). M) Mean fluorescence intensity (MFI) quantification of images obtained by immunofluorescence analysis on day 4. N) MFI quantification of images obtained by immunofluorescence analysis on day 11. Images represent one out of three independent experiments. Statistical significance was determined by one-way ANOVA with a Tukey post hoc test; ^*p < 0.05; ^**p < 0.01. Image magnification 10x.

PSEN1 E280A CSs present hyperphosphorylation of tau protein

It is well established that phosphorylation of tau occurs downstream of Aβ pathology in AD [37, 38]. Therefore, we assessed whether tau is phosphorylated in a time-dependent manner in mutant CSs. As shown in Fig. 3, no p-tau was observed in WT CSs (Fig. 3A, C) on Days 4 or 11, whereas p-tau was detected in PSEN1 E280A CSs only on Day 11 (Fig. 3B versus Fig. 3D and 3M versus 3N). Total tau was measured and used for protein normalization (Fig. 3E–H), and Hoechst staining was used to analyze the organoid cellularity (Fig. 3I–L).

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Fig. 3

PSEN1 E280A cerebral spheroids (CSs) show hyperphosphorylation of protein tau. After 4 and 11 days, WT PSEN1 and PSEN1 E280A CSs were double-stained as indicated in the figure with primary antibodies against phosphorylated (Ser²⁰²/Thr²⁰⁵) Tau (p-Tau) (red; A–D) and total Tau (t-Tau) (green; E–H). Nuclei were stained with Hoechst (blue; I–L). M) Mean fluorescence intensity (MFI) quantification of images obtained by immunofluorescence analysis on day 4. N) MFI quantification of images obtained by immunofluorescence analysis on day 11. Images represent one out of three independent experiments. Statistical significance was determined by one-way ANOVA with a Tukey post hoc test; ^**p < 0.01. Image magnification 10x.

PSEN1 E280A CSs show diminished ΔΨm, caspase 3 activity, and fragmented nuclei

We then wondered whether PSEN 1 E280A CSs show endogenous alterations in ΔΨm, active CASP-3 and nuclear fragmentation similar to those demonstrated in 2D PSEN 1 E280A human umbilical cord Wharton Jelly mesenchymal stromal cell-derived ChLNs [22]. Figure 4 shows that WT CSs displayed high ΔΨm, inactive CASP3 and intact nuclei (Fig. 4A, E). However, PSEN1 E280A CSs displayed typical features of apoptotic cells, including failure of ΔΨm, CASP3-positive signals, and broken nuclei (Fig. 4F, J). Similar observations were obtained by flow cytometry (Fig. 4K, L) and the TUNEL technique (Fig. 4M, Q, R, W).

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Fig. 4

Combined treatment with EGCG and aMT reestablish mitochondria membrane potential (ΔΨm), inactivate CASPASE-3 activation, and block nuclei fragmentation in PSEN1 E280A (CSs). The WT PSEN1 and PSEN1 E280A CSs were left without (UNT; A, F) or treated with 25μM EGCG (B, G) and 50μM aMT (C, H) alone or in combination (D, I) until day 11. CSs were triple stained as indicated in the figure with MitoTrackertrademark Red FM (red), 1X Vybrant TM FLICA (green), and Hoechst (blue). E) Mean fluorescence intensity (MFI) quantification of images obtained by immunofluorescence analysis of PSEN1 WT CSs. J) MFI quantification of images obtained by immunofluorescence analysis of PSEN1 E280A CSs. K, L) representative histograms of WT PSEN1 (K) and PSEN1 E280A CSs (L) treated with none (UNT) or with EGCG, aMT, and EGCG/aMT. M-P, R-V) The WT PSEN1 and PSEN1 E280A CSs were left without (UNT; M, R) or treated with EGCG (N, S), aMT (O, T), or in combination (P, V) for 11 days. Then, untreated or treated CS were TUNEL stained. Only untreated PSEN1 E280A CSs (R) show TUNEL-positive cells. Q, W) TUNEL reactivity quantification of images obtained by TUNEL technique analysis after 11 days. Histograms and images represent one out of three independent experiments. Statistical significance was determined by one-way ANOVA with a Tukey post hoc test; ^*p < 0.05; ^***p < 0.001. Image magnification 10x.

Combined treatment with EGCG and aMT increases ΔΨ_m and drastically reduces caspase 3 activation in PSEN1 E280A CSs

Previous work has shown that EGCG diminishes amyloid-induced cytotoxic effects on 2D PSEN1 E280A ChLNs derived from hUC-WJ MSCs [22] and that the hormone aMT acts as a neuroprotectant [30, 39]. Therefore, we determined the effect of polyphenols and hormones on mitochondrial functionality in CSs. As shown in Fig. 4, EGCG only (Fig. 4B), aMT only (Fig. 4C), or a combination of the two (Fig. 4D) induced neither alteration in the ΔΨ_m nor activation of the executer protein CASP3 in WT CSs (Fig. 4E) compared to untreated CSs (Fig. 4A, E). Remarkably, treatment with EGCG only (Fig. 4G), aMT only (Fig. 4H), or a combination of the two (Fig. 4I) significantly increased ΔΨ compared to untreated PSEN1 E280A CSs (Fig. 4F), and these treatments were equally efficient in reducing CASP3 activation in mutant CSs (Fig. 4E versus 4J). Similar results were obtained by flow cytometry (Fig. 4K, L) and the TUNEL technique (Fig. 4N–Q, S–W).

Combined treatment with EGCG and aMT dramatically diminishes iAβPPf and OS levels in PSEN1 E280A CSs

Based on the above observations, we were prompted to assess whether EGCG and aMT alone or in combination can reverse the neuropathologic features of FAD in mutant CSs. To determine this, WT CSs and PSEN1 E280A CSs were left untreated (used as control) or incubated with EGCG at 25μM, a pharmacological concentration of aMT (50μM) or a combination of EGCG and aMT. As illustrated in Fig. 5, EGCG, aMT, and EGCG/aMT were innocuous to WT CSs (Fig. 5A versus Fig. 5B–E). However, aMT (Fig. 5H, J) was significantly more effective than ECGC alone in preventing the accumulation of iAβPPf in PSEN1 E280A CSs (Fig. 5G, J), and treatment with a combination of the two reduced the level of iAβPPf in mutant CSs (Fig. 5I, J) to nearly control values, i.e., those found in WT CSs. In contrast, EGCG was much more effective than aMT in reducing the oxidation of the sensor protein DJ-1 (Fig. 5F versus Fig. 5G and 5J). The combination of the two drugs was even more effective than EGCG alone in reducing OS in mutant CSs (Fig. 5F versus Fig. 5I and J). Flow cytometry analysis yielded comparable results (Fig. 5K–T).

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Fig. 5

Combined treatment with EGCG and aMT reduce iAβPPf and OS in PSEN1 E280A CSs. The WT PSEN1 and PSEN1 E280A CSs were left without (UNT; A, F) or treated with 25μM EGCG (B, G) and 50μM aMT (C, H) alone or in combination (D, I) for 11 days. CSs were double-stained as indicated in the figure with antibodies against sAβPPf (red) and Ox-DJ-1 (green), and nuclei were stained with Hoechst (blue). E) Mean fluorescence intensity (MFI) quantification of images obtained by immunofluorescence analysis of PSEN1 WT CSs. J) MFI quantification of images obtained by immunofluorescence analysis of PSEN1 E280A CSs. K-N, P-S) Representative 2D density plot showing iAβPPf (y-axis) and ox-DJ-1 (x-axis) flow cytometry double analysis performed on untreated WT (K) and mutant CSs (P), or treated with EGCG only (L, Q), aMT only (M, R), and EGCG/ aMT (N, S). O, T) represent the quantitative analysis of the data from quadrant Q1 + Q2 for iAβPPf and Q2 + Q4 for ox-DJ-1. U-V) represent BTA-1 negative staining of a temporal cortex sample from a healthy individual (case #3648) and BTA-1 positive staining of a temporal cortex sample from a PSEN1 E280A individual (case #3396). Arrowheads show Aβ plaques, and asterisks show Aβ fibrils. W-AD) The WT PSEN1 and PSEN1 E280A CSs were left without (UNT; W, AA) or treated with EGCG (X, AB) and aMT (Y, AC) alone or in combination (Z, AD) for 11 days. Then, CSs were double stained with BTA-1 and propidium iodide as indicated in the figure. BTA-1 negative staining was observed in untreated or treated CSs. Except for Fig. 5U and 5V, 2D histograms and images represent one out of three independent experiments. Statistical significance was determined by one-way ANOVA with a Tukey post hoc test; ^***p < 0.001. Image magnification 10x.

To further confirm that the thwarting effect of the molecules was specifically on iAβPP and not on extracellular Aβ aggregates, we stained WT and mutant CSs with thioflavin BTA-1, a specific fluorescent thioflavin-T derivative that has high affinity for amyloid deposits and specifically labels β-amyloid aggregates. BTA-1 displays 50-fold higher affinity for Aβ aggregates than does thioflavin-T, selectively stains Aβ fibrils in postmortem brain tissue obtained from AD patients and crosses the blood-brain barrier. We also stained postmortem temporal cortical samples obtained from individuals with normal and E280A mutant PSEN1 as internal negative and positive controls, respectively. As expected, the mutant E280A temporal postmortem sample displayed typical amyloid plaques (Fig. 5V, arrowheads) and Aβ fibrils (Fig. 5V, asterisks), but such plaques were completely absent from the WT temporal postmortem sample (Fig. 5U). Interestingly, untreated or treated WT or mutant CSs showed neither BTA-1-positive staining for amyloid plaques nor Aβ fibrils (Fig. 5W-AD).

Simultaneously, we wanted to determine whether the reduction in DJ-1 oxidation via H₂O₂ activation might be due to antioxidant activity of EGCG and aMT when incubated with CSs. The ORAC assay (also known as the hydrogen atom transfer (HAT)-based assay) showed that EGCG, aMT and combined EGCG/aMT displayed high antioxidant activity, yielding values of 37,870.75±671.43μmol Trolox equivalents (TE)/g, 55,822.86±585.18μmol TE/g, and 130,894.34±2,874.21μmol TE/g, respectively. The mixture showed 3.35-fold and 2.34-fold increases in ORAC values, respectively.

Combined treatment with EGCG and aMT completely blocks phosphorylation of tau protein

We further evaluated whether exposure of CSs to polyphenols, hormones, or a combination of the two is able to decrease p-tau. As shown in Fig. 6, compared to untreated CSs (Fig. 6A, E), no p-tau was observed in WT CSs treated with EGCG (Fig. 6B, E), aMT (Fig. 6C, E), or EGCG/aMT (Fig. 6D, E). When mutant CSs (Fig. 6F) were exposed to these agents, both EGCG alone (Fig. 6G, J) and aMT alone (Fig. 6H, J) significantly diminished p-tau compared to the level found in untreated mutant CSs (Fig. 6J). Interestingly, treatment with combined EGCG/aMT completely eliminated p-tau (Fig. 6I, J). Flow cytometry yielded similar results (Fig. 6K–T).

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Fig. 6

Combined treatment with EGCG and aMT completely blocked the phosphorylation of protein tau. WT PSEN1 and PSEN1 E280A CSs were left without (UNT; A, B) or treated with 25μM EGCG (C, D) and 50μM aMT (E, F) alone or in combination (G, H) for 11 days. CSs were double-stained as indicated in the figure with antibodies against phosphorylated (Ser²⁰²/Thr²⁰⁵) Tau (p-Tau; red), total Tau (t-Tau; green), and nuclei were stained with Hoechst (blue). I) Mean fluorescence intensity (MFI) quantification of images obtained by immunofluorescence analysis of PSEN1 WT CSs. J) MFI quantification of images obtained by immunofluorescence analysis of PSEN1 E280A CSs. K-N, P-S) Representative 2D density plot showing t-tau (y-axis) and p-tau (x-axis) flow cytometry double analysis performed on untreated WT (K) and mutant CSs (P), or treated with EGCG only (L, Q), aMT only (M, R), or EGCG/ aMT (N, S). O, T) represent the quantitative analysis of the data from quadrant Q2. The 2D histograms and images represent one out of three independent experiments. Statistical significance was determined by one-way ANOVA with a Tukey post hoc test; ^**p < 0.01; ^***p < 0.001. Image magnification 10x.

When applied together with EGCG, aMT limits the beneficial effect of EGCG on Ca²⁺ influx in PSEN1 E280A CSs

Finally, we evaluated the effects of the two compounds on Ca²⁺ functionality in CSs exposed to acetylcholine (ACh). To this end, WT and mutant PSEN1 E280A CSs were exposed to ACh at 1 mM final concentration. As shown in Fig. 7, ACh stimulated a temporary increase in intracellular Ca²⁺ in WT CSs (Fig. 7A, ΔF/F = 0.165±0.06, mean duration = 10 s; n = 9 CSs imaged, N = 3 dishes Fig. 7E). Ca²⁺ influx into WT CSs after ACh addition was altered by EGCG (Fig. 7B), aMT (Fig. 7C), and by a combination of EGCG and aMT (Fig. 7D, E). In contrast, ACh did not affect intracellular Ca²⁺ in mutant PSEN1 CSs (Fig. 7F, ΔF/F = 0.00±0.02, mean duration = 10 s; n = 9 CSs imaged, N = 3 dishes, Fig. 7J). While EGCG dramatically restored Ca²⁺ influx in mutant CSs (Fig. 7G, ΔF/F = 0.33±0.01, mean duration = 10; n = 9 CSs imaged, N = 3 dishes, Fig. 7J), aMT did not restore Ca²⁺ influx in the mutant CSs (Fig. 7H, J), in contrast to its effect on untreated mutant CSs (Fig. 7F, J). Interestingly, administration of ECGG and aMT together decreased ACh-induced Ca²⁺ influx in PSEN1 E280A CSs (Fig. 7I; ΔF/F = 0.15±0.02, mean duration = 10 s; n = 9 CSs imaged, N = 3 dishes, Fig. 7J).

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Fig. 7

aMT lessens the effect of EGCG on Ca²⁺ influx in PSEN1 E280a CSs. The WT PSEN1 and PSEN1 E280A CSs were left without (UNT; A, F) or treated with 25μM EGCG (B, G) and 50μM aMT (C, H) alone or in combination (D, I) until day 11. Time-lapse images (0, 10, 20, 30, and 40 s) of Ca²⁺ fluorescence in CSs (n = 9 CSs imaged, N = 3 dishes) as a response to ACh treatment. ACh was puffed into the culture at 0 s (arrow). Then, the Ca²⁺ fluorescence of cells was monitored at indicated times. Color contrast indicates fluorescence intensity: dark blue < light blue < green < yellow < red. E) Normalized mean fluorescence signal (ΔF/ F) over time, indicating temporal cytoplasmic Ca²⁺ elevation in response to ACh treatment in PSEN1 WT CSs. J) Normalized mean fluorescence signal (ΔF/ F) over time, indicating temporal cytoplasmic Ca²⁺ elevation in response to ACh treatment in PSEN1 E280A CSs. Statistical significance was determined by two-way ANOVA with a Tukey post hoc test; ^**p < 0.01. Image magnification 10x.

Conclusion

Familial AD, most cases of which are caused by mutations in PSEN1, e.g., PSEN1 E280A, is a complex neurodegenerative malady in which progressive neuronal deterioration occurs, leading to loss of memory. It is now accepted that intracellular Aβ, rather than extracellular Aβ aggregation, is the likely cause of neuronal cell death. We have shown that early generation and accumulation of iAβPPf are responsible for initiating cellular events that lead to ROS (H₂O₂) production, hyperphosphorylation of tau, activation of proapoptotic proteins (e.g., CASP 3), alterations in Ca²⁺ influx, and apoptosis in ChLNs [22] and in CSs (this work). It is, therefore, reasonable to assume that drugs designed to eliminate Aβ-induced signaling cell death targets/or pathological signals may have cooperative (additive or synergistic) effects. Consistent with this view, combined use of EGCG and aMT has proven to be highly useful in almost completely reducing intracellular AβPPf, OS, p-tau, loss of ΔΨ_m, and apoptotic markers in PSEN1 E280A CSs. However, aMT and EGCG in combination were ineffective at restoring Ca²⁺ influx in mutant CSs because of their antagonistic action. Therefore, our present observations provide a cautionary note regarding the use of aMT to restore physiological functions associated with Ca²⁺ influx. However, this limitation might be overcome by delivering EGCG (e.g., [72]) and aMT (e.g., [73]) nanoparticles to specific brain neurons (e.g., cholinergic or pyramidal) that can be directly loaded with the cargo (EGCG and aMT) intracellularly. Further investigation is needed to validate the effectiveness of using combined EGCG and aMT in the early treatment of FAD patients carrying the PSEN1 E280A mutation.