Resveratrol Intervenes Cholesterol- and Isoprenoid-Mediated Amyloidogenic Processing of AβPP in Familial Alzheimer’s Disease

Abstract

Deterioration of cholesterol metabolism has recently been a frontier subject of investigation in the field of Alzheimer’s disease (AD). Though amyloid-β protein precursor (AβPP) primes the pathological cascade, changes in cholesterol levels and its intermediates, geranyl geranyl pyrophosphate and farnesyl pyrophosphate, is expected to have a different consequence on AβPP processing and amyloid-β (Aβ) generation. However, the use of statins (HMG-COA reductase inhibitor) has been widely implicated in slowing down the pathogenic progression of AD, while the epidemiological reports on its biological effect remains controversial. Considering this fact, the choice of drug that could maintain cholesterol homeostasis without altering its biosynthesis may yield a better therapeutic efficacy on AD. Thus, the present study focused on determining the influence of cholesterol and isoprenoids on amyloidogenic-cleavage of AβPP, in addition to resveratrol as a potent therapeutic drug in CHO-APPswe cell lines. High levels of cholesterol were found to enhance the maturation of AβPP and altered the expression and subcellular localization of ADAM10, BACE1, and PS1 thereby promoting Aβ generation, whereas high isoprenoids increased both maturation as well as amyloidogenic-cleavage of AβPP, which was evident through β-CTF production. Interestingly, the therapeutic efficacy of resveratrol maintained cholesterol homeostasis and reduced the amyloidogenic burden through its ability to enhance SIRT1 expression and thereby regulating differential expression of AD determinants.

Keywords

Alzheimer’s disease determinants amyloid-β protein precursor cholesterol geranyl geranyl pyrophosphate farnesyl pyrophosphate resveratrol SIRT1

INTRODUCTION

Alzheimer’s disease (AD) is an adult onset, progressive neurocognitive disorder that is responsible for the most common form of dementia [1, 2]. Worldwide, over 47 million people suffer from dementia [3], and this estimate is expected to exponentially increase every 20 years, reaching 74.7 million people by 2030 [4]. The prevalence and rate of progression of AD differs based on ethnicity and geographic locations. Thus, the genetic background in association with environmental factors appears to contribute to the development of AD [5, 6]. Several reports have shown that aging is one of the major risk factors for neurodegenerative diseases [7], which includes sporadic form of AD, whereas mutations within genes such as amyloid-β protein precursor (AβPP), presenilin 1 (PS1), and presenilin 2 (PS2) have been attributed to familial AD (FAD) [8 –10]. Among them, the specific accumulation of neurotoxic amyloid-β (Aβ) formed from the proteolytic cleavage of AβPP represents a major pathological step in the progression of AD [11, 12]. However, the genetic mutations are responsible for the accumulation of Aβ in FAD, while the identification of causative risk factors which increase the disease progression in sporadic AD is still controversial. This leads to the hypothesis of understanding the contributing factors involved in the pathogenesis of FAD and that could support the treatment of sporadic AD, which accounts for more than 95% of AD cases.

Dysregulation of cholesterol metabolism in the brain has been associated in the pathogenesis of AD [13 –15]. The possibility that abnormal cholesterol metabolism as a risk factor in the development of AD has also been supported by epidemiological studies [16 –18]. However, the molecular events by which cholesterol and its metabolites cause Aβ accumulation leading to the pathogenesis of AD are still poorly understood. Modulation of AβPP cleaving proteases such as α-secretase (A Disintegrin and metalloproteinase domain-containing protein 10, ADAM10), β-secretase (Beta-site amyloid precursor protein cleaving enzyme 1, BACE1), and γ-secretase complex (with PS1 at its active site) plays a vital role in the generation of Aβ [19]. Cholesterol is an essential component for generating lipid rafts, where most of the AD proteins, particularly AβPP, BACE1, and PS1, are associated. Hence, the alteration in raft components could change the configuration of either the enzyme or the substrate associated with the rafts, leading to an alteration in Aβ generation [20].

Another possibility for the shift from the non-amyloidogenic to the amyloidogenic pathway was suggested to be mediated by intermediates of cholesterol such as isoprenoids [viz., geranyl geranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)]. Isoprenoids are a complex class of biologically active lipids, which include dolichol, ubiquinone, FPP, and GGPP. Cholesterol intermediates such as GGPP and FPP have been found to play a vital role in the post-translational modification and trafficking of certain proteins, which function as molecular switches in numerous signaling pathways [21]. Despite these findings, the impact and causal role of cholesterol and its metabolites in AD remains controversial. Several studies reported the inconsistent results on the use of statins (HMG-COA reductase inhibitor), lipid-lowering drugs in treating AD [22]. Statin treatment is not only limited to cholesterol biosynthesis but also perturbs the intermediates (GGPP and FPP) that are involved in the process of protein prenylation [23]. The concentration of statins in brain of people administrated with these drugs may be very low, in order of few nanomolar concentrations. The contradictory results in epidemiological, cellular, and animal studies could be explained through different doses of statins that may have opposite effects [22]. This leads to the need for large-scale trials to document whether statins and cholesterol modification attenuates or worsen the progression of AD. However, an appropriate drug of choice in targeting AD-like conditions should pay particular attention to the maintenance of cholesterol homeostasis instead of inhibiting metabolic events. This is hypothesized to be achieved through natural supplements that work effectively in cholesterol regulation and one such interesting, yet explicable, drug of choice is resveratrol (RSV).

RSV (3,5,4’-trihydroxy-trans-stilbene) is a natural polyphenol produced in several plants, especially grape skins and seeds, berries, and peanuts and is a phytoalexin (against pathogens such as bacteria and fungi) [24, 25]. Several studies suggest that the RSV exerts neuroprotective and antioxidant properties [26] that might be relevant to treat age-associated diseases [27, 28]. RSV decreases the concentration of total cholesterol in hypercholesteremic rats [29]. Since RSV extends its protective mechanism by activating SIRT1, a homeostatic regulator [30], it was expected that SIRT1 may be efficient in regulating cholesterol homeostasis. Therefore, the current study utilized RSV to elucidate the interplay between cholesterol and isoprenoids in the amyloidogenic pathway, and the potential role of RSV on the regulation of cholesterol levels and its mediated alterations using CHO-APPswe cells as a FAD model. The generation of this knowledge could provoke new clues to challenge this complex neurodegenerative disorder.

MATERIALS AND METHODS

GGPP and FPP sodium salts were procured from Sigma Aldrich, and RSV was purchased from Cayman. Cell culture media were purchased from Himedia Laboratories Pvt Ltd., India. Lipofectamine 2000 and Geneticin (G418) were purchased from Invitrogen. All other chemicals were purchased from Himedia Laboratories Pvt Ltd, India unless otherwise specified. The antibodies used were anti-AβPP antibody (Cell Signaling), anti-Aβ antibody (Abcam), anti-Aβ₄₂ (Cell Signaling), anti-SIRT1 (Cell Signaling), anti-ADAM10 antibody (Santa Cruz), anti-BACE1 antibody (Cell Signaling), anti-PS1 antibody (Santa Cruz), anti-β-actin antibody (Cell Signaling), anti-GAPDH antibody (Sigma), and GFP-ER, GFP-Golgi, and Alexa Fluor (Invitrogen).

Cell culture, plasmid, transfection, stable cell lines, and treatment

CHO cells were procured from National Centre for Cell Science (NCCS), Pune, India. APPwt 695 and APPswe 695 plasmids were received as a kind gift from Dr. Dennis Selkoe (Brigham & Women’s Hospital, Boston, MA, USA). Stable cell lines were generated by transfecting APPwt695 and APPswe695 plasmids into CHO cells using Lipofectamine 2000 and the transfected colonies were selected by Geneticin (G418). These cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, and 100 U/ml penicillin/streptomycin. The transfection efficiency was confirmed by western blotting. The transfected cells showed significantly increased expression of AβPP (p < 0.001) (Supplementary Figure 1). Cells were maintained in reduced serum media while supplemented with lovastatin and seeded on poly L-lysine-coated 6-well plates at a density to obtain subconfluent cell monolayers at the time of drug treatment. For Aβ ELISAs, cells were plated on 12-well plates and the drug treatments were performed in triplicates. Cell viability remained 80% during drug treatment.

Experimental strategy for cholesterol dependent and isoprenoid dependent pathways in AβPP processing

The experimental conditions adopted for the current study include (Supplementary Figure 2) the following: Condition I: High cholesterol and normal isoprenoids (200 μM of cholesterol was supplemented); Condition II: Normal cholesterol and high isoprenoids (equal concentration of 10 μg/ml of GGPP and FPP were supplemented along with growth medium that contains FBS); Condition III: Normal cholesterol and low isoprenoids (exogenous cholesterol along with 5 μM lovastatin was supplemented in reduced serum medium), here lovastatin inhibits the intracellular cholesterol and isoprenoids biosynthesis but the cholesterol need was provided by exogenous supplementation; Condition IV: Normal cholesterol and normal isoprenoids (GGPP and FPP were supplemented along with lovastatin in reduced serum media). Lovastatin leads to inhibition of both isoprenoids and cholesterol biosynthesis but the exogenous isoprenoids supplementation can fulfil cholesterol and isoprenoids requirement; Condition V: low cholesterol and low isoprenoids (lovastatin supplementation in reduced serum inhibits both intracellular cholesterol and isoprenoids synthesis); Condition VI: High cholesterol and normal isoprenoids with RSV treatment (100 μM of RSV was treated based on the cytotoxicity assay, Supplementary Figure 3); Condition VII: High isoprenoids with RSV treatment; and Condition VIII: RSV alone.

Immunoblot analysis

Following treatment, the culture media were harvested, the cells were homogenized using RIPA lysis buffer (50 mM Tris–HCL, 150 mM Nacl, 1% triton X, 0.5% sodium deoxycholate, 0.1% SDS and 1 mM EDTA, 0.5 μg/ml of Leupeptin, and 0.5 μg/ml of Pepstatin), and the protein concentration was measured by Lowry’s method. 50 μg of protein was boiled for 5 min at 95°C in laemmli buffer and loaded on each lane of 12% SDS–PAGE at room temperature. The gels were transferred on to nitrocellulose membrane (0.4 μm, Pall Corporation). The membranes were then blocked in 5% BSA in phosphate-buffered saline with 0.1% Tween 20 (PBS-T) at room temperature (RT) for 2 h, and probed with the following primary antibodies diluted in PBS-T: anti-AβPP (1 : 1000, Cell Signaling), anti-Aβ (1 : 4000, Abcam), anti-SIRT1(1 : 1000, Cell Signaling), anti-BACE1(1 : 1000, Cell Signaling), anti-β-actin (1 : 1000, Cell Signaling), and anti-GAPDH (1 : 5000, Sigma) overnight at 4°C. Membranes were washed with PBS-T followed by subsequent incubation in secondary rabbit anti-mouse and goat anti-rabbit antibodies linked to horse peroxidase in blocking solution for 1 h at RT on a shaker. The membrane was alternatively washed 3 times for 10 min each with PBS-T and PBS, and the bands were visualized using an ECL kit. Finally, western blots were scanned and analyzed by Image J to provide quantitative values.

Quantification of cell-associated and secreted Aβ levels

Following drug treatments, the culture medium was collected, supplemented with protease inhibitor (Pepstatin and Leupeptin 0.5 μg/ml), and centrifuged at 12,000 rpm for 5 min at 4°C. 100 μl of the supernatant was used for total Aβ and Aβ₄₂ quantification by ELISA. To measure cell-associated Aβ₄₀ and Aβ₄₂, cells were washed twice with cold PBS, and cell pellets were solubilized in 125 μl of cold 5 M guanidine HCl and 50 mM Tris-HCl (pH 8.0). Following a brief sonication, samples were mixed at RT for 4 h, diluted into cold Dulbecco’s PBS with 5% BSA and 0.03% Tween 20 supplemented with protease inhibitor mixture (final guanidine HCl concentration of 0.1 M), and cleared by centrifugation at 12,000 rpm for 20 min at 4°C. 50 μl of supernatant was used to measure cell associated total Aβ₄₀ and Aβ₄₂ concentrations. The Aβ ELISA were performed as previously described by Englund et al. [31]. Briefly 96-well plates were coated with 100 μl of culture media or 50 μl of cell lysate was diluted in carbonate – bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. After washing the plates with PBS-T, wells were blocked with 200 μl of 1% BSA in PBS-T for 1 h at RT. The plates were again washed with PBS-T and 100 μl of primary antibodies Aβ (Abcam, 1 : 4000) and Aβ₄₂ (Cell Signaling, 1 : 1000) diluted in blocking solution were added and incubated for 1 h at RT. HRP-conjugated anti-rabbit diluted in blocking was used as secondary antibody and incubated for 1 h at RT. The plates were washed again, and 100 μl of 3, 3’, 5, 5’ tetra methyl benzidine (TMB) was added as the substrate and after color development, the reaction was stopped by adding 1 N sulphuric acid. The optical density (OD) was measured at 450 nm in a micro test ELISA reader. The concentration was calculated by standard curve with synthetic Aβ. Treatments were performed in triplicate, and the quantity of Aβ in each sample was measured in triplicate. Secreted total Aβ and Aβ₄₂ were represented as ng/ml±SEM. Cell associated total Aβ and Aβ₄₂ were represented as ng/mg (protein) ± SEM.

Total cholesterol level determination

For the lipid extraction, cells were pelleted and 2 : 1 chloroform:methanol was added to each tube. The mixture was vortexed for 20 min at RT, then the mixture was subjected to spin-win following, the fluid phase was collected, then it was allowed to air-dry. Cholesterol estimation was carried out by Zlatkis et al. [32] method. Cholesterol in acetic acid reacts with ferric chloride and sulphuric acid to produce a red color. The absorbance of red color solution is measured at 560 nm. To 0.1 of ml aliquot of total lipid extract 10 ml of ferric chloride reagent was added, mixed well and kept for 10 min at RT. It was then centrifuged for 10 min at 3000 rpm. 5 ml of the supernatant was pipetted out into a test tube and 3 ml of concentrated sulphuric acid was added and mixed well and the optical density was measured calorimetrically at 560 nm.

Immunocytochemistry

For the immunocytochemistry, cells were fixed with ice cold methanol (100%) for 15 min at RT. Then, the cells were washed with ice cold PBS, fixed cells were permeabilized with PBS containing 0.25% Triton–X 100 and blocked with 1% BSA in PBS containing 0.1% Triton-X 100 for 30 min. Immunostaining was performed with indicated concentration of primary antibodies anti-ADAM10 (1 : 100, Santa Cruz), anti-BACE1 (1 : 100, Cell Signaling), and anti-PS1 (1 : 100, Santa Cruz) in blocking solution for overnight (4°C), following 1 h incubation with secondary anti-rabbit alexa Fluor 594 antibody (1 : 100, Invitrogen) and mounted with glycerol: PBS (9 : 1).

For co-localization analysis of endoplasmic reticulum (ER) and Golgi expression of ADAM10, BACE1, and PS1, the cells were fixed and stained with ER-GFP and Golgi-GFP followed by indicated concentrations of primary antibodies anti-ADAM10 (1 : 100, Santa Cruz), anti-BACE1 (1 : 100, Cell Signaling), and anti-PS1 (1 : 100, Santa Cruz) in blocking solution for overnight (4°C) following 1 h incubation with secondary anti-rabbit alexa Fluor 594 antibody (1 : 100, Invitrogen) and mounted with glycerol: PBS (9 : 1). Samples were observed using a LSM (Zeiss) Confocal scanning microscope. Confocal images were acquired on an inverted laser scanning confocal microscope Carl Zeiss with 40X oil immersion lens. Images were taken using a ZEN 2011 Software and analyzed with Image J software.

Statistical analysis

All statistical analysis was performed using Graph Pad Prism software (Version 5.0) and analyzed by one way ANOVA followed by Tukey’s multiple comparison test and two-way ANOVA followed by Bonferroni post-hoc tests. The data were represented as Mean ±SEM for three independent experiments (n = 3). Values were considered to be statistically significant at ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

RESULTS

The source of the total cellular cholesterol and its intermediates (GGPP and FPP) are from endogenous cholesterol biosynthesis, endocytosis of exogenous cholesterol, and isoprenoids. In this condition, if the endogenous cholesterol biosynthesis is repressed by lovastatin (HMG-COA reductase inhibitor), the exogenous cholesterol will provide the cholesterol need inside the cell. The other cholesterol independent mechanism of statin is inhibition of isoprenoids which may have a significant impact not only on cholesterol metabolism but also on protein trafficking and targeting. Here, the isoprenoids needs are revoked by adding the exogenous addition of GGPP and FPP (10 μg/ml).

By using these properties, we have explored different experimental strategy to analyze the roles of cholesterol and isoprenoids on the AβPP pathway at various experimental conditions with and without RSV treatment. As an in vitro model, we employed CHO cells to express human APP695 containing Swedish mutation (CHO-APPswe) as a model system for FAD [33, 34].

Lovastatin inhibits intracellular cholesterol biosynthesis

The present study found that there was no significant alterations in the levels of cholesterol on CHO and CHO-APPswe cells. To confirm the uptake of exogenous cholesterol and to investigate the potency of lovastatin and RSV are same in both CHO (non-transfected) (Fig. 1A) and CHO-APPswe cells (Fig. 1B), cells were treated with lovastatin, RSV in the presence and absence of cholesterol and isoprenoids (GGPP and FPP) for 48 h, and analyzed for cholesterol levels. The supplementation of high cholesterol (Condition I) significantly (p < 0.01) increased cellular cholesterol concentration, whereas high isoprenoids condition (Condition II) displayed a slight increase in cholesterol levels but was not statistically significant when compared to control. Moreover, the treatment of lovastatin (Condition III–V) significantly decreased the levels of cholesterol (p < 0.001) in all three models when compared to the high cholesterol condition (Condition I). Interestingly, RSV treated conditions (viz., Condition VI–VIII) also exhibited significant (p < 0.001) decrease in the level of cholesterol when compared to the high cholesterol condition. We observed that the exogenous cholesterol increased the cellular cholesterol levels, whereas lovastatin (HMG-COA reductase inhibitor) inhibited cellular cholesterol biosynthesis and RSV has potentiated in regulating cholesterol levels in vitro.

Fig.1

Lovastatin inhibits the intracellular cholesterol synthesis. CHO or CHO-APPswe cells were treated with indicated concentrations of cholesterol, isoprenoids, lovastatin, and RSV as mentioned above for 48 h. Following treatment, the cells were harvested and the total cholesterol was estimated. A) Total cholesterol levels in CHO cells. B) Total cholesterol levels in CHO-APPswe cells. ^aComparison between cholesterol and isoprenoids treated conditions to the high cholesterol treated condition ^#Comparison of treated conditions to the control. Values represent mean ±SEM. Statistical significance was performed by one-way ANOVA followed by Tukey’s multiple comparison test, asterisks (^*) denote ^**p < 0.01; ^***p < 0.001.

Cholesterol and isoprenoids found to influence SIRT1 levels in in vitro conditions

SIRT1, being a homeostatic regulator, has been linked to various metabolic functions. Interestingly, the study on SIRT1 expression in CHO-APPswe control cells (Fig. 2A) revealed a significant (p < 0.01) decrease in its expression when compared to non-transfected CHO cells.

Fig.2

Expression of SIRT1 in control and experimental conditions. A) SIRT1 expression in CHO (non-transfected) and CHO-APPswe cells. B) SIRT1 expression in various experimental conditions. Cells were harvested and immunoblotted with anti-SIRT1 and anti-β actin or anti-GAPDH antibodies and analyzed by using ECL. ^#Comparison of treated groups with control. Values represents mean ± SEM. Statistical significance was performed by one-way ANOVA followed by Tukey’s multiple comparison test, asterisks (^*) denote ^**p < 0.01.

The effect of cholesterol and isoprenoids on SIRT1 expression (Fig. 2B) was found to be significantly increased (p < 0.01) in the high cholesterol, high isoprenoids (Condition I and II), and lovastatin treated conditions (Condition III and IV) when compared to control. Furthermore, the lovastatin alone treated condition (Condition V, low cholesterol and low isoprenoids) displayed decreased SIRT1 expression when compared to the high cholesterol condition, though the effect was not statistically significant.

RSV potentiated the activation of SIRT1 expression in control and experimental conditions

In a previous report using an in vivo model [29], it was found that RSV is a powerful activator for SIRT1. Expecting SIRT1 as a mediator for RSV’s cytoprotective mechanism, the CHO-APPswe cells were treated with cholesterol and isoprenoids in the presence of RSV (Condition VI–VIII) (Fig. 2B). The results displayed significant (p < 0.01) increase in the SIRT1 expression in all the RSV treated groups when compared to control. Thereby, it is speculated that the cytoprotective role of RSV was possibly mediated through the activation of SIRT1 in CHO-APPswe cells. Also, Vingtdeux et al. [34] demonstrated that SIRT1 activation is one of the main targets defined for the pharmacological effects of RSV.

Cholesterol and isoprenoids exhibited its influence over secreted total Aβ and Aβ₄₂ production

The presence of Aβ in CHO-APPswe cells was confirmed through ELISA (Supplementary Figure 4). Despite no considerable significance in the levels of cholesterol in non-transfected CHO and CHO-APPswe cells, the decreased SIRT1 expression and the presence of Aβ confirmed CHO-APPswe cells as a FAD-like model. Therefore, CHO (non-transfected) cells have been neglected for further experiments. To determine the influence of cholesterol and its intermediates, such as GGPP and FPP, on the expression of Aβ and Aβ₄₂, the cell culture media and cell lysate of CHO-APPswe cells were assessed for total Aβ and Aβ₄₂ using ELISA at different experimental conditions as mentioned in the methodology.

The presence of high cholesterol (Condition I) showed significantly increased (p < 0.001) secreted total Aβ and Aβ₄₂ in the medium (Fig. 3A, C), whereas high isoprenoids (Condition II) showed no significant change in secreted total Aβ and Aβ₄₂ when compared to control (CHO-APPswe) cells. In contrast, secreted total Aβ was significantly (p < 0.001) decreased in lovastatin treated conditions (III–V) when compared to the high cholesterol condition (Condition I) and control; however, Aβ₄₂ was significantly (p < 0.001) decreased when compared with the high cholesterol condition alone. The presence of lovastatin significantly decreased the production of intracellular cholesterol biosynthesis as evident in Fig. 1A, B, and it is thus hypothesized that the reduced Aβ levels could be a cholesterol dependent mechanism.

Fig.3

Cholesterol and isoprenoid dependent effects on secreted and cell associated total Aβ and Aβ₄₂. Cells were treated with indicated concentration of cholesterol, isoprenoids, lovastatin, and RSV as mentioned above for 48 h. Following treatments, secreted Aβ was measured from the cell culture medium whereas cell associated Aβ was measured by extraction with Guanidine HCl. (A) and (C) depict the secreted total Aβ and Aβ₄₂ in CHO-APPswe cells. (B) and (D) represent the cell associated total Aβ and Aβ₄₂ levels in CHO-APPswe cells. Secreted Aβ was represented as (ng/ml) ± SEM. Cell associated Aβ was represented as (ng/mg) ± SEM normalized to the protein content. ^aComparison between cholesterol and isoprenoids treated conditions to the high cholesterol treated condition. ^bComparison between the treated conditions to the high isoprenoids treated condition. ^#Comparison of treated groups with the control. Statistical significance was performed by one-way ANOVA followed by Tukey’s multiple comparison test, asterisks (^*) denote ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

High cholesterol deliberately decreased intracellular total Aβ and Aβ₄₂ in CHO-APPswe cells while increasing secretory Aβ

CHO-APPswe transfected cells treated with high cholesterol and high isoprenoids (Condition I and II) presented no significant change in total intracellular Aβ and Aβ₄₂ levels when compared to control (CHO-APPswe) cells (Fig. 3B, D). Interestingly, findings on low isoprenoids and normal cholesterol (Condition III) in the presence of lovastatin showed significantly increased levels of cell associated total Aβ (p < 0.001) and Aβ₄₂ (p < 0.001) when compared to control and high cholesterol condition (Condition I). The treatment of lovastatin along with exogenous cholesterol might have prevented the Aβ secretion to extracellular space by accumulating the Aβ in intracellular compartments. Further, the exogenous isoprenoids (Condition IV) administration showed significant increase in the total intracellular Aβ and no significant change in Aβ₄₂ when compared to the high cholesterol condition (Condition I). The lovastatin alone treated conditions (Condition V) displayed no significant change in either total Aβ or Aβ₄₂ levels when compared to control and high cholesterol conditions.

RSV prevents the cholesterol induced intracellular and extracellular form of total Aβ and Aβ₄₂

Strikingly, condition VI, VII, and VIII treated with RSV revealed that there were significant decreases in secreted total levels of Aβ (p < 0.001) and Aβ₄₂ (p < 0.01) when compared to the high cholesterol condition (Condition I) and control cells (Fig. 3A, C). Considering cell associated Aβ (Fig. 3B, D), the RSV treatment was found to regulate total Aβ and Aβ₄₂ both in the presence and in the absence of cholesterol and isoprenoids. Furthermore, role of RSV on Aβ reduction provoked us to understand its influence on AβPP cleavage patterns and cleavage enzymes in the presence of cholesterol and its intermediates. Hence, further experiments were carried out to analyze the cleavage pattern of AβPP under various experimental conditions.

High cholesterol and high isoprenoids enhanced AβPP maturation and its cleaved fragments yielding Aβ_40/42

The precise molecular mechanism underling AβPP cleavage in AD is not known. Several reports suggested the link between altered cholesterol homeostasis and AβPP metabolism [24, 33]. This led us to investigate the influence of cholesterol and its intermediates on AβPP cleavage pattern in CHO-APPswe cells.

The condition with high cholesterol (Condition I) (Fig. 4) showed significantly increased expression (p < 0.05) of AβPP when compared to control cells. Consistent with this, Condition II with high isoprenoids (Fig. 4) projected a higher intensity of AβPP expression with a significance of p < 0.01 and β-CTF (C99) with p < 0.001 when compared to control cells. Isoprenoids (GGPP and FPP) might be involved in the trafficking of AβPP to secretory compartments and thereby this could have increased the possibility of its cleavage as seen in the levels of β-CTF (Fig. 4).

Fig.4

Cholesterol and isoprenoids increased the full-length AβPP and CTF fragments. Following treatment, cells were harvested and cell lysates were prepared and the expression of AβPP and its corresponding CTFs were immunoblotted with anti-Aβ and analyzed by ECL. A) Expression pattern of AβPP, CTF-β, and CTF-α. B) Densitometry results of AβPP, CTF-β, and CTF-α. C) Ratio of CTF-β and CTF-α. Values represents mean ± SEM. Statistical significance was performed by two-way ANOVA followed by Bonferroni post-hoc tests, asterisks (^*) denotes ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Lovastatin treated conditions (Conditions III–V) (Fig. 4) displayed no significant alteration in the levels of full length AβPP and moreover significantly decreased the levels of CTFs (p < 0.05) when compared to control. These results imply that the high cholesterol and isoprenoids enhance AβPP and its processing, while depletion of cholesterol and isoprenoids leads to diminished AβPP processing and this was evident in CTFs formation (Fig. 4).

RSV modulates cholesterol and isoprenoids mediated amyloidogenic cleavage of AβPP

Treatment of RSV along with cholesterol or isoprenoids (Condition VI and VII) (Fig. 4) showed significantly increased AβPP expression and its CTFs as compared with control (CHO-APPswe) cells. Interestingly, RSV treated conditions increased α-CTF (C83) expression (p < 0.001) when compared to control (CHO-APPswe), high cholesterol, and high isoprenoids conditions (Condition I and II). Furthermore, RSV treatment alone (Condition VIII) showed decreased expression of AβPP when compared with cholesterol and isoprenoids treated conditions (Condition I, II, VI, and VII), and increased the accumulation of AβPP cleaved fragments resulting in decreased Aβ formation. The ratio of α-CTF and β-CTF was found to be significantly increased in RSV treated conditions (Fig. 4C). This result concludes that RSV prevented amyloidogenic cleavage of AβPP by promoting α-cleavage facilitating non-amyloidogenic pathway in CHO-APPswe cells irrespective of the presence of cholesterol and isoprenoids. However, these findings indicate the efficacy of RSV in promoting the non-amyloidogenic pathway of AβPP giving a positive note on action of RSV on AβPP cleaving enzymes that are considered as AD determinants.

Effect of cholesterol and isoprenoids on AD determinants

Since the cleavage of AβPP by BACE1 is the rate limiting step in Aβ generation, the expression of BACE1 gains considerable significance in targeting Aβ formation. To investigate the influence of cholesterol and isoprenoids on BACE 1 expression, CHO-APPswe cells were treated with various experimental conditions and analyzed using western blotting (Fig. 5). We found that there was no significant change in BACE1 in Conditions I–V when compared to control. From the previous results, it was found that the high cholesterol and high isoprenoids significantly increased Aβ (Fig. 3) and β-CTF formation (Fig. 4) and this might be due to increased amyloidogenic cleavage of AβPP. When corroborating the above observations with BACE1 expression, it is clear that cholesterol might have increased the proximity of AβPP to BACE1 compartments resulting in Aβ formation rather than altering its expression.

Fig.5

Effect of RSV on BACE1 expression in cholesterol and isoprenoids treated conditions. The expression of BACE1 was analyzed by western blotting. Following treatment, cells were harvested and cell lysates were prepared and immunoblotted with anti-BACE1 and analyzed by ECL. ^aComparison between cholesterol and isoprenoids treated conditions to the high cholesterol treated condition. ^bComparison between the treated conditions to the high isoprenoids treated condition. ^#Comparison of treated conditions to the control. Values represents mean ±SEM. Statistical significance was performed by one way ANOVA followed by Tukey’s multiple comparison test, asterisks (^*) denotes ^*p < 0.05; ^**p < 0.01.

ADAM10, BACE1, and PS1 are the active form of AβPP cleaving enzymes viz α-, β-, and γ-secretases and play a significant role in the progression of AD. With the suspicion of close proximity of AβPP to BACE1, the CHO-APPswe cells were treated with various experimental conditions and assessed for the expression of all the AD determinants (ADAM10, BACE1, and PS1) using confocal microscopy. ADAM10, which is involved in normal cleavage of AβPP, was shown to be significantly decreased (p < 0.05) in the high cholesterol condition (Condition I) (Fig. 6) with a slight increase in BACE1 and PS1 expression (Figs. 7, 8) when compared to control; however, it is not statistically significant. The isoprenoids (Condition II) did not show any significant alteration on AD determinants. On the other hand, lovastatin treatment (Condition III, IV, and V) showed a slight increase in ADAM10 expression (Fig. 6), which was not significant. The low cholesterol condition (Condition V) (Figs. 7, 8) exhibited a significant decrease in BACE1 and PS1 expression (p < 0.01 and p < 0.05 respectively) when compared to high cholesterol condition (Condition I). However, all these proteins are found to be similar when compared to that of control.

Fig.6

Effect of cholesterol and isoprenoids and the efficacy of RSV on ADAM10 expression. A) The expression of ADAM10 was analyzed by confocal microscopy. Magnification: 40X. Scale Bar: 10 μm. B) The intensity of ADAM10 measured by Image J. ^aComparison between cholesterol and isoprenoids treated conditions to the high cholesterol condition. ^bComparison between the treated conditions to the high isoprenoids condition. ^#Comparison of treated conditions to the control. Statistical significance was performed by one-way ANOVA followed by Tukey’s multiple comparison test, asterisks (^*) denote ^*p < 0.05; ^***p < 0.001.

Fig.7

Effect of cholesterol and isoprenoids and the efficacy of RSV on BACE1 expression. A) The expression of BACE1 was analyzed by confocal microscopy. Magnification: 40X. Scale Bar: 10 μm. B) The intensity of BACE1 was measured by Image J. ^#Comparison of treated conditions to the control. Statistical significance was performed by one-way ANOVA followed by Tukey’s multiple comparison test, asterisks (^*) denote ^*p < 0.05; ^**p < 0.01.

Fig.8

Effect of cholesterol and isoprenoids and the efficacy of RSV on PS1 expression. A) The expression of PS1 was analyzed by confocal microscopy. Magnification: 40X. Scale Bar: 10 μm. B) The intensity of PS1 was measured by Image J. ^aComparison between the treated conditions to the high cholesterol treated condition. ^bComparison between the treated conditions to the high isoprenoids condition. Statistical significance was performed by one-way ANOVA followed by Tukey’s multiple comparison test, asterisks (^*) denote ^*p < 0.05.

From the above observations, it is clear that expression patterns of ADAM10, BACE1, and PS1 are cholesterol dependent and is clearly evident from lovastatin alone supplemented condition (Condition V with low cholesterol) that significantly regulated the altered pattern of AβPP cleaving enzymes (Figs. 6–8).

Effect of high cholesterol on ER and Golgi expression of AβPP cleaving enzymes

To understand the synthesis and maturation pattern, the ER and Golgi expression of ADAM10, BACE1, and PS1 were analyzed in the presence of high cholesterol and RSV treatment on CHO-APPswe cells using confocal microscopy. Since isoprenoids treated conditions did not show any significant alterations, it has been neglected for these experiments. As a result, high cholesterol condition significantly increased the localization of ADAM10 in ER (0.53) but decreased its expression in the Golgi complex (0.42) (Fig. 9A, B). However, the weak localization of ADAM10 in the Golgi revealed that the presence of high cholesterol might have prevented the transport and maturation of ADAM10.

Fig.9

Effect of high cholesterol and RSV on ER and Golgi expression of ADAM10. CHO-APPswe cells were treated with cholesterol or RSV for 48 h. Following treatment, co-localization of ADAM10 in ER (A) and Golgi (B) were assessed by confocal microscopy. The co-localized areas are shown in the merged image. Pearson coefficient was analyzed by Fiji image J software. Magnification; 40X; Scale Bar: 10 μm.

Consistent with these findings, BACE1 showed significantly increased localization in the Golgi (colocalization coefficient-0.41) when compared to the ER (0.36) (Fig. 10A, B). Thus, it suggests that cholesterol plays a vital role in sequestrating BACE1 in the Golgi vesicles. Further, when compared to control cells, the presence of cholesterol was found to decrease co-localization coefficient and this may increase the possibility of mobilizing BACE1 to TGN or endocytic compartments where the amyloidogenic cleavage of AβPP takes place. The current study on the expression of PS1 that acts on both α- and β-CTF displayed increased localization of PS1 in Golgi (0.51) (Fig. 11A, B) with high cholesterol when compared with control. The intracellular cholesterol enrichment-mediated increased in BACE1 and PS1 in the Golgi compartments revealed that there was a significant increase in the transport of these key enzymes from ER to Golgi, therefore this represents an increased amount of matured form of BACE1 and PS1.

Fig.10

Effect of high cholesterol and RSV on ER and Golgi expression of BACE1. CHO-APPswe cells were treated with cholesterol or RSV for 48 h. Following treatment, co-localization of BACE1 in ER (A) and Golgi (B) were assessed by confocal microscopy. The co-localized areas are shown in the merged image. Pearson coefficient was analyzed by Fiji image J software: Magnification; 40X; Scale Bar: 10 μm.

Fig.11

Effect of high cholesterol and RSV on ER and Golgi expression of PS1. CHO-APPswe cells were treated with cholesterol or RSV for 48 h. Following treatment, co-localization of PS1 in ER (A) and Golgi (B) were assessed by confocal microscopy. The co-localized areas are shown in the merged image. Pearson coefficient was analyzed by Fiji image J software: Magnification; 40X; Scale Bar: 10 μm.

RSV rescued cholesterol mediated altered expression of AβPP cleaving enzymes

While treatment of RSV under experimental condition (Condition VIII) showed significant increases (p < 0.01) in ADAM10 expression (Fig. 6) when compared to the high cholesterol condition, BACE1 (Figs. 5, 7) displayed decreased expression in RSV treated conditions (Condition VI–VIII) and there was no significant change found in PS1 (Fig. 8) expression when compared to the high cholesterol condition (Condition I). Moreover, RSV treated CHO-APPswe cells indicated a significant increase in the expression and localization of ADAM10 (Fig. 9A, B) in both ER (0.60) and Golgi (0.49) when compared to control (CHO-APPswe) cells. The increased sequestration of ADAM10 from ER to Golgi revealed that RSV might increase the mature form of ADAM10 and this could be the reason for the increased formation of the α-CTF fragment of AβPP in the presence of RSV (Fig. 5). Furthermore, RSV treatment did not lead to a significant change in ER and Golgi expression of BACE1, while PS1 displayed increase expression in ER. However, there was no change in Golgi when compared to CHO-APPswe cells.

DISCUSSION

Aβ, being the principal component of amyloid plaques, is derived from the integral transmembrane protein AβPP. It is well documented that dysregulated cholesterol metabolism is associated with abnormal AβPP processing [36, 37] while the contribution of cholesterol and its metabolites in the formation of Aβ is an unrevealed mechanism. Sufficient availability of cholesterol is necessary for normal neuronal function, however, several epidemiological studies state that the elevated cholesterol level is one of the risk factors for AD [38 –40]. Although inhibition of cellular cholesterol by statins could play a beneficial role, cholesterol depletion in neurons impair synaptic vesicle exocytosis and decrease neuronal activity and neurotransmission, leading to dendritic spine and synapse degeneration [14, 41]. Thus, regulation of cholesterol homeostasis without affecting its normal function is beneficial in preventing disease progression, particularly in AD. RSV, a natural polyphenolic compound, exhibits antioxidant and anti-inflammatory functions, as well as a regulatory function on LDL receptor activity [42]. In line with this, it is expected that RSV maintains cholesterol homeostasis. Intriguingly, RSV regulated cholesterol levels in the experimental conditions of the current study (Fig. 1).

Since the current study focuses on the maintenance of cholesterol homeostasis and AβPP metabolism, the status of SIRT1, a homeostatic regulator of vital metabolic events, was examined and found to be significantly decreased in CHO-APPswe cells (Fig. 2A). This result is in line with the Lee et al. [43] report that states intracellular Aβ alters protein homeostasis, which is associated with the suppression of SIRT1 at the level of mRNA and protein expression. The current study on high cholesterol, high isoprenoids, and lovastatin treatment along with cholesterol or isoprenoids (Conditions I–IV), displayed significant increases in the expression of SIRT1 (Fig. 2B). This result suggests that conditions like high cholesterol might have triggered SIRT1 expression on an administration of acute concentrations of free cholesterol and isoprenoids that the cells could sense, thereby attempting cholesterol regulation in-vitro, whereas lovastatin, in the absence of cholesterol and isoprenoids, did not show any influence on SIRT1 levels. Further, the administration of RSV to experimental conditions significantly increased SIRT1 (Fig. 2B) and moreover regulated cholesterol levels as evidenced in Fig. 1A and B.

The current findings using in-vitro models showed that RSV could significantly increase SIRT1 expression. Liver X receptors are the substrate for SIRT1 and it regulates the function of liver X receptor α (LXRα), a nuclear receptor that functions as cholesterol sensor and regulates cholesterol and lipid homeostasis [44, 45]. LXR deacetylation by SIRT1 could be the possible mechanism behind the regulation of cholesterol homeostasis by RSV. Although our aim is not specific to deacetylation mechanisms that could be rendered by SIRT1, we focus on its effect on AβPP cleavage pattern that either ends up taking toxic pathways like an amyloidogenic pathway or non-toxic pathways resulting in Aβ₄₀ (a soluble fibril that benefits nervous system functioning).

The first evidence that cholesterol may impact Aβ production in the brain was provided in 1994, where Sparks et al. [46] demonstrated that dietary cholesterol increases amyloid production in rabbits. A large number of experiments performed using primary neurons or peripheral cell lines showed that an increase in cellular cholesterol levels enhances Aβ production [47 –49]. In this context, we analyzed the levels of secreted total Aβ and Aβ₄₂ under various experimental conditions, where high cholesterol significantly enhanced secreted total Aβ and Aβ₄₂ levels, while lovastatin treated conditions (Condition III–V) (Fig. 3A, C) significantly decreased Aβ formation. According to Shepardson et al. [50], cholesterol is able to bind Aβ and influence its aggregation state in the extracellular space. Increased interaction of Aβ with the cell membrane takes place under low cholesterol conditions and leads to its degradation; conversely high cholesterol prevents the association and leads to accumulation or aggregation at the extracellular space. Our results accord the same and confirmed that high cholesterol significantly influence secreted total Aβ and insoluble form of Aβ₄₂, while low cholesterol leads to decreased Aβ₄₂. These results imply that high cholesterol is one of the factors that might enhance the formation of insoluble Aβ₄₂.

Considering cell associated total Aβ and Aβ₄₂, exogenous cholesterol with lovastatin (Condition III) (Fig. 3B, D) significantly increased total Aβ and Aβ₄₂ formation with no significant change observed in other experimental conditions. Taking together the role of cholesterol in the cell associated total Aβ and Aβ₄₂, only the normal cholesterol levels in the presence of statin (and not the dysregulated cholesterol metabolism) resulted in increased level of cell-associated total Aβ and Aβ₄₂ when compared with control. This further supports the significance of cholesterol and metabolites in the formation of secreted Aβ. As reported by Cole et al. [32], statin treatment inhibited cholesterol and isoprenoids biosynthesis leading to accumulation of intracellular Aβ. The rate of degradation of Aβ in the cell lysate is more rapid than in the medium. Accumulation of intracellular Aβ due to the supplementation of lovastatin had the highest possibility of getting degraded. On the whole, the inhibition of the cholesterol biosynthetic pathway has a positive impact on attenuating Aβ generation and implies a substantial role of cholesterol metabolism in the progression of AD.

Based on the above results, we further intended to analyze the influence of cholesterol and its metabolites on the cleavage pattern of AβPP. The current findings demonstrated that the experimental conditions with high cholesterol and high isoprenoids (Condition I and II) enhance the maturation of AβPP (Fig. 4) that might tend to recycle AβPP by increasing its processing rate. Furthermore, Beel et al. [51] found that the propensity of C99 and full length AβPP to form stoichiometric complexes with cholesterol could modulate substrate binding and favors amyloidogenic pathway by promoting localization of AβPP/C99 to cholesterol-rich membrane domains and organelles where γ-secretase, and possibly β-secretase, reside. Prenylation is the vital function of GGPP and FPP [52], and thereby high isoprenoids may increase the trafficking of AβPP to BACE compartments through random prenylation thus promoting β-CTF production. Therefore, the possibility of increased prenylation of AβPP by GGPP and FPP might increase its proximity toward BACE1 compartments where the increased amyloidogenic cleavage of AβPP takes place.

Interestingly, lovastatin administration significantly reduced CTFs (Fig. 4) and Aβ (Fig. 3) formation. A recent report suggested that the treatment of Pitavastatin (PV) and Atorvastatin (AV) significantly reduced the expression levels of the mature form of AβPP and Thr668-phosphorylated AβPP (PAβPP), but not an immature form of AβPP [15, 24]. This is in line with our study which showed that the depletion of cholesterol and isoprenoids with lovastatin significantly reduced AβPP processing indicating possible effects of cholesterol and its metabolites on AβPP maturation and trafficking. In contrast, RSV treated conditions (Condition VI–VIII), in spite of promoting increased proteolytic cleavage of AβPP when compared to lovastatin treated and non-treated conditions, RSV was able to significantly promote non-amyloidogenic cleavage of AβPP resulting in α-CTF (C83) (Fig. 4) formation which precludes Aβ generation (Fig. 3). While inhibition of cholesterol and isoprenoids with lovastatin failed to increase α-CTF levels. According to Marambaud et al. [52], RSV acts by promoting proteasome-dependent intracellular degradation of the amyloid peptide. In line with these reports, our results imply that RSV treatment exhibited direct impact in attenuating total Aβ and Aβ₄₂ generation (Fig. 3). In addition to these results on increased cleavage of AβPP and CTFs in the presence of high cholesterol and high isoprenoids, we wanted to investigate the expression pattern of AβPP cleaving enzymes at various experimental conditions.

Kojro et al. [54] stated that cholesterol depletion below a critical concentration (about 60% of the initial quantity) caused significantly enhanced enzymatic activity of ADAM10 along with increased membrane fluidity. The present study using high cellular cholesterol (Condition I) reduced ADAM10 expression (Fig. 6) and increased β-secretase mediated cleavage resulting in the amyloidogenic processing of AβPP. On the contrary, RSV treated groups were able to regulate cholesterol mediated (Condition VI) decrease in ADAM10 expression. Particularly in Condition VIII (RSV alone), the expression of ADAM10 was significantly increased. Current findings revealed that expression of AD determinants viz., ADAM10, BACE1, and PS1, were cholesterol dependent (Figs. 6–8) and was regulated by depleting intracellular cholesterol (Condition V). Astonishingly, RSV proved to potentially regulate AβPP cleaving enzymes in both cholesterol dependent and independent experiments. Furthermore, to understand the influence of cholesterol and the efficacy of RSV in the synthesis and maturation of AβPP cleaving enzymes, the ER and Golgi expression of AβPP cleaving enzymes were analyzed in the presence of high cholesterol and RSV alone conditions.

The ER and Golgi expression of ADAM10 in the presence of high cholesterol displayed increased ADAM10 in ER and decreased expression in Golgi (Fig. 9A, B). This might be due to ER retention of ADAM10 in the presence of high cholesterol. According to Postina [55], Aβ production was reduced by decreasing intracellular cholesterol biosynthesis and simultaneous increasing ADAM10 expression. Conversely, high cholesterol increased the sequestration of BACE1 to Golgi (Fig. 10A, B) and decreased localization coefficient (0.41) and increased Aβ formation (Fig. 3), demonstrating that BACE1 could have mobilized to other compartments. The maturation of BACE1 increases the catalytic activity of the enzyme by at least two-fold over that of immature BACE1 [56]. Sun et al. [57] stated that the mature N-glycosylated form of BACE1 results in increased catalytic activity, leading to higher β-CTF production. On the whole, the present finding depicts that cholesterol significantly altered the localization of AβPP cleaving enzymes reflecting a differential expression pattern among the subcellular organelles. Moreover, the present findings showed increased levels of ADAM10 expression in ER and Golgi upon RSV treatment (Fig. 9A, B). This indicates that RSV might potentially increase the synthetic pathways of ADAM10 and further promoted maturation of ADAM10 possibly through the activation of SIRT1. This was reflected in other observations of AβPP cleavage pattern that displayed increased α-CTF formation and decreased Aβ generation (Figs. 3, 4). A direct and positive effect of RSV on AD pathology was found to be the activation of retinoic acid nuclear receptors that can activate ADAM10 gene transcription [58]. The transcription factor RA receptorβ, responsible for increasing ADAM10 expression, is regulated by nicotinamide adenine dinucleotide-dependent deacetylase (NAD⁺) SIRT1 [55].

According to Marwarha et al. [59], BACE1 protein levels and mRNA expression was decreased on administration of leptin and is proposed to be achieved by attenuating NFκB transcriptional activity. Since NFκB is a substrate of SIRT1, RSV supplementation might trigger inactivation of NFκB and thereby decreased BACE1 expression (Figs. 5, 7). Corroborating these results, RSV was found to significantly enhance the maturation of ADAM10 and raise the possibility of regulating the sequestration of BACE1 to the endocytic compartments in the presence of cholesterol.

In summary, the present study provides evidence that dysregulation of cholesterol metabolism is not only associated with age-related complications but also enhances the progression of the amyloidogenic processing of AβPP and Aβ generation in CHO-APPswe cells that mimics FAD. Targeting cholesterol homeostasis and its mediated alterations could be beneficial to slow down the disease progression in both the rare familial form and also in the sporadic form of AD. The present data supports that the activation of SIRT1 by RSV provides a new insight in regulating cholesterol dependent and independent amyloidogenic processing of AβPP (Fig. 12). Further work on the assessment of cholesterol metabolizing enzymes and the influence of SIRT1 activation in AD models will provide a clear strategy to develop SIRT1 as a therapeutic intervention to target AD-like fatal neurodegenerative diseases.

Fig.12

Scheme depicting the molecular mechanism of RSV in preventing cholesterol mediated AβPP processing. 1) RSV mediated SIRT1 expression in-vitro, regulates endogenous levels of cholesterol. 2) The treatment of RSV with cholesterol or isoprenoids (GGPP and FPP) depicted an increase in AβPP maturation and decreased amyloidogenic cleavage of AβPP. 3) RSV regulates exogenous cholesterol mediated altered expression and subcellular localization (ER and Golgi) of AD determinants such as ADAM10, BACE1, and PS1.

Footnotes

ACKNOWLEDGMENTS

This research was supported by Department of Science & Technology (DST), Technology Bhavan, New Mehrauli Road, New Delhi-110 016. Also, this research was partially supported by UGC through Junior Research Fellow (JRF) to Mohan Sathya. We would extend our gratitude to Dr. J. Tamilselvan, Anna university and Dr. Bharathiraja, University of Madras, Chennai for their support to carry out the transformation work. The instrumentation facility by the Department of Science & Technology (DST) under DST-FIST and DST-PURSE programmes are gratefully acknowledged. We would like to thank the Editor and anonymous reviewers for their comments to improve the manuscript.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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Resveratrol Intervenes Cholesterol- and Isoprenoid-Mediated Amyloidogenic Processing of AβPP in Familial Alzheimer’s Disease

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Cell culture, plasmid, transfection, stable cell lines, and treatment

Experimental strategy for cholesterol dependent and isoprenoid dependent pathways in AβPP processing

Immunoblot analysis

Quantification of cell-associated and secreted Aβ levels

Total cholesterol level determination

Immunocytochemistry

Statistical analysis

RESULTS

Lovastatin inhibits intracellular cholesterol biosynthesis

Cholesterol and isoprenoids found to influence SIRT1 levels in in vitro conditions

RSV potentiated the activation of SIRT1 expression in control and experimental conditions

Cholesterol and isoprenoids exhibited its influence over secreted total Aβ and Aβ42 production

High cholesterol deliberately decreased intracellular total Aβ and Aβ42 in CHO-APPswe cells while increasing secretory Aβ

RSV prevents the cholesterol induced intracellular and extracellular form of total Aβ and Aβ42

High cholesterol and high isoprenoids enhanced AβPP maturation and its cleaved fragments yielding Aβ40/42

RSV modulates cholesterol and isoprenoids mediated amyloidogenic cleavage of AβPP

Effect of cholesterol and isoprenoids on AD determinants

Effect of high cholesterol on ER and Golgi expression of AβPP cleaving enzymes

RSV rescued cholesterol mediated altered expression of AβPP cleaving enzymes

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Cholesterol and isoprenoids exhibited its influence over secreted total Aβ and Aβ₄₂ production

High cholesterol deliberately decreased intracellular total Aβ and Aβ₄₂ in CHO-APPswe cells while increasing secretory Aβ

RSV prevents the cholesterol induced intracellular and extracellular form of total Aβ and Aβ₄₂

High cholesterol and high isoprenoids enhanced AβPP maturation and its cleaved fragments yielding Aβ_40/42