Cyclosporin A protects against Lead neurotoxicity through inhibiting mitochondrial permeability transition pore opening in nerve cells

Accepted Manuscript Title: Cyclosporin A protects against Lead neurotoxicity through inhibiting mitochondrial permeability transition pore opening in nerve cells Author: Fang Ye Xiaoyi Li Fen Li Jianxin Li Wei Chang Jing Yuan Jun Chen PII: DOI: Reference:

S0161-813X(16)30208-X http://dx.doi.org/doi:10.1016/j.neuro.2016.10.004 NEUTOX 2087

To appear in:

NEUTOX

Received date: Revised date: Accepted date:

19-2-2016 22-9-2016 6-10-2016

Please cite this article as: Ye Fang, Li Xiaoyi, Li Fen, Li Jianxin, Chang Wei, Yuan Jing, Chen Jun.Cyclosporin A protects against Lead neurotoxicity through inhibiting mitochondrial permeability transition pore opening in nerve cells.Neurotoxicology http://dx.doi.org/10.1016/j.neuro.2016.10.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights： 1． PbAc increased the protein level of Cyp D and induced MPTP opening in SH-SY5Y and PC12 cells 2． CSA could ameliorate Pb-induced impairment of mitochondrial morphology and function in SH-SY5Y cells. 3． The nerves cell death caused by PbAc could be attenuated by CSA

1

Cyclosporin A protects against Lead neurotoxicity through inhibiting mitochondrial permeability transition pore opening in nerve cells Fang Yea,b, Xiaoyi Li c, Fen Lia,d, Jianxin Lie, Wei Changf, Jing Yuana,b, Jun Chena,b*

a

Department of Occupational and Environmental Health, School of Public Health, Tongji Medical

College, Huazhong University of Science and Technology, Wuhan, PR China. b

Ministry of Education Key Lab for Environment and Health, School of Public Health, Tongji

Medical College, Huazhong University of Science and Technology, Wuhan, PR China. c

Department of Immunology, Tongji Medical College, Huazhong University of Science and

Technology, Wuhan, PR China. d

Disease Control and Prevention Center of Xiqing, Tianjin, PR China.

e

Department of Internal medicine, Xiaonan Maternity and Child Health Hospital, Xiaogan, PR

China. f

Department of Public Health, Medical College, Wuhan University of Science and Technology,

Wuhan, PR China.

*

Corresponding author

Email: Jun Chen: [email protected]

2

Abstract Mitochondria play a key role in the process of lead (Pb)-induced impairment in nervous system. To further clarify the underlying mechanism of Pb neurotoxicity, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and cyclophilin D (CyPD), a component of MPT pore (MPTP), in Pb-induced mitochondrial apoptosis in nerve cells. In SH-SY5Y and PC12 cells, Cyclosporin A (CSA), a special inhibitor of CyPD, could alleviate cell death, lactate dehydrogenase (LDH) leakage and adenosine 5 triphosphate (ATP) decrease caused by PbAc. In the following experiments, we found PbAc increased the protein level of CyPD and induced MPT pore (MPTP) opening. When cells were pretreated with CSA to inhibit MPTP opening, the Pb-induced impairment of mitochondrial morphology (swelling and rupture) and the loss of mitochondria were attenuated. In addition, CSA obviously ameliorated the Pb-induced damage of mitochondrial function, such as reactive oxygen species (ROS) boost and mitochondrial membrane potential (MMP) collapse, as well as the release of cytochrome C (Cyto C) and apoptosis-inducing factor (AIF) from mitochondria. These beneficial effects could finally result in cell survival under Pb-exposure conditions. Furthermore, scavenging ROS also significantly abrogated MPTP opening and attenuated Pb neurotoxicity. Therefore, we found that MPT played an important role in Pb-induced mitochondrial damage and, ultimately, cell death. Our results provided a potential strategy for inhibiting PbAc neurotoxicity. However, due to the high Pb concentrations used in this study further investigations at Pb concentrations closer to human exposure are needed to verify the results. Key words: Pb, Neurotoxicity, CyPD, MPTP, CSA, ROS

1. Introduction 3

Lead (Pb) remains a widespread pollutant that causes significant public health problems despite the various preventative activities and treatment performed to reduce Pb emissions and toxicity. Several studies have pointed out that Pb can impair virtually every organ system, notably the central nervous system (Garza et al., 2006). Pb can affect different biological processes in the nervous system, including metal transport, neurotransmitter storage and release processes, energy metabolism, and genetic regulation (Garza et al., 2006). Furthermore, it causes the loss and death of brain neurons (Chao et al., 2007). Exposure to low levels of Pb has been associated with learning impairment, behavioral abnormalities, impaired cognitive functions, and decreased hearing in humans and experimental animals. Although several studies have been devoted to investigating the neurotoxic effects of Pb, the precise molecular mechanisms behind the subtle toxic effects of Pb remain largely unknown. Given that mitochondria play an essential role in controlling cell fate, death, or survival, this study focused on the role of mitochondrial permeability transition (MPT) and cyclophilin D (CyPD) in Pb neurotoxicity (He et al., 2000). In most vertebrates, mitochondria are central players in most apoptotic pathways (Green and Kroemer, 2004). The increase in MPT mainly induced by MPTP opening can serve as a checkpoint to determine the fate of cells (Kroemer et al., 2007). However, the precise components of MPTP have not been clarified, recent works indicate that MPTP may be comprised by F1F0-ATPase and spastic paraplegia 7 (SPG7), and regulated by voltage-dependent anion channel (VDAC) and CyPD (Izzo et al., 2016). MPTP acts as a high-conductance channel across the mitochondrial membrane, whose open–closed transition is required for maintaining normal mitochondrial functions, such as adenosine 5 triphosphate (ATP) production and the maintenance of an electrochemical gradient for respiration (Giorgio et al., 2010). When cells undergo stress, such as reactive oxygen species (ROS) and calcium overload, the MPTP will be opened, which is normally accompanied by the collapse of the mitochondrial membrane potential (MMP), mitochondrial swelling, and outer mitochondrial membrane (OMM) rupture (Tsujimoto and Shimizu, 2007). The aberrant MPTP opening will liberate intermembrane mitochondrial pro-apoptotic proteins, including Cyto C and apoptosis-inducing factor (AIF), into the cytosol, thereby triggering necrosis or apoptosis in cells. Once in the cytosol, cytochrome C (Cyto C) can activate a cascade of caspases, which propagate apoptotic signals, and finally induce caspase-dependent apoptosis. Other apoptogenic factors, such as AIF and endonuclease G 4

(EndoG), will be translocated into the nucleus and cleave nuclear DNA, triggering caspase-independent apoptosis (Bernardi, 1999). In fact, among those proteins composing MPTP, most of the attention has been focused on CyPD in the matrix because of its importance in controlling MPTP opening and cell death on the basis of multiple genetic and biochemical studies (Giorgio et al., 2010). Therefore, we focused on CyPD, one of the seven major cyclophilin isoforms (Lee and Kim, 2010). In general cases, CyPD is activated by an elevated Ca2+ level in the intra mitochondria; Ca2+ binds directly to the binding site of divalent metal (Me2+) on the matrix side of the MPTP and induces its opening (Mazzeo et al., 2009; Toman and Fiskum, 2011). Conditions that favor protein oxidation may also promote MPTP opening, which acts as a redox sensor (Linard et al., 2009; Tsujimoto and Shimizu, 2007). Mitochondria isolated from mice lacking ppif-/-, the gene encoding for mitochondrial CyPD, were more resistant to MPTP induction than wild-type mice, as well as exhibited MPTP opening at higher Ca2+ levels and less cell death in response to oxidative stress (Baines et al., 2005; Nakagawa et al., 2005). Baines et al. found that CyPD overexpression would induce mitochondrial swelling, MPTP opening, and spontaneous cardiac apoptosis, whereas mice lacking cardiac CyPD were protected from cell death induced by ischemia/reperfusion (Baines et al., 2005). In aging models, CyPD expression was enhanced with advancing age, which is concomitant with high oxidative stress and increased apoptotic DNA fragmentation (Marzetti et al., 2008). Pb can concentrate in the mitochondria and damage the organelle, thereby degrading energy metabolism and favoring the generation of ROS (Lidsky and Schneider, 2003). In addition, Pb2+ can function as a potent Ca2+ agonist and bind to the internal metal (Me2+) binding site (He et al., 2000). The disturbance of Ca2+ homeostasis and the induction of oxidative stress are mostly discussed in Pb toxicity; these two aspects are also the two most important activators of CyPD-induced MPTP opening (Garza et al., 2006; Patrick, 2006a). In consideration of the two major toxicological characteristics of Pb, we hypothesized that CyPD plays an important role in the process of MPTP opening induced by Pb in the nervous system. Several studies have observed that Pb could promote MPTP opening and MMP collapse (He et al., 2000). Liu et al. found that CyPD participated in MPTP opening during Pb exposure in rat proximal tubular (rPT) cells (Liu et al., 2015). Low Pb levels could reduce VDAC transcription and expression, resulting in reduced cellular ATP levels (Prins et al., 2010). However, these studies do not clarify the precise 5

mechanism, and more detailed studies are needed. In current study, we applied Cyclosporin A (CSA), a compound that effectively interacts with CyPD and blocks MPTP opening to investigate the interactions among Pb, CyPD, and MPT, (Matas et al., 2009). CSA has been widely used in organ transplantation as a well-known immunosuppressive drug and it possesses neuroprotective properties in traumatic brain injury (TBI) and ischemic stroke models. Several studies demonstrated that the protective effects of CSA by inhibiting cell death are probably caused by its function of blocking MPTP opening and maintaining the MMP (Lulic et al., 2011; Osman et al., 2011). Human SH-SY5Y and Rattus PC12 cells were used as relevant in vitro model systems for primary neuronal cells because they stop dividing, grow long neuritis, and show changes in cellular composition associated with neuronal differentiation in response to treatment with the nerve growth factor and retinoic acid, respectively (Prins et al., 2010). Current study contains three parts. Firstly, the protective effects of CSA against Pb neurotoxicity were observed in both SH-SY5Y and PC12 cells. Then, the effects on transcription and expression of CyPD and MPT were detected in Pb-exposed SH-SY5Y and PC12 cells. Finally, the mechanism of CSA protection against Pb neurotoxicity was explored in SH-SY5Y cells.

2. Method and materials 2.1 Materials Lead acetate (PbAc), N-acetyl-L-cysteine (NAC), and CSA were purchased from Sigma–Aldrich (St. Louis, MO, USA). DMEM/F12, Hank’s balanced salt solution, and fetal bovine serum were acquired from GIBCO (Invitrogen, Carlsbad, CA, USA). Deionized water was produced with an ultrapure water purification system.

2.2 cell culture Human neuroblastoma SH-SY5Y cells obtained from the American Type Culture Collection (ATCC, CA, USA) were maintained and subcultured in DMEM/F12 medium supplemented with 10% FBS，1% non-essential amino acids, 100 U/mL penicillin and 100 U/mL streptomycin at 37°C in a humidified 5% CO2 atmosphere. Well-differentiated rat pheochromocytoma (PC12) cells induced by nerve growth factor were obtained from Shanghai Institutes for Biological Sciences, 6

Chinese Academy of Cell Resource Center (Shanghai, China). Cells were maintained and subcultured in DMEM/F12 medium supplemented with 10% FBS，1% non-essential amino acids, 100 U/mL penicillin and 100 U/mL streptomycin at 37°C in a humidified 5% CO2 atmosphere. Both of Cells were seeded on 6-well plates for western blot, real time PCR, cell apoptosis and ROS production analysis. To analyze the cell viability and LDH leakage, cells were grown onto 96-well plates.

2.3 Cell viability assay Cell viability was assessed with the Cell Counter Kit-8 (CCK-8) assay (Dojindo Laboratories., Japan) according to the manufacturer’s instruction. SH-SY5Y cells (ATCC, CA, USA) or PC12 cells (Chinese Type Culture Collection, Shanghai, China) were seeded on a 96-well plate and left to attach overnight. After the indicated treatments, 10 μM of the CCK-8 solution was dissolved by serum-free medium and added to each well of the plate. The cells were incubated for 1 h in the incubator, and the absorbance at 450nm was quantified on an automated microplate reader (Synergy 2, Bio-Tec, CA, USA).

2.4 lactate dehydrogenase (LDH) assay LDH assay (Beyotime Biotechnology Co., China) was performed with a microplate reader (Synergy 2, Bio-Tec, CA, USA) according to the manufacturer’s instructions. SH-SY5Y or PC12 cells were seeded on a 96-well plate and left to attach overnight. After treatment with PbAc, the 96-well plate was centrifuged at 400 rpm for 5 min. Subsequently, 100 µL of the cell supernatant was transferred to a fresh plate, added with 100 µL of the reaction solution, and incubated for 30 min at 37 °C before the absorbance at 490 nm was read.

2.5 Annexin V-PI assay Pb-induced apoptosis was determined by flow cytometry with the Annexin V-FITC Apoptosis Detection Kit (Biovision, CA, USA) as described by the manufacturer. Briefly, SH-SY5Y cells (2.5×105 cells/well) were seeded into 6-well plates. At the end of the treatment, the cells were collected by centrifugation at 1000 rpm for 5 min and washed twice with ice-cold phosphate-buffered saline (PBS). The cells were resuspended in 500 μL of binding buffer and 7

stained with Annexin V-FITC solution (5 μL) and propidine iodide (PI) solution (5 μL) for 15 min at room temperature in the dark. The samples were then analyzed by flow cytometry (LSRⅡ,Becton Dickinson, San Jose, CA, USA). A total of 10 000 cells were analyzed for each sample. The percentage distributions of early apoptotic (FITC+PI-) and late apoptotic (FITC+PI+) were calculated for comparison.

2.6 Hoechst staining Hoechst 33342 staining (Life Technology, Carlsbad, CA, USA) was applied to assess the morphological changes of treated cells. After SH-SY5Y cells were treated with PbAc, 10 μg/ml of Hoechst 33342 medium were added and incubated for 10 min, then cells were washed three times with PBS before counted under fluorescence microscope (BX43, Olympus, Tokyo, Japan). A total of 200 cells were randomly selected to count the apoptotic cells (chromatin condensation, nucleic fragmentation) within each batch of experiments, which were performed in triplicate.

2.7 Transmission electron microscopy SH-SY5Y cells were pretreated with or without 0.2 μM CSA for 1 h and exposed to 25 μM PbAc for 24 h. A total of 1×107 cells were fixed in 2.0% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h at room temperature and scraped with a cell scraper. Ultrathin sections of osmium-stained SH-SY5Y cells were prepared. Imaging was performed with a Tecnai G2 20 TWIN (FEI, USA) electron microscope.

2.8 ROS, MMP, and mitochondrial mass assay Flow cytometry was used to analyze intracellular ROS, MMP, and mitochondria mass by means of fluorescence probe. SH-SY5Y or PC12 cells were seeded into plates at the end of the treatment. The medium was replaced with fresh serum-free medium containing 10 μg/ml of 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime Biotechnology Co., China), 10 μg/ml of Rhodamine 123 (Life Technology, Carlsbad, CA, USA), or 100 nM of Mitotracker Deep Red (Life Technology, Carlsbad, CA, USA). The cells were incubated for 20 min at 37 °C in the dark and washed thrice with serum-free medium. Collected cells were then washed twice with cold PBS and immediately analyzed by flow cytometry (LSRⅡ, Becton Dickinson, San Jose, CA, 8

USA). The channels for ROS, MMP, and mitochondria mass measurement were DCFH-DA, FITC and APC, respectively. A total of 10 000 cells were analyzed for each sample, and the mean fluorescence intensity was obtained.

2.9 ATP assay The cellular ATP levels for each group were determined by an ATP Bioluminescence Assay Kit (Beyotime Biotechnology Co., China). Cells were seeded in a 6-well plate and left overnight. After the indicated treatment, cells were lysed and centrifuged at 12 000rpm for 5 min at 4 °C. The supernatants were obtained. For ATP determinations, 100 µL of working solution was added to 20 µL of sample in the 96-well plate, and the luminescence measurement was immediately started on an automated microplate reader (Synergy 2, Bio-Tec, CA, USA). Measurements from all samples were normalized to the protein concentration.

2.10 MPT assay MPTP opening was assessed by the calcein-AM/cobalt method according to (Katoh et al., 2002), and SH-SY5Y and PC12 cells were seeded in the 6-well plates. After the indicated treatment, cells were washed with PBS and loaded with 1 µM calcein-AM (Life Technology, CA, USA) for 20 min at 37 °C in the serum-free medium, followed by additional incubation with 1 mM CoCl2 for 1 h. After quenching, cells were washed using a serum-free medium and collected in PBS. The fluorescence was immediately analyzed by flow cytometry (LSR Ⅱ ,Becton Dickinson, San Jose, CA, USA). A total of 10 000 cells were analyzed for each sample and the mean fluorescence intensity (MFI) was obtained.

2.11 Real Time-PCR RNA was extracted from SH-SY5Y or PC12 cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The quality and quantity of RNA were measured by NanoDrop 1000 (Thermo Scientific, USA). Approximately 2 μg of extracted total RNA was used for cDNA synthesis with the Ominiscript RT Kit (Thermo Scientific, USA) in accordance with the manufacturer’s instructions. RT-PCR was performed in 10 µL containing 100 nM primers (Table 1) purchased from Invitrogen (CA, USA) and SYBR Green PCR Master 9

Mix (Life Technology, CA, USA). Amplification was conducted in an ABI 7900HT sequence detection system. The PCR conditions were 95 °C for 2 min, 40 cycles at 95 °C for 15 s, and 60 °C for 60 s, then the method of melting curve was added in the end. The mRNA expression was normalized to human GAPDH or rat GAPDH mRNA expression by the comparative cycle threshold method. The identity and purity of the amplified product was checked by analyzing the melting curve at the end of amplification.

2.12 Western blot analysis Total proteins were extracted with an ice-cold RIPA buffer (Beyotime Biotechnology Co., China) containing a protease inhibitor cocktail (Roche, Germany). As previously reported (Ye et al., 2015), to prepare the cytoplasmic and nuclear proteins, the cells or tissues were first lysed or homogenized in cytoplasmic protein lysis buffer [10 mM HEPES-NaOH (pH 7.9), 10 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2% NP-40] on ice. The lysates were ultracentrifuged at 10 000 rpm for 10 min at 4 °C, and the supernatants were collected as cytoplasmic protein. The pelleted nuclei were resuspended in nuclear protein lysis buffer (20 mM HEPES-NaOH, 420 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% NP-40). After 40 min of incubation at 4 °C, the lysates were centrifuged, and the supernatants containing the nuclear proteins were obtained. Mitochondrial fractions of the SH-SY5Y cells were isolated with the Mitochondria Isolation Kit (Thermo Scientific, USA) according to the manufacturer’s recommendation. Protein concentrations were determined by the BCA protein assay kit (Pierce, Rockford, IL, USA) as described by the manufacturer. Equivalent amounts of protein were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose (NC) membranes (Millipore Co., Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in TBST [10 mM Tris–HCl (pH 7.6), 0.1% Tween 20)] for 1 h at room temperature, followed by overnight incubation at 4 °C with one of the following primary antibodies: Cyto C, caspase 3 (Santa Cruz, CA, USA), CyPD (Abcam., CA, USA), AIF, COX IV (Cell Signaling Technology, CA, USA), β-actin, or proliferating cell nuclear antigen (PCNA) (Sungene Biotech, Tianjin, China). The immunoblots were incubated with the species-appropriate secondary antibody conjugated with horseradish peroxidase (Abbkine, CA, USA) for 1 h at room temperature. The membranes were developed with an electrochemiluminescence kit (Pierce, Rockford, IL, USA) according to the manufacturer’s 10

protocol. The signals were detected by a chemiluminescence detection system (Systemgen, England). Density of the immunoreactive bands was analyzed through ImageJ 1.41 (National Institutes of Health, USA).

2.13 Statistical analysis All experiments were repeated at least thrice. Data were expressed as mean ± SEM. The results were analyzed by one-way ANOVA with post hoc Dunnett’s test or LSD’s test. A p value of <0.05 was taken to be significant.

3. Results 3.1 PbAc-induced cell death and ATP loss could be attenuated by CSA in SH-SY5Y and PC12 cells Firstly, we found that CSA could ameliorate PbAc-induced cell death and ATP loss. The analysis of cell viability revealed obvious cell death after PbAc exposure, the cell viabilitys in SH-SY5Y and PC12 cells were 61% and 58% at the concentration of 25 μM PbAc, respectively. Preincubation with CSA could result in efficient protection by inhibiting cell death, raising the cell viability to 84% and 80% (Figs. 1A–B). Consistent with the results of cell viability, the LDH leakage induced by PbAc exposure was partly prevented by pretreatment with CSA (Figs. 1C–D). In addition, cellular ATP in SH-SY5Y and PC12 cells were obviously decreased in a PbAc concentration-dependent manner and CSA could significantly prevent ATP loss (Figs.1E–F). These results indicated that Pb-induced cell death and ATP loss could be attenuated by CSA pretreatment, which results in cell survival.

3.2 CSA inhibited the PbAc-induced MPTP opening and MMP collapse in both SH-SY5Y and PC12 cells Following our hypothesis that CyPD participated in the PbAc-induced MPTP opening, we detected the protein levels of CyPD in SH-SY5Y and PC12 cells by using western blot analysis. The results in Figs. 2A–B showed that the protein level of CyPD was increased after PbAc treatment in a concentration-dependent manner in both cell lines; 25 μM of PbAc could induce the 11

two- to threefold increase in CyPD protein levels. However, the mRNA levels of CyPD did not change at all (Figs. 2C–D), indicating that the PbAc-induced CyPD increase was probably via post-translation regulation. After incubation of both cell lines with PbAc, MPTP opening was detected by staining with calcein. When cells were exposed to 25 μM PbAc, the calcein fluorescence rapidly decreased during the first 6 h (~50% loss) and still remained low after 24 h in both cell lines (Figs. 3A–B). Pretreatment with 0.2 μM CSA would significantly inhibit the calcein fluorescence depression induced by PbAc. Consistent with the results of MPTP opening, CSA could also inhibit the MMP collapse induced by PbAc in the SH-SY5Y and PC12 cells (Figs.3C–D). These results indicated that CyPD might be involved in the PbAc-induced MPTP opening and MMP collapse and suggested that CSA possibly showed protective effects against PbAc neurotoxicity.

3.3 PbAc-induced MPTP opening and ROS production could promote each other To illustrate the relationship of ROS production and MMP collapse induced by PbAc, ROS was detected by DCFH in SH-SY5Y cells. In Fig. 4A–B, PbAc could drastically increase ROS accumulation for 6 and 24 h exposure, but pretreatment with CSA would significantly inhibit ROS production. Therefore, inhibiting MPTP opening by CSA was an effective way to reduce oxidative stress. . In fact, ROS could also induce MPTP opening and MMP collapse (Robertson et al., 2009). Fig. 4C showed that inhibiting ROS accumulation by NAC, a ROS scavenger, would attenuate the calcein fluorescence loss caused by PbAc. Higher cell viability was also observed in the groups incubated with NAC in Pb-exposed SH-SY5Y cells (Fig. 4D). ROS accumulation and the PbAc-induced MPTP opening could mutually promote each other and induce cell death synergistic.

3.4 CSA could attenuate the structural damage and mass loss of mitochondria caused by PbAc in SH-SY5Y cells The PbAc-induced impairment of mitochondrial morphological structure in SH-SY5Y cells was examined using transmission electron microscopy. As shown in Fig. 5A, PbAc caused mitochondrial swelling and rupture, and few mitochondria survived after PbAc exposure for 24 h. However, when pretreated by CSA, the mitochondrial damage was ameliorated. In addition, the 12

analysis of fluorescence by staining Mitotracker Deep Red from cells exposed to PbAc showed that nearly half of mitochondrial mass was lost after 24 h; however, this event did not happen at 6 h compared with the control group (Figs. 5B–C). Incubation with CSA could attenuate the process of mitochondrial damage and retain the mitochondria in cells. PbAc could obviously cause the mitochondrial damage, but the inhibition of MPTP opening by CSA would partially attenuate these processes.

3.5 CSA maintained the apoptosis-associated Cyto C and AIF in the mitochondria and inhibited caspase 3 activation. As shown in Figs. 6A and 6C, under the stimulation of PbAc, the levels of Cyto C and AIF decreased in the mitochondria, whereas the Cyto C in the cytoplasm and the AIF in the nucleus significantly increased in a concentration-dependent manner in SH-SY5Y cells. Cyto C in the cytoplasm activated caspase 3, thereby inducing caspase-dependent apoptosis (Fig. 6B). Meanwhile, AIF in the nucleus caused DNA damage and initiated caspase-independent apoptosis. Given that MPT played a key role in the process, the inhibition of MPTP by CSA could obviously restrain the release of Cyto C and AIF from mitochondria. The inhibition of pro-apoptosis factors release from mitochondria would attenuate Pb-induced cell death. Hoechst staining in SH-SY5Y cells showed that incubation with CSA would significantly protect against Pb-induced nuclear pyknosis and fragmentation (Fig. 7A). The PbAc-induced activation of cell apoptosis could also be attenuated by CSA, thereby decreasing the rate of total apoptosis detected by AV-PI in SH-SY5Y cells from 28% to 13% (Fig. 7 B).

4. Discussion Pb-induced cell death or nerve cell death specifically have been described in plenty of studies and we also observed the damage in SH-SY5Y and PC12 cells in current study (Li et al., 2014; Sun et al., 1999). However, the precise mechanisms are obscure, several studies conclude that Pb induces cell death mainly through mitochondrial damage and the subsequent initiation of mitochondria-associated apoptosis or necrosis (Patrick, 2006a, b; Xu et al., 2006). In current study, our results further revealed that MPTP mediated the significant impairment of Pb-induced mitochondrial morphology and function. This toxic effect was attenuated by the CSA-induced 13

inhibition of MPTP opening in nerve cells. Pb could increase the CyPD protein level but not the mRNA level, and then induce MPTP opening, thereby suggesting that post-translation regulation mediated the process in Pb-treated nerve cells. The increase of CyPD is probably caused by the association of CyPD with the mitochondrial inner membrane and the conformational changes of the ANT-inducing MPTP opening (Mazzeo et al., 2009). In addition, we explored the effect of Pb-induced ROS overproduction in the process of MPTP opening because ROS accumulation is regarded as the chief mechanism of Pb toxicity (Patrick, 2006a). The scavenging of ROS in cells by NAC mostly, but not completely, inhibited MPTP opening and MMP collapse, which attenuated the subsequent cell death. ROS mainly mediated the Pb-induced MPTP opening, but the effect of mimicking Ca2+ activity should not be ignored and needs to be further investigated. In current study, we observed the Pb-induced impairment of the mitochondrial morphological structure and mitochondrial mass, which may be important parts of cell apoptosis and/or necrosis (Golstein and Kroemer, 2007). The mitochondrial swelling and rupture in different Pb-exposed cell types would lead to the release of apoptosis-associated proteins, including Cyto C and AIF, thereby resulting in cell apoptosis (Liu et al., 2012). The mechanism of mitochondrial morphological impairment proposed by Marchlewicz et al is attributed to the Pb-induced burst of ROS production (Marchlewicz et al., 2004). However, we proposed that the CyPD-mediated MPTP opening accounted for the impairment. The application of CSA would inhibit the ROS burst and attenuate mitochondrial damage. The results of flow cytometry experiment detecting fluorescence of Mitotracker Deep Red revealed that Pb induced the loss of mitochondria, thereby suggesting that treatment with Pb caused severe damage to mitochondria in nerve cells. Normally, mitochondria are replaced every two to four weeks in the brain, heart, liver, and kidney (Menzies and Gold, 1971). When mitochondria suffer impairment induced by environmental stress, such as nutrient deprivation (Zhu et al., 2007), mitochondrial autophagy would be activated to dispose of damaged mitochondria and prevent cell death. This self-protective response could limit the impairment induced by environmental stress (Zhang et al., 2008). We observed that the inhibition of MPTP opening would alleviate the damage to the mitochondrial structure and maintain the mitochondrial mass. Two explanations may account for these results: (1) the less damaged mitochondria induced less mitophagy, or (2) MPTP participated in mitophagy or both. Carreira et 14

al demonstrated that the inhibition of CyPD by CSA could cause the failure to induce autophagy by starvation (Carreira et al., 2010). However, the precise mechanism of mitophagy as mediated by MPTP is still not clear and needs further investigation. The mitochondria could act as a pivotal decision center for several types of cell death responses, including apoptosis and necrosis. This organelle releases cell death-promoting factors from the intermembrane space into the cytosol and boosts ROS production (Martinou and Green, 2001). We found that MPTP opening mediated the translocation of Cyto C and AIF from the mitochondria to the cytosol. In addition, the Pb-induced cell death, including apoptosis and necrosis, could be attenuated by inhibition of the MPTP opening with CSA. The release of Cyto C and AIF from mitochondria required the OMM opening, whereas the CyPD normally induced the MPTP opening in IMM. AIF is translocated into the mitochondrial intermembrane space (IMS) to form the inner-membrane-anchored mature form (AIFmit). During apoptosis, it is further generated to the mature soluble form (AIFsol). AIFsol is released to the cytoplasm in response to specific death signals, and translocated to the nucleus, where it induces nuclear apoptosis in a caspase-independent manner (Boujrad et al., 2007). Therefore, the decrease of CyPD protein level would clearly not affect the release of proteins in the mitochondrial intermembrane space; this theory involving the Bax subfamily has been demonstrated (Baines et al., 2005; Martinou and Green, 2001). Another mechanism for the permeabilization of the OMM during apoptosis has emerged in literature. The MPTP opening caused by Ca2+ or ROS would induce small solutes (<1.5 kD); consequently, water enters the mitochondrial matrix, the mitochondria swell, and the OMM ruptures, thereby allowing the release of proteins from the intermembrane space (Martinou and Green, 2001). The current and previous results of electron microscopy confirmed this mechanism (Baranowska-Bosiacka et al., 2013). CSA is a CyPD inhibitor that could significantly attenuate the Pb-induced impairment of mitochondrial morphology. We hypothesized that both mechanisms might occur concurrently during Pb-induced cell apoptosis because the inhibition of Bax-mediated OMM opening could also benefit cell survival under Pb exposure (Baines et al., 2005). ROS is an important factor for cell survival and proliferation; ROS accumulation is also regarded as the major event during Pb exposure (Flora et al., 2012). The mitochondria are the major sites of ROS formation, and MPTP may be involved in the process. MPTP opening could 15

disturb the electron flow and oxidative phosphorylation, resulting in increased ROS production because a burst in ROS formation is observed in the presence of high matrix concentrations of Ca2+ (Di Lisa et al., 2001). Consistent with previous results, we also found that Pb caused ROS accumulation and CSA could significantly inhibit the burst of ROS formation (Bustos et al., 2015; He et al., 2000). ROS accumulation is a proposed cause of energy metabolism impairment and DNA alteration, including fragmentation, rearrangements, deletions, and point mutations (Ercal et al.,

2001).

Meanwhile,

ROS

could

induce

MPTP

opening

and

then

activate

mitochondria-dependent cell apoptosis. Therefore, ROS formation and MPTP opening could promote each other, but the question of which is upstream or downstream is difficult to establish. Notably, the inhibition of either event could attenuate the Pb toxicity and result in cell survival. Energy metabolism is an important function of mitochondria and is vulnerable to stress. ATP is mainly synthesized in mitochondria, any structural or functional alteration in the mitochondrial membrane could lead to impaired ATP production. Consistent with some studies, we observed that Pb exposure caused ATP reduction in a concentration-dependent manner in nerve cells (Baranowska-Bosiacka et al., 2011; Verma et al., 2005). The inhibition of MPTP by CSA could partially prevent the ATP reduction, thereby suggesting that MPTP played a role in ATP production. Given that CSA could ameliorate the mitochondrial morphology and mitochondrial mass, as well as inhibit the ROS burst against Pb neurotoxicity, these beneficial effects might improve the energy status. The precise mechanism of CSA benefiting for APT production need to be further clarified. Our results provide the evidence that CSA could benefit for Pb-exposed cell through inhibiting MPTP opening in vitro. However, due to the other effects of CSA including inhibition of mitochondrial fission by inhibiting the calcium-activated phosphatase calcineurin (PP2B), the application of gene modification, such as knockdown or knockout of CyPD, should be performed in the further experimental studies (Cereghetti et al., 2008). Moreover, the protective effect of CSA against Pb should also be examined in animal models to further conform the conclusion.

5. Conclusion In summary, Pb could cause impairment of mitochondrial morphology (mitochondria swelling, and loss of mitochondria) and mitochondrial function (ATP loss, ROS boost, and release 16

of Cyto C and AIF), thereby resulting in cell apoptosis. MPTP opening is induced by Pb mainly through ROS production; the open MPTP mediated these impairments. By contrast, the inhibition of MPTP opening by abolishing the activity of CyPD via treatment with CSA could obviously attenuate the Pb-induced mitochondrial damage and ameliorate mitochondrial function. Scavenging ROS also significantly abrogated MPTP opening and further attenuated Pb neurotoxicity. Therefore, we thought that MPTP might be the checkpoint of Pb-induced mitochondrial damage, which would greatly affect cell fate. However, because of the high PbAc concentrations used in this study, this mechanism needs confirmation with PbAc concentrations closer to human exposure and in more physiologically relevant study models. Conflicts of interest statement There are no conflicts of interest for any of the authors Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant NO. 81273019 and 81072265 ).

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Figures Figure 1. The loss of ATP induced by PbAc could be partly reversed by CSA in SH-SY5Y and PC12 cells. The SH-SY5Y and PC12 cells pretreated with CSA or DMSO were exposed to various concentrations of PbAc (5, 25 and 125μM) for 24h, then the cell viability (A-B), LDH release (C-D) and ATP level (E-F) were detected and normalized to cell protein concentration. *P<0.05 and ** P<0.01 represent significant differences compared with the control group. # P<0.05 and ##P<0.05 represent significant differences between two groups with or wthout CSA treatment at the same concentration of PbAc .

Figure 2. PbAc increased the protein level of CyPD in both SH-SY5Y and PC12 cells. The SH-SY5Y (A) and PC12 (B) cells were treated with various concentrations of PbAc (5, 25 and 125μM) for 24h, then the whole cell protein were extracted and the protein levels of CyPD were detected by western blot, respectively. When SH-SY5Y (C) or PC12 (D) cells were exposed to PbAc (25μM) for various time (1,3, 6, 12 or 24h), the mRNA levels of CyPD were detected by RT-PCR. * P<0.05 and ** P<0.01 represent significant differences compared with the control group.

Figure 3. CSA inhibited PbAc-induced MPTP opening and MMP collapse. The SH-SY5Y (A) and PC12 (B) cells pretreated with 0.2μM CSA or DMSO for 1h were exposed to 25μM PbAc for 6h and 24h, the MPTP opening was assessed by flow cytometry using the calcein-AM/cobalt method. The MMP level of SH-SY5Y (C) and PC12 (D) cells were detected using Rhodamine 123. Data represent the Mean ± SEM of the results. * P<0.05 ,** P<0.01 and *** P<0.001 represent significant differences compared with the control group. # P<0.05 ,## P<0.01 and ### P<0.001 represent significant differences between two groups with or wthout CSA treatment at the concentration of 25 μM PbAc .

Figure 4. PbAc-induced MPTP opening and ROS production could promote each other. (A-B) The SH-SY5Y cells pretreated with 0.2μM CSA or DMSO for 1h were exposed to 25μM PbAc for 6h and 24h, the ROS production was assessed by flow cytometry. (C) MPTP opening was assessed in PbAc-exposed cells pretreated with or without 5mM NAC for 1h, the calcein fluorescence 21

represent the level of MPTP opening (D). * P<0.05 ,** P<0.01 and *** P<0.001 represent significant differences compared with the control group. # P<0.05 ,## P<0.01 and ### P<0.001 represent significant differences between two groups with different pretreatment but with the same exposure to PbAc .

Figure 5. CSA could attenuate the mitochondria structure damage and the loss of mitochondria mass caused by PbAc in SH-SY5Y cells. (A) The SH-SY5Y cells pretreated with or without 0.2μM CSA for 1h were exposed to 25μM PbAc for 24h, then the transmission electron microscopy was applied to detect the change of mitochondrial morphological structure. White arrows represented the healthy mitochondria and black arrows represented the damaged mitochondria. (B-C) the mitochondrial mass level in PbAc-treated SH-SY5Y cells pretreated with CSA or DMSO was analyzed by staining Mitotracker Deep Red. ** P<0.01 represents significant differences compared with the control group. # P<0.05 and ## P<0.01 represent significant differences between two groups with or wthout CSA treatment .

Figure 6. CSA prevented the apoptosis-associated Cyto C and AIF releasing from mitochondria and inhibited caspase 3 activation in SH-SY5Y cells. The SH-SY5Y cells pretreated with CSA or DMSO for 1h were exposed to various concentrations of PbAc (5, 25 and 125) for 24h, the proteins of cytoplasma, mitochondria and nucleus were extracted respectively. Then the protein level of Cyto C in cytoplasm and mitochondria (A), cleaved caspase 3 in cytoplasm (B) and AIF in mitochondria and nucleus (C) were detected. * P<0.05 and ** P<0.01 represent significant differences compared with the control group. # P<0.05 and ## P<0.01 represent significant differences between two groups with or wthout CSA treatment at the same concentration of PbAc.

Figure 7. The cell death caused by PbAc could be attenuated by CSA in SH-SY5Y cells. After CSA-pretreated SH-SY5Y cells were exposed to 25μM PbAc for 24h, Hochest staining (A) and Annexin-V/PI staining (B) were performed to detect cell death. * P<0.05 and ** P<0.01 represent significant differences compared with the control group. # P<0.05 and ## P<0.01 represent significant differences between two PbAc-treated groups with or wthout CSA. 22

GENE

Primer sequence

Human CyPD

F

5’-GGA GCG AGT TGG TCG AAT TG -3’

（mRNA）

R

5’-TCC CAG TCG TGT GTC CAA TG -3’

Human GAPDH

F

5’-CTG ACT TCA ACA GCG ACA CC-3’

（mRNA）

R

5’-TGC TGT AGC CAA ATT CGT TGT -3’

Rat CyPD

F

5’-CTG CTC GGT CTG CTC TCC-3’

（mRNA）

R

5’-TGC CTT TAA CTC CAG CAC CA-3’

Rat GAPDH

F

5’-CAA GTT CAA CGG CAC AGT CAA-3’

（mRNA）

R

5’-TGG TGA AGA CGC CAG TAG ACT C-3’

Table. 1 The primer sequence

24

Graphical Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7