Combinatorial Treatment Effects in a Cell Culture Model of Alzheimer’s Disease

Abstract

Background: Alzheimer’s disease (AD) is the leading cause of dementia, and as its prevalence increases, so does its detrimental impact on society. The currently available therapies have limited efficacy, leaving AD patients on an irrevocably fatal path of this disease.

Objective: The purpose of this study was to test efficacy of a novel combinatorial treatment approach to alleviate AD-like pathology.

Methods: We selected four naturally occurring compounds and used them in different combinations to test their effect on AD-like pathology. Employing a well-established cell culture AD model system, we evaluated levels of several diverse biomarkers associated with a number of cellular pathways associated with AD. The readouts included: amyloid-β peptides, anti-inflammatory and anti-apoptotic proteins, oxidative enzymes, and reactive oxygen species.

Results: Using this approach, we demonstrated that the compounds delivered in combination had higher efficacy than individual treatments. Specifically, we observed significant reduction in levels of the amyloid-β peptides, as well as pro-inflammatory proteins and reactive oxygen species. Similarly, delivery of compounds in combination resulted in an increased expression of anti-apoptotic proteins and anti-oxidative enzymes. Collectively, these modifications in AD pathology biomarkers reflect a promising therapeutic and preventive strategy to combat this disease.

Conclusion: The above findings support a novel therapeutic approach to address a currently unmet medical need, which would benefit not only AD patients and their caregivers, but also society as a whole.

Keywords

Alzheimer’s disease combinatorial treatment (–)-epigallocatechin-3-gallate melatonin multi-target N2aneuroblastoma cells resveratrol vitamin B12

INTRODUCTION

There is currently no cure for the estimated five million Americans living with Alzheimer’s disease (AD) [1], and these numbers are predicted to increase rapidly in the coming years. The FDA-approved treatments are few, and they are beset by limited efficacy and significant side effects [2]. The challenge of developing an effective therapy stems from the fact that AD has a complex, multi-faceted pathology that involves a number of cellular pathways, and includes overproduction of the amyloid-β (Aβ) peptide leading to aggregation and formation of toxic Aβ species, inflammation, oxidative stress, and tau protein abnormalities [3 –6]. As each of these events can be a target for therapeutic and/or preventive treatments, one of the strategies is to use a set of compounds that collectively address various aspects of the AD pathology.

To that effect, we selected four naturally occurring compounds that have been scientifically shown to diminish progression of AD in humans and/or in animal models [7 –9], with the prediction that their concerted action would translate into efficacy exceeding that of individual components. The combinations included: vitamin B12 (B), resveratrol (R), (–)-epigallocatechin-3-gallate, or EGCG (E), and melatonin (M). Previous studies have shown that vitamin B12, resveratrol, EGCG, and melatonin individually diminish the severity of a spectrum of AD pathologies [8 , 11], as they target cellular processes and pathways essential to the progression of AD. Briefly: (1) vitamin B12 has been shown to modify the self-association pathway of the Aβ peptide resulting in reduction of toxicity, decrease production of the Aβ peptide, and impede abnormal phosphorylation of tau and formation of neurofibrillary tangles [12]; (2) resveratrol has been shown to function through anti-oxidative pathways and processes leading to diminished production of toxic Aβ species [13 –15]; (3) EGCG has been demonstrated to reduce Aβ levels and modulate aggregation/toxicity of the Aβ peptide [16 –21]; and (4) melatonin has been implied in anti-inflammatory/anti-oxidative processes and processing of tau, in addition to its ability to directly diminish formation of toxic amyloid species [22 –29].

To determine efficacy of the combinatorial approach, we treated N2a murine neuroblastoma cells engineered to overproduce the human Aβ peptide (N2a/APP_swe) [30], and characterized their response using a number of biomarkers associated with AD. This well-established cell culture model of AD, used successfully in similar studies by others [28 , 32], enabled us to compare the combinatorial approach efficacy to reduce the AD pathology relative to single compound delivery. The four compounds were tested individually (B, R, E, and M), in combinations of three (BRE, BRM, and REM), or all four (BREM). As readouts, we used previously established “signature” markers associated with processes linked to AD progression, which encompass amyloid production, apoptosis, inflammation, and oxidation [33 –40]. Specifically, the readouts included: (1) Aβ peptide levels (Aβ_1 - 40 and Aβ_1 - 42); (2) cell survival-promoting proteins (BCL-2 and BCL-XL); (3) amyloid-β protein precursor (AβPP) processing enzymes (BACE1 and ADAM10); (4) pro-inflammatory proteins (IL-6 and TGF-β1); (5) anti-oxidative proteins (GPX-1, CAT, and SOD1); and (6) reactive oxygen species (ROS).

MATERIALS AND METHODS

Cell culture

Mouse neuroblastoma (N2a) cells transfected with a human gene containing AD Swedish mutations (N2a/APP_swe) [30] were a gift from Dr. Sangram Sisodia (University of Chicago, USA). They were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin/streptomycin (Gibco), and 0.2 mg/ml G418, and kept in a hydrated, 5% CO₂ environment at 37°C. Cells were passaged every 3-4 days, at approximately 80% confluence and kept between 2 and 10 passages for all experiments. Cells were detached with 0.25% Trypsin-EDTA (Gibco) and briefly centrifuged, at 200 g, prior to sub-culturing. Immediately prior to compound treatment the growth medium was removed from the culture dish and the cells were gently washed in 1X phosphate buffered saline (PBS, Gibco). The PBS was aspirated and vehicle or compound-containing DMEM was added to the cells. This ensured that there was no pre-existing Aβ in the medium and any subsequent differences were detected without bias.

Compound solubilization

Each of the three compounds: resveratrol, melatonin, and EGCG were stored at –20°C and vitamin B12 was stored at 4°C in a desiccator. All compounds were prepared fresh immediately prior to use. Resveratrol, melatonin, and EGCG (Cayman Chemical Company; purity ≥ 98%) were dissolved in 100% ethanol to a stock concentration of 100 mM. Vitamin B12 (Sigma-Aldrich; purity ≥ 98%) was dissolved in supplemented DMEM to a stock concentration of 10 mM. Each compound was subsequently diluted with supplemented DMEM to 10 μM (resveratrol), 30 μM (melatonin), 20 μM (EGCG), and 40 μM (vitamin B12), as these were shown in preliminary studies to represent optimal concentrations. The control (Con) contained 0.06% ethanol diluted in DMEM.

Immunoblotting

N2a/APP_swe cells were sub-cultured into 6 cm² dishes (Nunc), approximately 2.2×10⁶ cells/dish. 48 h later cells were treated with the compounds, as indicated, placed at 37°C, 5% CO₂, and harvested 12 h later. In brief, cells were placed on ice and washed once with ice-cold PBS and harvested in ice-cold PBS, followed by centrifugation for 5 min at 1500 g at 4°C. The PBS was removed and cell pellet immediately frozen on dry ice. Pellets were homogenized with a Pellet Pestle (Kontes) in extraction buffer [41]: 120 mM NaCl, 20 mM HEPES pH 7.4, 0.1% Triton X-100, 3 mM Sodium β-glycerophosphate, 1 mM dithiothreitol (DTT), 1 protease tablet (Thermo Scientific), and 1 mM phenylmethanesulfonyl fluoride (PMSF) added prior to use. Homogenized pellets were centrifuged for 5 min at 12,000 g at 4°C. The supernatant was removed and added to 2X sample buffer: 100 mM Tris pH 6.8, 4% SDS, 20% glycerol, 5% mercaptoethanol, 5 mM EDTA pH 8, 0.1 mg/ml bromophenol blue, and boiled at 95°C for 3 min. The preparations were briefly centrifuged, and loaded onto an SDS PAGE gel.

Gels were run at 180 V for 1 h and transferred to nitrocellulose membrane using a semi-dry transfer method (BioRad), at 23 V for 26 min. Membranes were blocked with Odyssey blocking buffer (LI-COR) at room temperature for 30 min prior to antibody incubation. The membranes were incubated with the primary antibody for 2 h at room temperature, with shaking. Primary antibodies: Anti-Catalase (CAT), Sigma (mouse); anti-glutathione peroxidase I (GPX1), Thermo Fisher (rabbit); anti-superoxide dismutase (SOD1), (rabbit); anti-BCL2, Cell Signaling (50E3, rabbit); anti-BCL-XL, Cell Signaling (54H6, rabbit); anti-ADAM10, Millipore (rabbit); anti-actin, Sigma (A2066, rabbit). Each membrane was washed in Tris buffered saline 0.1% Tween-20 (TBST 3×5 min). The respective secondary antibody was added for 1 h at room temperature, with shaking: Goat anti-rabbit IRDye 800CW, or donkey anti-mouse IRDye 680LT (LI-COR). Membranes were washed in TBST 5×5 min, and imaged using the Odyssey infra-red imaging system (LI-COR). Quantification was performed using the Odyssey application software version 3.0 (LI-COR).

Quantification of reactive oxygen species (ROS)

N2a/APP_swe cells were sub-cultured, as indicated, into a clear-bottom black-coated 96-well plate (Nunc), at approximately 1.25×10⁴ cells/well. 48 h later cells were treated with the compounds in supplemented DMEM, as indicated, and placed at 37°C, 5% CO₂. 12 h later the medium was removed and replaced with phenol red-free DMEM without any supplements, but including 25 μM 2^′,7^′-Dichlorodihydrofluorescein diacetate (ATT Bioquest). The cells were returned to 37°C, 5% CO_2, for 30 min then washed once with PBS and fresh supplement free, phenol-red free DMEM was added. Cells were immediately analyzed for fluorescence emission: excitation 485 nm and emission528 [28].

ELISAs

N2a/APP_swe cells were sub-cultured into 24-well plates (Nunc) as previously described at a density of approximately 5×10⁴ cells/well. 48 h later cells were treated with the compounds, as indicated, and placed at 37°C, 5% CO₂. 12 h later the medium was removed, aliquoted, and placed immediately on dry ice, and stored at –80°C until use.

For the determination of secreted Aβ_1 - 40 and Aβ_1 - 42, ELISA kits (Invitrogen, Cat # KHB3482 and KHB3442, respectively) were used, with the assays carried out according to the manufacturers’ instructions, but using supplemented DMEM for sample and standard dilutions. The plates were immediately assayed for absorbance at 450 nm.

IL-6 and TGF-β1, in the culture medium, were assayed according the manufacturer’s instructions, using mouse ELISA kits (Affymetrix/eBiosciences, Cat # 88-7064-22 and 88-7324-22, respectively). The final chromogenic reaction was stopped with 1 M H₃PO₄. The plates were immediately assayed for absorbance at 450 nm.

Statistical analysis

Data were normalized to the control group in each data set. Individual data points in each experiment were divided by the control group average and multiplied by 100, including the control group. This allowed for a normalized average of the data±standard error of the mean (SEM), including the control group. Statistical data analyses were performed using a Kruskal-Wallis test, followed by Dunn’s multiple comparison test. p-values <0.05 were considered significant. Data are presented as mean±standard error of the mean (SEM). GraphPad Prism^® software was used for all analyses.

RESULTS

Amyloid-β peptide, or Aβ_1 - 42, was used as the initial readout in order to determine the optimum experimental conditions, namely duration of treatment and concentration of the four compounds. Aβ_1 - 42 was chosen as it is regarded as one of the main determinants in AD neurological degeneration [35, 36] and has been shown to interact with, or diminish in levels by, the compounds used in this study [9 , 42]. We assayed melatonin and vitamin B12 treatments over three time points, 6 h, 12 h, and 24 h, and using three concentrations, which were based on effective concentrations reported in the literature [43, 44] and Supplementary Figure 1. We selected a 12 h incubation, as well as 30 μM melatonin and 40 μM vitamin B12 for further investigation, as these conditions resulted in the greatest reduction in Aβ_1 - 42 expression (Supplementary Figure 1B). The 6 h and 24 h melatonin and vitamin B12 treatments did not result in an appreciable change in Aβ_1 - 42 (Supplementary Figure 1A,C). To determine the optimal dose of EGCG and resveratrol, we used the incubation time of 12 h, and used three different concentrations (Supplementary Figure 2). The greatest reduction with EGCG was with 20 μM, and this value was selected for subsequent study (Supplementary Figure 2A). The optimal concentration of resveratrol was determined to be 10 μM, as it produced the greatest reduction in Aβ_1 - 42 released into the medium (Supplementary Figure 2B). None of the EGCG or resveratrol doses resulted in a significant reduction in Aβ_1 - 42.

Given the optimal incubation time and concentration for each compound, we treated the N2a/APP_swe cells with individual compounds and in combinations of three, or all four. In each of the combinations tested, there was a significant reduction in Aβ_1 - 40 in the medium, by approximately 20%, as well as with resveratrol alone (Fig. 1A). However, with the exception of EMB, only the compounds in combination had a significant effect on lowering levels of Aβ_1 - 42, which again resulted in a 20% reduction (Fig. 1B).

To further evaluate the combinatorial approach, we determined the effect of the four compounds on enzymes associated with suppression of oxidative stress: CAT, SOD1, and GPX-1. In each case, the individual compounds had no effect on enzyme expression, yet there was a significant increase when the compounds were used in combination (Fig. 2). Catalase levels were significantly increased with REM, RMB, and REB combinations, approximately 1.5-fold, over the control (Fig. 2A). Likewise, SOD1 and GPX-1 expression was significantly increased, by approximately 1.5- and 2-fold, respectively (Fig. 2B, C). In addition to anti-oxidative enzymes, a broad spectrum of ROS was measured using the fluorescent dye 2^′,7^′-dichlorodihydrofluorescein diacetate [28]. ROS, such as superoxide and hydrogen peroxide, are by-products of normal aerobic metabolism. At low levels they are utilized as cell signaling molecules, however higher ROS concentrations are detrimental [45]. Each combination treatment resulted in reducing ROS levels by almost 20%, whereas the individual compounds had no significant effect (Fig. 2D).

Previous reports have suggested that the progression of AD is associated with a change in anti-apoptotic enzymes [46, 47]. Therefore, we treated N2a/APP_swe cells with the compounds in combination and tested for their effect on expression of BCL-2 and BCL-XL, both anti-apoptotic enzymes (Fig. 3). Though there was no significant change in expression with the individual compounds with either enzyme, there was a trend toward induction. In combination, REB and RMB resulted in a small, yet significant, increase in BCL-2, whereas RMB increased BCL-XL expression approximately 2-fold over baseline (Fig. 3A and B, respectively). Similarly, we looked at the expression of two AβPP-processing enzymes, BACE1 and mature-ADAM10 (Supplementary Figure 3). After incubation with the compounds in combination, there was no difference in the expression of either enzyme. There was a slight inhibition of BACE1 expression with EGCG and EMB, but not significant. Of note, mature-ADAM10 expression appeared to increase with all treatment conditions, although none was significant (Supplementary Figure 3B).

The release of pro-inflammatory cytokines IL-6 and TGF-β1, into the extracellular medium after combination drug treatment, was characterized. Combinatorial treatment produced a significant reduction in IL-6 expression, by 30–40%, with EMB and BREM (Fig. 4A). Only one individual compound (EGCG) significantly inhibited IL-6 release, by approximately 30% (Fig. 4A). Vitamin B12, REB, and EMB inhibited TGF-β1 by approximately 35%, with no effect by RMB or REM (Fig. 4B). The combination of all four compounds produced the greatest inhibition, over 40% (Fig. 4B).

The above results are summarized in Table 1. The numbers indicate statistical p-values for each compound, individually and in combination. Each read-out is grouped according to its role in AD pathology, as well as literature-based expected outcomes with treatment.

DISCUSSION

AD is characterized by progressive memory loss and cognitive dysfunction, eventually leading to complete dependence for care and an inevitable death [35]. The World Alzheimer’s Disease report from 2015 estimates that 47 million people are currently living with dementia worldwide, with the cost of $818 billion [48], and these numbers are climbing at an alarming rate as our society ages. Even so, there is still no cure and the emerging therapies are few [49, 50]. Therefore, the primary purpose of our work was to ascertain the effectiveness of a combinatorial approach to reducing AD biomarkers. We used four naturally occurring compounds (vitamin B12, resveratrol, EGCG, and melatonin), previously shown to beneficially modulate molecular pathways implicated in AD, including amyloid production, apoptosis, inflammation, and oxidation, as described below.

Vitamin B12 has long been recognized as an important player in maintenance of cognitive health [7 , 51–53]. A 2-year study with patients identified as having mild cognitive impairment, B vitamin supplements (B12, B6, and folate) reduced brain atrophy in regions associated with cognitive decline and AD pathology [8]. Vitamin B12, B6, and folate are involved in homocysteine (Hcy) metabolism that has been implicated in AD progression [54], though increased Hcy levels are not observed in all AD patients [55]. Furthermore, a lack of B vitamins was found to promote tau phosphorylation, which is associated with AD progression, through upregulation of GSK3β and downregulation of protein phosphatase 2A [12]. In the cell culture system used in this study, tau/phospho-tau produced unreliable results, thus was not included in the biomarker analysis (Bienkiewicz and Olcese, unpublished data). For the purpose of our studies, we chose vitamin B12 as the most appropriate compound to use, as there is some evidence that long-term folate supplements are deleterious to health [56], and long-term exposure to vitamin B6 can lead to peripheral neuropathy [57].

Resveratrol, or 3,4^′,5-trihydroxystilbene, is a polyphenol found in many foods. Though isolated more than 60 years ago, only recently have its potential benefits become apparent. Resveratrol’s effects are thought to be wide-ranging, having been described as anti-cancer, anti-inflammatory, cardio-protective, and effective in diabetic neuropathy pain management [58]. Furthermore, resveratrol is hypothesized to function through anti-oxidative pathways leading to diminished production of toxic Aβ species [59]. To date there are a number of ongoing clinical trials regarding resveratrol and AD (ClinicalTrials.gov), yet with results to bedetermined.

Polyphenols are natural substances present in food and beverages obtained from plants, fruits and vegetables, including olive oil, red wine, and tea. The polyphenol (–)-epigallocatechin-3-gallate, or EGCG, is highly abundant in green tea, and the active compound attributed to its anticancer, antioxidant and anti-inflammatory properties [60]. Recent studies have provided direct evidence that EGCG modulates Aβ fibril formation through binding to the unfolded polypeptide, and prevents the formation of amyloid oligomers and protofibrils [61, 62]. Further, EGCG provides a neuroprotective role through the chelation of metal ions (iron, zinc, and copper), which are associated with toxic Aβ species [63] and the inhibition of oxidative stress [60].

Melatonin, or N-acetyl-5-methoxytryptamine, is synthesized in the pineal gland from its tryptophan precursor. It has been implicated in a range ofphysiological functions [64, 65] and has been associated with anti-inflammatory/anti-oxidative processes and tau processing [66]. Melatonin production is under circadian control and is synthesized primarily at night. Circadian dysregulation is common amongst AD patients [67], and a decline in melatonin levels often parallels the neurological progression of AD [68, 69]. In a recent study, exposure of the rat pineal to the Aβ peptide induced a significant inflammatory response [70]. This resulted in decreased melatonin production and decreased binding to its MT₁ and MT₂ receptors [70]. In addition, melatonin has been shown to directly diminish the formation of toxic amyloid species [9, 22].

The above descriptions of vitamin B12, resveratrol, EGCG, and melatonin reflect a large body of scientific evidence demonstrating importance of these compounds in various aspects of AD pathology. The exact mechanisms involved have not been fully elucidated, but it is quite certain that, even with the same “end point” (for instance, reduced levels of the Aβ peptide), the cellular processes through which this is accomplished may be either shared or distinct for each compound. For instance, reduction of toxic Aβ peptide levels has been shown to involve direct binding of vitamin B12 (Bienkiewicz, unpublished data), resveratrol, EGCG, or melatonin to diverse Aβ forms, utilizing different epitopes, resulting in protective outcomes [16–18 , 62]. An additional mechanism to diminish Aβ peptide levels involves modification of the AβPP processing via regulation of β-secretase (BACE1), glycogen synthase kinase-3 (GSK-3), protein kinase C (PKC), and disintegrin and metalloproteinase domain-containing protein 10 and 17 (ADAM10 and ADAM17), as shown for vitamin B12, resveratrol, EGCG, and melatonin [12 , 71–74]. When tested in our system, BACE1 levels did not significantly change, and mature-ADAM10 levels showed an upward trend indicating reduced production of amyloidogenic Aβ for compounds tested either individually or in combination (Supplementary Figure 3). Our combinatorial approach, however, did result in significantly decreased levels of both Aβ_1 - 40 and Aβ_1 - 42, as shown in Fig. 1. Of particular significance is the reduction of the latter, as Aβ_1 - 42 self-associates more readily and is considered to be more toxic [35, 36]. REM and EMB significantly reduced levels of Aβ_1 - 40, and four out of five combinations tested (REM, REB, RMB, and BREM) reduced levels of Aβ_1 - 42, demonstrating increased efficacy relative to individualtreatments.

Another complex and key factor in AD’s etiology is inflammation [4 , 75]. Previous reports linked vitamin B12, resveratrol, EGCG, and melatonin to reduced neuroinflammation, as reflected by levels of a number of cytokines, including interleukin 6 (IL-6), transforming growth factor (TGF-β1), TNFα, and IL-1β [27 , 76–82]. In this study, we probed levels of IL-6 and TGF-β1, as they have been linked specifically to progression of AD [75 , 83–87]. Significant reduction in levels of IL-6 was observed for resveratrol, EMB, and BREM, whereas TGF-β1 levels decreased significantly with vitamin B12, REB, EMB, and BREM (Fig. 4). Thus, the highest impact on reducing inflammation in our AD model system, as reflected by IL-6 and TGF-β1 levels, was achieved with the use of all four compounds delivered jointly.

Similarly to the processes described above, the anti-oxidative effects evoked by vitamin B12, resveratrol, EGCG, and melatonin engage multiple mechanisms. All four compounds function as free radical scavengers and/or modulators of the levels of anti-oxidative enzymes, including SOD, GPX, and CAT [88], in addition to their ability to regulate inflammatory cytokines that contribute to oxidative stress [89]. Vitamin B12, in its reduced state, is almost as effective at scavenging superoxide radicals as superoxide dismutase [90]. Likewise, melatonin and resveratrol are effective at reducing ROS production [28, 91], and EGCG is thought to reduce cellular ROS through its ability to chelate iron [92]. Previous in vitro studies have yielded mixed outcomes when compounds were used in combination. For example, melatonin tested with high doses of resveratrol or EGCG, was either antagonistic or protective, respectively [93]. In our cell culture AD model, the four compounds in combination effected a significant decrease in the ROS levels (Fig. 2D), and an increase in anti-oxidative enzymes (Fig. 2A-C), demonstrating potentiated anti-oxidative efficacy of vitamin B12, resveratrol, EGCG, and melatonin when delivered in combination.

Previous studies employing cell culture models of AD have identified apoptosis as a characteristic feature of progression of AD. One of the commonly probed apoptotic biomarkers in the context of AD are B-cell lymphoma proteins (BCL-2 and BCL-XL) [94 –96]. When tested in our system, the levels of these anti-apoptotic markers showed an upward trend for all treatments, but a significant rise in levels of BCL-2 and BCL-XL was only observed with the combinatorial approach (Fig. 3).

Collectively, our findings demonstrate the advantage of using vitamin B12, resveratrol, EGCG, and melatonin in combination as a strategy to mitigate deleterious effects of the multi-factorial cellular assaults that characterize AD pathology. It is also apparent that no single compound is effective across the range of biomarkers tested on its own. Joint delivery then would conceivably potentiate the efficacy of treatment, not merely by duplicating the actions of another compound, but by concertedly targeting multiple cellular targets to achieve overall increase in efficacy. Although the “cocktail” approach to disease treatment is often beneficial, this was not obvious in the case of the four compounds used here. Outcome predictions and direct comparisons with findings by others are often hampered by diversity in experimental design and model systems used. These variables include diverse cell lines and transgenic mouse strains, acute versus long-term treatments, and measurements of protein or mRNA levels that do not always correspond. Also, the compounds tested do not always evoke the same effect. For instance, in contrast to our findings, resveratrol and EGCG elicit a pro-apoptotic response in cancer therapeutics [97, 98], and EGCG and melatonin can be antagonistic when delivered in combination [93]. One also needs to acknowledge that this study describes an acute treatment, with compounds delivered for 12 h, within a cell culture system. As such, it does not recapitulate the chronic nature of the AD. It does, however, provide a valuable model system to determine the validity of potential compounds that could be used in the chronic AD treatment to deliver a positive therapeutic response.

In summary, this study demonstrates that a combinatorial treatment delivers a multi-targeted and more comprehensive coverage, thus increasing the overall treatment efficacy and alleviating the key aspects of the AD pathology.

CONCLUSIONS

The findings of this study provide evidence for a novel approach toward the AD treatment and prevention. We used four naturally occurring compounds with known efficacy in modulating a number of signature AD biomarkers, and characterized their combinatorial effect on key aspects of AD progression: Aβ levels, inflammatory cytokines, cell survival proteins, oxidative enzymes expression, and oxidative species production. Our results indicate that vitamin B12, resveratrol, EGCG, and melatonin used in combination are more efficacious than individual treatments at alleviating AD pathology. Thus, this cell culture study sets the stage for testing of the BREM combinatorial approach in an animal model, which would expand the biomarker analysis to include histological analyses of brain tissue and neurological/behavioral/cognition assessments of AD progression. The ultimate goal is to test the combinatorial treatment in clinical setting and deliver the resulting therapeutic agent to the aid of AD patients.

Footnotes

ACKNOWLEDGMENTS

We thank Dr. Sangram Sisodia for providing us with the N2a/APP_Swe cell line, Dr. Richard Nowakowski and Don Rosenkoetter for helpful discussions, and Dr. Cathy Levenson’s laboratory for the BCL-2 and BCL-XL antibodies. These studies were supported by Alzyn, LLC and Department of Biomedical Sciences, Florida State University.

Authors’ disclosures available online ().

Appendix

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