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SIRT3 activator Honokiol attenuates β-Amyloid by modulating amyloidogenic pathway

  • Sindhu Ramesh ,

    Contributed equally to this work with: Sindhu Ramesh, Manoj Govindarajulu

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

  • Manoj Govindarajulu ,

    Contributed equally to this work with: Sindhu Ramesh, Manoj Govindarajulu

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Writing – original draft

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

  • Tyler Lynd,

    Roles Data curation, Investigation, Methodology, Writing – original draft

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

  • Gwyneth Briggs,

    Roles Investigation

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

  • Danielle Adamek,

    Roles Investigation, Writing – review & editing

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

  • Ellery Jones,

    Roles Investigation, Writing – review & editing

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

  • Jake Heiner,

    Roles Investigation, Writing – review & editing

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

  • Mohammed Majrashi,

    Roles Investigation, Methodology

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

  • Timothy Moore,

    Roles Writing – review & editing

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

  • Rajesh Amin,

    Roles Resources, Writing – review & editing

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

  • Vishnu Suppiramaniam,

    Roles Resources, Writing – review & editing

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

  • Muralikrishnan Dhanasekaran

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    dhanamu@auburn.edu

    Affiliation Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States of America

SIRT3 activator Honokiol attenuates β-Amyloid by modulating amyloidogenic pathway

  • Sindhu Ramesh, 
  • Manoj Govindarajulu, 
  • Tyler Lynd, 
  • Gwyneth Briggs, 
  • Danielle Adamek, 
  • Ellery Jones, 
  • Jake Heiner, 
  • Mohammed Majrashi, 
  • Timothy Moore, 
  • Rajesh Amin
PLOS
x

Abstract

Honokiol (poly-phenolic lignan from Magnolia grandiflora) is a Sirtuin-3 (SIRT3) activator which exhibit antioxidant activity and augment mitochondrial functions in several experimental models. Modern evidence suggests the critical role of SIRT3 in the progression of several metabolic and neurodegenerative diseases. Amyloid beta (Aβ), the precursor to extracellular senile plaques, accumulates in the brains of patients with Alzheimer’s disease (AD) and is related to the development of cognitive impairment and neuronal cell death. Aβ is generated from amyloid-β precursor protein (APP) through sequential cleavages, first by β-secretase and then by γ-secretase. Drugs modulating this pathway are believed to be one of the most promising strategies for AD treatment. In the present study, we found that Honokiol significantly enhanced SIRT3 expression, reduced reactive oxygen species generation and lipid peroxidation, enhanced antioxidant activities, and mitochondrial function thereby reducing Aβ and sAPPβ levels in Chinese Hamster Ovarian (CHO) cells (carrying the amyloid precursor protein-APP and Presenilin PS1 mutation). Mechanistic studies revealed that Honokiol affects neither protein levels of APP nor α-secretase activity. In contrast, Honokiol increased the expression of AMPK, CREB, and PGC-1α, thereby inhibiting β-secretase activity leading to reduced Aβ levels. These results suggest that Honokiol is an activator of SIRT3 capable of improving antioxidant activity, mitochondrial energy regulation, while decreasing Aβ, thereby indicating it to be a lead compound for AD drug development.

Introduction

Alzheimer’s disease is a neurodegenerative disease characterized by a decline in cognition due to morphological and functional alterations to neurons. Pathologically, it is characterized by abnormal accumulation of extracellular senile plaques consisting of amyloid beta (Aβ), and intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein [1]. Epidemiological evidence shows that patients with type 2 diabetes mellitus have an increased risk of developing Alzheimer’s disease. This can be attributed to altered glucose metabolism, impaired insulin signaling, and insulin resistance [24]. Insulin resistance (IR) results in reduced glucose uptake and utilization, which compromises cell energy and homeostatic functions, thereby promoting oxidative stress and mitochondrial dysfunction. This energy deficiency results in the disruption of the neuronal cytoskeleton and synaptic connection [5,6]. Interestingly, brain insulin resistance has been known to accelerate the accumulation of Aβ and plaque formation in the brain by enhancing amyloidogenic processing of the amyloid precursor protein [7]. In addition, high insulin levels tend to inhibit Aβ degradation, thereby increasing amyloid accumulation which leads to neurodegeneration and irreversible cognitive dysfunction [8].

Mitochondria play a crucial role in the normal functioning of neurons and synapses by supplying constant energy in the form of ATP. Deficits in energy metabolism lead to increased oxidative stress and endoplasmic reticulum stress thereby promoting mitochondrial dysfunction. Oxidative stress results in the generation of excessive reactive oxygen species (ROS) and reactive nitrogen species (RNS) [912], which promotes the formation of lipid peroxides and damages the RNA, DNA, and proteins. Moreover, ROS can up regulate the expression of APP, β and γ-secretase to generate Aβ deposition, and fibrilization [5, 13, 14]. This Aβ in turn interacts with various mitochondrial proteins, disrupting the electron transport chain and increasing reactive oxygen species, thereby decreasing the levels of ATP [1517]. Therefore, oxidative stress and mitochondrial dysfunction may be significantly implicated in the development and progression of Alzheimer’s disease [18].

Sirtuins are a family of proteins that act predominantly as nicotinamide adenine dinucleotide (NAD)-dependent deacetylases, causing post-translational modifications in target proteins to regulate their function. Seven sirtuin family members exist, out of which SIRT3, SIRT4, and SIRT5 localize exclusively within mitochondria while the reminder of the sirtuins are localized within the cytoplasm and nucleus [19]. Acetylation causes proteins required for proper mitochondrial function to malfunction which leads to oxidative stress. These abnormalities are prevented by SIRT3 due to its deacetylating properties [2023]. In addition, SIRT3 has been shown to act as a pro-survival factor that plays an essential role in protecting neurons experiencing excitotoxicity [24]. Until recently, the only means to achieve high intracellular levels of SIRT3 was through calorie restriction and endurance exercise [2528]. However, Honokiol [2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol] has recently been considered to be a pharmacological activator of SIRT3 and known to modulate the pathologies of AD [29]. Honokiol, by binding to SIRT3 causes increased expression of AMPK-5' adenosine monophosphate-activated protein kinase, which plays a crucial role in cellular energy homeostasis. Additionally, it is known to increase the expression of PGC-1α. Furthermore, SIRT3 is a downstream target gene of PGC-1α and SIRT3 mediates the PGC-1α effects on cellular ROS production and mitochondrial biogenesis [30]. Honokiol has also been shown to possess non-adipogenic partial PPAR-γ agonistic activity [31]. PPAR-γ activity is known to promote glucose and lipid metabolism, oxidative phosphorylation, and mitochondrial biogenesis by increasing the expression of PGC-1α, a master regulator of mitochondrial biogenesis [32, 33]. PGC-1α decreases Aβ generation and increases non-amyloidogenic sAPPα levels by reducing the β-APP cleaving enzyme (BACE1 or β-secretase) gene transcription via PPAR-γ-dependent mechanism and directly through SIRT3 [34, 35].

Aβ is a proteolytic product of the amyloid-β precursor protein (APP) and is generated through sequential cleavages by enzymes called β- and γ-secretases. During this amyloidogenic processing, β-secretase first cleaves the type I transmembrane APP protein to generate an extracellular fragment known as sAPPβ and a membrane-associated carboxyl terminal fragment known as APP β-CTF. APP β-CTF is then cleaved by γ-secretase to release Aβ. Alternatively, APP can be subjected to a non-amyloidogenic processing and cleaved by α-secretase within the Aβ domain. α-secretase-mediated cleavage precludes Aβ generation and generates an extracellular domain of APP known as sAPPα instead [36,37]. β-cleavage of APP is the first and rate-limiting step in Aβ production. The transmembranous aspartic protease β-site APP cleaving enzyme 1 (BACE1) has been identified as the essential β-secretase in vivo [38]. The level and activity of BACE1 are found to be elevated in postmortem brain of sporadic AD patients [39, 40], suggesting a causative role of BACE1 in AD.

In this study, we hypothesize that Honokiol, a SIRT3 activator suppress oxidative stress, enhance mitochondrial functions and modulate Aβ levels by inhibiting BACE1 activity. PS70 cell lines (Chinese Hamster Ovarian cells expressing Swedish mutant APP (APPswe) and wild type human PSEN1 were used in this study. The swedish mutant APP (APPswe) has been shown to induce early AD-like histopathology with dispersed deposits of Aβ and aberrant tau protein expression [41, 42]. The PSEN1 gene and its protein are part of the γ-secretase complex which play a crucial role in processing APP and is known to increase Aβ levels [43]. The net effect of these two genes is increased secretion of Aβ by the cell which aids in studying the effect of Honokiol on the amyloidogenic pathway. Various other studies have employed APP-CHO cells to study and validate the amyloidogenic pathway [4446]. Therefore, this study aimed at elucidating the molecular mechanisms and signaling pathways by which Honokiol modulate Aβ levels in PS70 cells.

Materials and methods

Cell culture

PS70 cell lines (Chinese Hamster Ovary cells–CHO expressing Swedish mutant APP (APPswe) and wild type human PSEN1) was a kind gift from Dr. Raj Amin. Cells were grown in DMEM (VWR, USA) supplemented with 10% fetal bovine serum (FBS; Biosciences, USA), 100U/ml penicillin (Corning, USA) and 100μg/ml streptomycin (Corning, USA) in a humidified atmosphere of 5% CO2/95% air at 37°C. The cells were cultured in the presence of G418 (200 μg/ml, Invitrogen) and puromycin (7.5 μg/ml, ThermoFischer Scientific) to maintain selection for the expression plasmid. The cells were plated at an appropriate density according to each experimental scale.

Treatment strategies

Honokiol was purchased from Cayman chemicals, USA. Regarding the cell viability assay, different doses of Honokiol (0.5, 1, 2, 5, 10 and 20μM) were incubated with PS70 cells for 2 different times (24 and 48 hours) periods in the presence of insulin 10nM and serum. However, based on cell viability results and to elucidate the molecular mechanisms of action, PS70 cells were treated with Honokiol (5 and 10μM) for 24 and 48 hours. Insulin (10nM) was used as a positive control. To establish insulin resistance (IR) in PS70 cells, we used high concentrations of insulin (10nM) for both 24 and 48 hours [47]. Insulin-degrading enzyme (IDE) is involved in clearance of Aβ in the brain as both insulin and Aβ are catabolized by IDE [48]. In presence of high insulin, IDE is diverted to degrade insulin, consequently allowing APP-Aβ accumulation [49, 50]. IDE is thought to be a link connecting hyperinsulemia, IR, and AD [51, 52].

Cell viability assay

PS70 cells were seeded in 96-well plates with 1000 cells/well in culture medium and following their fixation, cells were treated with Honokiol (0.5, 1, 2, 5, 10 and 20μM concentrations) for 24 and 48 hours. Cell viability was assessed using the PrestoBlue® assay (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. Absorbance was measured by a spectrophotometer (BioTek, Winooski, VT, USA). The results were evaluated as percent of control and calculated as mean±SEM. Furthermore, microscopic imaging was performed on the PS70 cells to validate the cell viability and study the morphological changes seen at various concentrations.

Determination of ROS generation

Reactive oxygen species generation was estimated spectrofluorometerically by conversion of the non-fluorescent chloromethyl-DCF-DA (2′,7′-dichlorofluorescin diacetate, DCF-DA, VWR, USA) to fluorescent DCF at an excitation wavelength of 492 nm and an emission wavelength of 527 nm. The generation of ROS was measured, normalized to total protein content and reported as relative fluorescence intensity/mg protein. The fluorimetric reading was measured with BioTek Synergy HT plate reader (BioTek, VT, USA). Results were expressed as percentage change from the control [53].

Measurement of mitochondrial ROS levels

Mitochondrial ROS levels were measured using mitochondrial specific dihydro- 244 rhodamine (DHR) indicator purchased from Biotium. DHR is an uncharged non-fluorescent ROS indicator that accumulates in the mitochondria and becomes oxidized to the cationic rhodamine123, which exhibits a green fluorescence. The PS70 cell lines were stained according to the manufacture’s protocol. Fluorescence was measured using a multispectral-fluorescent plate reader (Bio-Tek) at excitation/emission wavelengths (λEx/ λEm) at 505/ 534 nm. [54].

Estimation of lipid peroxidation

Colorimetric assay procedure using thiobarbituric acid was used to quantify the lipid peroxide content. The index of lipid peroxidation was estimated by measuring the malondialdehyde (MDA) content in the form of thiobarbituric acid reactive substances (TBARS). TBARS was measured in the plate reader at 532 nm and calculated as TBARS formed per mg protein. Results were expressed as percentage change from the control [55, 56].

Assay of superoxide dismutase (SOD) activity

SOD activity was measured spectrophotometrically following the Marklund and Marklund method, using pyrogallol as substrate at 420 nm. Results were expressed as percentage control [57].

Estimation of catalase activity

Catalase activity was determined spectrophotometrically where the degradation of hydrogen peroxide is measured at 240 nm [58, 59]. Results were expressed as percentage control.

Glutathione peroxidase assay

Spectrophotometric estimation of glutathione peroxidase was performed according to the method of Lawrence and Burk [60]. The activity was calculated as glutathione (μmol) oxidized/mg total protein.

Glutathione reductase activity

Glutathione Reductase assay was performed spectrophotometrically using glutathione reductase assay kit (Cayman Chemicals, no. 703202). Values were based on a standard curve and calibrated to the total levels of protein concentrations.

Mitochondrial Complex-I activity

Mitochondrial Complex-I activity (NADH dehydrogenase activity) was assessed based on the NADH oxidation. Oxidation of NADH by the NADH dehydrogenase was measured spectrophotometrically at 340nm. Results were expressed as percentage control [61].

Mitochondrial Complex-IV activity

Complex-IV activity was based on the Cytochrome-c oxidation. The Cytochrome-C oxidation was measured spectrophotometrically at 550 nm and the Complex IV activity was expressed as cytochrome-C oxidized/mg protein [62].

Mitochondrial membrane potential assay

The mitochondrial membrane potential through microplate assay was measured in 96 well plate utilizing tetramethylrhodamine ethyl ester (TMRE) according to the manufacturer’s instructions (TMRE; Biotium, no.70016). TMRE florescent intensity (Ex: 549nm, Em: 575nm) was measured by a BioTek Synergy HT plate reader (BioTek, VT, USA). The results were expressed as percentage change from the control. In addition, imaging of the mitochondrial membrane potential was evaluated using fluorescence microscope with the fluorescent dye tetramethylrhodamine methyl ester TMR.

Western blot analysis

Conditioned media from treated cells were assayed for sAPPα, sAPPβ and secreted Aβ by Western blot. PS70cells were lysed in RIPA buffer (Roche, USA) and equal protein amounts of cell lysates were analyzed by Western blot. Each sample was denatured at 95°C for 5minutes before loading onto freshly prepared 10% SDS-PAGE gel for protein separation. Separated proteins on SDS-PAGE were transferred onto polyvinylidene fluoride membrane. Non-specific binding sites on the membranes were blocked with 5% fat-free milk in Tris-buffered saline plus 0.1% Tween-20 (TBST) at pH 7.4. The membranes were incubated overnight at 4°C with specific antibody constituted in 5% BSA in TBST. Primary antibodies used in this study included: AMPK (#2532), phospho-AMPK Thr172 (#2535), CREB (#4820) from CST; Anti-SIRT3 antibody (ab86671), Anti-PGC1α (ab54481), Anti-beta Amyloid 1–42 antibody (ab12267), Anti-beta Amyloid 1–40 antibody [BDI350] (ab20068), Anti-beta Actin antibody (ab8227), Anti-GAPDH (ab8245), Anti-ADAM10 antibody [EPR5622] (ab124695) from abcam; APP C-terminal antibody pAb751/770 (EMD Biosciences, La Jolla, CA, USA); Anti-BACE1 monoclonal antibody (MAB5308), anti-ADAM10 polyclonal antibody and Anti phospho-CREB (pAb06-519) from Merck Millipore; 6E10 (against sAPPα and β-CTF) and anti-sAPPβ antibodies from Covance. Membranes were then washed with TBST (3X, each for 10 min) and incubated with species dependent Goat Anti-Rabbit (H+L) IgG DyLight550 conjugated secondary antibodies (Invitrogen™) for 60 min at room temperature. Membranes were again washed three times for 10 minutes with TBST after incubation with each antibody. After washing, membranes were analyzed in FluorChem® system Imaging. Band densities for each sample were normalized to their respective β-actin or GAPDH signal and reported as percentage control.

α-secretase activity assay

The activity of α-secretase in cells was measured by using InnoZyme TACE Activity Kit (Millipore), following the manufacturer’s protocols.

β-secretase activity assay

β-site-APP cleaving enzyme (BACE) or β-Secretase activity was determined fluorimetrically with a commercially available β- Secretase activity kit (Biovison, California, USA) according to the manufacturer’s instructions. Beta-secretase activity was represented as relative fluorescence unit per mg of total protein.

Aβ ELISA assay

After treatment, conditioned media from the treated and untreated cells were collected to detect secreted Aβ1–40 and Aβ1–42. The Aβ1–40 and Aβ1–42 concentrations were quantified using ELISA kits following the manufacturer's protocol. The optical densities of each well at 450 nm were read on a microplate reader (Biotek FLx800, USA)] and the sample Aβ1–40 and Aβ1–42 concentrations were determined by comparison with the Aβ1–40 and Aβ1–42 standard curves. All readings were in the linear range of the assay.

Protein estimation

Protein quantification was determined using the Thermo Scientific Pierce 660 nm Protein Assay reagent kit (Pierce, Rockford, IL).

Statistical analysis

All data are expressed as means ± SEM. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by an appropriate post-hoc test including Tukey's and Dunnett's method (p< 0.05 was considered to indicate statistical significance). All statistical analyses were performed using the Prism-V software (La Jolla, CA, USA).

Results

Effect of Honokiol on PS70 cell viability

The effect of Honokiol treatment on the PS70 cell viability was assessed using Prestoblue® assay: PS70 cells were treated with various concentrations of Honokiol (0.5, 1, 2, 5, 10 and 20μM) for 24 and 48 hours. The control cells were treated with DMSO. As shown in (Fig 1A), when exposed to Honokiol concentrations of up to 10μM, there was no statistically significant change in the viability of PS70 cells as compared to the control (n = 12: p < 0.05). However, a significant decrease in cell viability was observed with 20μM Honokiol treatment (n = 12: p < 0.05). The microscopic images further validated the above findings by clearly showing (Fig 1B) no cell death up to 10μM concentrations of Honokiol. Similar to the Prestoblue® assay, cells treated with Honokiol (20μM) showed a relatively higher proportion of dead cells as compared to the control. Moreover, there were significant morphological changes observed with 20μM treatment. Honokiol (20μM) induced extensive shrinkage and fragmentation of PS70 cells, suggesting extensive cell death. Interestingly, we found both time dependent and concentration dependent effects of Honokiol on cell viability. Consequently, based on the results obtained, two highest concentrations of Honokiol (5 and 10μM) at which no cell death was noted were used in the subsequent experiments to elucidate the molecular mechanisms of Honokiol.

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Fig 1. Effect of honokiol on PS70 cells.

(A) PS70 cells were treated with various concentrations of honokiol (0, 1, 2, 5, 10 and 20μM) for 24 hours and 48 hours and then analyzed by Prestoblue cell viability assay. DMSO (0.1%) was used as the vehicle for honokiol. Data are expressed in terms of percent of control cells (non-honokiol-treated) as the means ± SE. ***P< 0.001 vs. vehicle-treated (control) cells. (B) Morphological changes in PS70 cells at 24h and 48 h following treatment with Honokiol (0, 1, 2, 5, 10 and 20μM). Scale bar = 100μm.

https://doi.org/10.1371/journal.pone.0190350.g001

Honokiol increases the activity of antioxidant enzymes in PS70 cells

A defense mechanism of the cell is to promote antioxidant expression and activity, which protects against highly reactive oxy or nitro radicals and their harmful toxic effects. We therefore investigated the effect of Honokiol on the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and glutathione reductase (GR). These are the key antioxidant enzymes that play a significant role in scavenging toxic free radicals. Honokiol (5 and 10μM) significantly increased the activity of SOD (1.2 and 1.6- fold, Fig 2A), GPX (1.1 and 1.4-fold, Fig 2B), CAT (1.3 and 1.5-fold, Fig 2C) and GRX (1.2 and 1.4-fold, Fig 2D) as compared to the control at 24 hours (n = 6, p < 0.05). Honokiol had similar effect on the activity of antioxidant enzymes at 48 hours. Insulin (10nM) significantly decreased the activity of SOD, CAT, GPX and GRX at 24 hours (Fig 2A–2D, n = 6, p < 0.05). At 48 hours, Insulin (10nM) significantly decreased the activity of SOD, and CAT only (Fig 2A–2D, n = 6, p < 0.05).

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Fig 2. Honokiol increases antioxidant enzymes on PS70 cells.

Effect of Honokiol (5 and 10 μM) and Insulin (10nM) on the activities of the antioxidant enzymes were assessed on PS70 cells over a 24 and 48 hours. Activity of (A) SOD, (B) GPx (C) CAT and (D) GR activity were determined as described above. The results are expressed as mean ± SEM (n = 6, #/*p<0.1, ##/**p<0.01, and ###/***p< 0.001 compared with the control). Data with multiple comparisons were analyzed using ANOVA with Dunnett's Multiple Comparison Test. [SOD, superoxide dismutase; GPx, glutathione-peroxidase; CAT, Catalase; GR, Glutathione Reductase].

https://doi.org/10.1371/journal.pone.0190350.g002

Honokiol scavenges reactive oxygen species and inhibits lipid peroxidation in PS70 cells

The generation of reactive oxygen species (ROS) triggers oxidative stress and induces irreversible oxidation of lipids and proteins, which has lethal effects on cells viability leading to cell death. Therefore, ROS-induced lipid peroxidation was also investigated in the present study. With respect to the DCF based ROS assay, Honokiol (5 and 10μM) significantly decreased the ROS generation at 24 hours by (14% and 40%) and 48 hours by (29% and 56%) as compared to the control (Fig 3A, n = 6, p <0.05).Insulin (10nM) significantly increased the generation of ROS (43% and 52%) at both the time point (Fig 3A, n = 6, p < 0.05). DHR fluorescent dye was used to further validate the effect of Honokiol and insulin on ROS generation. DHR fluorescent assay yielded similar results as compared to the DCF assay on the ROS generation (Fig 3B, n = 6, p < 0.05). Due to the increase in ROS at 24 and 48 hours, insulin (10nM) significantly increased lipid peroxidation (33 and 52%) as compared to the control (Fig 3C, n = 6, p < 0.05). Since, Honokiol (5 and 10μM) significantly scavenged the ROS; it resulted in decreased lipid peroxide formation by (29% and 36%) at 24 hours and by (30% and 40%) at 48 hours (Fig 3C, n = 6, p < 0.05).

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Fig 3. Honokiol scavenges ROS and inhibits lipid peroxidation in PS70 cells.

PS70 cells were incubated with Insulin (10nM) and Honokiol (5 and 10 μM). ROS generation was assayed using DCF dye and measured with a spectrophotometer (A) and mitochondrial ROS was measured using DHR assay(B). Lipid peroxidation was measured with a spectrophotometer using the TBARS method (C). The results are expressed as mean ± SEM (#/*p<0.1, ##/**p<0.01, and ###/***p< 0.001 compared with the control). Data with multiple comparisons were analyzed using ANOVA with Dunnett's Multiple Comparison Test (n = 6).

https://doi.org/10.1371/journal.pone.0190350.g003

Honokiol improves mitochondrial bioenergetics

To explore the effects of Honokiol on mitochondrial bioenergetics and to understand the molecular processes of mitochondrial function, we evaluated the effects of Honokiol on Complex-I, Complex-IV activity and mitochondrial membrane potential. Honokiol (5 and 10μM) notably improved mitochondrial bioenergetics, as demonstrated by significant increase in Complex-I (29% and 45%) and Complex-IV (43% and 74%) at 24 hours. Similar results with Complex I (52% and 57%) and with Complex IV (61% and 86%) were noted at 48 hours (Fig 4A and 4B, n = 6, p < 0.05). Likewise, Honokiol (5 μM and 10μM) also increased the mitochondrial membrane potential significantly (13% and 39%) at 24 hours and (21% and 35%) at 48 hours. (Fig 4C, n = 6, p < 0.05). Insulin (10nM) significantly inhibited Complex-I activity at 24 and 48 hours (24% and 25%) and Complex-IV activity by (32% and 33%) (Fig 4A–4C, n = 6, p < 0.05). Similar results were noted with insulin (10nM) on MMP activity at both 24 and 48 hours.

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Fig 4. Honokiol improve mitochondrial bioenergetics.

Cell lysate analyzed for activities of mitochondrial electron transport chain complex -I and Complex IV activity. The specific activities of complexes I and IV were normalized with respect to specific activities in their corresponding control groups (A and B). All samples are averages ± SEM (n = 6) and (**p<0.01, and ***p< 0.001 compared with the control. Mitochondrial membrane potential measured at 24 and 48 hours (C) following staining of cells with TMRE dye and detected using a fluorescence microscope. Magnification, ×20. Average fluorescence intensity.

https://doi.org/10.1371/journal.pone.0190350.g004

Honokiol treatment reduces Aβ secretion

To study whether Honokiol can affect Aβ generation, we measured total Aβ levels by western blot and the results showed that Honokiol (5 and 10μM) significantly decreased total intracellular Aβ (48% and 61%) at 24 hours (Fig 5A). At the same concentration range, Honokiol also reduced total secreted Aβ. Insulin (10nM) showed significantly increased levels of Amyloid-beta levels compared to control. Next, we performed the concentration-response effect of Honokiol (0, 0.2, 0.5, 1, 2, 5, 10 and 15μM) on the generation of Aβ-42. As shown in Fig 5B, there was a dose-dependent decrease in the generation of Aβ-42 in the media by Honokiol. Lower doses (0.1–1μM) of Honokiol had no significant effect. However, there was a significant decrease at 2μM (28%) and a robust decrease at 5 and 10μM Honokiol (40% and 60% respectively). These results clearly confirm that Honokiol can dose dependently decrease Aβ-42 production in this cell-based model. Furthermore, to validate our findings, previous other studies using Honokiol have shown the cytoprotective and neuroprotective effects at 5 and 10μM dose [63]. When PS70 cells were treated with Honokiol (5 and 10μM) for 24 h, levels of Aβ40 and Aβ42 (Fig 5C) in conditioned media were markedly decreased in a dose-dependent manner. On the contrary, Insulin 10nM increased the levels of both Aβ40 and Aβ42 respectively.

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Fig 5. Honokiol treatment reduced Aβ secretion.

PS70 cells were treated with DMSO (negative control), Insulin (10nM) or indicated doses of Honokiol for 24h. (A) TsAβ (Total secreted Aβ) and TiAβ (total intracellular Aβ) levels were then analyzed by WB using respective antibodies. The Western blots shown are representative of at least three independent experiments. Densitometric quantification was performed and expressed as percentage change. (B) Concentration response curve of Honokiol shows a dose dependent reduction of Aβ42 production by Honokiol. (C) ELISA measurements of secreted Aβ40 and Aβ42 in conditioned-medium collected from DMSO, Insulin and Honokiol treated PS70 cells. The Aβ results are represented as the mean±SEM of nanograms of Aβ40 or Aβ42 normalized to the amount of total protein [mg] extracted from the cells in the corresponding well. These results are representative of four independent experiments with n = 3 for each condition. (One-way ANOVA followed by Dunnett’s post hoc test, n = 3, **: p< 0.01, ***: p< 0.001).

https://doi.org/10.1371/journal.pone.0190350.g005

Honokiol modulates amyloidogenic pathway

Because β-secretase-mediated APP processing is the first step leading to Aβ generation, we studied whether Honokiol affects β-secretase. To ascertain this possibility, we carried out a cell-based assay to measure the β-secretase activity and found that Honokiol dramatically inhibited β-secretase activity by (26% and 44%) at 24 hours and (37% and 60%) at 48 hours (Fig 6A).These results indicate that Honokiol at 10μM exhibited IC50 activity towards β-secretase. Western Blot analysis to detect protein expression showed significantly decreased expression of β-secretase at both the doses (Fig 6B). Since there is another possibility that Honokiol inhibits APP β-processing and Aβ generation through promoting α-secretase activity, we also assayed the activity of TACE, a major α-secretase in PS70 cells treated with Honokiol. We found that Honokiol did not affect TACE activity (Fig 6E), suggesting that Honokiol does not affect α-secretase activity. Honokiol treatment dose-dependently decreased the secreted level of sAPPβ, an amino-terminal fragment of APP generated by β-secretase cleavage by 48% and 40% respectively (Fig 6C). Consistently, the level of APP β-CTF (a carboxyl-terminal fragment of APP generated by β-secretase cleavage) was also decreased upon Honokiol treatment by 23% and 40% (Fig 6D). These results suggest that Honokiol inhibits β-cleavage of APP. In addition, Honokiol (10μM) increased the level of secreted sAPPα, the major extracellular fragment of APP released by α-secretase cleavage (Fig 6C). Moreover, we found that Honokiol treatment did not affect protein levels of APP, and α-secretase ADAM10 (Fig 6D). These results suggest that Honokiol reduces APP amyloidogenic processing not through affecting α but through β-secretase levels [64].

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Fig 6. Honokiol treatment reduces amyloidogenic pathway by inhibits β-secretase activity and reducing APP β-CTF and sAPPβ levels.

PS70 cells were treated with DMSO (negative control), Insulin (10nM) or indicated doses of Honokiol for 24h. (A) Cell lysates were assayed for β-secretase activity by using a commercial kit from Biovision and subjected to comparison. (B) Cell lysates were processed and examined for BACE expression in Western blots with anti-BACE1 antibodies. β-Actin was used as a loading control. (C) Conditioned media and (D) cell lysates were analyzed by WB using respective antibodies. The Western blots shown are representative of at least three independent experiments. Densitometric quantification was performed and expressed as percentage change. (E) PS70 cells treated with DMSO, insulin 10nM, Honokiol (5 & 10μM) and α-secretase inhibitor TAPI-1 (10μM) for 24 hours. Cell lysates were assayed for α-secretase activity for comparison (One-way ANOVA followed by Dunnett’s post hoc test, n = 3, *: p<0.05, **: p< 0.01, ***: p< 0.001).

https://doi.org/10.1371/journal.pone.0190350.g006

Honokiol increases SIRT3 and activates AMPK-CREB-PGC1α pathway

To confirm that high insulin levels predispose to increased Aβ formation, we used increasing concentrations of Insulin (0, 1, 5 and 10nM) on PS70 cells and found that Aβ levels (normalized to β-actin) increased significantly (1.12-, 1.28- and 1.46-fold) compared to control (Fig 7A). Both doses (5 and 10μM) of Honokiol increased SIRT3 levels by nearly twofold and optimum SIRT3 activation was found to be at 24h (Fig 7B). Furthermore, we explored the molecular signaling pathway related to the reduction of Aβ levels by Honokiol. Honokiol (5μMand 10μM) increased the phosphorylation of AMPK by (1.37- and 1.5- fold) compared to total AMPK at 24 hours. Similarly, phosphorylation of CREB was increased by (1.42- and 1.61-fold) with respect to total CREB (Fig 7C). These phosphorylation changes of AMPK and CREB in turn are found to increase the levels of PGC1α. Similarly, we found a statistically significant increase in the levels of PGC1α (1.75) fold at 10μM normalized to GADPH, but no effect was noticed at 5μM (Fig 7C, n = 3, p < 0.05). Insulin (10nM) decreased but did not show a statistically significant change in the phosphorylation of AMPK, CREB and PGC1α.

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Fig 7. Effect of Honokiol on SIRT3 activation and AMPK-CREB-PGC1α pathway.

Effect of increasing concentrations of insulin (0, 1, 5 and 10nM) on TsAβ (Total secreted Aβ) along with the densitometric analysis of band intensity normalized to β-Actin. (B) Representative Western blot of SIRT3 performed on whole cell lysates from PS70 cells exposed to either vehicle (DMSO) or 5 and 10μM Honokiol for 24 and 48 h. The graph displays the densitometric analysis of band intensity of the SIRT3 normalized to the corresponding GADPH level, used as loading control. (C) Effect of Insulin 10nM and Honokiol (5 and 10μM) on p-AMPK/AMPK ratio. Representative Western blot of total AMPK and phosphorylated-AMPK (p-AMPK) levels, total CREB and phosphorylated-CREB (p-CREB) levels and PGC-1α performed on whole cell lysates from PS70 cells exposed to either vehicle, Insulin or Honokiol for 24 h. The graph displays the statistical analysis of the p-AMPK/AMPK and p-CREB/CREB ratio calculated by densitometric analysis of band intensity normalized to the corresponding β-Actin used as loading control; PGC-1α normalized to corresponding GAPDH level, used as loading control. Data, means ± SEM are expressed as percentage of vehicle-treated control; n = 3 under each condition. Significance was calculated with Student's t test, *p < 0.05, vs. vehicle-treated cells.

https://doi.org/10.1371/journal.pone.0190350.g007

Discussion

In this study, we report that Honokiol, an activator of SIRT3 attenuated oxidative stress and beta amyloid secretion in PS70 cells, in addition to improving mitochondrial function. High dose insulin treatment caused increased ROS levels, decrease in mitochondrial functions and increased formation of beta amyloid. Honokiol counteracted these effects by activating SIRT3 and by increasing in AMPK, CREB and PGC1α protein levels thereby causing reduction in beta-secretase activity. Compelling evidence has shown that Honokiol, a SIRT3 activator, expresses many beneficial effects in neurodegenerative diseases [65]. However, there are very few studies that have elucidated the novel mechanisms of SIRT3-mediated decrease in Aβ production. To the best of our knowledge this is the first report describing an activator of SIRT3 capable of improving mitochondrial function, and blocking the beta secretase activity thereby decreasing beta amyloid secretion.

Insulin resistance (IR) is an important risk factor for Alzheimer’s disease and causes an increase in age-related memory impairment [66, 67]. Presence of insulin receptors in the hippocampus and the medial temporal cortex indicate that insulin is known to influence memory and learning [68]. Optimal cerebral insulin levels augment memory and synaptic plasticity in the hippocampus and areneuroprotective [68, 69]. On the contrary, insulin resistance characterized by high insulin levels has been associated with increased levels of reactive oxygen species [70]. High insulin levels promote increased Aβ deposition and tau protein phosphorylation [7173]. In this study, we further validated the cellular effects of hyperinsulinemia on cognitive impairment. High dose insulin, representing IR, showed an increase in ROS and lipid peroxidation; decrease in the activity of antioxidants; decrease in phosphorylated AMPK, pCREB, and PGC-1α expression. These deficits resulted in decreased mitochondrial functions, increased BACE, and increased Aβ in the PS70 cells. Thus, our results concur with the existing literature showing that hyperinsulinemia can enhance Aβ production.

Oxidative damage has been known to occur at a very early stage of Alzheimer’s disease even prior to Aβ plaque formation and the onset of symptoms [7476]. Several cellular changes caused by oxidative stress have been related to Aβ plaque formation and pathophysiological events of Alzheimer’s disease [77]. Increased ROS occurs due to an imbalance between pro-oxidants (ROS, RNS, superoxide anion, hydroxyl radicals, and hydrogen peroxide) and antioxidants (GSH, GPX, CAT, GRx, SOD). In addition, ROS leads to deficits in membrane integrity, oxidation of mitochondrial proteins, damage to the mitochondrial respiratory chain, changes in mitochondrial membrane permeability and structure and increased permeability of the plasma membrane to Ca2+[78]. Down-regulation in antioxidant defense mechanisms and elevated ROS generation leads to oxidative stress-mediated neurodegeneration [79].

Exposure of polyunsaturated fatty acids to ROS leads to the production of toxic lipid peroxidation products. Similarly, an increase in the levels of lipid peroxidation was observed in Aβ-induced rat hippocampal cells, due to depletion of antioxidants and increased pro-oxidants [80]. Furthermore, Honokiol has been shown to exert beneficial effects on Aβ-induced toxicity in PC12 cells by inhibiting oxidative stress through reduction of ROS production, intracellular Ca2+ elevation, and caspase-3 activity [81]. In this study, we have reported that Honokiol treatment significantly increased enzymatic antioxidant activities, decreased ROS generation, and decreased lipid peroxidation in PS70 cells. Oxidative stress subsequently leads to impairment of mitochondrial dysfunction [82], which leads to Aβ formation and Aβ induced neurotoxicity [8386]. At the mitochondrial level, complex I and complex IV seem to be specifically targeted; tau pathology mainly impairs complex I activity and Aβ impairs complex IV activity [87]. Importantly, mitochondrial dysfunction and reduced bioenergetics occur early in pathogenesis and precede the development of plaque formation [88]. Interestingly, hyperinsulinemia has also shown to decrease mitochondrial functions [89]. Our results showed that Honokiol increases the activities of Complex I and IV and increased the mitochondrial membrane potential thereby indicating that it enhances the mitochondrial function.

Furthermore, AMPK is a kinase considered to be a metabolic sensor which is implicated in the regulation of IR and Aβ pathology [90]. Evidence shows that activation of AMPK decreases the production levels of Aβ and AMPK activators like resveratrol have been shown to increase the lysosomal clearance of Aβ [91, 92]. In addition, AMPK enhances mitochondrial biogenesis by inducing PGC-1α transcription and by phosphorylating PGC-1α at threonine-177 and serine-538 [93]. This increased PGC-1α has been shown to decrease BACE and Aβ production. Honokiol increased the phosphorylation of AMPK in a dose-dependent manner and in the same concentration range, increased the phosphorylation of CREB. Together, these results indicate that one of the primary effects of Honokiol is to target AMPK to increase its phosphorylation at Thr-172 and to promote its activation. Furthermore, downstream of AMPK, there is increased phosphorylation of CREB which promotes the activation of PGC-1α. In turn, PGC-1α reduces the activity of β-secretase; reducing Aβ generation through a PPAR-γ-dependent mechanism [94, 95]. Alternatively, SIRT3 is known to directly up regulate the expression of PGC-1α, which increases SIRT3 gene expression [96]. In our study, Honokiol increased the protein levels of AMPK, CREB and PGC-1α thereby decreasing Aβ.

Interestingly, Honokiol had a major role in modulating amyloidogenic pathway. Honokiol had no effect on total APP levels, protein levels of α-secretase ADAM10 and cell based TACE activity, indicating that Honokiol does not affect α-secretase. In contrast, Honokiol treatment decreased protein levels of β-secretase BACE1 and reduced BACE1 enzyme activity, as well as both sAPPβ and APP β–CTF levels, indicating that Honokiol reduces Aβ generation probably through inhibiting β-secretase activity. Hence, we found a modest increase in sAPPα release. Since, γ-secretase complex is part of downstream signaling of both amyloidogenic and non-amyloidogenic pathway, we did not investigate the effect of Honokiol on γ-secretase.

Together, our results demonstrate that Honokiol can reduce Aβ generation in vitro thereby opening avenues for it to be a lead compound for AD drug development.

Conclusion

Honokiol, a dual SIRT3 activator and PPAR-γ agonist, attenuated the markers of oxidative stress, improved cellular antioxidant defense systems, and altered the AMPK pathway, leading to enhanced mitochondrial functions thereby having a modulatory effect on amyloidogenic pathway and eventually decreasing Aβ levels (Fig 8). Overall, these findings demonstrate a potential mitochondrial protective and Aβ reducing effect of Honokiol in PS70 cells. This mechanistic study of Honokiol to suppress pro-oxidative pathways, improve mitochondrial function, and reduce Aβ production prompts further in vitro studies on neuronal cell lines and in vivo studies to elucidate the neuroprotective effects of Honokiol in AD. Identifying these functions of Honokiol and their relations to AD will give rise to therapeutic avenues where new concepts can be developed to find an effective treatment.

In the mitochondria, Honokiol binds to SIRT3 and increases the level of SIRT3 through a positive feedback mechanism. Increased levels of SIRT3 enhances mitochondrial biogenesis thereby promoting mitochondrial function and attenuates Amyloid beta levels by acting through AMP-CREB-PGC1α pathway. In the nucleus, increased PGC1α levels promote mitochondrial biogenesis and attenuate amyloid beta levels.

Acknowledgments

PS70 cell lines [Chinese Hamster Ovary cells–CHOexpressing Swedish mutant APP (APPswe) and wild type human PSEN1] were kindly provided by Dr. Rajesh Amin.

References

  1. 1. Kumar A, Singh A, Ekavali. A review on Alzheimer’s disease pathophysiology and its management: an update. Pharmacol Reports [Internet]. 2015 Apr [cited 2017 Jun 11];67(2):195–203. Available from: http://linkinghub.elsevier.com/retrieve/pii/S1734114014002886
  2. 2. Talbot K, Wang H-Y. The nature, significance, and glucagon-like peptide-1 analog treatment of brain insulin resistance in Alzheimer’s disease. Alzheimer’s Dement [Internet]. 2014 Feb [cited 2017 Jun 12];10(1):S12–25. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24529520
  3. 3. Tolppanen A-M, Solomon A, Soininen H, Kivipelto M. Midlife vascular risk factors and Alzheimer’s disease: evidence from epidemiological studies. J Alzheimers Dis [Internet]. 2012 [cited 2017 Jun 12];32(3):531–40. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22842867 pmid:22842867
  4. 4. Steen E, Terry BM, Rivera EJ, Cannon JL, Neely TR, Tavares R, et al. Impaired insulin and insulin-like growth factor expression and signaling mechanisms in Alzheimer’s disease—is this type 3 diabetes? J Alzheimers Dis [Internet]. 2005 Feb [cited 2017 Jun 12];7(1):63–80. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15750215 pmid:15750215
  5. 5. de la Monte SM, Tong M. Brain metabolic dysfunction at the core of Alzheimer’s disease. Biochem Pharmacol [Internet]. 2014 Apr 15 [cited 2017 Jun 12];88(4):548–59. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24380887 pmid:24380887
  6. 6. de la Monte SM. Brain insulin resistance and deficiency as therapeutic targets in Alzheimer’s disease. Curr Alzheimer Res [Internet]. 2012 Jan [cited 2017 Jun 12];9(1):35–66. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22329651 pmid:22329651
  7. 7. Craft S. Insulin Resistance Syndrome and Alzheimer Disease: Pathophysiologic Mechanisms and Therapeutic Implications. Alzheimer Dis Assoc Disord [Internet]. 2006 Oct [cited 2017 Jun 12];20(4):298–301. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17132977 pmid:17132977
  8. 8. Grasso G, Mielczarek P, Niedziolka M, Silberring J. Metabolism of Cryptic Peptides Derived from Neuropeptide FF Precursors: The Involvement of Insulin-Degrading Enzyme. Int J Mol Sci [Internet]. 2014 Sep 22 [cited 2017 Jun 12];15(9):16787–99. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25247577 pmid:25247577
  9. 9. HOYER S, LANNERT H. Inhibition of the Neuronal Insulin Receptor Causes Alzheimer-like Disturbances in Oxidative/Energy Brain Metabolism and in Behavior in Adult Rats. Ann N Y Acad Sci [Internet]. 1999 Nov [cited 2017 Jun 12];893(1 OXIDATIVE/ENE):301–3. Available from: http://doi.wiley.com/10.1111/j.1749-6632.1999.tb07842.x
  10. 10. de la Monte SM, Wands JR. Chronic gestational exposure to ethanol impairs insulin-stimulated survival and mitochondrial function in cerebellar neurons. Cell Mol Life Sci [Internet]. 2002 May [cited 2017 Jun 12];59(5):882–93. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12088287 pmid:12088287
  11. 11. Hoyer S, Lee SK, Löffler T, Schliebs R. Inhibition of the neuronal insulin receptor. An in vivo model for sporadic Alzheimer disease? Ann N Y Acad Sci [Internet]. 2000 [cited 2017 Jun 12];920:256–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11193160 pmid:11193160
  12. 12. De la Monte SM. Contributions of Brain Insulin Resistance and Deficiency in Amyloid-Related Neurodegeneration in Alzheimer’s Disease. Drugs [Internet]. 2012 Jan 1 [cited 2017 Jun 12];72(1):49–66. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22191795 pmid:22191795
  13. 13. Smith IF, Boyle JP, Green KN, Pearson HA, Peers C. Hypoxic remodelling of Ca2+ mobilization in type I cortical astrocytes: involvement of ROS and pro-amyloidogenic APP processing. J Neurochem [Internet]. 2004 Feb [cited 2017 Jun 12];88(4):869–77. Available from: http://www.ncbi.nlm.nih.gov/pubmed/14756807 pmid:14756807
  14. 14. Tamagno E, Guglielmotto M, Aragno M, Borghi R, Autelli R, Giliberto L, et al. Oxidative stress activates a positive feedback between the γ- and β-secretase cleavages of the β-amyloid precursor protein. J Neurochem [Internet]. 2007 Nov 14 [cited 2017 Jun 12];0(0):071115163316002–??? Available from: http://www.ncbi.nlm.nih.gov/pubmed/18005001
  15. 15. Musiek ES, Holtzman DM. Three dimensions of the amyloid hypothesis: time, space and “wingmen.” Nat Neurosci [Internet]. 2015 May 26 [cited 2017 Jun 12];18(6):800–6. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26007213 pmid:26007213
  16. 16. Manczak M, Anekonda TS, Henson E, Park BS, Quinn J, Reddy PH. Mitochondria are a direct site of Aβ accumulation in Alzheimer’s disease neurons: implications for free radical generation and oxidative damage in disease progression. Hum Mol Genet [Internet]. 2006 Mar 16 [cited 2017 Jun 12];15(9):1437–49. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16551656 pmid:16551656
  17. 17. Mungarro-Menchaca X, Ferrera P, Morán J, Arias C. β-Amyloid peptide induces ultrastructural changes in synaptosomes and potentiates mitochondrial dysfunction in the presence of ryanodine. J Neurosci Res [Internet]. 2002 Apr 1 [cited 2017 Jun 12];68(1):89–96. Available from: http://doi.wiley.com/10.1002/jnr.10193 pmid:11933053
  18. 18. Pappolla MA, Chyan YJ, Omar RA, Hsiao K, Perry G, Smith MA, et al. Evidence of oxidative stress and in vivo neurotoxicity of beta-amyloid in a transgenic mouse model of Alzheimer’s disease: a chronic oxidative paradigm for testing antioxidant therapies in vivo. Am J Pathol [Internet]. 1998 Apr [cited 2017 Jun 12];152(4):871–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9546346 pmid:9546346
  19. 19. Michishita E, Park JY, Burneskis JM, Barrett JC, Horikawa I. Evolutionarily Conserved and Nonconserved Cellular Localizations and Functions of Human SIRT Proteins. Mol Biol Cell [Internet]. 2005 Jul 19 [cited 2017 Jun 12];16(10):4623–35. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16079181 pmid:16079181
  20. 20. Hirschey MD, Shimazu T, Goetzman E, Jing E, Schwer B, Lombard DB, et al. SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation. Nature [Internet]. 2010 Mar 4 [cited 2017 Jun 12];464(7285):121–5. Available from: http://www.nature.com/doifinder/10.1038/nature08778 pmid:20203611
  21. 21. Ahn B-H, Kim H-S, Song S, Lee IH, Liu J, Vassilopoulos A, et al. A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proc Natl Acad Sci U S A [Internet]. 2008 Sep 23 [cited 2017 Jun 12];105(38):14447–52. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18794531 pmid:18794531
  22. 22. Hallows W, Albaugh B, Denu J. Where in the cell is SIRT3?—functional localization of an NAD + -dependent protein deacetylase. Biochem J [Internet]. 2008 Apr 15 [cited 2017 Jun 12];411(2):e11–3. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18363549 pmid:18363549
  23. 23. Shi T, Wang F, Stieren E, Tong Q. SIRT3, a Mitochondrial Sirtuin Deacetylase, Regulates Mitochondrial Function and Thermogenesis in Brown Adipocytes. J Biol Chem [Internet]. 2005 Apr 8 [cited 2017 Jun 12];280(14):13560–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15653680 pmid:15653680
  24. 24. Kim SH, Lu HF, Alano CC, Zheng J, Zhang J. Neuronal Sirt3 Protects against Excitotoxic Injury in Mouse Cortical Neuron Culture. Combs C, editor. PLoS One [Internet]. 2011 Mar 1 [cited 2017 Jun 12];6(3):e14731. Available from: http://dx.plos.org/10.1371/journal.pone.0014731 pmid:21390294
  25. 25. Someya S, Yu W, Hallows WC, Xu J, Vann JM, Leeuwenburgh C, et al. Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-Related Hearing Loss under Caloric Restriction. Cell [Internet]. 2010 Nov 24 [cited 2017 Jun 12];143(5):802–12. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21094524 pmid:21094524
  26. 26. Qiu X, Brown K, Hirschey MD, Verdin E, Chen D. Calorie Restriction Reduces Oxidative Stress by SIRT3-Mediated SOD2 Activation. Cell Metab [Internet]. 2010 Dec 1 [cited 2017 Jun 12];12(6):662–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21109198 pmid:21109198
  27. 27. Palacios OM, Carmona JJ, Michan S, Chen KY, Manabe Y, III JLW, et al. Diet and exercise signals regulate SIRT3 and activate AMPK and PGC-1α in skeletal muscle. Aging (Albany NY) [Internet]. 2009 Aug 15 [cited 2017 Jun 12];1(9):771–83. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20157566
  28. 28. Heilbronn LK, Ravussin E. Calorie restriction and aging: review of the literature and implications for studies in humans. Am J Clin Nutr [Internet]. 2003 Sep [cited 2017 Jun 12];78(3):361–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12936916 pmid:12936916
  29. 29. Woodbury A, Yu SP, Wei L, García P. Neuro-modulating effects of honokiol: a review. Front Neurol [Internet]. 2013 [cited 2017 Jun 12];4:130. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24062717 pmid:24062717
  30. 30. Kong X, Wang R, Xue Y, Liu X, Zhang H, Chen Y, et al. Sirtuin 3, a New Target of PGC-1α, Plays an Important Role in the Suppression of ROS and Mitochondrial Biogenesis. Deb S, editor. PLoS One [Internet]. 2010 Jul 22 [cited 2017 Jun 12];5(7):e11707. Available from: http://dx.plos.org/10.1371/journal.pone.0011707 pmid:20661474
  31. 31. Atanasov AG, Wang JN, Gu SP, Bu J, Kramer MP, Baumgartner L, et al. Honokiol: A non-adipogenic PPARγ agonist from nature. Biochim Biophys Acta—Gen Subj [Internet]. 2013 Oct [cited 2017 Jun 12];1830(10):4813–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23811337
  32. 32. Puigserver P, Spiegelman BM. Peroxisome Proliferator-Activated Receptor-γ Coactivator 1α (PGC-1α): Transcriptional Coactivator and Metabolic Regulator. Endocr Rev [Internet]. 2003 Feb [cited 2017 Jun 12];24(1):78–90. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12588810 pmid:12588810
  33. 33. Austin S, St-Pierre J. PGC1α and mitochondrial metabolism—emerging concepts and relevance in ageing and neurodegenerative disorders. J Cell Sci [Internet]. 2012 Nov 1 [cited 2017 Jun 12];125(21):4963–71. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23277535
  34. 34. Katsouri L, Parr C, Bogdanovic N, Willem M, Sastre M. PPARγ co-activator-1α (PGC-1α) reduces amyloid-β generation through a PPARγ-dependent mechanism. J Alzheimers Dis [Internet]. 2011 [cited 2017 Jun 12];25(1):151–62. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21358044 pmid:21358044
  35. 35. Qin W, Haroutunian V, Katsel P, Cardozo CP, Ho L, Buxbaum JD, et al. PGC-1α Expression Decreases in the Alzheimer Disease Brain as a Function of Dementia. Arch Neurol [Internet]. 2009 Mar 1 [cited 2017 Jun 12];66(3):352–61. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19273754 pmid:19273754
  36. 36. Zheng H, Koo EH. Biology and pathophysiology of the amyloid precursor protein. Mol Neurodegener [Internet]. 2011 [cited 2017 Dec 6];6:27. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098799/pdf/1750-1326-6-27.pdf pmid:21527012
  37. 37. Zhang Y, Thompson R, Zhang H, Xu H. APP processing in Alzheimer’s disease. Mol Brain [Internet]. 2011 Jan 7 [cited 2017 Dec 6];4(1):3. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21214928
  38. 38. Vassar R., Bennett B. D., Babu-Khan S., Kahn S., Mendiaz E. A., Denis P., … Citron M. (1999). Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science (New York, N.Y.), 286(5440), 735–41. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10531052
  39. 39. Yang L.-B., Lindholm K., Yan R., Citron M., Xia W., Yang X.-L., Shen Y. (2003). Elevated beta-secretase expression and enzymatic activity detected in sporadic Alzheimer disease. Nature Medicine, 9(1), 3–4. https://doi.org/10.1038/nm0103-3 pmid:12514700
  40. 40. Li R., Lindholm K., Yang L.-B., Yue X., Citron M., Yan R., … Shen Y. (2004). Amyloid beta peptide load is correlated with increased beta-secretase activity in sporadic Alzheimer’s disease patients. Proceedings of the National Academy of Sciences of the United States of America, 101(10), 3632–7. https://doi.org/10.1073/pnas.0205689101 pmid:14978286
  41. 41. Shin J-Y, Yu S-B, Yu U-Y, Ahnjo S-M, Ahn J-H. Swedish mutation within amyloid precursor protein modulates global gene expression towards the pathogenesis of Alzheimer’s disease. BMB Rep [Internet]. 2010 Oct 31 [cited 2017 Aug 7];43(10):704–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21034535 pmid:21034535
  42. 42. Haass C, Lemere CA, Capell A, Citron M, Seubert P, Schenk D, et al. The Swedish mutation causes early-onset Alzheimer’s disease by beta-secretase cleavage within the secretory pathway. Nat Med [Internet]. 1995 Dec [cited 2017 Aug 7];1(12):1291–6. Available from: http://www.ncbi.nlm.nih.gov/pubmed/7489411 pmid:7489411
  43. 43. Duff K, Eckman C, Zehr C, Yu X, Prada C-M, Perez-tur J, et al. Increased amyloid-β42(43) in brains of mice expressing mutant presenilin 1. Nature [Internet]. 1996 Oct 24 [cited 2017 Jun 13];383(6602):710–3. Available from: http://www.ncbi.nlm.nih.gov/pubmed/8878479 pmid:8878479
  44. 44. Obregon D, Hou H, Deng J, Giunta B, Tian J, Darlington D, et al. Soluble amyloid precursor protein-α modulates β-secretase activity and amyloid-β generation. 2012 [cited 2017 Aug 7]; Available from: https://www.nature.com/articles/ncomms1781.pdf?proof=true pmid:22491325
  45. 45. Deng J, Hou H, Giunta B, Mori T, Wang Y-J, Fernandez F, et al. Autoreactive-Aβ antibodies promote APP β-secretase processing. J Neurochem [Internet]. 2012 Mar [cited 2017 Aug 7];120(5):732–40. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22188568 pmid:22188568
  46. 46. Hahn S, Brüning T, Ness J, Czirr E, Baches S, Gijsen H, et al. Presenilin-1 but not amyloid precursor protein mutations present in mouse models of Alzheimer’s disease attenuate the response of cultured cells to γ-secretase modulators regardless of their potency and structure. J Neurochem [Internet]. 2011 Feb 1 [cited 2017 Aug 7];116(3):385–95. Available from: http://doi.wiley.com/10.1111/j.1471-4159.2010.07118.x pmid:21091478
  47. 47. Thomson MJ, Williams MG, Frost SC. Development of insulin resistance in 3T3-L1 adipocytes. J Biol Chem [Internet]. 1997 Mar 21 [cited 2017 Jun 12];272(12):7759–64. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9065437 pmid:9065437
  48. 48. Qiu WQ, Walsh DM, Ye Z, Vekrellis K, Zhang J, Podlisny MB, et al. Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J Biol Chem [Internet]. 1998 Dec 4 [cited 2017 Jun 12];273(49):32730–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9830016 pmid:9830016
  49. 49. Ling Y, Morgan K, Kalsheker N. Amyloid precursor protein (APP) and the biology of proteolytic processing: relevance to Alzheimer’s disease. Int J Biochem Cell Biol [Internet]. 2003 [cited 2017 Jun 12];35(11):1505–35. Available from: http://www.sciencedirect.com/science/article/pii/S135727250300133X pmid:12824062
  50. 50. Schuh AF, Rieder CM, Rizzi L, Chaves M, Roriz-Cruz M. Mechanisms of brain aging regulation by insulin: implications for neurodegeneration in late-onset Alzheimer’s disease. ISRN Neurol [Internet]. 2011 [cited 2017 Jun 12];2011:306905. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22389813 pmid:22389813
  51. 51. Williamson R, McNeilly A, Sutherland C. Insulin resistance in the brain: An old-age or new-age problem? Biochem Pharmacol [Internet]. 2012 Sep [cited 2017 Jun 12];84(6):737–45. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0006295212003504 pmid:22634336
  52. 52. QIU W, FOLSTEIN M. Insulin, insulin-degrading enzyme and amyloid-β peptide in Alzheimer’s disease: review and hypothesis. Neurobiol Aging [Internet]. 2006 Feb [cited 2017 Jun 12];27(2):190–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16399206 pmid:16399206
  53. 53. Ahuja M, Buabeid M, Abdel-Rahman E, Majrashi M, Parameshwaran K, Amin R, et al. Immunological alteration & toxic molecular inductions leading to cognitive impairment & neurotoxicity in transgenic mouse model of Alzheimer’s disease. Life Sci [Internet]. 2017 [cited 2017 Jun 13];177:49–59. Available from: http://www.sciencedirect.com/science/article/pii/S0024320517300875 pmid:28286225
  54. 54. Mouli S, Nanayakkara G, AlAlasmari A, Eldoumani H, Fu X, Berlin A, et al. The Role of Frataxin in Doxorubicin Mediated Cardiac Hypertrophy. Am J Physiol—Hear Circ Physiol [Internet]. 2015 Jul 24 [cited 2017 Jun 12];309(5):ajpheart.00182.2015. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26209053
  55. 55. Wills ED. Mechanisms of lipid peroxide formation in tissues Role of metals and haematin proteins in the catalysis of the oxidation of unsaturated fatty acids. Biochim Biophys Acta—Lipids Lipid Metab [Internet]. 1965 Apr [cited 2017 Jun 12];98(2):238–51. Available from: http://linkinghub.elsevier.com/retrieve/pii/0005276065901189
  56. 56. Thrash B, Karuppagounder SS, Uthayathas S, Suppiramaniam V, Dhanasekaran M. Neurotoxic Effects of Methamphetamine. Neurochem Res [Internet]. 2010 Jan 21 [cited 2017 Jun 12];35(1):171–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19697126 pmid:19697126
  57. 57. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem [Internet]. 1974 Sep 16 [cited 2017 Jun 12];47(3):469–74. Available from: http://www.ncbi.nlm.nih.gov/pubmed/4215654 pmid:4215654
  58. 58. Muralikrishnan D, Mohanakumar KP. Neuroprotection by bromocriptine against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity in mice. FASEB J [Internet]. 1998 Jul [cited 2017 Jun 12];12(10):905–12. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9657530 pmid:9657530
  59. 59. Beers RF, Sizer IW. A SPECTROPHOTOMETRIC METHOD FOR MEASURING THE BREAKDOWN OF HYDROGEN PEROXIDE BY CATALASE*. 2017 [cited 2017 Jun 12]; Available from: http://www.jbc.org/
  60. 60. Lawrence RA, Burk RF. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun [Internet]. 1976 Aug 23 [cited 2017 Jun 12];71(4):952–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/971321 pmid:971321
  61. 61. Ramsay RR, Salach JI, Dadgar J, Singer TP. Inhibition of mitochondrial NADH dehydrogenase by pyridine derivatives and its possible relation to experimental and idiopathic parkinsonism. Biochem Biophys Res Commun [Internet]. 1986 Feb 26 [cited 2017 Jun 12];135(1):269–75. Available from: http://www.ncbi.nlm.nih.gov/pubmed/3485428 pmid:3485428
  62. 62. Wharton DC, Tzagoloff A. [45] Cytochrome oxidase from beef heart mitochondria. In 1967 [cited 2017 Jun 12]. p. 245–50. Available from: http://linkinghub.elsevier.com/retrieve/pii/0076687967100487
  63. 63. Xian Y.-F., Ip S.-P., Mao Q.-Q., & Lin Z.-X. (2016). Neuroprotective effects of honokiol against beta-amyloid-induced neurotoxicity via GSK-3β and β-catenin signaling pathway in PC12 cells. Neurochemistry International, 97, 8–14. https://doi.org/10.1016/j.neuint.2016.04.014 pmid:27131736
  64. 64. Choi I. S., Lee Y.-J., Choi D.-Y., Lee Y. K., Lee Y. H., Kim K. H., … Hong J. T. (2011). 4-O-methylhonokiol attenuated memory impairment through modulation of oxidative damage of enzymes involving amyloid-β generation and accumulation in a mouse model of Alzheimer’s disease. Journal of Alzheimer’s Disease: JAD, 27(1), 127–41. https://doi.org/10.3233/JAD-2011-110545 pmid:21799245
  65. 65. Akagi M, Matsui N, Akae H, Hirashima N, Fukuishi N, Fukuyama Y, et al. Nonpeptide neurotrophic agents useful in the treatment of neurodegenerative diseases such as Alzheimer’s disease. J Pharmacol Sci (Internet). 2015 Feb (cited 2017 Jun 12);127(2):155–63. Available from: http://linkinghub.elsevier.com/retrieve/pii/S1347861315000043 pmid:25727952
  66. 66. Luchsinger JA, Tang M-X, Shea S, Mayeux R. Hyperinsulinemia and risk of Alzheimer disease. Neurology (Internet). 2004 Oct 12 (cited 2017 Jun 12);63(7):1187–92. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15477536 pmid:15477536
  67. 67. Schrijvers EMC, Witteman JCM, Sijbrands EJG, Hofman A, Koudstaal PJ, Breteler MMB. Insulin metabolism and the risk of Alzheimer disease: the Rotterdam Study. Neurology (Internet). American Academy of Neurology; 2010 Nov 30 (cited 2017 Jun 12);75(22):1982–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21115952 pmid:21115952
  68. 68. Zhao W-Q, Chen H, Quon MJ, Alkon DL. Insulin and the insulin receptor in experimental models of learning and memory. Eur J Pharmacol (Internet). 2004 Apr 19 (cited 2017 Jun 12);490(1–3):71–81. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15094074 pmid:15094074
  69. 69. Van Der Heide LP, Kamal A, Artola A, Gispen WH, Ramakers GMJ. Insulin modulates hippocampal activity-dependent synaptic plasticity in a N-methyl-d-aspartate receptor and phosphatidyl-inositol-3-kinase-dependent manner. J Neurochem (Internet). 2005 Jun 17 (cited 2017 Jun 12);94(4):1158–66. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16092951 pmid:16092951
  70. 70. Tangvarasittichai S. Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes mellitus. World J Diabetes [Internet]. 2015 Apr 15 [cited 2017 Aug 3];6(3):456. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25897356 pmid:25897356
  71. 71. Calvo-Ochoa E, Arias C. Cellular and metabolic alterations in the hippocampus caused by insulin signalling dysfunction and its association with cognitive impairment during aging and Alzheimer’s disease: studies in animal models. Diabetes Metab Res Rev (Internet). 2015 Jan (cited 2017 Jun 12);31(1):1–13. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24464982 pmid:24464982
  72. 72. Farris W, Mansourian S, Chang Y, Lindsley L, Eckman EA, Frosch MP, et al. Insulin-degrading enzyme regulates the levels of insulin, amyloid β-protein, and the β-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci (Internet). 2003 Apr 1 (cited 2017 Jun 12);100(7):4162–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12634421 pmid:12634421
  73. 73. Chen Z, Zhong C. Decoding Alzheimer’s disease from perturbed cerebral glucose metabolism: Implications for diagnostic and therapeutic strategies. Prog Neurobiol (Internet). 2013 Sep (cited 2017 Jun 12);108:21–43. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23850509 pmid:23850509
  74. 74. Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj EK, et al. Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol (Internet). 2001 Aug (cited 2017 Jun 12);60(8):759–67. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11487050 pmid:11487050
  75. 75. Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol (Internet). 2007 Jan (cited 2017 Jun 12);39(1):44–84. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16978905 pmid:16978905
  76. 76. Moreira PI, Carvalho C, Zhu X, Smith MA, Perry G. Mitochondrial dysfunction is a trigger of Alzheimer’s disease pathophysiology. Biochim Biophys Acta—Mol Basis Dis (Internet). 2010 Jan (cited 2017 Jun 12);1802(1):2–10. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19853658
  77. 77. Anekonda TS, Reddy PH. Can herbs provide a new generation of drugs for treating Alzheimer’s disease? Brain Res Rev (Internet). 2005 Dec 15 (cited 2017 Jun 12);50(2):361–76. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16263176 pmid:16263176
  78. 78. Guo, Sun L, Chen X, Zhang D. Oxidative stress, mitochondrial damage and neurodegenerative diseases. Neural Regen Res [Internet]. 2013 [cited 2017 Aug 3];8(21):2003. Available from: http://www.nrronline.org/article.asp?issn=1673-5374;year=2013;volume=8;issue=21;spage=2003;epage=2014;aulast=Guo pmid:25206509
  79. 79. Gemma C, Vila J, Bachstetter A, Bickford PC. Oxidative Stress and the Aging Brain: From Theory to Prevention (Internet). Brain Aging: Models, Methods, and Mechanisms. 2007 (cited 2017 Jun 12). Available from: http://www.ncbi.nlm.nih.gov/pubmed/21204345
  80. 80. Keller JN, Dimayuga E, Chen Q, Thorpe J, Gee J, Ding Q. Autophagy, proteasomes, lipofuscin, and oxidative stress in the aging brain. Int J Biochem Cell Biol (Internet). 2004 Dec (cited 2017 Jun 12);36(12):2376–91. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15325579 pmid:15325579
  81. 81. Hoi CP, Ho YP, Baum L, Chow AHL. Neuroprotective effect of honokiol and magnolol, compounds from Magnolia officinalis, on beta-amyloid-induced toxicity in PC12 cells. Phyther Res (Internet). 2010 Aug 19 (cited 2017 Jun 12);24(10):1538–42. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20878707
  82. 82. Pope S, Land JM, Heales SJR. Oxidative stress and mitochondrial dysfunction in neurodegeneration; cardiolipin a critical target? Biochim Biophys Acta—Bioenerg [Internet]. 2008 Jul [cited 2017 Aug 3];1777(7–8):794–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18420023
  83. 83. Swerdlow RH, Burns JM, Khan SM. The Alzheimer’s disease mitochondrial cascade hypothesis: Progress and perspectives. Biochimica et Biophysica Acta—Molecular Basis of Disease. 2014. p. 1219–31.
  84. 84. Fukui H, Moraes CT. The mitochondrial impairment, oxidative stress and neurodegeneration connection: reality or just an attractive hypothesis? Trends in Neurosciences. 2008. p. 251–6. pmid:18403030
  85. 85. Picone P, Nuzzo D, Caruana L, Scafidi V, Di Carlo MD. Mitochondrial dysfunction: Different routes to Alzheimer’s disease therapy. Oxidative Medicine and Cellular Longevity. 2014.
  86. 86. Castellani R, Hirai K, Aliev G, Drew KL, Nunomura A, Takeda A, et al. Role of mitochondrial dysfunction in Alzheimer’s disease. J Neurosci Res (Internet). 2002;70(3):357–60. Available from: http://dx.doi.org/10.1002/jnr.10389 pmid:12391597
  87. 87. Leuner K, Kurz C, Guidetti G, Orgogozo J-M, Müller WE. Improved mitochondrial function in brain aging and Alzheimer disease—the new mechanism of action of the old metabolic enhancer piracetam. Front Neurosci (Internet). Frontiers; 2010 (cited 2017 Jun 12);1:44. Available from: http://journal.frontiersin.org/article/10.3389/fnins.2010.00044/abstract
  88. 88. Hauptmann S, Scherping I, Dröse S, Brandt U, Schulz KL, Jendrach M, et al. Mitochondrial dysfunction: An early event in Alzheimer pathology accumulates with age in AD transgenic mice. Neurobiol Aging (Internet). 2009 Oct (cited 2017 Jun 12);30(10):1574–86. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18295378 pmid:18295378
  89. 89. Montgomery MK, Turner N. Mitochondrial dysfunction and insulin resistance: an update. Endocr Connect [Internet]. 2015 Mar 1 [cited 2017 Jun 13];4(1):R1–15. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25385852 pmid:25385852
  90. 90. Yang T-T, Shih Y-S, Chen Y-W, Kuo Y-M, Lee C-W. Glucose regulates amyloid β production via AMPK. J Neural Transm (Internet). 2015 Oct 13 (cited 2017 Jun 12);122(10):1381–90. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26071020 pmid:26071020
  91. 91. Lin C-L, Cheng Y-S, Li H-H, Chiu P-Y, Chang Y-T, Ho Y-J, et al. Amyloid-β suppresses AMP-activated protein kinase (AMPK) signaling and contributes to α-synuclein-induced cytotoxicity. Exp Neurol (Internet). 2016 Jan (cited 2017 Jun 12);275:84–98. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26515689 pmid:26515689
  92. 92. Vingtdeux V, Giliberto L, Zhao H, Chandakkar P, Wu Q, Simon JE, et al. AMP-activated protein kinase signaling activation by resveratrol modulates amyloid-beta peptide metabolism. J Biol Chem [Internet]. 2010 Mar 19 [cited 2017 Jun 13];285(12):9100–13. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20080969 pmid:20080969
  93. 93. Jager S, Handschin C, St.-Pierre J, Spiegelman BM. AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1α Proc Natl Acad Sci (Internet). 2007 Jul 17 (cited 2017 Jun 12);104(29):12017–22. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17609368 pmid:17609368
  94. 94. Katsouri L, Lim YM, Blondrath K, Eleftheriadou I, Lombardero L, Birch AM, et al. PPARγ-coactivator-1α gene transfer reduces neuronal loss and amyloid-β generation by reducing β-secretase in an Alzheimer’s disease model. Proc Natl Acad Sci U S A (Internet). National Academy of Sciences; 2016 Oct 25 (cited 2017 Jun 12);113(43):12292–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27791018 pmid:27791018
  95. 95. Katsouri L, Parr C, Bogdanovic N, Willem M, Sastre M. PPARγ co-activator-1α (PGC-1α) reduces amyloid-β generation through a PPARγ-dependent mechanism. J Alzheimers Dis (Internet). 2011 (cited 2017 Jun 12);25(1):151–62. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21358044 pmid:21358044
  96. 96. Pillai VB, Samant S, Sundaresan NR, Raghuraman H, Kim G, Bonner MY, et al. Honokiol blocks and reverses cardiac hypertrophy in mice by activating mitochondrial Sirt3. Nat Commun [Internet]. 2015 Apr 14 [cited 2017 Aug 3];6:6656. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25871545 pmid:25871545