Parkin overexpression attenuates Aβ-induced mitochondrial dysfunction in HEK293 cells by restoring impaired mitophagy

Journal Pre-proof Parkin overexpression attenuates Aβ-induced mitochondrial dysfunction in HEK293 cells by restoring impaired mitophagy

Hongmei Wang, Ting Zhang, Xuhua Ge, Jingjiong Chen, Yuwu Zhao, Jianliang Fu PII:

S0024-3205(20)30069-2

DOI:

https://doi.org/10.1016/j.lfs.2020.117322

Reference:

LFS 117322

To appear in:

Life Sciences

Received date:

12 November 2019

Revised date:

13 January 2020

Accepted date:

13 January 2020

Please cite this article as: H. Wang, T. Zhang, X. Ge, et al., Parkin overexpression attenuates Aβ-induced mitochondrial dysfunction in HEK293 cells by restoring impaired mitophagy, Life Sciences(2020), https://doi.org/10.1016/j.lfs.2020.117322

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© 2020 Published by Elsevier.

Journal Pre-proof Parkin overexpression attenuates Aβ-induced mitochondrial dysfunction in HEK293 cells by restoring impaired mitophagy Hongmei Wanga,1, Ting Zhanga,1, Xuhua Geb, Jingjiong Chena, Yuwu Zhaoa,*, Jianliang Fua,* a

Department of Neurology, Shanghai Jiao Tong University Affiliated Sixth People’s

Hospital, Shanghai, China b

Department of general medicine, Yangpu Hospital Affiliated to Tongji University, Shanghai, China * Corresponding authors at: Department of Neurology, Shanghai Jiao Tong University

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Affiliated Sixth People’s Hospital, No. 600 Yishan Road, Shanghai 222003, China. E-mail address: [email protected] (Y. Zhao), [email protected] (J. Fu) These authors contributed equally to this work.

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ABSTRACT

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Aims: Mitochondrial dysfunction is an early prominent feature of Alzheimer’s disease (AD). In the present study, we sought to investigate whether defective mitophagy is

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tightly related to amyloid-β (Aβ)-induced mitochondrial dysfunction. Main methods: Immunofluorescence, western blot and transmission electron

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microscopy were used to examine mitophagy. Mitochondrial membrane potential was assessed using the JC-1 dye. Mitochondrial ROS was detected using MitoSOX™ Red

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staining.

Key findings: Aβ induced mitochondrial dysfunction in HEK293 cells. Moreover, Aβ induced an increase in parkin translocation to mitochondria and led to a drastic reduction in cytosolic parkin. Furthermore, Aβ-treated cells displayed a microtubule-associated protein 1 light chain 3 (LC3) punctate pattern and elevated mitochondrial LC3-II levels, suggesting the upregulation of mitophagy. Notably, Aβ induced the accumulation of mitochondrial p62, which was associated with impaired mitophagy. In addition, Aβ-treated cells exhibited fragmented or swollen mitochondria with severely decreased cristae. We then investigated whether overexpression of parkin could protect cells against Aβ-induced mitochondrial dysfunction. Interestingly, parkin overexpression inhibited Aβ-induced mitochondrial dysfunction. Besides, parkin overexpression increased cytosolic and mitochondrial parkin levels as well as mitochondrial LC3-II levels in Aβ-treated cells. Additionally, parkin overexpression reversed the accumulation of p62 in mitochondria, indicating

Journal Pre-proof that parkin overexpression restored impaired mitophagy in Aβ-treated cells. Importantly, parkin overexpression remarkably reversed Aβ-induced mitochondrial fragmentation. Significance: Our data demonstrate that overexpression of parkin ameliorates impaired mitophagy and promotes the removal of damaged mitochondria in Aβ-treated cells, indicating that upregulation of parkin-mediated mitophagy may be a potential strategy for the therapy of AD. Keywords: Alzheimer’s disease; Amyloid-β; Mitochondrial dysfunction; Mitophagy;

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Parkin 1. Introduction

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Alzheimer’s disease (AD) is a common neurodegenerative disorder that is

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characterized clinically by progressive deterioration of cognitive functions, memory loss, motor impairments, and behavioral changes. The two major hallmarks of the

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disease are intracellular neurofibrillary tangles (NFTs) and extracellular amyloid-β (Aβ) plaques. In addition, growing evidence suggests that mitochondrial dysfunction

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plays a critical role in the pathogenesis of AD. Indeed, the accumulation of Aβ not only triggers mitochondrial dysfunction but also contributes to the decline in synaptic

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plasticity and cognition. Besides, impairment of mitochondrial energy metabolism, abnormal mitochondrial morphology, decreased mitochondrial membrane potential

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(Δψm), and reduced adenosine triphosphate (ATP) levels have been found in patients with AD [1,2]. Moreover, impaired mitochondrial electron transport chain (ETC) enzymatic activities are associated with AD pathogenesis. A decline in complex I activity was observed in the brain of transgenic AD mouse model [3]. Emmerzaal et al. [4] found that complex II activity was increased in APP/PS1 mice. In addition, Aβ42 has been shown to decrease complex III activity without affecting complex I and II activities in SY5Y cells [5]. Furthermore, postmortem brains of patients with AD exhibit a decline in respiratory capacity [6] as well as excessive mitochondrial fragmentation. Recently, a decrease of complex IV staining associated with plaques has been observed in a mouse model of AD [7]. Consistent with this finding, brain mitochondria isolated from Thy-1 AβPPSL mice display reduced complex IV levels, Δψm, and ATP levels along with increased accumulation of intracellular Aβ [8]. Interestingly, complex IV activity is decreased in AD brain [6,9]. Besides, senescent or damaged mitochondria are sources of reactive oxygen species (ROS). In fact,

Journal Pre-proof damaged mitochondria are correlated with enhanced ROS production in many neurodegenerative disorders including AD. Overproduction of ROS damages proteins, lipids, and nucleic acids, contributing to the development of progressive neurodegeneration associated with AD. Mitophagy, a selective autophagy for eliminating damaged mitochondria, is an essential component of mitochondrial quality control (MQC). The cytosolic E3 ubiquitin ligase parkin and its regulatory kinase PINK1 are key factors in mitophagy. It is believed that parkin is autoinhibited under basal conditions. Upon mitochondrial damage and subsequent mitophagy activation, parkin is recruited to the outer

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mitochondrial membrane (OMM) and ubiquitylates multiple OMM proteins, leading

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to the removal of damaged or dysfunctional mitochondria. More recently, it has been reported that synaptosomal mitochondria from the AD mouse model (5xFAD mice)

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exhibit a dramatic increase in parkin-mediated mitophagy [10]. Moreover, enhanced parkin-mediated mitophagy has been observed in mutant hAPP neurons and AD

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patient brains [11]. Nevertheless, increased mitophagy accompanied by depletion of cytosolic parkin is confirmed in late-stage AD brain, indicating that mitophagy is

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impaired in AD [12]. Thus, efficient clearance of damaged mitochondria by autophagy may be a potential therapeutic strategy for AD. Parkin overexpression has

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been shown to ameliorate Aβ levels as well as plaque deposition in lentiviral models of intracellular Aβ. Importantly, parkin overexpression not only reduces caspase

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activity but also prevents mitochondrial dysfunction and oxidative stress [13]. Although increasing evidence suggests that mitochondrial dysfunction plays a prominent role in the pathogenesis of AD. The detailed mechanisms underlying Aβ-induced mitochondrial dysfunction are still elusive. The purpose of this study was to investigate the role of parkin-mediated mitophagy in protection against Aβ-induced mitochondrial dysfunction. Firstly, we found that Aβ reduced ATP levels, diminished activities of complex I, II, and IV, decreased Δψm, and increased mitochondrial ROS production, confirming that Aβ induced mitochondrial dysfunction in HEK293 cells. In addition, malondialdehyde (MDA) was enhanced in the presence of Aβ. Together, these results indicated that Aβ triggered mitochondrial dysfunction in HEK293 cells. Currently, parkin translocation to mitochondria is often used to display activation of mitophagy. Our results showed that parkin translocation to mitochondria was dramatically increased in Aβ-treated cells, indicating that parkin was recruited to dysfunctional mitochondria in the presence of Aβ. Similarly, Aβ enhanced LC3

Journal Pre-proof punctate pattern and elevated mitochondrial LC3-II levels, confirming the augmentation of mitophagy. These data suggested that Aβ induced mitophagy in HEK293 cells. The adaptor protein p62/SQSTM1 is important for eliminating damaged organelle by autophagy and its accumulation is a marker of dysfunctional autophagy [14]. In the current study, p62/SQSTM1 was robustly accumulated in mitochondrial fractions from Aβ-treated cells. In parallel, cytosolic parkin was evidently reduced in Aβ-treated cells. These results suggested that Aβ induced an increase in impaired mitophagy. Therefore, excessive amounts of damaged mitochondria could not be

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efficiently eliminated by impaired mitophagy. In agreement with mitochondrial dysfunction, abnormal mitochondrial morphology was observed in Aβ-treated cells.

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Compared with control, mitochondria in Aβ-treated cells exhibited fragmented or

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swollen shape with severe loss of cristae. Additionally, the number of mitochondria was significantly increased in Aβ-treated cells, suggesting that Aβ induced

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mitochondrial fragmentation. These findings indicated that Aβ-induced mitochondrial dysfunction was accompanied by mitochondrial abnormalities. Importantly,

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transmission electron microscopy revealed that Aβ-treated cells had some autophagosomes containing mitochondria, whereas untreated cells had scarce

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autophagosomes containing mitochondria, supporting that Aβ induced impaired mitophagy in HEK293 cells.

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In view of the significance of parkin-mediate mitophagy in mitochondrial clearance, we then investigated whether parkin overexpression protected HEK293 cells against Aβ-induced mitochondrial dysfunction. Compared with empty vector-transfected control cells treated with Aβ, parkin-overexpressing cells exposed to Aβ displayed improved mitochondrial functions such as increased cellular ATP levels, elevated activities of complex I, II, and IV, enhanced Δψm, and reduced mitochondrial ROS. In addition, parkin overexpression abolished the enhancement of MDA triggered by Aβ. Interestingly, parkin overexpression significantly reversed Aβ-induced depletion of cytosolic parkin. Besides, following Aβ treatment, enhanced LC3

punctate

pattern

and

mitochondrial

LC3-II

levels

were

found

in

parkin-overexpressing cells relative to empty vector-transfected control cells. In line with these results, parkin overexpression inhibited the accumulation of mitochondrial p62 induced by Aβ, indicating that parkin overexpression induced an increase in efficient

mitophagy.

Consistent

with

improved

mitochondrial

functions,

Journal Pre-proof overexpression of parkin obviously recovered Aβ-induced abnormal mitochondrial morphology. Parkin-overexpressing cells treated with Aβ exhibited oval or round mitochondria with clear cristae, whereas empty vector-transfected control cells displayed fragmented or swollen mitochondria with disrupted cristae in the presence of Aβ, suggesting that parkin overexpression had protective effects on Aβ-induced mitochondrial abnormalities including mitochondrial fragmentation. Notably, transmission electron microscopy revealed that parkin-overexpressing cells exposed to Aβ had abundant autophagosomes containing mitochondria compared with empty vector-transfected control cells treated with Aβ. These results demonstrated that

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overexpression of parkin enhanced efficient mitophagy and promoted the removal of

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damaged mitochondria in Aβ-treated cells.

Taken together, our findings demonstrated that parkin overexpression restored

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impaired mitophagy and partially attenuated mitochondrial dysfunction in Aβ-treated HEK293 cells. Thus, upregulation of parkin-mediated mitophagy may be a potential

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strategy for the therapy of AD.

2.1. Antibodies and reagents

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2. Materials and methods

Human Aβ1-42 peptide, DAPI, antibodies against microtubule-associated protein

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light chain 3 (LC3), and β-actin were purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies against VDAC1, p62, and parkin were acquired from Abcam

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(Cambridge, MA, USA). Anti-mouse and anti-rabbit secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), Lipofectamine 2000, and fetal bovine serum were acquired from Invitrogen (Carlsbad, CA, USA). Plasmids pCMV-HA-parkin and pCMV-HA empty vector were obtained from Hunan Fenghui Biotechnology Co., Ltd. (Hunan, China). 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine

iodide

(JC-1),

MitoSOX, Alexa Fluor 488, and Alexa Fluor 594 were purchased from Molecular Probes (Eugene, OR, USA). Enhanced chemiluminescence reagents were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). 2.2. Cell culture and transfection HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) in a humid, 5% CO2, 37°C incubator. Cells were transfected with pCMV-HA-parkin plasmid or pCMV-HA empty vector using Lipofectamine 2000, according to the manufacturer’s instructions.

Journal Pre-proof At 24 h post-transfection, the cells were treated with Aβ. 2.3. Preparation of oligomeric Aβ Oligomeric Aβ1-42 was prepared according to a published protocol [15]. The Aβ1-42 peptide was initially dissolved to 1 mM in hexafluoroisopropanol and aliquoted into microcentrifuge tubes. Subsequently, Hexafluoroisopropanol was discarded under vacuum in a Speed Vac centrifuge. For oligomer preparation, the peptide film was dissolved in dimethyl sulfoxide (DMSO) to obtain a concentration of 5 mM. The resuspended peptide was then diluted in F12 medium to a concentration of 100 μM, and further incubated for 24 h at 4°C. Finally, the preparation was centrifuged at 14,

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000 g for 10 minutes at 4°C to remove insoluble aggregates and the supernatant

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containing soluble Aβ oligomers was used. 2.4. Measurement of ATP levels

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ATP levels were measured using an ATP Bioluminescence Assay Kit (Promega)

with four replicates per experiment.

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following the manufacturer’s instructions. All experiments were repeated three times

2.5. Measurement of respiratory chain complex activities

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The activities of Complex I were measured as the rotenone sensitive rate of NADH oxidation in the presence or absence of 5μM rotenone [16]. The reaction mixture

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contained 25 mM KH2PO4, 5 mM MgCl2, 0.5 mM KCN, 1 mM NaN3, 2 mg/ml BSA, 2.4μg/mL antimycin, and 0.24 mM CoQ1, 0.15 mM NADH. The absorbance was

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monitored at 340 nm. Complex II activity was determined by following the reduction of DCPIP (2, 6-dichlorophenolindophenol) at 600 nm [5,16]. The reaction mixture contained 50 mM KH2PO4, 5 mM KCN, 2 mg/ml BSA, 20 mM succinate, 5 μM rotenone, 4 mM antimycin A, and 50 μM DCPIP. Complex III specifc catalytic activity was measured by monitoring the reduction of cytochrome c [16,17]. The reaction mixture contained 50 mM KH2PO4, 0.5 mM KCN, 1 mM NaN3, 2 mg/ml BSA, 0.12 mM cytochrome c, 5 mM NADH, and 5 μM rotenone. The absorbance was measured at 550 nm. Complex IV activity was determined by measuring the oxidation of cytochrome c at 550 nm in 1 ml of buffer solution containing 25 mM KH2PO4 (pH 7.4), 0.45 mM dodecyl maltoside, and 15 μM reduced cytochrome c as previously described [18,19]. Activities were expressed as μmol/min/mg protein. All experiments were repeated three times with four replicates per experiment. 2.6. Mitochondrial membrane potential Mitochondrial membrane potential was assessed using the JC-1 dye. Cells were

Journal Pre-proof incubated with 10 μg/ml JC-1 for 15 minutes. Mitochondrial depolarization was indicated by the increase in red fluorescence (aggregates) accompanied with loss of green fluorescence (monomers). Images were taken with a fluorescence microscope and quantitative analysis of fluorescence intensity was performed using ImageJ analysis software. In each experiment, more than 100 cells were detected. The experiments were repeated three times. 2.7. Mitochondrial superoxide production Mitochondrial superoxide production was measured using the MitoSOX™ Red reagent. Cells were incubated with 5 μM MitoSOX™ Red for 10 minutes and images

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were captured with a fluorescence microscope. Fluorescent intensity was analyzed

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using ImageJ analysis software. In each experiment, more than 100 cells were investigated. The experiments were repeated three times.

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2.8. Malondialdehyde (MDA) levels

Levels of MDA were detected using a commercial assay kit (Jiancheng

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Bioengineering Institute, Nanjing, China) according to the manufacturer’s manual. All experiments were repeated three times with four replicates per experiment.

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2.9. Cytosolic and mitochondrial fractionation

Cytosolic and mitochondrial fractions were isolated using a cell mitochondrial

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isolation kit (Beyotime, Jiangsu, China). Fractions were analyzed by western blot. 2.10. Immunofluorescence

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Cells were first fixed with 4% paraformaldehyde for 10 minutes and washed twice with PBS. After fixation, cells were permeabilized with 0.2% Triton X-100 for 15 minutes and blocked in 5% goat serum in PBS for 1 hour at room temperature. Cells were then incubated with (parkin or LC3) primary antibodies overnight at 4 °C. Next, the cells were detected with Alexa 488 or 594 conjugated secondary antibodies in blocking buffer for 1 hour at room temperature. Nuclei were stained with DAPI and images were acquired using a fluorescence microscope. The experiments were repeated at least three times. 2.11. Transmission electron microscopy (TEM) Cells were fixed in 2% gluteraldehyde for 2 hours. After a thorough rinsing in phosphate-buffered saline (PBS), samples were post fixed in 1% osmium tetroxide and subsequently dehydrated with a graded series of alcohol. Samples were then embedded in Epon Resin 618. Ultrathin sections were obtained with a microtome, stained with uranyl acetate and lead citrate. Images were observed using a Philips

Journal Pre-proof CM120 transmission electron microscope. 2.12. Western blotting analysis Cells were lysed in RIPA buffer with protease inhibitors. Samples were run on SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature, and then incubated with primary antibodies overnight at 4°C. The next day, membranes were rinsed for 30 minutes in TBST and incubated with the appropriate HRP-conjugated secondary antibodies for 1 h. Finally, the blots were exposed to enhanced chemiluminescence detection kit.

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2.13. Statistical analysis

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Data are expressed as means ± SEM. Differences between two groups were analyzed using 2-tailed Student's t-test. A p value < 0.05 was considered statistically

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significant.

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3. Results

3.1. Aβ induces mitochondrial dysfunction in HEK293 cells

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Mitochondrial dysfunction has been linked to the pathogenesis of AD. Indeed, mitochondria are the major ATP producers in the cell. We first examined

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mitochondrial function by measuring ATP levels in Aβ-treated HEK293 cells. As shown in Fig. 1A, ATP levels were significantly decreased in Aβ-treated cells

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compared with control. In addition, Aβ has been reported to reduce respiratory chain complex activities. Therefore, we evaluated respiratory chain complex activities in response to Aβ. Compared with control, reduced activities of complex I, II, and IV were observed in Aβ-treated cells (Fig. 1B, C, and E). However, Aβ treatment did not affect complex III activity in HEK293 cells (Fig. 1D). Given that mitochondrial damage leads to the loss of ΔΨm, we then measured ΔΨm in the presence of Aβ. Consistent with decreased ATP levels and diminished activities of complex I, II, and IV, ΔΨm was obviously reduced in Aβ-treated cells (Fig. 1F). Excessive production of ROS is closely linked to mitochondrial dysfunction. Furthermore, mitochondrial ROS was detected using MitoSOX™ Red staining. Mitochondrial ROS production was significantly increased in Aβ-treated cells (Fig. 1G) as compared with control. Finally, MDA (a marker of lipid peroxidation) was detected to monitor mitochondrial function (Fig. 1H). Interestingly, Aβ remarkably enhanced the levels of MDA. Together, these results showed that Aβ induced mitochondrial dysfunction in HEK293 cells.

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3.2. Aβ decreases cytosolic parkin levels and impairs mitophagy in HEK293 cells Next, we investigated the effects of Aβ on mitophagy in HEK293 cells. Parkin translocation to mitochondria is often adopted to display activation of mitophagy. Our results showed that parkin translocation to mitochondria was robustly increased in Aβ-treated cells, whereas parkin displayed a pattern of diffuse staining in untreated cells (Fig. 2A), indicating that parkin was recruited to damaged mitochondria in the presence of Aβ. LC3 is a marker protein for autophagy. LC3-I is cleaved by autophagy-related protein 4 (Atg4) and conjugated to phosphatidylethanolamine to

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form LC3-II, which accumulates in autophagosome membranes. Therefore, LC3-II is

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a useful marker of autophagic compartments [20,21]. Moreover, when autophagy is elicited, LC3 relocates from a diffuse pattern to a punctate autophagosomal

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membrane-bound pattern. Hence, LC3 immunofluorescence staining was performed using LC3-specific antibody. Interestingly, LC3 appeared as a diffused pattern in

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untreated cells, whereas increased punctate distribution of LC3 was detected in Aβ-treated cells (Fig. 2B). Additionally, p62 is recruited to ubiquitin-labeled

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mitochondria, and promotes mitophagy by directly binding to LC3 [22]. Besides, p62 is widely used as a marker of autophagic degradation and the accumulation of p62 is

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correlated with impaired autophagy [23,24]. We subsequently evaluated the levels of parkin, LC3-II, and p62 in cytosolic and mitochondrial fractions from HEK293 cells

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by immunoblotting. Compared with control, Aβ slightly increased cytosolic LC3-II levels and remarkably decreased cytosolic parkin levels (Fig. 2C). However, no significant changes were observed in cytosolic p62 levels between control and Aβ-treated cells. Strikingly, mitochondrial levels of parkin, LC3-II and p62 were increased in Aβ-treated cells (Fig. 2D). Notably, Aβ reduced cytosolic levels of parkin, triggered parkin translocation to mitochondria and the accumulation of mitochondrial p62. Taken together, these results suggested that Aβ impaired mitophagy in HEK293 cells.

3.3. Ultrastructural abnormalities of mitochondria and impaired mitophagy in Aβ-treated HEK293 cells Recent evidence indicates that mitochondria in AD brains exhibit abnormal morphology, such as fragmented with decreased size, swollen shape with disorganized cristae, and reduced matrix volume [1,7,25-28]. Therefore, we then observed whether

Journal Pre-proof mitochondrial dysfunction was accompanied by abnormal mitochondrial morphology in Aβ-treated cells. Mitochondrial morphology was evaluated by transmission electron microscopy. Healthy mitochondria in untreated cells appeared oval or round with intact cristae, whereas damaged mitochondria in Aβ-treated cells exhibited fragmented or swollen shape with severe loss of cristae (Fig. 3A). Indeed, most mitochondria in Aβ-treated cells were fragmented with reduced size. Compared to control cells, the number of mitochondria was significantly increased in Aβ-treated cells (Fig. 3B). These results demonstrated that Aβ induced abnormal mitochondrial morphology in HEK293 cells. Additionally, transmission electron microscopy

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indicated that the number of autophagosomes containing mitochondria (white arrows) was increased in Aβ-treated cells compared to untreated cells (Fig. 3A and B),

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confirming that Aβ induced defective mitophagy in HEK293 cells. 3.4. Parkin overexpression attenuates Aβ-induced mitochondrial dysfunction in

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HEK293 cells

It has been shown that parkin is essential for removal of damaged mitochondria by

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mitophagy. Besides, parkin translocation to mitochondria plays an important role in mitophagy. Based on the results that decreased cytosolic parkin is accompanied by

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mitochondrial dysfunction in Aβ-treated cells. Parkin-overexpressing cells were then used in this study. As shown in supplementary Fig. S1, parkin overexpression

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increases exogenous parkin levels in HEK293 cells. We observed the effects of parkin overexpression on mitochondrial function in the presence of Aβ. HEK293 cells were transfected with pCMV-HA-parkin plasmid or pCMV-HA empty vector for 24h and then treated with 10 µM Aβ for 24 h. Compared with empty vector-transfected control cells treated with Aβ, ATP levels (Fig. 4A), activities of complex I, II, and IV (Fig. 4B, C, and E) were increased in parkin-overexpressing cells exposed to Aβ. However, overexpression of parkin did not affect complex III activity in Aβ-treated cells (Fig. 4D). Besides, overexpression of parkin elevated ΔΨm (Fig. 4F) and diminished mitochondrial ROS production (Fig. 4G) in Aβ-treated cells. Additionally, after Aβ treatment,

significantly

decreased

levels

of

MDA

were

found

in

parkin-overexpressing cells relative to empty vector-transfected control cells (Fig. 4H). These results suggested that Aβ-induced mitochondrial dysfunction was rescued by parkin overexpression.

Journal Pre-proof 3.5. Aβ-induced impaired mitophagy is inhibited by parkin overexpression We subsequently observed whether parkin overexpression attenuated Aβ-induced mitochondrial dysfunction by upregulating parkin-mediated mitophagy and promoting the removal of damaged mitochondria. On the one hand, the fluorescence intensity of parkin was increased in parkin-overexpressing cells treated with Aβ when compared with empty vector-transfected control cells in response to Aβ (Fig. 5A). On the other hand, following Aβ-treatment, enhanced LC3 punctate pattern was found in parkin-overexpressing cells compared with empty vector-transfected control cells (Fig. 5B). Additionally, overexpression of parkin elevated the levels of parkin both in

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cytosolic and mitochondrial fractions from Aβ-treated cells (Fig. 5C and D). Besides, mitochondrial LC3-II levels were obviously enhanced in parkin-overexpressing cells

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exposed to Aβ when compared with empty vector-transfected control cells treated

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with Aβ. However, no significant differences in cytosolic LC3-II levels were observed between control+Aβ cells and parkin+Aβ cells (Fig 5C). These results suggested that

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overexpression of parkin increased efficient mitophagy in Aβ-treated cells. Importantly, overexpression of parkin remarkably increased cytosolic and

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mitochondrial parkin levels as well as inhibited the accumulation of mitochondrial p62 in Aβ-treated cells (Fig. 5C and D), confirming that parkin overexpression

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restored impaired mitophagy triggered by Aβ treatment.

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3.6. Parkin overexpression reverses mitochondrial abnormalities as well as promotes efficient mitophagy in Aβ-treated HEK293 cells To further confirm the effects of parkin overexpression on mitochondrial morphology and mitophagy in Aβ-treated cells, mitochondrial morphology and autophagosomes containing mitochondria were measured by transmission electron microscopy. We found that empty vector-transfected control cells treated with Aβ displayed fragmented or swollen mitochondria with disrupted cristae, whereas parkin-overexpressing cells appeared oval or round mitochondria with clear cristae in the presence Aβ (Fig. 6A). Moreover, overexpression of parkin reversed Aβ-induced mitochondrial fragmentation. Compared to empty vector-transfected control cells treated with Aβ, the number of mitochondria was significantly reduced in parkin-overexpressing cells upon treatment with Aβ (Fig. 6B). Following Aβ treatment, abundant autophagosomes containing mitochondria were found in parkin-overexpressing cells compared with empty vector-transfected control cells (Fig.

Journal Pre-proof 6A and B), indicating that overexpression of parkin remarkably promoted efficient mitophagy in Aβ-treated cells.

3.7. Proposed mechanism for impaired mitophagy and mitochondrial dysfunction in Aβ-treated HEK293 cells Exposure to Aβ induces an increase in the number of damaged mitochondria, reduces cytosolic parkin levels and enhances mitochondrial p62 levels associated with impaired mitophagy, resulting in mitochondrial dysfunction in HEK293 cells. However, overexpression of parkin partially restores Aβ-induced insufficient removal

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of damaged mitochondria and inhibits mitochondrial dysfunction by promoting

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parkin-mediated mitophagy (Fig. 7).

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4. Discussion

Mitochondrial impairment is commonly found in Alzheimer disease. Growing

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evidence suggests that mitochondrial dysfunction is an early pathological feature of AD [29]. Impaired mitochondrial electron transport chain enzymatic activities have

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been implicated in the pathogenesis of AD [3-6,30,31]. Moreover, decreased ΔΨm and ATP levels as well as increased mitochondrial oxidative stress have been observed

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in AD brain [32-34]. Previous evidence suggests that Aβ impairs mitochondrial transport, disrupts mitochondrial electron transport chain, decreases ΔΨm, enhances

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ROS levels, and causes mitochondrial damage [31,35-37]. Consistent with these observations, our results showed that Aβ attenuated ATP levels, reduced activities of complex I, II, and IV, and decreased ΔΨm in HEK293 cells. Similarly, mitochondrial ROS was enhanced in the presence of Aβ, indicating that Aβ impaired mitochondrial functions. Besides, increased ROS production results in peroxidation of lipids and release of toxic lipid aldehydes. We found that Aβ significantly elevated the levels of MDA in comparison to control. Taken together, these results suggested that Aβ induced mitochondrial dysfunction in HEK293 cells, which was consistent with other previous studies showing an increased mitochondrial dysfunction in AD patient and transgenic AD mouse model [30,31,33,34]. Mitophagy is a selective autophagy for removing damaged mitochondria and serves as a form of mitochondrial quality control. It is a key cellular mechanism to maintain mitochondrial integrity and function. Interestingly, recent data suggest that mitophagy is increased in the brains of patients with AD and mouse models of AD.

Journal Pre-proof Moreover, parkin-mediated mitophagy is a critical factor in the efficient elimination of damaged mitochondria. Notably, the recruitment of parkin to the depolarized mitochondria triggers the selective degradation of damaged mitochondria. Indeed, parkin-mediated mitophagy plays a critical role in AD pathogenesis. A recent study shows that mitochondrial fractions from AD brains exhibit increased levels of parkin and LC3-II. In addition, a progressive reduction in parkin along with robustly enhanced expression of p62 has been observed in the brains from AD patients [11]. Besides, parkin translocation to mitochondria is found in mutant hAPP neurons, whereas parkin remains diffusely cytosolic in control cells [11]. LC3-II may be

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recruited to the depolarized mitochondria by p62 in a parkin-dependent manner.

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Importantly, p62 is a multifunctional protein acting as an adaptor protein for mitophagy. P62 binds to ubiquitinated mitochondria, interacts with LC3, mediates the

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cargo engulfment into autophagosomes [38], and promotes the recruitment of damaged mitochondria to the phagophore. In addition, stimulation of autophagy flux

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decreases p62, whereas impaired autophagic clearance leads to p62 accumulation [23,24].

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Furthermore, we investigated the effects of Aβ on mitophagy in HEK293 cells. Parkin translocation to mitochondria was increased in Aβ-treated cells, whereas

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diffused cytosolic parkin was shown in untreated cells, supporting that parkin was recruited to damaged mitochondria in the presence of Aβ. In line with the result of

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parkin, LC3 punctate pattern was elevated in Aβ-treated cells. We subsequently evaluated the levels of parkin, LC3-II, and p62 in mitochondria and cytosol by immunoblotting. Parkin levels were obviously reduced in cytosolic fractions from Aβ-treated cells, whereas cytosolic LC3-II levels were slightly increased in Aβ-treated cells when compared with control. However, Aβ failed to alter cytosolic p62 levels. Notably, levels of pakin, LC3-II, and p62 were increased in mitochondrial fractions from Aβ-treated cells. Interestingly, Aβ remarkably enhanced the accumulation of mitochondrial p62 as well as promoted the depletion of cytosolic parkin, suggesting that Aβ triggered impaired mitophagy. Furthermore, impaired mitophagy results in the accumulation of damaged mitochondria. Eventually, excessive numbers of damaged mitochondria contribute to the overproduction of ROS, causing synaptic loss and neuronal damage in AD. In addition to mitochondrial dysfunction, abnormal mitochondrial morphology has been shown in neurons from AD brains [7,27,28]. AD brains display abnormal

Journal Pre-proof accumulation of altered mitochondria including decreased length, increased fragmentation, reduced matrix volume, and broken internal membrane cristae [1,7,26-28]. Therefore, we measured mitochondrial morphology by transmission electron microscopy. As a result, mitochondria in untreated cells exhibited oval or round with intact cristae. Conversely, mitochondria in Aβ-treated cells showed fragmented or swollen shape with obviously decreased cristae. These results indicated that Aβ induced abnormal mitochondrial morphology, which is associated with mitochondrial dysfunction. Additionally, Aβ-treated cells had some autophagosomes containing mitochondria, whereas untreated cells had scarce autophagosomes

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containing mitochondria, confirming that Aβ triggered impaired mitophagy. Thus,

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efficient elimination of damaged mitochondria through mitophagy may be a promising therapeutic strategy for AD treatment.

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Many studies showed that parkin overexpression promoted mitophagy to degrade damaged mitochondria, leading to the inhibition of ROS. Furthermore, we

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investigated the protective mechanism of parkin overexpression against Aβ-induced mitochondrial dysfunction. Our results showed that following Aβ treatment, enhanced

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ATP levels, elevated activities of complex I, II, and IV, and increased ΔΨm were observed in parkin-overexpressing cells compared with empty vector-transfected

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control cells. In addition, parkin overexpression obviously reduced mitochondrial ROS production in Aβ-treated cells. Similarly, parkin overexpression attenuated the

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enhanced levels of MDA induced by Aβ. Taken together, these results demonstrated that overexpression of parkin inhibited Aβ-induced mitochondrial dysfunction. We then detected whether parkin overexpression inhibited Aβ-induced mitochondrial dysfunction by upregulating parkin-mediated mitophagy. Our data showed that parkin fluorescence intensity was enhanced in parkin-overexpressing cells following Aβ treatment, when compared to empty vector-transfected control cells. Moreover, overexpression of parkin increased LC3 punctate pattern in Aβ-treated cells. Besides, parkin overexpression elevated the levels of parkin both in mitochondria and cytosol. Additionally, parkin overexpression obviously increased the expression levels of mitochondrial LC3-II and abolished the accumulation of mitochondrial p62 induced by Aβ treatment, indicating that parkin overexpression induced an increase in efficient mitophagy. Since parkin overexpression improved mitochondrial function, we then observed the effects of parkin overexpression on mitochondrial morphology in Aβ-treated cells.

Journal Pre-proof Following Aβ treatment, most parkin-overexpressing cells appeared oval or round mitochondria with clear cristae, whereas empty vector-transfected control cells exhibited fragmented or swollen mitochondria with severely decreased cristae. Importantly, overexpression of parkin remarkably reversed Aβ-induced mitochondrial fragmentation. These results indicated that overexpression of parkin inhibited Aβ-induced abnormal mitochondrial morphology associated with mitochondrial dysfunction. Furthermore, the number of autophagosomes containing mitochondria was increased in parkin overexpressing cells treated with Aβ compared with empty vector-transfected

control

cells

following

Aβ

treatment,

confirming

that

of

overexpression of parkin remarkably promoted efficient mitophagy in Aβ-treated

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cells. 5. Conclusion

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In summary, our data demonstrated that parkin overexpression remarkably reversed the depletion of cytosolic parkin, partially restored impaired mitophagy,

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leading to efficient elimination of damaged mitochondria in Aβ-treated HEK293 cells. Therefore, activation of parkin-mediated mitophagy by genetic or pharmacological

Acknowledgements

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interventions may have therapeutic relevance for the treatment of AD.

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This work was supported by grants from the National Natural Science Foundation of China (81672243, 31771185, 81871103, 81870952) and Medical Professional Cross

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Research Fund Project of Shanghai Jiao Tong University (YG2015MS14). Conflict of interest

The authors declare no conflicts of interest.

Figure legends

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Fig. 1. Mitochondrial dysfunction in Aβ-treated HEK293 cells. (A-H) HEK293 cells were untreated or treated with 10 µM Aβ for 24 h, then adenosine triphosphate (ATP) levels, activities of complex I, II, III, and IV, mitochondrial membrane potential (Δψm), mitochondrial reactive oxygen species (ROS), and malondialdehyde (MDA) levels were measured. Mitochondrial depolarization is indicated by a decrease in the JC-1 aggregates (red)/JC-1 monomers (green) fluorescence intensity ratio. Aβ significantly decreased ATP levels (n=3, P=0.0219), diminished activities of complex I (n=3, P=0.0322), complex II (n=3, P=0.0066), and complex IV (n=3, P=0.0413), reduced ΔΨm (n=100 cells per group, P=0.0156), increased mitochondrial ROS (n=100 cells per group, P=0.0439), and enhanced MDA levels (n=3, P=0.0308) in HEK293 cells. Scale bars: (F) 20 μm, (G) 20 μm. Data were analyzed using using 2-tailed Student's t-test. *P < 0.05, **P < 0.01.

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Fig. 2. Increased recruitment of parkin and LC3-II to mitochondria in Aβ-treated HEK293 cells. (A) HEK293 cells were untreated or treated with 10 µM Aβ for 24 h,

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and then stained with parkin (red) antibody. DAPI (blue) was used to visualize the nuclei. Compared with control, Aβ triggered parkin translocation to mitochondria in HEK293 cells. Scale bar, 20 µm. (B) HEK293 cells were untreated or treated with 10 µM Aβ for 24 h, and then stained with LC3 (green) antibody. DAPI (blue) was used to visualize the nuclei. Compared with control, Aβ increased LC3 punctate pattern in HEK293 cells. Scale bar, 20 µm. (C-D) HEK293 cells were untreated or treated with 10 µM Aβ for 24 h, and then cytosolic and mitochondrial fractions were isolated. Protein levels of parkin, p62, and LC3-II were analyzed by western blotting. VDAC1 and β-actin were used as the loading controls for mitochondrial and cytosolic fractions, respectively. The band intensity was quantified by using ImageJ software. Values represent mean ± SEM from western blot analyses of three independent experiments. Compared with control, Aβ slightly increased cytosolic LC3-II levels (n=3, P=0.0412) and remarkably decreased cytosolic parkin levels (n=3, P=0.0075). In addition, Aβ robustly increased mitochondrial parkin (n=3, P=0.0324), LC3-II (n=3,

Journal Pre-proof P=0.0078) and p62 (n=3, P=0.0164) levels in HEK293 cells. Data were analyzed

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using using 2-tailed Student's t-test. *P < 0.05, **P < 0.01.

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Fig. 3. Abnormal mitochondrial morphology and impaired mitophagy in Aβ-treated

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HEK293 cells. (A) HEK293 cells were untreated or treated with 10 µM Aβ for 24 h and then mitochondrial morphology was measured by transmission electron

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microscopy. Normal mitochondria in control cells were oval or round with intact cristae, whereas mitochondria in Aβ-treated cells displayed fragmented or swollen

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with severely decreased cristae. Transmission electron microscopy revealed that Aβ-treated cells had more autophagosomes containing mitochondria than untreated

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cells. White arrow indicates autophagosome containg mitochondria. Scale bar, 1 µm. (B) Quantification of mitochondrial number (n=30 cells scored for number of mitochondria in 3 independent experiments, P=0.0082) and autophagosomes containing mitochondria (n=30 cells scored for number of autophagosomes in 3 independent

experiments,

P=0.0146).

The

number

of

mitochondria

and

mitophagosomes was counted manually. Data were analyzed using using 2-tailed Student's t-test. *P < 0.05, **P < 0.01.

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Fig. 4. Parkin overexpression prevents Aβ-induced mitochondrial dysfunction in HEK293 cells. (A-H) HEK293 cells were transfected with pCMV-HA-parkin plasmid or empty vector (control) for 24 h and then treated with Aβ (10 µM) for 24 h. Following various treatments, ATP levels, activities of complex I, II, III, and IV, Δψm, mitochondrial ROS, and MDA levels were measured. Compared with empty vector-transfected control cells treated with Aβ, elevated ATP levels (n=3, P=0.0355), enhanced activities of complex I (n=3, P=0.0199), complex II (n=3, P=0.0130), and complex IV (n=3, P=0.0037), increased Δψm (n=100 cells per group, P=0.0426), reduced mitochondrial ROS (n=100 cells per group, P=0.0091), and decreased MDA levels (n=3, P=0.0185) were detected in parkin-overexpressing cells following Aβ treatment. Scale bars: (F) 20 μm, (G) 20 μm. Data were analyzed using using 2-tailed Student's t-test. *P < 0.05, **P < 0.01.

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Fig. 5. Parkin overexpression restores Aβ-induced impaired mitophagy in HEK293

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cells. (A) HEK293 cells were transfected with pCMV-HA-parkin plasmid or empty vector (control) for 24 h and then treated with Aβ (10 µM) for 24 h. Following various treatments, cells were stained with pakin (red) antibody. DAPI (blue) was used to visualize the nuclei. The expression of parkin staining was significantly increased in parkin-overexpressing cells following Aβ treatment, when compared to empty vector-transfected control cells. Scale bar, 20 µm. (B) HEK293 cells were transfected with pCMV-HA-parkin plasmid or empty vector (control) for 24 h and then treated with Aβ (10 µM) for 24 h. Cells were then stained with LC3 (green) antibody. DAPI (blue) was used to visualize the nuclei. Compared with empty vector-transfected control

cells

treated

with

Aβ,

LC3

punctate

pattern

was

elevated

in

parkin-overexpressing cells exposed to Aβ. Scale bar, 20 µm. (C-D) HEK293 cells were transfected with pCMV-HA-parkin plasmid or empty vector (control) for 24 h and then treated with Aβ (10 µM) for 24 h. Cytosolic and mitochondrial fractions were isolated. Protein levels of parkin, p62, and LC3-II were then analyzed by

Journal Pre-proof western blotting. VDAC1 and β-actin were used as the loading controls for mitochondrial and cytosolic fractions, respectively. The band intensity was quantified by using ImageJ software. Values represent mean ± SEM from western blot analyses of three independent experiments. Overexpression of parkin significantly elevated the levels of cytosolic parkin (n=3, P= 0.0088) and mitochondrial parkin (n=3, P=0.0072), mitochondrial LC3-II (n=3, P=0.0208) as well as reduced the accumulation of mitochondrial p62 (n=3, P= 0.0084) in Aβ-treated cells. Data were analyzed using

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using 2-tailed Student's t-test.*P < 0.05, **P < 0.01.

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Fig. 6. Parkin overexpression inhibits abnormal mitochondrial morphology and enhances efficient mitophagy in Aβ-treated HEK293 cells. (A) HEK293 cells were transfected with pCMV-HA-parkin plasmid or empty vector (control) for 24 h and then treated with Aβ (10 µM) for 24 h. Mitochondrial morphology was then measured by transmission electron microscopy. Following Aβ treatment, mitochondria in empty vector-transfected control cells appeared fragmented or swollen with severe loss of cristae, whereas mitochondria in parkin overexpressing cells displayed oval or round with distinct cristae. The number of autophagosomes containing mitochondria was obviously increased in parkin-overexpressing cells following Aβ treatment, when compared to empty vector-transfected control cells. White arrows indicate autophagosomes containing mitochondria. Scale bar, 1µm. (B) Quantification of mitochondrial number (n=30 cells scored for number of mitochondria in 3 independent experiments, P=0.0237) and autophagosomes containing mitochondria (n=30 cells scored for number of autophagosomes in 3 independent experiments,

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P=0.0201). Data were analyzed using using 2-tailed Student's t-test. *P < 0.05.

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Fig. 7. Schematic diagram showing the signaling pathways leading to Aβ-induced

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impaired mitophagy and mitochondrial dysfunction. Aβ treatment results in depletion of cytosolic parkin and impaired mitophagy in HEK293 cells. However,

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restoring impaired mitophagy.

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Conflict of interest The authors declare no conflicts of interest. Author contributions

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HW, YZ, and JF designed the study, HW and TZ performed the experiments, XG and JC analyzed the data, HW and TZ wrote the paper. All authors approved the final manuscripts.

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Graphical abstract