Neurochem Res DOI 10.1007/s11064-016-1837-9

ORIGINAL PAPER

Lycopene Prevents Amyloid [Beta]-Induced Mitochondrial Oxidative Stress and Dysfunctions in Cultured Rat Cortical Neurons Mingyue Qu1 • Zheng Jiang2 • Yuanxiang Liao1 • Zhenyao Song3 • Xinzhong Nan1

Received: 4 October 2015 / Revised: 26 December 2015 / Accepted: 15 January 2016 Ó Springer Science+Business Media New York 2016

Abstract Brains affected by Alzheimer’s disease (AD) show a large spectrum of mitochondrial alterations at both morphological and genetic level. The causal link between b-amyloid (Ab) and mitochondrial dysfunction has been established in cellular models of AD. We observed previously that lycopene, a member of the carotenoid family of phytochemicals, could counteract neuronal apoptosis and cell damage induced by Ab and other neurotoxic substances, and that this neuroprotective action somehow involved the mitochondria. The present study aims to investigate the effects of lycopene on mitochondria in cultured rat cortical neurons exposed to Ab. It was found that lycopene attenuated Ab-induced oxidative stress, as evidenced by the decreased intracellular reactive oxygen species generation and mitochondria-derived superoxide production. Additionally, lycopene ameliorated Ab-induced mitochondrial morphological alteration, opening of the mitochondrial permeability transition pores and the consequent cytochrome c release. Lycopene also improved mitochondrial complex activities and restored ATP levels in Ab-treated neuron. Furthermore, lycopene prevented mitochondrial DNA damages and improved the protein level of mitochondrial transcription factor A in

mitochondria. Those results indicate that lycopene protects mitochondria against Ab-induced damages, at least in part by inhibiting mitochondrial oxidative stress and improving mitochondrial function. These beneficial effects of lycopene may account for its protection against Ab-induced neurotoxicity. Keywords Alzheimer’s disease  b-Amyloid  Lycopene  Mitochondria  Neurons Abbreviations AD Alzheimer’s disease Ab b-Amyloid ROS Reactive oxygen species mtDNA Mitochondrial DNA mPTP Mitochondrial permeability transition pores DCFH-DA Dichlorofluorescin diacetate NRFU Normalized relative fluorescence units THF Tetrahydrofuran BHT Butylated hydroxytoluene COX I Cytochrome c oxidase subunit I COX IV Cytochrome c oxidase subunit IV ND6 NADH dehydrogenase subunit 6 GAPDH Glyceraldehyde-3-phosphate dehydrogenase

Mingyue Qu and Zheng Jiang contributed equally to this work. & Yuanxiang Liao [email protected] 1

Center for Diseases Prevention and Control of the Rocket Force of PLA, Beijing 100094, People’s Republic of China

2

Affiliated Hospital of Academy of Military Medical Sciences, Beijing 100071, People’s Republic of China

3

Center for Diseases Prevention and Control of the Air Force of PLA, Beijing 100076, People’s Republic of China

Introduction Alzheimer’s disease (AD) is characterized by the presence of neurofibrillary tangles, senile plaques and loss of synapses. Accumulating evidence suggest that amyloidbeta peptide (Ab), the major component of senile plaques, is toxic to the mitochondria and implicates this organelle in

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the pathogenesis of AD [1]. Previous studies demonstrate that Ab can enter mitochondria and interact with mitochondrial proteins, such as amyloid-beta binding alcohol dehydrogenase (ABAD) and cyclophilin D, leading to impairment of mitochondrial membrane permeability, disruption of electron transport chain, and leakage of reactive oxygen species (ROS) [2, 3]. Conversely, the oxidative stress resulting from excessive mitochondrial ROS production can cause further damage to mitochondrial function and exacerbate ROS production, forming a vicious cycle [4]. One of the consequences of mitochondrial damage is mitochondrial DNA (mtDNA) damage [5]. MtDNA is more vulnerable to oxidative attack than nuclear DNA because of its proximity to the respiratory chain and lack of protective histone-like proteins and introns. MtDNA damage is reflected by the presence of mtDNA mutation, by a decline in mtDNA copy numbers or mtDNA transcript levels [6]. Each of these types of mtDNA damage can disturb energy metabolism and aggravate ROS generation by encoding the deficient critical proteins for the respiratory chain in mitochondria. Lycopene, a phytochemical belonging to carotenoid family, is naturally synthesized by tomatoes and other fruits with red color. Lycopene has shown diverse and remarkable bio-activities, including anti-oxidation, antiinflammation, and anti-cancer. Recently, the role of lycopene in neuroprotection is emerging. It is reported that lycopene mediates its neuroprotective effects in 3-nitropropionic acid induced Huntington’s disease [7] and rotenone or MPTP induced Parkinson’s disease [8–10]. In addition, lycopene provides protections against ischemia/ reperfusion-induced brain injury in Mongolian gerbils by inducing an increase in SOD activity and inhibiting apoptosis [11]. These findings suggest that lycopene may be a promising therapeutic candidate for treating neurologic disease. One recent epidemiological study shows that high serum levels of lycopene are associated with a lower risk of AD mortality in adults [12]. Furthermore, laboratory research results demonstrate that lycopene abrogates nuclear factor kappa B activation and proinflammatory cytokines expression [13], attenuates mitochondrial oxidative damage and restores brain-derived neurotrophic factor level in Ab treated rats [14]. Our previous study also demonstrates that lycopene inhibits trimethyltin-induced mitochondria-dependent apoptotic pathway in cultured rat hippocampal neurons [15], methylmercury-induced mitochondrial dysfunction in rat cerebellar granule neurons [16], and Ab-induced oxidative damage in cultured rat cortical neurons [17]. We therefore hypothesize that the underlying mechanism of lycopene versus oxidative stress and apoptosis is directly linked to the protection of mitochondria. To address this, we extended our previous report to investigate the protective effect of lycopene on neuronal

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mitochondrial function in cultured rat cortical neurons by using an established paradigm of cellular injury induced by Ab.

Materials and Methods Cell Culture and Drug Treatment Newborn Sprague-Dawley rats were purchased from the Animal Center of Academy of Military Medical Sciences. All experimental procedures were performed in accordance with the guidelines of the Animal Care Committee. The primary cultured rat cortical neurons were prepared as described in our previous study [17]. Ab1–42 (Sigma-Aldrich) was solubilized in distilled water at a concentration of 5 mM, incubated in a capped vial at 37 °C for 7 days to form aggregates and stored at -20 °C until use. Lycopene (Sigma-Aldrich) was dissolved in Tetrahydrofuran (THF) containing 0.025 % butylated hydroxytoluene (BHT) to avoid formation of peroxides. Immediately before the experiment, THF-lycopene aliquots were added to culture medium to the indicated concentration. The amount of THF in culture medium was not [0.1 % (v/v), a concentration that did not affect the assays as evidenced by comparisons with vehicle-free control medium. For every experiment, lycopene was applied 4 h prior to the treatment with Ab1–42 (10 lM), and was also present in the medium during the incubation period with Ab1–42. Detection of Intracellular and Mitochondrial ROS Intracellular ROS level was detected by the dichlorofluorescin diacetate (DCFH-DA; Beyotime, Nanjing, China) assay. In brief, after the indicated treatments, neurons cultured in 96-well plates (1 9 104 cells per well) were incubated with DCFH-DA for 20 min at 37 °C in dark. After incubation, cells were washed with pre-warmed PBS and signal of fluorescence was read at 488 nm for excitation and 525 nm for emission with a fluorescence microplate reader (Tecan, Mannedorf, Switzerland). Mitochondria-derived ROS was detected using MitoSOX Red staining (Invitrogen). After the indicated treatments, neurons cultured in 96-well plates (1 9 104 cells per well) were incubated with culture medium containing 5 lM MitoSOX Red for 10 min at 37 °C in dark. After incubation, cells were washed with pre-warmed PBS and signal of fluorescence was immediately quantified with a microplate reader. MitoSOX Red excitation was measured at 510 nm, and emission was measured at 580 nm. The data of the treatment groups were expressed as a percentage of that of control cells.

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Analysis of Mitochondrial Fragmentation Mitochondrial morphology was observed by the fluorogenic probe, MitoTracker Red (Invitrogen). Briefly, after the indicated treatment, neurons cultured in 96-well plates (1 9 104 cells per well) were loaded with 20 nM MitoTracker Red for 20 min at 37 °C in dark. After incubation, MitoTracker Red were removed and cells were washed with Hanks’ balanced salt solution, then cultures were fixed at 4 °C with 4 % formaldehyde in PBS for 30 min. Fluorescent images were captured using a Leica confocal laser scanning microscope (TCS SP2, Germany). Determination of Mitochondrial Permeability Transition Pores (mPTP) Opening The mPTP opening was assessed using a calcein-cobalt based mPTP assay kit (Genmed Scientifics) strictly according to the manufacturer’s protocol. In short, after the indicated treatments, neurons cultured in 24-well plates (5 9 104 cells per well) were washed with Reagent A (Hanks’ Balanced Salt Solution with sodium bicarbonate, calcium, magnesium, HEPES, L-glutamine and succinate), incubated with Reagent B (calcein AM) and Reagent C (cobalt dichloride; 1:50; 500 ll per well) at 37 °C for 20 min, then washed twice with Reagent A again. The intensity of fluorescence was measured using a microplate reader with excitation and emission wavelengths of 488 and 505 nm, respectively. The fluorescent signals were normalized to total protein concentration assessed using the Bradford assay. Results were expressed as normalized relative fluorescence units (NRFU; U/mg protein). Western Blot Analysis Western blot analysis was used for determining the cytosolic levels of cytochrome c and the mitochondrial levels of Tfam. Preparation of cytosolic or mitochondrial fractions was achieved using a cytosol/mitochondria fraction isolation kit according to the manufacturer’s instructions (Sigma-Aldrich). Protein concentrations were determined using the Bradford assay. For Western blotting, equal amounts of the protein were separated by SDSPAGE. Following protein transfer to nitrocellulose membrane, the membranes were blocked with Odyssey blocking buffer (LICOR) for 1 h. The membranes were then incubated with antibodies against the proteins of interest (cytochrome c from Cell Signaling Technology and Tfam from Abcam) overnight at 4 °C. b-actin and COX IV were used as the loading control for cytosolic and mitochondrial fractions, respectively. For the secondary antibody, IRDyeÒ 800 Donkey Anti-Rabbit antibody and IRDyeÒ 700 Donkey Anti-Mouse IgG antibody (LI-COR) were

used. The fluorescent signals were detected and quantified by Odyssey infrared imaging system (LI-COR). Measurements of Mitochondrial Complex Activities Mitochondria were isolated from cortical neurons using a Mitochondria Isolation Kit (Sigma-Aldrich) according to the manufacturer’s instruction. The assays for activities of Complex I (NADH-ubiquinone oxidoreductase), Complex II (succinate dehydrogenase), Complex III (ubiquinol-cytochrome c oxidoreductase), Complex IV (Cytochrome c oxidase) were performed as described in our previous study [16]. Protein concentration was measured using the Bradford assay and the mitochondrial complex activities were normalized by dividing them by protein concentration and expressing as a ratio to control. Measurement of Cellular ATP Levels Intracellular ATP levels were detected using an ATP Determination Kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s protocol. Briefly, after the indicated treatments, neurons in 6-well plates (1 9 106 cells per well) were collected and resuspended in the reaction buffer containing 1 mM dithiothreitol, 0.5 mM luciferin and 12.5 lg/ml luciferase. After gentle mixing, the readings for the above mixtures were taken with the Luminometer (Turner Designs, Sunnyvale, CA). ATP levels were calculated using an ATP standard curve. Results were expressed as nmol/mg protein. Measurement of 8-OHdG Contents To analyze the 8-OHdG contents in mitochondria, mtDNA was isolated from the cortical neurons using a Qiagen Plasmid Midi kit, as previously described [18]. MtDNA was digested with nuclease P1 and Escherichia coli alkaline phosphatase. 8-OHdG contents were measured using high-performance liquid chromatography (HPLC) with an electrochemical detection system as reported. And deoxyguanine were measured using a UV monitor coupled to the HPLC system. Results were expressed as 8-OHdG/ 106 dG. Measurement of mtDNA Copy Numbers and mtDNA Transcript Levels We applied quantitative real-time PCR to detect mtDNA copy numbers and mtDNA transcript levels as described in our previous study [16]. Samples were analyzed on the iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). PCR was carried out as follows: denature at 94 °C for 5 min followed up with thirty-five

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cycles of PCR (94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s). Total cellular DNA was collected for mtDNA copy number analysis by using E.Z.N.A.TM Tissue DNA Kit (Omega, Atlanta, GA, USA). We compared relative amounts of the mtDNA with nuclear DNA content. The mtDNA amplicons were generated from cytochrome c oxidase subunits I (COX I) and NADH dehydrogenase subunit 6 (ND6) encoded by mtDNA. Primers were designed according to the mtDNA sequence of rat. The nuclear amplicon was generated by amplification of the bactin segment, which was selected as the internal standard. The primers used were as follows: COX I, forward 50 -CC ACTTCGCCATCATATTCGTAGG-30 , reverse 50 -TCTG AGTAGCGTCGTGGTATTCC-30 ; ND6, forward 50 -TCA CCCAGCTACCACCATCATTC-30 , reverse 50 -CACTGA GGAGTACCCAGAGACTTG-30 ; b-actin, forward 50 -CC ACACCCGCCACCAGTTC-30 , reverse 50 -CCACACCCG CCACCAGTTC-30 . The threshold cycle number (Ct) values of b-actin and the mtDNA were detected for each individual quantitative PCR run. The -ddCt (mtDNA to bactin) represented the mtDNA copy number in cells. Total RNA was extracted for mtRNA transcript quantifications using the RNeasy Kit and cDNA was prepared using Omniscript RT Kit (Qiagen). The primers for COX I and ND6 were used as mentioned above. Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was chosen as the internal standard. The primers used were as follows: GAPDH, forward 50 -AACCTGCCAAGTATGATGA-30 , reverse 50 -TGTTGCTGTAGCCGTATT-30 . Ct values of GAPDH and mtDNA transcripts were detected for each individual quantitative PCR run. The -ddCt (mtDNA transcripts to GAPDH) represented the mtDNA transcripts in cells. Statistical Analysis Values are expressed as mean ± SD. Each experiment was repeated at least four times. All analysis was carried out with GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA). Differences between groups were analyzed by ANOVA, followed by post hoc least significant difference (LSD) tests. P \ 0.05 was considered statistically significant.

Results Lycopene Alleviated Ab-Induced Mitochondrial Oxidative Stress To investigate the effects of lycopene on Ab-induced oxidative stress, cortical neurons were pretreated with

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lycopene at different doses (0.1, 0.5, 1, 2 or 5 lM) for 4 h before Ab addition. The intracellular ROS levels were measured 12 h after Ab (10 lM) insult. It was found that lycopene significantly inhibited the increase of intracellular ROS level induced by Ab insult in a dose-dependent manner (Fig. 1a). In addition, time-course analysis indicated that the intracellular ROS level significantly increased to 1.59, 2.13, 2.68, and 2.31 fold of that in controls after cortical neurons were exposed to 10 lM Ab for 3, 6, 12, and 24 h, respectively. However, pretreatment with 2 lM lycopene successfully suppressed the Ab-induced intracellular ROS accumulation at all four toxin treatment times (Fig. 1b). Furthermore, we observed similar protective effects by MitoSOX Red assay that lycopene pretreatment reduced mitochondria-derived ROS generation induced by Ab in a dose- and time-dependent manner (Fig. 1d). These results indicated a beneficial effect of lycopene on the mitochondrial oxidative stress induced by Ab and intrigued us to further investigated whether maintaining mitochondrial function account for the protective effects of lycopene against Ab-induced neurotoxicity. Lycopene Prevented Ab-Induced Mitochondrial Fragmentation and Membrane Permeability Transition Mitochondrial morphology was observed by MitoTracker Red staining. Mitochondria displayed typical elongated filamentous morphology in control group, whereas many filamentous mitochondria converted to small and round organelles following 12 h Ab treatment. However, the morphology of mitochondria was more natural in the lycopene pretreated group (Fig. 2a). These results indicated that lycopene prevented mitochondrial fragmentation in Ab-treated cortical neurons. Multiple studies have found that mitochondrial permeability transition, resulting from the opening of mPTP, is a key factor in the damage to neurons caused by Ab neurotoxicity. The effect of lycopene on Ab-induced mPTP opening was assayed using the calcein-cobalt quenching method. As shown in Fig. 2b, Ab treatment caused an obvious reduction in mitochondrial fluorescence compared to control groups (P \ 0.01), indicating cobalt quenching of casein in the inner mitochondrial matrix, consistent with mPTP opening. However, a relatively higher NRFU was detected in Ab-treated cortical neurons pretreated with lycopene (P \ 0.05), indicating less casein quenching and suggesting that lycopene significantly inhibited mPTP activation. Mitochondrial membrane permeability transition may initiate apoptosis by releasing pro-apoptotic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytoplasm. Accordingly, cytochrome c

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Fig. 1 Effects of lycopene on Ab-induced mitochondrial oxidative stress. a and c Dose-dependent effects of lycopene on intracellular and mitochondrial ROS levels. Lycopene was applied to neurons at 0.1, 0.5, 1, 2, or 5 lM. After preincubation for 4 h, the cultures were then challenged with 10 lM of Ab for 12 h in the continued presence of lycopene. b and d Time-dependent effects of lycopene on intracellular and mitochondrial ROS levels. Cortical neurons were

pretreated with 2 lM lycopene for 4 h, and then exposed to 10 lM Ab for 3, 6, 12, or 24 h. The intracellular and mitochondrial ROS levels were measured using DCFH-DA and MitoSOX Red, respectively. Results are presented as a percentage of the control group (set to 100 %). *P \ 0.05, **P \ 0.01 versus control group, #P \ 0.05, ## P \ 0.01 versus Ab-treated group. Values are mean ± SD, n = 4

release was measured as the ratio of cytochrome c in cytoplasmic and mitochondrial fractions. As shown in Fig. 2c, d, Ab treatment largely increased the ratio of the relative quantity of cytochrome c in the cytosolic fraction to that in the mitochondrial pellet, indicating the release of cytochrome c from mitochondria to cytosol (P \ 0.01). However, lycopene pretreatment significantly reduced the release of cytochrome c in response to Ab (P \ 0.05).

were efficiently prevented by 2 lM lycopene pretreatment. These results suggested the potential of lycopene to preserve the integrity of mitochondrial respiratory chain. Subsequently, intracellular ATP content was determined for analyzing the effect of lycopene on mitochondrial energy metabolism in Ab-treated cortical neurons. As described in Fig. 3e, ATP contents were significantly reduced from 22.8 nmol/mg protein in the control group to 11.0 nmol/mg protein in Ab-treated cortical neurons (*P \ 0.05). However, the Ab-induced reduction in ATP content was significantly inhibited by lycopene pretreatment (#P \ 0.05).These findings provided evidence that lycopene efficiently attenuated Ab-induced mitochondrial dysfunction.

Lycopene Prevented Ab-Induced Mitochondrial Respiratory Dysfunction To investigate whether the lycopene-mediated protection is related to maintaining mitochondrial respiratory function, the activities of individual mitochondrial complexes in mitochondria isolated from cortical neurons were detected. As shown in Fig. 3, treatment of cortical neurons with 10 lM Ab for 12 h resulted in significant reductions in the activities of the mitochondrial respiratory chain enzymes (complexes I, II, III and IV), however, these reductions

Lycopene Prevented Ab-Induced mtDNA Damage Effects of Ab exposure on mtDNA content were independently analyzed by two coding sequences of mtDNA (COX I and ND6) using quantitative real-time PCR. Ab

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Fig. 2 Effects of lycopene on Ab-induced mitochondrial fragmentation and mitochondrial membrane permeability transition. a Representative confocal images of cortical neurons stained with MitoTracker Red. Cortical neurons were pretreated with vehicle or 2 lM lycopene for 4 h and further treated with 10 lM of Ab for 12 h, mitochondria were then loaded with MitoTracker Red probe. The morphological changes of mitochondria were observed with a Leica confocal laser scanning microscope. Scale bar 10 lm. b Lycopene prevented Ab-induced mPTP opening. After the indicated treatment as described in (a), the mPTP opening was detected using the calceincobalt quenching method. Results were presented as normalized

relative fluorescence units (NRFU; U/mg protein). c Representative Western blot showing the expression of cytochrome c in the cytosolic and mitochondrial fractions. d Quantitative analysis of cytochrome c release from mitochondria into the cytosol. The relative quantity of cytochrome c in the cytosolic fractions and mitochondrial fractions were normalized to b-actin and COX IV, respectively. Cytochrome c release was then estimated by the ratio of the relative quantity of cytochrome c in the cytosolic fraction to that in the mitochondrial fraction. *P \ 0.05, **P \ 0.01 versus control group, #P \ 0.05, ## P \ 0.01 versus Ab-treated group. Values are mean ± SD, n = 6 (Color figure online)

treatment for 12 h caused reductions of COX I and ND6 mtDNA copy numbers, while these reductions in mtDNA copy numbers induced by Ab were predominantly prevented by 2 lM lycopene pretreatment (Fig. 4a, b). Similarly, the transcript levels of COX I and ND6 in Ab-treated cortical neurons were markedly decreased and also these decreases in mtDNA transcripts prevented by lycopene pretreatment (Fig. 4c, d). To evaluate the oxidative damage of mtDNA that was induced by Ab treatment, we performed HPLC analysis to

detect the levels of 8-OHdG in mitochondria. As shown in Fig. 4e, HPLC analysis revealed that there was a significant increase in the level of 8-OHdG in mitochondria of the Ab treatment group compared to those of the control and lycopene groups (P \ 0.01), and 2 lM lycopene pretreatment strongly prevented this type of mtDNA oxidative damage (Fig. 4e). Mitochondrial transcription factor A (Tfam) plays a key role in the maintenance of mtDNA integrity. To further explore the underlying mechanism of mtDNA damage, we

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Fig. 3 Effects of lycopene on Ab-induced mitochondrial respiratory dysfunction. Effects of lycopene on electron transport chain complex activity: complex I (a), complex II (b) complex III (c) and complex IV (d). Cortical neurons were pretreated with vehicle or 2 lM lycopene for 4 h and further treated with 10 lM Ab for 12 h. Activities of mitochondrial complex enzyme were then detected by measuring the rate of oxidation of different substrates in mitochondria

isolated from cortical neurons. The mitochondrial complex activities were expressed as percentage of control. e Effects of lycopene on Abinduced reduction in ATP content. The intracellular ATP content was determined using an ATP Determination Kit. The results are expressed as nmol/mg protein. *P \ 0.05, **P \ 0.01 versus control group, #P \ 0.05, ##P \ 0.01 versus Ab-treated group. Values are mean ± SD, n = 9

measured the mitochondrial levels of Tfam by western blot analysis. The protein expression of Tfam in mitochondria was markedly reduced in Ab-treated cortical neurons compared with control. This reduction was prevented by lycopene pretreatment (Fig. 4f).

Discussion It is widely accepted that mitochondrial dysfunction and oxidative stress are implicated in the pathogenesis of AD [19]. Our previous studies demonstrated that lycopene

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Neurochem Res b Fig. 4 Effects of lycopene on Ab-induced mtDNA damage. a, b,

c and d Lycopene prevented Ab-induced reductions in mtDNA copy numbers and transcript levels. Cortical neurons were pretreated with vehicle or 2 lM lycopene for 4 h and further treated with 10 lM of Ab for 12 h. MtDNA copy number (a and b) and mtDNA transcript levels (c and d) were detected by quantitative real-time PCR. Two specific fragments of mtDNA, COX I and ND6, were designed for the quantification. The mtDNA content and mtDNA transcript levels were normalized to the internal control, b-actin and GAPDH, respectively. e Lycopene prevented the increase in the level of 8-OHdG in mitochondria. Cortical neurons were treated as described above, mtDNA in exposed neurons was extracted and digested to deoxynucleotides using nuclease P1 and alkaline phosphatase for HPLC analysis, then the level of 8-OHdG of mitochondria for each experimental group was determined by HPLC using an electrochemical detection system. 8-OHdG amount was expressed as the number of 8-OHdG molecules per 106 dG, determined simultaneously with a UV monitor coupled to the HPLC system. f Representative Western blot showing the expression of mitochondrial Tfam levels and quantitative analysis of the Tfam normalized to the internal control, COX IV. Results are presented as percentages of the control group, which is set at 100 %. *P \ 0.05, **P \ 0.01 versus control group, # P \ 0.05, ##P \ 0.01 versus Ab-treated group. Values are mean ± SD, n = 4

could ameliorate apoptosis and oxidative damage triggered by Ab in a cellular model. In addition, lycopene inhibited mitochondrial membrane potential collapse and restored Bax/Bcl-2 levels, suggesting that lycopene may sustain mitochondrial function under Ab-induced cellular stress [17]. However, the molecular pathways mediating this neuroprotective effect were unclear. In the present study, the mechanistic basis for lycopene-mediated neuroprotection against Ab toxicity was investigated. In light of the central role of mitochondria in AD pathogenesis, we tested the effect of lycopene on several parameters indicative of mitochondrial function in Ab-treated primary cultured rat cortical neurons. Our observation showed that lycopene pretreatment significantly prevented Ab-induced intracellular ROS accumulation and mitochondria-derived superoxide production. In addition, lycopene ameliorated Abinduced mitochondrial morphological alteration, opening of mPTP and the consequent cytochrome c release. Lycopene also prevented Ab-induced decrease of mitochondrial complex enzyme activities and decline of ATP generation. Furthermore, lycopene prevented mitochondrial DNA damages and improved the protein level of Tfam in mitochondria. Based on these finding, we proposed that the ability of lycopene to prevent Ab-induced neurotoxicity is closely related to inhibiting mitochondrial oxidative stress and improving mitochondrial function. Oxidative stress is recognized as an early event in neurodegenerative process in AD and plays a key role in Ab-induced cell death. Previous studies demonstrate that treatment of neuronal cells with Ab causes oxidative stress by various pathways. Mitochondria is a major source of

ROS production and one of the primary targets for Ab. Ab can enter mitochondria and interact with mitochondrial proteins, leading to disruption of electron transport chain and consequent ROS production [20]. Other sources apart from mitochondria also contribute to total ROS generation by Ab. Ab can increase ROS generation through forming an Ab-metal ion complex. Ab and copper ions form an Abmetal ion complex, which produces ROS when coupling Fenton reaction [21]. In addition, Ab can increase oxidative stress through mechanisms involving NMDA receptors and nitric oxide synthase [22]. Our investigation showed that exposure of cortical neurons to Ab caused intracellular ROS accumulation concomitant with mitochondria-derived superoxide production. However, lycopene pretreatment significantly prevented the Ab-induced mitochondrial oxidative stress. Considering that mitochondria is one of the main sources of Ab-induced ROS generation, it seems reasonable to identify the effects of lycopene on mitochondrial integrity and bioenergetic function. mPTP is a non-selective pore spanning the inner and outer mitochondrial membranes. Under pathological conditions, the high conductance mPTP can open, resulting in mitochondrial swelling and release of apoptosis-promoting factors, such as cytochrome c, from mitochondria into the cytosol, leading to cell damage and apoptosis. Increasing evidence suggest that mPTP opening is a key event in Ab-induced neurodegeneration [4]. Consistent with these findings, our results found that Ab treatment caused alteration in mitochondrial morphology, opening of mPTP and release of cytochrome c, indicating that the intoxication led to mitochondrial permeability transition. However, this Ab-induced mitochondrial dysfunction was prevented by lycopene pretreatment. We further investigated the effects of lycopene on mitochondrial bioenergetic function. Mitochondrial respiration is carried out by a series of concurrent protein complexes located in the mitochondrial inner membrane and matrix. Therefore, the impairment of mitochondrial respiratory chain enzymes may decrease the efficiency of oxidative phosphorylation. Indeed, Ab can directly inhibit key enzymes, resulting in disruption of mitochondrial function, which contributes to the deficiency of energy metabolism seen in Alzheimer’s disease [23]. Our results confirmed that Ab treatment resulted in mitochondrial respiratory dysfunction as showed by the decreased activities of mitochondrial enzyme complexes and reduced ATP production. In contrast, lycopene pretreatment prevented the Ab-induced mitochondrial respiratory dysfunction. The results indicated that the improvement in the activities of key enzymes probably contributed to the maintenance by lycopene of mitochondrial active respiration. This finding is consistent with a previous study that chronic administration of lycopene treatment restores the mitochondrial

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respiratory enzyme activities in Ab treated rats [14]. A recently published report from our study also strengthens the fact that lycopene restored mitochondrial enzyme complex activity [16]. The integrity of mtDNA is critical for normal mitochondrial function. Once mtDNA is damaged, the encoding of critical proteins for the respiratory chain becomes deficient, amplifying ROS production and mitochondrial dysfunction. In turn, mitochondrial dysfunction enhances ROS generation and exacerbates oxidative damage to the mtDNA. Chronic mtDNA damage and mitochondrial dysfunction induced by increased ROS have been proved to contribute to the development of AD [24]. In addition, the extracellular Ab has been shown to cause obvious damage to mtDNA through oxidative stress [25]. In our study, there was a significant increase in 8-OHdG in mitochondria after Ab exposure. Concomitant with this alteration, the mtDNA copy numbers and mtDNA transcript levels also decreased after Ab treatment. Furthermore, we also found that lycopene was able to prevent these Ab-induced mtDNA damages. The results were consistent with our and other previous studies that lycopene can protect mtDNA from oxidative damage in human skin subjected to ultraviolet radiation [26], rat cerebellar granule neurons incubated with methylmercury [16], and SH-SY5Y cells incubated with 1-methyl-4-phenylpyridinium iodide [10]. Tfam is a nuclear-encoded high-mobility group (HMG) box protein that binds upstream of the light-strand and heavy-strand of mtDNA. It has multiple pathophysiologic roles in mtDNA maintenance, including nucleoid formation, mtDNA stabilization and mtDNA transcription. Previous studies have demonstrated that extracellular amyloid causes a reduction in Tfam expression [27] and Tfam overexpression can protect mitochondria against Ab-induced oxidative damage [18], indicating that Tfam may be involved in the pathogenesis of AD. Here, we demonstrated that Ab decreased the protein level of mitochondrial Tfam, which was prevented by lycopene pretreatment. Thus, the protective role of lycopene on mtDNA may be due to stabilization of mitochondrial Tfam. This point of view is supported by a recent study that lycopene prevents mitochondrial DNA oxidative damage induced by ischemia/ reperfusion-injury [28]. Lycopene is the most potent singlet oxygen scavenger among the carotenoids with 11 linearly arranged conjugated double bonds. Previous studies revealed that lycopene protects against 3-nitropropionic acid-induced mitochondrial dysfunction and apoptosis in a Huntington’s disease-like model [7] and rotenone-induced oxidative stress and neurobehavioral deficits in rat [8], underscoring its broad neuroprotective efficacy. Similar to these studies, our present study also demonstrated the protective potential of lycopene against mitochondrial oxidative damage and

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dysfunction in amyloid b-treated neurons. In contrast to the results of our study, Choi and Lee [29] revealed that lycopene induces apoptosis in Candida albicans through ROS production and mitochondrial dysfunction. This study suggested that lycopene induces ROS accumulation, which causes mitochondrial dysfunction and triggers DNA damage, thereby contributing to G2/M cell cycle arrest and apoptosis in Candida albicans. These findings seem inconsistent with our observations that lycopene suppressed ROS generation and inhibited apoptosis. The discrepancies may be caused by differences in the structure of mammalian cells and fungi. This issue needs further research to be finally clarified. In summary, the present results confirm and extend our previous investigations that lycopene possesses the ability to prevent Ab-induced mitochondrial oxidative stress and dysfunctions in cultured rat cortical neurons. These therapeutic mechanisms may enable lycopene to attenuate neuronal apoptosis and aid in the treatment of neurodegenerative disease. Acknowledgments We are particularly grateful to Dr. Fanzheng Yang (Central university of Finance and Economics) for her critical review and valuable suggestions that improved the quality of this manuscript. Compliance with Ethical Standards Conflict of interest of interest.

The authors declare that they have no conflict

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