Green Tea Epigallocatechin-3-Gallate (EGCG) and ...

Journal of Alzheimer’s Disease 26 (2011) 507–521 DOI 10.3233/JAD-2011-101629 IOS Press

507

Green Tea Epigallocatechin-3-Gallate (EGCG) and Other Flavonoids Reduce Alzheimer’s Amyloid-Induced Mitochondrial Dysfunction Natasa Dragicevica,b , Adam Smithc,d,e , Xiaoyang Linf , Fang Yuanf,g , Neil Copesa , Vedad Delica , Jun Tanc,d,e,h,i , Chuanhai Caoa,e,f , R. Douglas Shytlec,e,i and Patrick C. Bradshawa,∗ a Department

of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL, USA b Department of Radiology, University of South Florida, Tampa, FL, USA c Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, College of Medicine, University of South Florida, Tampa, Florida, USA d Neuroimmunology Laboratory, Department of Psychiatry & Behavioral Medicine, University of South Florida, Tampa, FL, USA e Department of Molecular Pharmacology and Physiology, College of Medicine, University of South Florida, Tampa, FL, USA f Byrd Alzheimer’s Center and Research Institute, Tampa, FL, USA g Third Military Medical University, Chongqing, China h Department of Psychiatry and Neurosciences, University of South Florida, Tampa, FL, USA i Silver Child Development Center, Department of Psychiatry and Behavioral Medicine, University of South Florida, Tampa, FL, USA Handling Associate Editor: Russell Swerdlow

Accepted 21 April 2011

Abstract. Amyloid-! (A!)-induced mitochondrial dysfunction may play a role in the onset and progression of Alzheimer’s disease (AD). Therefore, therapeutics targeted to improve mitochondrial function could be beneficial. Plant-derived flavonoids have shown promise in improving certain AD phenotypes, but the overall mechanism of action(s) through which flavonoids protect from AD is still unknown. To identify flavonoids and other natural products that may correct amyloid-induced mitochondrial dysfunction, 25 natural products were screened for their ability to restore altered mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, or ATP levels in neuroblastoma cells expressing mutant amyloid-! protein precursor (A!PP). Epigallocatechin-3-gallate (EGCG) and luteolin were identified as the top two mitochondrial restorative compounds from the in vitro screen. EGCG was further tested in vivo to determine its effects on brain mitochondrial function in an A!PP/PS-1 (presenilin 1) double mutant transgenic mouse model of AD. EGCG treatment restored mitochondrial respiratory rates, MMP, ROS production, and ATP levels by 50 to 85% in mitochondria isolated from the hippocampus, cortex, and striatum. The results

∗ Correspondence to: Patrick C. Bradshaw, Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave.; BSF 218, Tampa, FL, USA. Tel.: +813 974 6180; Fax: +813 974 1614; E-mail: [email protected].

ISSN 1387-2877/11/$27.50 © 2011 – IOS Press and the authors. All rights reserved

508

N. Dragicevic et al. / EGCG Improves Mitochondrial Function in AD

of this study lend further credence to the notion that EGCG and other flavonoids, such as luteolin, are ‘multipotent therapeutic agents’ that not only reduce toxic levels of brain A!, but also hold the potential to protect neuronal mitochondrial function in AD. Keywords: Adenosine triphosphate, Alzheimer’s disease, EGCG, flavonoids, membrane potential, mitochondrial, polyphenols, reactive oxygen species, respiration

INTRODUCTION Alzheimer’s disease (AD) is a progressive neurodegenerative disorder linked to increased oxidative stress [1–4] and is associated with pathological features including amyloid-! (A!) plaques and neurofibrillary tangles. These features are caused by altered amyloid-! protein precursor (A!PP) proteolysis and tau hyperphosphorylation, respectively [5–11]. Therefore, discovering therapeutics that decrease oxidative stress and/or reduce the proteolytic processing of A!PP into toxic A! peptides have remained a primary focus of AD research. The dominant hypothesis driving drug development in AD for nearly twenty years is known as the ‘amyloid hypothesis’ and maintains that the disease occurs when A! accumulates and reaches toxic levels in the brain. The central implication of such an idea is that if A! levels are reduced, the disease might be slowed, halted or even prevented if treatment is started early enough. An intense search for small natural molecules that slow the progression or even reverse AD pathology have been inspired by epidemiological studies of dietary habits associated with protection against neurodegeneration and other aging-related health problems. For example, The Kame Study, found that regular consumption of fruit juice reduced the risk of developing dementia in a population-based prospective study of 1836 Japanese Americans in King County, Washington [12]. With the ‘amyloid hypothesis’ of AD in mind, various polyphenols (found in many fruits and vegetables) have been identified that are potentially anti-amyloidogenic [13–15]. One of these compounds, the most abundant green tea flavonoid, epigallocatechin-3-gallate (EGCG), has been found to protect neurons against A!-mediated toxicity and increase secreted levels of sA!PP" in A!PP transfected cells [16]. Some members of our research group were the first to report that administration of EGCG decreased A! levels and plaques via promotion of the non-amyloidogenic "-secretase proteolytic pathway in “Swedish” mutant A!PP (A!PPsw ) overexpressing mice [14]. A follow-up study by the same group

later reported that chronic EGCG in drinking water also provided cognitive benefits in AD transgenic mice [17]. Although we have indentified other flavonoids, luteolin and diosmin, with anti-amyloidogenic effects, both in vitro and in vivo [18, 19], it is becoming increasingly clear that flavonoids are ‘multipotent therapeutic agents’, with multiple therapeutic targets across multiple disease states [20, 21]. This is a very important point to emphasize considering that more than a dozen single-target anti-amyloidogenic drugs have now failed in Phase II and III human clinical trials [22]. Therefore, it is likely that future efforts to identify therapeutic agents or even cocktails for AD will necessitate the characterization of multiple therapeutic targets from any one approach. While the anti-amyloidogenic effects of flavonoids appear to play a role in their therapeutic effects observed in AD transgenic mice, other neurochemical and pharmacological mechanisms may contribute to their overall potential therapeutic benefits in treating AD. One such target area of interest is modulation of neuronal mitochondrial function. Mitochondrial dysfunction, apoptosis, and disruption of Ca2+ homeostasis are pathophysiological processes known to occur in AD brain [23–29]. Mitochondrial dysfunction in AD is in part characterized by increased reactive oxidative species generation, altered Krebs cycle enzyme activities, and decreased cytochrome c oxidase activity [27, 30–32]. These events may result from intracellular A! accumulation as evidenced by the dysfunction caused by A! administration to isolated mitochondria [33]. Increased intracellular A! levels may also facilitate mitochondrial permeability transition opening [34], a key event in cell death. Therefore, intracellular A! has the potential to directly disrupt mitochondrial function and contribute to the metabolic deficiencies and loss of neuronal function observed in AD patient brains [35]. In addition, other AD-associated proteins including tau, and a proteolytic product of ApoE4, also cause some degree of mitochondrial dysfunction [36, 37], but the extent of damage caused by these proteins at the organelle level has not been fully explored. Thera-


peutic applications targeted to improve mitochondrial function in AD are therefore very promising and could aid in improvement of both pathological and cognitive aspects of the disease. Because of the reported therapeutic effects of a limited number of flavonoids on mitochondrial function in neurodegenerative disease models [20, 21, 38], as well as the evidence for neuronal mitochondrial dysfunction in AD, the focus of the present study was to screen various flavonoids and other natural compounds for their potential to restore mitochondrial function in neuroblastoma cells overexpressing human A!PPsw gene. Based on these in vitro studies, a top compound was administered to an AD transgenic mouse model and mitochondrial function was determined in different brain regions.

509

esterases and oxidized to fluorescent dichlorofluorescein (DCF). Fluorescence measurements were read using a Biotek Synergy 2 microplate reader. Mitochondrial membrane potential (MMP) determination in cells Cells were cultured until confluency in black, clear bottom 96 well microplates. MMP was determined following incubation of the cells with 1 #M JC-1 (excitation filter 530/25, emission filter 590/35) for 30 min. ATP level determination in cells

Murine neuroblastoma N2a cells were stably transfected with the human A!PP-695 gene harboring the “Swedish” mutation (A!PPsw ), as detailed in [39], and were obtained from those authors. The cells were grown in a medium containing high (4.5 g/L) glucose Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum in 5% CO2 at 37◦ C. Cells were treated with different polyphenols or natural compounds at a final concentration of 1 #M for 48 h. Experiments were performed in triplicate.

An ATP determination kit containing d-luciferin, luciferase [40 #l of a 5 mg/ml solution in 25 mM Tris·acetate, pH 7.8, 0.2 M ammonium sulfate, 15% (vol/vol) glycerol, and 30% (vol/vol) ethylene glycol], dithiothreitol (DTT), ATP, and a reaction buffer (10 ml of 500 mM tricine buffer, pH 7.8, 100 mM MgSO4 , 2 mM EDTA, and 2 mM sodium azide) was purchased from Invitrogen. The reagents and reaction mixture were combined according to the supplied protocol. Firefly luciferase was added to the luciferin-containing ATP-Glo™ assay solution in a ratio of 1 #L to 100 #L (25 #L luciferase for 2.5 mL of the ATP-Glo™ assay solution). The ATP-Glo™ Detection Cocktail was prepared immediately before each use according to the manufacturer’s directions. The relative luminescence activity was recorded using a 96 well white microplate in a Biotek Synergy 2 microplate reader.

Cell respiratory studies

Isolation of brain mitochondria from mice

Cells grown until confluency on a 10 cm plate were trypsinized and suspended in 0.35 mL of phosphate buffered saline in a Clark type oxygen electrode (Strathkelvin Instruments, MT200A chamber, Glasgow, UK) as in [40]. The maximal respiratory rate was obtained following the addition of 10 #M carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation.

All experimental protocols involving animals were approved by University of South Florida Animal Use and Care Committee. PSA!PP (A!PPsw, PSEN1dE9) transgenic mice further referred to as A!PP/PS1 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). In the present study, A!PP/PS1 EGCG treated (n = 6), A!PP/PS1 non-treated (n = 6), and littermate NT controls (n = 6) were doubly housed. EGCG was administered to mice at a concentration of 10 mg/mL in their drinking water as in [17]. Following a 5.5 month oral application of EGCG (37.1 ± 1.6 (mean ± stand. dev.) mg/kg/day), animals were euthanatized using CO2 asphyxiation and decapitated at 9.5 months of age as previously described [41]. Brain mitochondrial isolation was performed using a standard procedure [42]. First, brains were quickly removed and placed on ice. Brain regions of interest were carefully dissected following anatomical guidelines and

MATERIALS AND METHODS Cell culture

Cell reactive oxygen species (ROS) generation studies Cells were cultured until confluency in black, clear bottom 96 well microplates. ROS generation was determined following incubation of the cells for 20 min in the presence of 25 #M 2# ,7# -dichloro dihydrofluorescein diacetate (excitation filter 485/20, emission filter 528/20). This compound is cleaved by intracellular

510


placed in a glass Dounce homogenizer containing five times the volume of isolation buffer (215 mM mannitol, 75 mM sucrose, 0.1% BSA, 1 mM EGTA, 20 mM HEPES (Na+ ), pH 7.2). Following homogenization, a low-speed spin (1,300 × g for 5 min) to remove unbroken cells and nuclei was performed. The supernatant was carefully placed in fresh tubes, topped off with isolation buffer, and spun down again at 13,000 × g for 10 min. The supernatant was discarded and the resultant mitochondrial pellets were suspended in 500 #L of isolation buffer with 1 mM EGTA (215 mM mannitol, 75 mM sucrose, 0.1% BSA, 20 mM HEPES (Na+ ), pH 7.2) and 0.1% digitonin (in DMSO) was added to the pellets to disrupt the synaptosomes. After 5 min, samples were brought to a final volume of 2 ml using isolation buffer containing 1 mM EGTA and centrifuged at 13,000 × g for 15 min. Next the pellets were resuspended in isolation buffer without EGTA (75 mM sucrose, 215 mM mannitol, 0.1% BSA, and 20 mM HEPES with the pH adjusted to 7.2 using KOH) and was centrifuged at 10,000 g for 10 min. The final mitochondrial pellet was suspended in isolation buffer without EGTA to yield a final protein concentration of approximately 10 mg/mL and immediately stored on ice. To normalize the results, the protein concentrations were determined with all the samples on the same microwell plate using a BCA protein assay kit. For all mitochondrial analyses, the brain areas of interest from 3 mice of the same genotype were combined to form a single homogenate that was then assayed in triplicate. We performed the experiment twice (n = 2) with 6 EGCG treated A!PP/PS-1 mice, 6 control A!PP/PS-1 mice, and 6 nontransgenic mice. Respiratory measurements on isolated mitochondria

Reactive oxygen species production from isolated mitochondria Mitochondrial ROS production was measured following incubation of isolated mitochondria with 25 #M 2# ,7# -dichlorodihydrofluorescein diacetate for 20 min and then the DCF fluorescence (excitation filter 485/20 nm, emission filter 528/20 nm) was read as previously described [41]. In short, 100 #g (0.8 mg/ml final concentration) of isolated mitochondria were added to 120 #l of KCl-based respiration buffer (see above) with 5 mM pyruvate and 2.5 mM malate added as respiratory substrates and 25 #M 2# ,7# dichlorodihydrofluorescein diacetate. Mitochondrial ROS production in the presence of oligomycin (to increase ROS production) or FCCP (to decrease ROS production) were performed to ensure measurement values were within the range of the indicator. Membrane potential measurements on isolated mitochondria A 200 #M stock solution of JC-1 (5,5# ,6,6# -tetra chloro-1,1# ,3,3# -tetraethylbenzimidazolylcarbocyanine iodide) was made using DMSO as the solvent. The assay buffer contained mitochondrial isolation buffer with the addition of 5 mM pyruvate and 5 mM malate. 150 #l of assay buffer and 20 #l (1.2 mg/ml final concentration) of mitochondria were added to the wells of a 96 well black, clear bottom microplate (Corning) followed by the addition of 1 #M JC-1 and mixed gently. The microplate was covered with aluminum foil and left at room temperature for 20 min before reading. Fluorescence (excitation 530/25 nm, emission 590/35 nm) was then measured. ATP level determination in isolated mitochondria

The respiratory function of mitochondria was measured using a miniature Clark type oxygen electrode (Strathkelvin MT200A chamber). 100 #g (0.3 mg/ml final concentration) of mitochondria were suspended in a sealed, constantly stirred and thermostatically controlled chamber at 37◦ C containing 350 #l of respiration buffer (125 mM KCl, 1 mM MgCl2 , 2 mM KH2 PO4 , 5 mM pyruvate, 2.5 mM malate, 500 #M EGTA, 20 mM HEPES, pH 7.0) at 37◦ C. State III respiration was assessed by the addition of 200 #M ADP. State IV respiration was achieved by addition of 1 #M oligomycin. Maximal respiration was assessed by addition of 1 #M FCCP. The respiratory control ratio (RCR) was determined by dividing the rate of oxygen consumption for state III by that of state IV.

ATP was determined in isolated mitochondria similarly as described above in the cells. Mitochondria from brain regions of interest were isolated following standard protocol (see above). For these experiments, ATP standard curves were run in the range of 0.5 to 50 #M. ATP levels from the mitochondrial samples were quantified, the relative luminescence activity was recorded, and translated into ATP concentrations using the calibration curves. Brain Aβ + AβPP ELISA The left brain hemispheres from the A!PP/PS1 mice were homogenized and stored as previously described


[14]. Quantitation of detergent-soluble and insoluble (extracted from brain homogenate using a 5 M guanidine buffer) A! + A!PP species was performed according to published methods [13]. 6E10 (capture antibody) was coated at 2 #g/mL in PBS into 96-well immunoassay plates for 24 h at 4◦ C. The plates were washed with 0.05% Tween 20 in PBS five times and blocked with blocking buffer (PBS with 1% BSA, 5% horse serum) for 2 h at room temperature. Conditioned medium or A! standards were added to the plates and incubated overnight at 4◦ C. Following three washes, biotinylated antibody, 4G8 (0.5 #g/mL in PBS with 1% BSA) was added to the plates and incubated for 2 h at room temperature. After 5 washes, streptavidinhorseradish peroxidase (1 : 200 dilutions in PBS with 1% BSA) was added to the 96-wells for 30 min at room temperature. Tetramethylbenzidine (TMB) substrate was added to the plates and incubated for 15 min at room temperature. 50 #L of stop solution (2 N N2 SO4 ) was added to each well of the plates. The optical density of each well was determined using a microplate reader measuring absorbance at 450 nm. Isolation of mitochondria from cells Mitochondria were isolated from N2a cells stably expressing A!PPsw as done in [40]. Cell and isolated mitochondrial total Aβ + AβPP ELISA N2a cells stably expressing A!PPsw were treated with 1 #M EGCG for 24 h. Cells, isolated mitochondria from these cells, or hippocampal mitochondria were stored at −80◦ C and then used for total A! + A!PP ELISA. Briefly, a 96 well plate was coated with 50 #l of 6E10 at 2.5 #g/ml overnight in CBC buffer (15 mm Na2 CO3 35 mm NaHCO3 , pH 9.6), and washed with wash buffer (PBS with 0.05% Tween 20). Samples (and standards) and biotinylated 4G8 antibody (1 : 5000 dilution) were loaded on the antibodypre-coated plate and incubated overnight at 4◦ C. Horseradish peroxidase-conjugated avidin was added after washing. Substrates were added for the colorimetric reaction, and then stopped with sulfuric acid. The optical density was obtained and sample concentrations were calculated according to the standard curve. Cell and mitochondria Western blots Western blots of N2a cells stably expressing A!PPsw and isolated mitochondria from these cells

511

were performed using the 6E10 antibody. This antibody detects full length A!PP as well as any proteolytic fragments containing amino acids 1-16 of A!. RESULTS To determine if flavonoids or other natural products reduce or prevent amyloid-induced mitochondrial dysfunction in AD models, N2a neuroblastoma cells stably expressing A!PPsw (N2a-A!PPsw ) were treated with one of 25 natural compounds for 48 h. Three separate assays were performed to determine overall mitochondrial function. These assays included measurements of MMP, ROS production, and cellular ATP levels. Mitochondrial function was also monitored in treated and non-treated non-transfected N2a cells as a control. The results are shown in Table 1. The flavonoids luteolin, epigallocatechin gallate (EGCG), apigenin, naringenin, baicalein, hesperitin, diosmetin, flavone, flavanone, methylflavone, aminoflavone, methoxyflavone, and diosmin were some of the top compounds that restored mitochondrial function. The non-flavonoid ferulic acid was also extremely effective at protecting mitochondrial function. Roughly three quarters of the compounds tested at least partially restored mitochondrial function at the 1 #M concentration administered. This concentration was chosen for all compounds in the screen, because it led to a near complete restoration of mitochondrial function for the top compound (luteolin) from the screen. Data examining the restoration of MMP showed that 100 nM luteolin was only 20% as effective as the 1 #M chosen concentration (data not shown). The effect of the top two compounds (luteolin and EGCG), as well as four other mitochondrialprotective flavonoids (apigenin, naringenin, diosmin, and flavone), on the cellular oxygen consumption rate of N2a-A!PPsw cells was also determined. The results of these 6 compounds on the four assays broadly reflecting mitochondrial function are compared in graphical format in Fig. 1 and Fig. 2. Mitochondria from N2a-A!PPsw cells showed greatly reduced maximal respiratory rates compared to nontransfected N2a control cells (Fig. 1A). The respiratory rate data corresponded well with the data obtained from the other assays of mitochondrial function. Luteolin and EGCG nearly completely restored the respiratory rate, while the four other flavonoids tested restored the respiratory rate by over fifty percent. The MMP was then monitored in N2a-A!PPsw cells (Fig. 1B). The MMP reflects both electron transport chain function and inner

512


Table 1 The effect of flavonoids and other natural products on ROS production, MMP, and ATP levels in N2a and N2a-A!PPsw neuroblastoma cells. Arrows were placed in front of the values statistically different than the control (p < 0.05) indicating the direction of change N2a-A!PPsw cells

N2a cells

% of untreated cells

% of untreated cells

Compound

ROS

MMP

ATP

ROS MMP

ATP

luteolin EGCG baicalein hesperitin diosmetin ferulic acid apigenin naringenin flavone methylflavone aminoflavone methoxyflavone flavanone diosmin methyl gallate quercetin methyl cinnamate catechin acacetin chrysin inosine homocysteine dopamine taurine isoamylsalicylate

↓ 40 ↓ 51 ↓ 34 ↓ 30 ↓ 37 ↓ 35 ↓ 57 ↓ 54 ↓ 32 ↓ 34 ↓ 39 ↓ 42 ↓ 37 ↓ 72 ↓ 42 ↓ 40 ↓ 54 ↓ 52 ↓ 86 ↓ 69 ↓ 75 100 108 ↓ 64 ↑ 115

↑ 202 ↑ 188 ↑ 204 ↑ 136 ↑ 186 ↑ 177 ↑ 144 ↑ 139 ↑ 129 ↑ 136 ↑ 154 ↑ 150 ↑ 143 ↑ 124 ↑ 143 ↑ 121 ↑ 121 ↑ 189 98 111 102 ↓ 86 ↓ 70 106 96

↑ 444 ↑ 384 ↑ 344 ↑ 370 ↑ 320 ↑ 345 ↑ 349 ↑ 304 ↑ 247 ↑ 250 ↑ 246 ↑ 260 ↑ 260 ↑ 330 ↑ 180 ↑ 138 ↑ 145 108 ↑ 136 98 100 110 92 ↓ 92 94

↓ 70 ↓ 74 ↓ 78 ↓ 77 ↓ 85 ↓ 72 ↓ 80 ↓ 78 ↓ 82 ↓ 84 ↓ 90 96 ↓ 88 ↓ 84 ↓ 93 ↓ 89 ↓ 88 104 ↓ 75 ↓ 90 100 102 102 ↓ 90 103

↑ 149 ↑ 140 109 109 ↑ 125 ↑ 130 ↑ 135 ↑ 132 109 110 ↑ 116 ↑ 118 ↑ 112 ↑ 122 110 ↑ 132 104 99 ↑ 128 ↑ 118 102 100 99 97 99

↑ 142 ↑ 138 ↑ 112 100 ↑ 118 ↑ 125 ↑ 119 ↑ 122 106 102 ↑ 117 ↑ 121 ↑ 112 ↑ 115 110 ↑ 118 105 102 ↑ 140 100 101 98 97 100 96

membrane permeability. N2a-A!PPsw cells had a significantly decreased MMP that increased following treatment with flavonoids. Luteolin and EGCG treatment restored MMP close to control N2a levels while apigenin and naringenin were more effective than diosmin and flavone. Mitochondria produce the majority of cellular ROS in brain, so the rate of ROS reflects the efficiency of mitochondrial function (Fig. 2A). A!PPsw expression caused a large increase in ROS production, which could be decreased by flavonoid treatment. The six chosen flavonoids were all successful in decreasing ROS production, with luteolin and EGCG being most effective. Apigenin and naringenin were more effective than flavone and diosmin. Diosmin only slightly reduced A!-mediated ROS production. Mitochondria produce roughly 90% of the total cellular ATP in neurons. Therefore, ATP levels were monitored in the N2a-A!PPsw cells as a measure of mitochondrial function (Fig. 2B). N2a-A!PPsw cells had greatly decreased ATP levels that were restored by the flavonoids nearly mirroring the effect observed for respiratory rates and MMP. However, in this assay,

.

.

Fig. 1. Maximal respiratory rates and MMP of N2a-A!PPsw cells treated with natural products. N2a-A!PPsw cells were grown in the presence of 1 #M of selected compounds for 48 h. Lut. = luteolin, Apig. = apigenin, Nari. = Naringenin, Dios. = diosmin, Flav. = flavone. A) Increase in maximal O2 consumption in N2a-A!PPsw cells treated with compounds when compared to N2aA!PPsw cells treated with vehicle control (*p < 0.05) Maximal O2 consumption was determined in the presence of 10 #M FCCP. B) Increase in MMP in N2a-A!PPsw cells treated with compounds when compared to untreated N2a-A!PPsw cells (*p < 0.05) Results presented are the means ± S.E. from three independent experiments (n = 3).

apigenin was slightly more efficacious, increasing ATP levels almost to the same extent as EGCG. From the four cell-based assays that were used to monitor different parameters of mitochondrial function, ECGC was identified as one of the two most protective compounds in our small chemical screen. Therefore a time course of its restorative action on the MMP (Fig. 3A) and ROS production (Fig. 3B) was performed. EGCG required roughly 24 h to exert a complete effect on the MMP, while roughly 48 h was required for EGCG to exert its maximal restorative effect on ROS production. Therefore, the increased ROS generation in N2a-A!PPsw cells may be a downstream effect of the decreased MMP. To determine if EGCG was protecting mitochondria by altering A!PP levels or A! levels, we performed


.

513

.

.

.

Fig. 2. ROS production and ATP levels in N2a-A!PPsw cells treated with natural products. N2a-A!PPsw cells were grown in the presence of 1 #M of select compounds for 48 h. Lut. = luteolin, Apig. = apigenin, Nari. = Naringenin, Dios. = diosmin, Flav. = flavone. A) Decrease in ROS production in N2a-A!PPsw cells treated with select compounds compared to N2a-A!PPsw cells treated with vehicle control (*p < 0.05). B) Increase in ATP levels in N2a-A!PPsw cells treated with select compounds compared to N2a-A!PPsw cells treated with vehicle control (*p < 0.05).

a Western blot of A!PP and oligomeric A! levels in isolated mitochondria or whole cell extracts using the 6E10 antibody that recognizes an epitope in the A! region of A!PP. No differences were found in the whole cell (Fig. 4A) or mitochondrial (Fig. 4B) levels of A!PP or in the levels of several oligomeric species of A! present on the blot (data not shown). Independent of EGCG treatment we observed a much higher concentration of low molecular weight A! oligomers in the mitochondrial extracts than in the whole cell extracts (Fig. 4 C). In a previous work [14], we found that concentrations of EGCG of at least 5 #M were required to decrease the secreted levels of A! from the N2a-A!PPsw cells. Therefore, not surprisingly, at the low 1 #M EGCG concentration that restores mitochondrial function, we also found no decrease in total A! + A!PP levels by Western blot from whole cells or isolated mitochondria (Fig. 4D). In fact EGCG

Fig. 3. Time course of the restorative effect of EGCG on MMP and ROS production in N2a-A!PPsw cells. A) N2a-A!PPsw cells were treated with 1 #M EGCG for the indicated amounts of time and the A) MMP or (B) rate of ROS production was measured. Values were expressed as the % restoration from untreated N2a-A!PPsw cell levels (0%) to untreated N2a cell levels (100%).

treatment resulted in a slight increase in whole cell A! + A!PP levels. Therefore, EGCG can restore mitochondrial function without decreasing mitochondrial A! or A!PP levels. Based on the cell studies and our previous experience with the compound, we chose to administer EGCG to A!PP/PS1 mice in their drinking water for 5.5 months and then assay the function of isolated brain mitochondria. The average EGCG consumption was monitored and was found to be 37.1 ± 1.6 mg/kg/day. This was slightly lower than our target dose of 50 mg/kg/day that we have previously shown to reduce A! plaques in the brain and improve cognitive function in these mice [17]. After animal sacrifice and brain mitochondrial isolation, maximal respiratory rates (Figs. 5A, 6A, and 7A), ROS production (Figs. 5B, 6B, and 7B), MMP (Figs. 5 C, 6 C, and 7 C), and mitochondrial ATP levels (Figs. 5D, 6D, and 7D) were measured in hippocampal (Fig. 5A–D), striatal (Fig. 6A–D), and cortical (Fig. 7A-D) mitochondria. The respiratory control ratio (RCR) was also calculated. A 40-55% restoration of the RCR occurred following EGCG treatment (Figs. 5A, 6A, and 7A). In mitochondria from the hippocampus and cortex, EGCG administration restored respiratory rates, MMP, ROS production

514


.

.

.

.

Fig. 4. EGCG did not decrease the mitochondrial or whole cell levels of A! or A!PP in cultured cells at the concentration used to restore mitochondrial function. N2a-A!PPsw cells were grown for 24 h in the presence or absence of 1 #M EGCG. A Western blot of A!PP was performed on (A) whole cell or (B) isolated mitochondrial extracts. (C) The Western blot of whole cell and mitochondrial lysates from untreated cells shows a large amount of low molecular weight A! oligomers in the mitochondrial lysate compared to the whole cell lysate. Equal amounts of protein were run in the whole cell and isolated mitochondrial lanes. (D) An ELISA of total A! + A!PP was performed on cell and mitochondrial extracts. EGCG treatment increased A! + A!PP levels in the whole cell extract compared to untreated control (*p < 0.05).

rates, and ATP levels by 70–85%. EGCG similarly restored the respiratory rate and ATP levels in the striatum, but the MMP and ROS production rates in this brain region were slightly less affected, only being restored by 50–60%. We then measured total A! + A!PP levels in isolated hippocampal mitochondria (Fig. 8). EGCG treatment lowered these levels by roughly 50 percent. We then measured both soluble and insoluble A! + A!PP in the total brain (Fig. 9). A combined 18% reduction was measured. This included a 13% reduction (p = 0.003) in insoluble levels.

DISCUSSION Twenty-five natural compounds, mostly flavonoids, were screened for the ability to restore mitochondrial function in N2a-A!PPsw cells. Roughly three-quarters of the compounds provided a significant improvement in mitochondrial function. Luteolin, EGCG, apigenin, naringenin, diosmin, and flavone were some of the top compounds with luteolin and EGCG nearly completely restoring mitochondrial function. In the cells, EGCG was found to improve mitochondrial function without decreasing A! or A!PP levels. To determine


515

.

.

.

Fig. 5. Analysis of isolated hippocampal mitochondrial function from A!PP/PS1 mice treated with EGCG. A) Maximal respiratory rate (oxygen consumption) was determined in the presence of 1 #M FCCP. EGCG treatment increased maximal respiratory rates compared to control A!PP/PS-1 hippocampal mitochondria (*p < 0.05). The respiratory control ratio (RCR) mean ± SEM is also shown in each bar. B) MMP in A!PP/PS-1 hippocampal mitochondria was increased compared to control A!PP/PS-1 hippocampal mitochondria (*p < 0.05). C) ROS Production in A!PP/PS-1 hippocampal mitochondria was decreased following EGCG treatment (*p < 0.05). D) ATP levels were increased by EGCG treatment in hippocampal mitochondria compared to untreated controls (*p < 0.05).

if EGCG could improve mitochondrial function in an AD mouse model, it was administered orally to A!PP/PS1 mice for 5.5 months. Following treatment, brain mitochondria were isolated and assayed, and a significant restoration of mitochondrial function by EGCG treatment was found. The restorative effect was quite dramatic, restoring parameters of mitochondrial function by 40–85%. A portion of this restoration of brain mitochondrial function may result from the large EGCG-induced decrease in mitochondrial A! + A!PP

levels. However, these results must be interpreted cautiously as a low number of mice were used in the study and the small size of some of the brain regions studied necessitated the pooling of brain regions from more than one animal prior to mitochondrial isolation and functional analysis. The studies performed in this report are quite novel in that they are the first to compare the effectiveness of a wide range of natural products in a cell model of a neurological disease. Many studies have been performed

516


.

.

.

.

Fig. 6. Analysis of isolated striatal mitochondria from A!PP/PS1 mice treated with EGCG. (A) Maximal respiratory rate and RCR, (B) MMP, (C) ROS production, and (D) ATP levels in mitochondria from A!PP/PS1 mice were partially restored by EGCG treatment (*p < 0.05).

using individual flavonoids and other natural products in numerous disease models, but it is impossible to compare the relative effectiveness of each compound from these studies. Since mitochondrial dysfunction is a common theme in many disorders of aged individuals, it is possible that the top flavonoids identified in this report will also be the top flavonoids for maintaining mitochondrial function in other aging-related diseases. The study of mitochondrial function as an endpoint for the EGCG mouse feeding studies is also unique in this report. We had previously reported that EGCG treatment decreases amyloid plaques and improves cognitive dysfunction in AD mice [14, 17]. We attributed this improvement to the increase in brain

"-secretase activity following EGCG treatment. In this report, we have discovered further protective actions of EGCG. These functions include the stabilization of mitochondrial function in the presence of A!PP and A! in vitro and the decrease in mitochondrial A! + A!PP levels in vivo. There was a near complete restoration of mitochondrial function in A!PP expressing cells in culture by a low concentration (1 #M) of EGCG without affecting cell or mitochondrial A!PP or A! levels. The complete molecular mechanism of how this occurs has yet to be identified, but it may involve ROS scavenging and/or a stabilization of the electron transport chain. The reason why decreased A! + A!PP levels were found in brain and isolated


.

.

.

.

517

Fig. 7. Analysis of isolated cortical mitochondria from A!PP/PS1 mice treated with EGCG. (A) Maximal respiratory rate and RCR, (B) MMP, (C) ROS production, and (D) ATP levels in mitochondria from A!PP/PS1 mice were partially restored by EGCG treatment (*p < 0.05).

brain mitochondria from EGCG treated mice and not in EGCG treated cells likely reflects either a higher concentration of EGCG in brain or the longer duration of the treatment. It is of therefore of interest to determine if either a lower dose or a much shorter duration of EGCG treatment would also restore mitochondrial function in the brains of the AD mice. Mitochondria have been proposed to act as central organelles in the regulation of aging and age related neurodegeneration because they control cellular energy status, ROS production, and apoptosis, all of which are important in determining lifespan [43]. Mitochondria constitute the major source of superoxide and other ROS within most tissues, generating

approximately 85-90% of total cellular superoxide [44]. Mitochondrial electron transport in aged tissues is less efficient, which impairs ATP synthesis and results in increased oxidant production [45]. The steady-state levels of oxidatively damaged molecules are dependent both on net ROS formation and clearance of damaged molecules [46]. Antioxidant defenses also decline with age [47], making mitochondria even more vulnerable to oxidative injury. The resultant mitochondrial decay may eventually cause inadequate energy production and/or the loss of mitochondrial and cellular calcium homeostasis. Such changes could result in cellular apoptosis and also lead to the loss of cognitive function accompanying brain aging [48].

518


Fig. 8. Total mitochondrial A! + A!PP levels from the hippocampus of A!PP/PS1 and NT mice. Mitochondria from EGCG treated A!PP/PS1 mice had lower A! + A!PP levels than untreated A!PP/PS1 mice (*p < 0.05).

Fig. 9. Soluble and insoluble brain A! + A!PP levels of A!PP/PS1 mice treated with EGCG. Soluble and insoluble A! + A!PP levels are presented to show the effects of EGCG treatment in A!PP/PS1 mice (n = 6) versus the untreated control A!PP/PS1 mice (n = 2). Data is shown as pg (A! + A!PP) per mg total protein ± SEM for the respective fraction. EGCG treatment resulted in a 23% reduction in soluble A! + A!PP (p = 0.15) and a significant 13% reduction in insoluble A! + A!PP load (*p < 0.01).

Familial AD-linked mutant A!PP overexpression in mouse brain has been shown to cause increased mitochondrial ROS production [49, 50], decreased MMP, decreased respiratory rates [33], and altered mitochondrial morphology [51]. A! and A!PP are both partially localized to brain mitochondria and likely involved in these manifestations [52, 53]. Mitochondrial dysfunction and increased oxidative stress occur as early events in mutant A!PP transgenic mice, even before the onset

of insoluble A! plaque pathology [54–56]. This A!induced oxidative stress even elicits a compensatory increase in expression of several mitochondrial genes Including ATPase-6 [57], which could also possibly contribute to the downstream events in AD progression such as synaptic loss and cognitive impairment. A! also causes mitochondrial fragmentation and reduced neurite outgrowth, which can both be suppressed by mitochondrial targeted antioxidants, such as MitoQ and the cell-permeable SS31 peptide [58]. Therefore, mitochondrial antioxidants represent a promising strategy for AD treatment. Plant polyphenols have been suggested as potential anti-aging and neuroprotective compounds as evidenced by several epidemiological studies. Based on their chemical structure flavonoids can be divided into two groups: anthocyanins and anthoxanthins. Anthocyanins are water-soluble pigments found in flowers and fruits and are responsible for the dark color of fruits. Anthoxanthins are colourless and comprise flavans, flavanols, flavanones, flavanonols, flavones, flavonols and isoflavones. Among the non-flavonoid polyphenols, resveratrol (trans-3, 40, 5trihydroxystilbene, present in grapes and in red wine), and curcumin (diferuloyl methane), the yellow component of turmeric are of most well known and have been shown to have beneficial effects in AD animal models [59, 60]. The role of polyphenols in the protection of the aging brain depends on the ability of these compounds to cross the blood–brain barrier, which tightly controls the influx in the brain of metabolites and nutrients as well as of drugs. Studies have shown that polyphenols present in the rodent diet have a limited absorption, but can later be detected in various brain regions important for learning and memory [61]. Green tea catechins are brain permeable [62], which after oral administration and enzymatic conversion, can be found in the brain as epicatechin glucuronide and 3# -O-methyl epicatechin glucuronide [63]. Several groups have demonstrated the antioxidant property of catechins [64, 65] and demonstrated the beneficial effect of epigallocatechin gallate (EGCG) against oxidative stress-induced damage in the brain [66]. EGCG has been shown to prevent changes in superoxide dismutase expression caused by neuronal oxidative stress [67–69], increase gammaglutamyl cysteine ligase expression, the rate limiting step in glutathione synthesis [38], and also increase the protein levels of the beta subunit of ATP synthase [70]. Other studies have found that EGCG is able to decrease oxidative stress-mediated mitochondrial deterioration in aged brain [71]. It has been shown that 90–95% of cellu-


lar EGCG accumulates in mitochondria in neurons [68]. This localization may be responsible for the remarkable mitochondrial protection afforded by this compound. These studies suggest an important role of EGCG as an antioxidant and protector of mitochondrial function in brain not only in AD, but also in other diseases and conditions associated with mitochondrial oxidative stress. However, in some neuronal culture models, such as human SY5Y cells, ECGC causes apoptosis at concentrations greater than 10 #M and can potentiate cell death caused by mitochondrial electron transport chain inhibitors [72]. Whether or not this toxicity at high concentrations occurs in the brain has not been well studied. Therefore, therapies combining moderate doses of EGCG with other drugs or natural compounds may prove to be optimal. In addition to its roles as an antioxidant and decreasing A! generation, ECGC has been shown to possess other beneficial mechanisms of action. EGCG directly binds and inhibits the toxic oligomerization of A! and instead induces the formation of unstructured nontoxic oligomers [73]. EGCG also acts as a fairly potent chelator of ionic iron and copper [74]. This activity is very important because free iron and copper are increased in AD brain and contribute to increased ROS production. Lastly EGCG is an important inducer of three neuroprotective signal transduction pathways, including the MAP kinase (MAPK), protein kinase C (PKC), and phosphatidylinositol-3-kinase (PI-3 kinase)-Akt pathways [74]. EGCG has been shown to induce the neuroprotective activity of ERK, JNK, and p38 using the MAPK pathway. Whether ECGC works through any of these or other pathways to protect mitochondrial function from A!-induced damage is not yet known. In regards to other mitochondrial protective pathways, we did not detect any difference in the mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1"), a master mitochondrial regulator of gene expression, following EGCG treatment to N2a-A!PPsw cells (data not shown). In summary, administration of EGCG had marked beneficial effects on mitochondria in mouse and cell models of AD. Part of this effect may occur through the antioxidant properties of EGCG, while another part may result from a stabilization of electron transport chain (ETC) activity in the presence of A!PP and A!. Future work aims to determine if EGCG directly binds ETC proteins, alters their expression level, or alters their post-translational modification. The mechanisms by which other flavonoids such as luteolin enhance mitochondrial function will also be addressed.

519

ACKNOWLEDGMENTS We would like to thank Robert Buzzeo for help with the ATP determination and Dr. Paula Bickford for helpful discussion and providing flavonoids for testing. Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=863). REFERENCES [1]

[2]

[3]

[4] [5]

[6]

[7] [8]

[9] [10] [11]

[12] [13] [14]

Montine KS, Olson SJ, Amarnath V, Whetsell WO, Jr, Graham DG, Montine TJ (1997) Immunohistochemical detection of 4-hydroxy-2-nonenal adducts in Alzheimer’s disease is associated with inheritance of APOE4. Am J Pathol 150, 437-443. Smith CD, Carney JM, Starke-Reed PE, Oliver CN, Stadtman ER, Floyd RA, Markesbery WR (1991) Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proc Natl Acad Sci U S A 88, 1054010543. Smith MA, Richey PL, Taneda S, Kutty RK, Sayre LM, Monnier VM, Perry G (1994) Advanced Maillard reaction end products, free radicals, and protein oxidation in Alzheimer’s disease. Ann N Y Acad Sci 738, 447-454. Pappolla MA, Omar RA, Kim KS, Robakis NK (1992) Immunohistochemical evidence of oxidative [corrected] stress in Alzheimer’s disease. Am J Pathol 140, 621-628. Alonso A, Zaidi T, Novak M, Grundke-Iqbal I, Iqbal K (2001) Hyperphosphorylation induces self-assembly of tau into tangles of paired helical filaments/straight filaments. Proc Natl Acad Sci U S A 98, 6923-6928. Funamoto S, Morishima-Kawashima M, Tanimura Y, Hirotani N, Saido TC, Ihara Y (2004) Truncated carboxylterminal fragments of beta-amyloid precursor protein are processed to amyloid beta-proteins 40 and 42. Biochemistry 43, 13532-13540. Goedert M (1996) Tau protein and the neurofibrillary pathology of Alzheimer’s disease. Ann N Y Acad Sci 777, 121-131. Golde TE, Eckman CB, Younkin SG (2000) Biochemical detection of Abeta isoforms: implications for pathogenesis, diagnosis, and treatment of Alzheimer’s disease. Biochim Biophys Acta 1502, 172-187. Huse JT, Doms RW (2000) Closing in on the amyloid cascade: recent insights into the cell biology of Alzheimer’s disease. Mol Neurobiol 22, 81-98. Sambamurti K, Greig NH, Lahiri DK (2002) Advances in the cellular and molecular biology of the beta-amyloid protein in Alzheimer’s disease. Neuromolecular Med 1, 1-31. Selkoe DJ, Yamazaki T, Citron M, Podlisny MB, Koo EH, Teplow DB, Haass C (1996) The role of APP processing and trafficking pathways in the formation of amyloid beta-protein. Ann N Y Acad Sci 777, 57-64. Dai Q, Borenstein AR, Wu Y, Jackson JC, Larson EB (2006) Fruit and vegetable juices and Alzheimer’s disease: the Kame Project. Am J Med 119, 751-759. Marambaud P, Zhao H, Davies P (2005) Resveratrol promotes clearance of Alzheimer’s disease amyloid-beta peptides. J Biol Chem 280, 37377-37382. Rezai-Zadeh K, Shytle D, Sun N, Mori T, Hou H, Jeanniton D, Ehrhart J, Townsend K, Zeng J, Morgan D, Hardy J, Town T, Tan J (2005) Green tea epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and

520

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]

N. Dragicevic et al. / EGCG Improves Mitochondrial Function in AD reduces cerebral amyloidosis in Alzheimer transgenic mice. J Neurosci 25, 8807-8814. Yang F, Lim GP, Begum AN, Ubeda OJ, Simmons MR, Ambegaokar SS, Chen PP, Kayed R, Glabe CG, Frautschy SA, Cole GM (2005) Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J Biol Chem 280, 5892-5901. Levites Y, Amit T, Mandel S, Youdim MB (2003) Neuroprotection and neurorescue against Abeta toxicity and PKC-dependent release of nonamyloidogenic soluble precursor protein by green tea polyphenol (-)-epigallocatechin-3gallate. FASEB J 17, 952-954. Rezai-Zadeh K, Arendash GW, Hou H, Fernandez F, Jensen M, Runfeldt M, Shytle RD, Tan J (2008) Green tea epigallocatechin-3-gallate (EGCG) reduces beta-amyloid mediated cognitive impairment and modulates tau pathology in Alzheimer transgenic mice. Brain Res 1214, 177-187. Parker-Athill E, Luo D, Bailey A, Giunta B, Tian J, Shytle RD, Murphy T, Legradi G, Tan J (2009) Flavonoids, a prenatal prophylaxis via targeting JAK2/STAT3 signaling to oppose IL-6/MIA associated autism. J Neuroimmunol 217, 20-27. Rezai-Zadeh K, Ehrhart J, Bai Y, Sanberg PR, Bickford P, Tan J, Shytle RD (2008) Apigenin and luteolin modulate microglial activation via inhibition of STAT1-induced CD40 expression. J Neuroinflammation 5, 41. Darvesh AS, Carroll RT, Bishayee A, Geldenhuys WJ, Van der Schyf CJ (2010) Oxidative stress and Alzheimer’s disease: dietary polyphenols as potential therapeutic agents. Expert Rev Neurother 10, 729-745. Mandel SA, Amit T, Kalfon L, Reznichenko L, Youdim MB (2008) Targeting multiple neurodegenerative diseases etiologies with multimodal-acting green tea catechins. J Nutr 138, 1578S-1583 S. Sabbagh MN (2009) Drug development for Alzheimer’s disease: where are we now and where are we headed? Am J Geriatr Pharmacother 7, 167-185. Beal MF (1998) Mitochondrial dysfunction in neurodegenerative diseases. Biochim Biophys Acta 1366, 211-223. Manfredi G, Beal MF (2000) The role of mitochondria in the pathogenesis of neurodegenerative diseases. Brain Pathol 10, 462-472. Mattson MP (1997) Cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives. Physiol Rev 77, 1081-1132. Offen D, Elkon H, Melamed E (2000) Apoptosis as a general cell death pathway in neurodegenerative diseases. J Neural Transm Suppl, 153-166. Reddy PH, Beal MF (2005) Are mitochondria critical in the pathogenesis of Alzheimer’s disease? Brain Res Brain Res Rev 49, 618-632. Sims NR (1996) Energy metabolism, oxidative stress and neuronal degeneration in Alzheimer’s disease. Neurodegeneration 5, 435-440. Tabira T (2002) [Significance of intracellular A beta in Alzheimer’s disease]. Nihon Shinkei Seishin Yakurigaku Zasshi 22, 175-180. Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787-795. Maurer I, Zierz S, Moller HJ (2000) A selective defect of cytochrome c oxidase is present in brain of Alzheimer disease patients. Neurobiol Aging 21, 455-462. Parker WD, Jr, (1991) Cytochrome oxidase deficiency in Alzheimer’s disease. Ann N Y Acad Sci 640, 59-64.

[33] [34]

[35] [36]

[37]

[38]

[39]

[40] [41] [42]

[43] [44] [45]

[46] [47] [48]

[49]

Casley CS, Canevari L, Land JM, Clark JB, Sharpe MA (2002) Beta-amyloid inhibits integrated mitochondrial respiration and key enzyme activities. J Neurochem 80, 91-100. Parks JK, Smith TS, Trimmer PA, Bennett JP, Jr, Parker WD, Jr, (2001) Neurotoxic Abeta peptides increase oxidative stress in vivo through NMDA-receptor and nitric-oxide-synthase mechanisms, and inhibit complex IV activity and induce a mitochondrial permeability transition in vitro. J Neurochem 76, 1050-1056. Beal MF (2005) Mitochondria take center stage in aging and neurodegeneration. Ann Neurol 58, 495-505. Chang S, ran Ma T, Miranda RD, Balestra ME, Mahley RW, Huang Y (2005) Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity. Proc Natl Acad Sci U S A 102, 18694-18699. David DC, Hauptmann S, Scherping I, Schuessel K, Keil U, Rizzu P, Ravid R, Drose S, Brandt U, Muller WE, Eckert A, Gotz J (2005) Proteomic and functional analyses reveal a mitochondrial dysfunction in P301 L tau transgenic mice. J Biol Chem 280, 23802-23814. Kim CY, Lee C, Park GH, Jang JH (2009) Neuroprotective effect of epigallocatechin-3-gallate against beta-amyloidinduced oxidative and nitrosative cell death via augmentation of antioxidant defense capacity. Arch Pharm Res 32, 869-881. Thinakaran G, Teplow DB, Siman R, Greenberg B, Sisodia SS (1996) Metabolism of the “Swedish” amyloid precursor protein variant in neuro2a (N2a) cells. Evidence that cleavage at the “beta-secretase” site occurs in the golgi apparatus. J Biol Chem 271, 9390-9397. Frezza C, Cipolat S, Scorrano L (2007) Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc 2, 287-295. Brown MR, Geddes JW, Sullivan PG (2004) Brain regionspecific, age-related, alterations in mitochondrial responses to elevated calcium. J Bioenerg Biomembr 36, 401-406. Dragicevic N, Mamcarz M, Zhu Y, Buzzeo R, Tan J, Arendash GW, Bradshaw PC (2010) Mitochondrial amyloidbeta levels are associated with the extent of mitochondrial dysfunction in different brain regions and the degree of cognitive impairment in Alzheimer’s transgenic mice. J Alzheimers Dis 20(Suppl 2), S535-S550. Wallace DC (2005) A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 39, 359-407. Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82, 47-95. Hagen TM, Yowe DL, Bartholomew JC, Wehr CM, Do KL, Park JY, Ames BN (1997) Mitochondrial decay in hepatocytes from old rats: membrane potential declines, heterogeneity and oxidants increase. Proc Natl Acad Sci U S A 94, 3064-3069. Trifunovic A, Larsson NG (2008) Mitochondrial dysfunction as a cause of ageing. J Intern Med 263, 167-178. Sanz N, Diez-Fernandez C, Alvarez A, Cascales M (1997) Age-dependent modifications in rat hepatocyte antioxidant defense systems. J Hepatol 27, 525-534. Sastre J, Pallardo FV, Pla R, Pellin A, Juan G, O’Connor JE, Estrela JM, Miquel J, Vina J (1996) Aging of the liver: age-associated mitochondrial damage in intact hepatocytes. Hepatology 24, 1199-1205. Li F, Calingasan NY, Yu F, Mauck WM, Toidze M, Almeida CG, Takahashi RH, Carlson GA, Flint Beal M, Lin MT, Gouras GK (2004) Increased plaque burden in brains of APP mutant MnSOD heterozygous knockout mice. J Neurochem 89, 1308-1312.

N. Dragicevic et al. / EGCG Improves Mitochondrial Function in AD [50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

Smith MA, Hirai K, Hsiao K, Pappolla MA, Harris PL, Siedlak SL, Tabaton M, Perry G (1998) Amyloid-beta deposition in Alzheimer transgenic mice is associated with oxidative stress. J Neurochem 70, 2212-2215. Sirk D, Zhu Z, Wadia JS, Shulyakova N, Phan N, Fong J, Mills LR (2007) Chronic exposure to sub-lethal beta-amyloid (Abeta) inhibits the import of nuclear-encoded proteins to mitochondria in differentiated PC12 cells. J Neurochem 103, 1989-2003. Mungarro-Menchaca X, Ferrera P, Moran J, Arias C (2002) beta-Amyloid peptide induces ultrastructural changes in synaptosomes and potentiates mitochondrial dysfunction in the presence of ryanodine. J Neurosci Res 68, 89-96. Gillardon F, Rist W, Kussmaul L, Vogel J, Berg M, Danzer K, Kraut N, Hengerer B (2007) Proteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque deposition. Proteomics 7, 605-616. Devi L, Prabhu BM, Galati DF, Avadhani NG, Anandatheerthavarada HK (2006) Accumulation of amyloid precursor protein in the mitochondrial import channels of human Alzheimer’s disease brain is associated with mitochondrial dysfunction. J Neurosci 26, 9057-9068. Eckert A, Hauptmann S, Scherping I, Rhein V, Muller-Spahn F, Gotz J, Muller WE (2008) Soluble beta-amyloid leads to mitochondrial defects in amyloid precursor protein and tau transgenic mice. Neurodegener Dis 5, 157-159. Manczak M, Anekonda TS, Henson E, Park BS, Quinn J, Reddy PH (2006) Mitochondria are a direct site of A beta accumulation in Alzheimer’s disease neurons: implications for free radical generation and oxidative damage in disease progression. Hum Mol Genet 15, 1437-1449. Reddy PH, McWeeney S, Park BS, Manczak M, Gutala RV, Partovi D, Jung Y, Yau V, Searles R, Mori M, Quinn J (2004) Gene expression profiles of transcripts in amyloid precursor protein transgenic mice: up-regulation of mitochondrial metabolism and apoptotic genes is an early cellular change in Alzheimer’s disease. Hum Mol Genet 13, 1225-1240. Manczak M, Mao P, Calkins MJ, Cornea A, Reddy AP, Murphy MP, Szeto HH, Park B, Reddy PH (2010) Mitochondria-targeted antioxidants protect against amyloidbeta toxicity in Alzheimer’s disease neurons. J Alzheimers Dis 20(Suppl 2), S609-S631. Begum AN, Jones MR, Lim GP, Morihara T, Kim P, Heath DD, Rock CL, Pruitt MA, Yang F, Hudspeth B, Hu S, Faull KF, Teter B, Cole GM, Frautschy SA (2008) Curcumin structure-function, bioavailability, and efficacy in models of neuroinflammation and Alzheimer’s disease. J Pharmacol Exp Ther 326, 196-208. Vingtdeux V, Giliberto L, Zhao H, Chandakkar P, Wu Q, Simon JE, Janle EM, Lobo J, Ferruzzi MG, Davies P, Marambaud P (2010) AMP-activated protein kinase signaling activation by resveratrol modulates amyloid-beta peptide metabolism. J Biol Chem 285, 9100-9113. Andres-Lacueva C, Shukitt-Hale B, Galli RL, Jauregui O, Lamuela-Raventos RM, Joseph JA (2005) Anthocyanins in

[62]

[63]

[64]

[65] [66]

[67]

[68]

[69] [70]

[71]

[72] [73]

[74]

521

aged blueberry-fed rats are found centrally and may enhance memory. Nutr Neurosci 8, 111-120. Mandel S, Weinreb O, Reznichenko L, Kalfon L, Amit T (2006) Green tea catechins as brain-permeable, non toxic iron chelators to “iron out iron” from the brain. J Neural Transm Suppl, 249-257. Abd El Mohsen MM, Kuhnle G, Rechner AR, Schroeter H, Rose S, Jenner P, Rice-Evans CA (2002) Uptake and metabolism of epicatechin and its access to the brain after oral ingestion. Free Radic Biol Med 33, 1693-1702. Chen L, Yang X, Jiao H, Zhao B (2003) Tea catechins protect against lead-induced ROS formation, mitochondrial dysfunction, and calcium dysregulation in PC12 cells. Chem Res Toxicol 16, 1155-1161. Sastre J, Borras C, Garcia-Sala D, Lloret A, Pallardo FV, Vina J (2002) Mitochondrial damage in aging and apoptosis. Ann N Y Acad Sci 959, 448-451. Srividhya R, Jyothilakshmi V, Arulmathi K, Senthilkumaran V, Kalaiselvi P (2008) Attenuation of senescenceinduced oxidative exacerbations in aged rat brain by (-)-epigallocatechin-3-gallate. Int J Dev Neurosci 26, 217223. Lee SJ, Lee KW (2007) Protective effect of (-)epigallocatechin gallate against advanced glycation endproducts-induced injury in neuronal cells. Biol Pharm Bull 30, 1369-1373. Schroeder EK, Kelsey NA, Doyle J, Breed E, Bouchard RJ, Loucks FA, Harbison RA, Linseman DA (2009) Green tea epigallocatechin 3-gallate accumulates in mitochondria and displays a selective antiapoptotic effect against inducers of mitochondrial oxidative stress in neurons. Antioxid Redox Signal 11, 469-480. Yin ST, Tang ML, Su L, Chen L, Hu P, Wang HL, Wang M, Ruan DY (2008) Effects of Epigallocatechin-3-gallate on lead-induced oxidative damage. Toxicology 249, 45-54. Weinreb O, Amit T, Youdim MB (2008) The application of proteomics for studying the neurorescue activity of the polyphenol (-)-epigallocatechin-3-gallate. Arch Biochem Biophys 476, 152-160. Srividhya R, Zarkovic K, Stroser M, Waeg G, Zarkovic N, Kalaiselvi P (2009) Mitochondrial alterations in aging rat brain: effective role of (-)-epigallo catechin gallate. Int J Dev Neurosci 27, 223-231. Chung WG, Miranda CL, Maier CS (2007) Epigallocatechin gallate (EGCG) potentiates the cytotoxicity of rotenone in neuroblastoma SH-SY5Y cells. Brain Res 1176, 133-142. Ehrnhoefer DE, Bieschke J, Boeddrich A, Herbst M, Masino L, Lurz R, Engemann S, Pastore A, Wanker EE (2008) EGCG redirects amyloidogenic polypeptides into unstructured, offpathway oligomers. Nat Struct Mol Biol 15, 558-566. Weinreb O, Amit T, Mandel S, Youdim MB (2009) Neuroprotective molecular mechanisms of (-)-epigallocatechin-3gallate: a reflective outcome of its antioxidant, iron chelating and neuritogenic properties. Genes Nut 10, 10.