Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue
- Yoichi Imaizumi1,
- Yohei Okada1, 2,
- Wado Akamatsu1,
- Masato Koike3,
- Naoko Kuzumaki1,
- Hideki Hayakawa4,
- Tomoko Nihira4,
- Tetsuro Kobayashi5,
- Manabu Ohyama5,
- Shigeto Sato6,
- Masashi Takanashi6,
- Manabu Funayama6, 7,
- Akiyoshi Hirayama8,
- Tomoyoshi Soga8,
- Takako Hishiki9,
- Makoto Suematsu9,
- Takuya Yagi10,
- Daisuke Ito10,
- Arifumi Kosakai10,
- Kozo Hayashi11,
- Masanobu Shouji11,
- Atsushi Nakanishi11,
- Norihiro Suzuki10,
- Yoshikuni Mizuno12,
- Noboru Mizushima13,
- Masayuki Amagai5,
- Yasuo Uchiyama3,
- Hideki Mochizuki4, 14,
- Nobutaka Hattori6, 7 and
- Hideyuki Okano1Email author
© Imaizumi et al.; licensee BioMed Central Ltd. 2012
Received: 19 September 2012
Accepted: 2 October 2012
Published: 6 October 2012
Parkinson’s disease (PD) is a neurodegenerative disease characterized by selective degeneration of dopaminergic neurons in the substantia nigra (SN). The familial form of PD, PARK2, is caused by mutations in the parkin gene. parkin-knockout mouse models show some abnormalities, but they do not fully recapitulate the pathophysiology of human PARK2.
Here, we generated induced pluripotent stem cells (iPSCs) from two PARK2 patients. PARK2 iPSC-derived neurons showed increased oxidative stress and enhanced activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. iPSC-derived neurons, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis. Although PARK2 patients rarely exhibit Lewy body (LB) formation with an accumulation of α-synuclein, α-synuclein accumulation was observed in the postmortem brain of one of the donor patients. This accumulation was also seen in the iPSC-derived neurons in the same patient.
Thus, pathogenic changes in the brain of a PARK2 patient were recapitulated using iPSC technology. These novel findings reveal mechanistic insights into the onset of PARK2 and identify novel targets for drug screening and potential modified therapies for PD.
Parkin is a causative gene of autosomal recessive juvenile Parkinson’s disease (PARK2). It encodes a component of an E3 ubiquitin ligase involved in mitochondrial homeostasis[1–5]. Parkin deficiency is thought to result in aberrant ubiquitination and compromised mitochondrial integrity, leading to neuronal dysfunction and degeneration. Several PARK2 mouse models exist, but they do not replicate all of the pathogenic changes seen in human PARK2 neurons; thus, these models do not fully account for the molecular mechanisms of PD[6–9]. A recent report demonstrated that there is a defect in dopamine (DA) utilization in PARK2 induced pluripotent stem cell (iPSC)-derived neurons. However, it is not known whether neuronal homeostasis is disrupted in PARK2 patients. Furthermore, studies have yet to demonstrate whether the phenotype of PD-specific iPSC-derived neurons recapitulates the in vivo phenotype of the corresponding cell donor. To address these questions, we generated iPSCs from two PARK2 patients (PA and PB). In PARK2 iPSC-derived neurons, but not PARK2 fibroblasts or iPSCs, abnormal mitochondrial morphology and aberrant tubulovesicular structures adjacent to the Golgi were observed, as was increased oxidative stress. Although α-synuclein accumulation and Lewy body (LB) formation are very rare in PARK2 patients[1, 12, 13], we observed pathological changes and prominent LB formation, including the accumulation of α-synuclein, in postmortem brain tissue from one of the donor patients (PA). However, we obtained autopsied brain tissue from the father of donor PB, who carried the same parkin deletion as PB, and observed no evidence of LB formation or α-synuclein-positive cells. Consistent with these observations in postmortem brain tissue, increased α-synuclein accumulation was clearly observed in PA iPSC-derived neurons in vitro, but not in PB iPCS-derived neurons. These results are the first demonstration of pathogenic changes in the brain of a PARK2 patient that were recapitulated using iPSC technology. Our findings also provide mechanistic insights into PARK2 pathophysiology.
Results & discussion
Generation of PARK2 iPSCs
PA and PB patient information
Age of onset
Mutation of parkin
Exon 2–4 homozygous deletions
Exon 6, 7 homozygous deletions
The PARK2 iPSCs expressed pluripotent hESC markers (Figure 1A and Additional file2A-C) and formed teratomas containing all three germ layers (Additional file2D). All of the retroviral transgenes were silenced in each clone (Additional file2E). The iPSCs derived from PA and PB retained the corresponding homozygous parkin deletions and exhibited genomic stability (Figure 1B-D; Additional file3A and B; and Table 1). All of the clones differentiated into neurons, including tyrosine hydroxylase (TH)-positive neurons, through a process of embryoid body and neurosphere formation (Figure 1A). Thus, all of the lines were successfully reprogrammed into a pluripotent state and were suitable for further analysis.
Increased oxidative stress accompanied by activation of the Nrf2 pathway in PARK2 iPSC-derived neurons
The Nrf2 pathway plays a cytoprotective role under conditions of ROS accumulation. Recent studies show that activation of the Nrf2 pathway reduces oxidative stress and provides partial protection from MPTP-mediated neurotoxicity. Elevated Nrf2 expression was observed in the postmortem brain of a PD patient. These data suggest a putative link between the Nrf2 pathway and PD, and prompted a closer investigation of this signaling pathway in control and PARK2 iPSC-derived neurons[19–21]. The expression of Nrf2 pathway proteins, such as Nrf2 and NADH quinone oxidoreductase (NQO1), was significantly increased in PARK2 iPSC-derived neurons (Figure 2D and E). These data are in line with previous reports[19–21], and suggest that the Nrf2 cytoprotective pathway may be activated in PARK2 iPSC-derived neurons to prevent further damage from oxidative stress. Taken together, these data demonstrated an increased level of oxidative stress accompanied by activation of the Nrf2 pathway in PARK2 neurons.
Abnormal mitochondrial morphology and impaired mitochondrial turnover in PARK2 iPSC-derived neurons
PARKIN is involved in the mitochondrial fission/fusion system and is recruited to depolarized mitochondria to promote mitophagy[5, 26–29]. In iPSC-derived neurons containing a mutation in PINK1 (a protein kinase upstream of PARKIN), PARKIN is not recruited appropriately to mitochondria. We hypothesized that PARKIN-deficient human neurons would show aberrant removal of depolarized mitochondria. To examine the turnover of damaged mitochondria, we treated iPSC-derived neurons with carbonyl cyanide m-chlorophenyl hydrazine (CCCP), which triggers the loss of mitochondrial membrane potential and results in the removal of damaged mitochondria. The intensity of TMRE, a mitochondrial membrane potential-dependent dye, clearly decreased in both control and PARK2 iPSC-derived neurons treated with CCCP, which indicated a reduced mitochondrial membrane potential in both sets of neurons (Additional file6). To determine the extent to which the damaged mitochondria were eliminated, we measured the area of the IMM after CCCP treatment. Compared with untreated cells, there was a dramatic loss of IMM area in the treated control neurons, but not in the treated PARK2 neurons (Figure 3C, left four columns; Figure 3D, black bars). To assess whether lysosomes were involved in the CCCP-induced elimination of mitochondria, we treated cells with Bafilomycin (Baf) A1, an inhibitor of the vacuolar type H(+)-ATPase. Baf A1 attenuated the CCCP-dependent reduction in the IMM area in control neurons (Figure 3C, right four columns; Figure 3D, white bars). To confirm that the abnormal turnover of damaged mitochondria was characteristic of neuronal cells, PARK2 fibroblasts and undifferentiated iPSCs were treated with CCCP. CCCP-treated PARK2 fibroblasts and undifferentiated iPSCs exhibited the same mitochondrial dynamics as CCCP-treated control cells (Additional file5C-E). Together, these data indicated aberrant degradation of mitochondria damaged by CCCP treatment in PARK2 iPSC-derived neurons.
These results support a recently proposed working model for PD, in which damaged mitochondria accumulate due to a disruption in PARKIN-mediated mitochondrial quality control. The electron microscopy data, which showed a mixture of abnormal and normal mitochondria, indicated that PARKIN-mediated mitochondrial quality control is compromised, even in young PARK2 iPSC-derived neurons. In these cells, residual normal mitochondria may have compensated for the damaged ones. Thus, while our findings suggest that the PARKIN-dependent mechanisms that regulate mitochondrial homeostasis are disrupted in PARK2 cells, further detailed analyses are required to fully understand the mechanism underlying this disruption and the implications for PD.
Patient-specific accumulation of α-synuclein in PARK2 iPSC-derived neurons and its correlation with LB formation
LBs are pathological neuronal inclusions composed principally of α-synuclein. They are typically associated with PD and certain forms of dementia[1, 13, 31]. Although LBs are generally thought to be absent from PARK2 patients[1, 13, 31], rare cases of LB formation in the brains of PARK2 patients have been reported recently[12, 32, 33]. The PARKIN protein co-localizes with LBs in some patients with sporadic PD, and a functional interaction between PARKIN and α-synuclein is indicated by both in vitro and in vivo findings[35–37]. These results suggest that PARKIN-pathway may contribute to LB formation in PD patients.
LB type pathology in PA patient’s postmortem brain
LB type pathology
The obvious LB-formation was observed in the postmortem brain of PA patient, who showed a late onset at 61 years, corresponding to the enhanced α-synuclein accumulation in the iPSC-derived neurons from the same patient. Thus, it is likely that early-stage LB formation was recapitulated in vitro in iPSC-derived neurons. Furthermore, the present findings are consistent with recent work by several groups, which suggest that the age of onset of PARK2 in patients with LB formation (41 on average) is later than in patients without LB formation (below 40)[12, 32, 33]. The earlier onset in patient PB (at 28 years) than in PA (at 61 years) would be consistent with the finding of lower α-synuclein accumulation in PB iPSC-derived neurons compared with PA iPSC-derived neurons. On the other hand, and in contrast to the observations of brain tissue from PA, analysis of brain tissue from the father of patient PB, in whom the onset of PD was 39 years of age, revealed no evidence of LB formation (Figure 4D). Importantly, PA iPSC-derived neurons showed significantly more α-synuclein accumulation than PB iPSC-derived neurons (Figure 5A and C). These results suggest that the extent of α-synuclein accumulation is an important factor in LB formation. Then, how can we explain the difference of α-synuclein accumulation between PA and PB patients-derived neuronal cells? It is possible that PA is a rare example of PARK2 complicated by sporadic PD. Although both PA and PB iPSCs showed a normal SNCA gene copy number, it is possible that PA-derived cells acquired an unknown gene mutation relating to LB formation. Thus, we cannot rule out the possibility that other factors may affect LB formation in PARK2 patients. Further analyses will be required to identify these putative factors. Although iPSC clones from sporadic and familial PD patients were recently established[17, 30, 39–42], this report is the first to demonstrate that the phenotype of PD-specific iPSC-derived neurons replicates the in vivo phenotype seen in postmortem brain tissue from the corresponding cell donor.
In summary, dysfunctional neuronal homeostasis (characterized by increased oxidative stress and activation of the Nrf2 pathway), impaired mitochondrial function, and increased α-synuclein accumulation were observed in PARK2 iPSC-derived neurons. These results indicate that PARK2-associated phenotypes may appear soon after, or possibly even before, the onset of PARK2. Detailed analyses of PARK2 iPSC-derived neurons, particularly mature neurons, to determine the time course of LB accumulation and synaptic dysfunction will be of great interest. Such analyses will further our understanding of the pathogenesis of PARK2 as well as sporadic PD. The ultimate goal is the development and application of novel preventative therapies for PD.
Materials & methods
Isolation of human skin fibroblasts and generation of iPSCs
For control A, human dermal fibroblasts (HDFs) from the facial dermis of a 36-year-old Caucasian female (Cell Applications Inc.) were used to establish iPSCs (201B7; Passage 20–29, YA9; Passage 15–24). The 201B7 iPSCs were kindly provided by Dr. Yamanaka. A skin-punch biopsy from a healthy 16-year-old Japanese female obtained after written informed consent (Keio University School of Medicine) was used to generate the control B iPSCs (WD39; Passage 8–17). PA iPSCs (PA1, 9, and 22; Passage 10–19) and PB iPSCs (PB1, 2, 18, and 20; Passage 8–17) were generated from a 71-year-old Japanese female patient and a 50-year-old Japanese male patient, respectively, using the same methods used to generate control B iPSCs. The maintenance of HDFs, lentiviral production, retroviral production, infection, stem cell culture and characterization, and teratoma formation were performed as described previously[14, 15]. All of the experimental procedures for skin biopsy and iPS production were approved by the Keio University School of Medicine Ethics committee (Approval Number: 20-16-18) and Juntendo University School of Medicine Ethics committee (Approval Number: 2012068). hESCs (KhES-1; Passage 29–38 (kindly provided by Dr. Norio Nakatsuji) were cultured on feeder cells in iPS culture media.
In vitro differentiation of human iPSCs
Neural differentiation of iPSCs was performed as previously described with slight modifications (Okada et al., manuscript in preparation). Briefly, iPSC colonies were detached from feeder layers and cultured in suspension as EBs for about 30 days in bacteriological dishes. EBs were then enzymatically dissociated into single cells and the dissociated cells cultured in suspension in serum-free media (MHM) for 10 to 14 days to allow the formation of neurospheres. Neurospheres were passaged repeatedly by dissociation into single cells followed by culture in the same manner. Typically, neurospheres between passages 3 and 8 were used for analysis. For terminal differentiation, dissociated or undissociated neurospheres were allowed to adhere to poly-L-ornithine- and fibronectin-coated coverslips and cultured for 10 days.
Immunocytochemical analysis of iPSCs and neurons
For immunocytochemical analysis, cells were fixed with phosphate buffered saline (PBS) containing 4% paraformaldehyde (PFA) for 30 min at room temperature (RT). The cells were analyzed by immunofluorescence staining using antibodies to the following proteins: β-III-tubulin (1:1000, Sigma), NANOG (1:100, ReproCELL), OCT3/4 (1:200, Santa Cruz Biotechnology), SSEA-4 (1:200, Millipore), TRA-1-60 (1:200, Millipore), TH (1:100, Millipore), α-synuclein (1:500, Invitrogen), pα-synuclein (1:1000, Wako), cleaved-Caspase3 (1:500, Cell Signaling) and ComplexIII (C-III)-core I (1:200, Invitrogen). Cells were washed with PBS after incubation with the primary antibody, followed by incubation with an Alexa Fluor 488-, Alexa Fluor 555-, or Alexa Fluor 647-conjugated secondary antibody (1:500, Invitrogen). Images were obtained using Apotome (Zeiss) or LSM-710 confocal (Zeiss) microscopes.
PCR amplification of genomic DNA
Reverse transcription (RT)-PCR
RNA isolation and reverse transcription (RT)-PCR were performed as previously described. The amount of cDNA was normalized to β-actin mRNA. Real-time RT-PCR was performed on a ABI PRISM Sequence detection System 7900HT (Applied BioSystems) using SYBR premix ExTaq (Takara). Primers for the detection of Oct4, the transgenes Oct4-tg, Sox2-tg, Klf4-tg and c-Myc-tg, and MAO-A, and -B have been previously described[10, 15].
To assess teratoma formation, iPSCs were injected into the testis of 8-week-old NOD/SCID mice (OYG International) as previously described. Eight weeks after transplantation, tumors were dissected and fixed with 4% PFA in PBS. Paraffin-embedded tissue was sectioned and stained with H&E. Images were obtained using a BZ-9000 (Keyence) microscope.
Genomic DNA was restricted, labeled, and purified using the Agilent Oligo CGH Microarray Kit (Agilent Technologies) according to the manufacturer’s protocol. Labeled genomic DNA was processed for hybridization on a 4x 180K microarray (Agilent Technologies). Processing was performed as instructed by the manufacturer. The genomic analysis was performed using Agilent Genomic Workbench ver. 6.0 software (Agilent Technologies).
Reduced GSH levels were measured according to the kit manufacturer’s protocol (GSH-Glo Glutathione Assay; Promega). Chymotrypsin-like proteasome activity was measured using a Cell-Based Proteasome-Glo Assay according to the manufacturer’s instructions (Promega). Briefly, neural cells (1.0 × 104) derived from neurospheres were seeded in triplicate into a white 96-well plate (Nunc). Prepared reagent (100 μl) was added to each well. After incubation for 10 min at RT, luminescence intensity was recorded. ROS levels were determined by measuring DCFH-DA fluorescence (Invitrogen). Briefly, neurons were incubated with 5 μM DCFH-DA and Hoechst (1:2000) for 30 min at 37°C, after which they were washed with PBS and then incubated in differentiation media. Fluorescence was measured by an In Cell Analyzer 2000 system (GE Healthcare Biosciences).
Differentiated neurons were harvested in MAPK lysis buffer containing proteinase inhibitor, and protein concentrations were measured by BCA assay (Thermo Scientific). Samples were diluted to yield equivalent protein concentrations and then 4 μg was denatured by the addition of 4X sample buffer (Invitrogen) supplemented with β-mercaptoethanol followed by boiling. Samples (7 μl/lane) were loaded onto a 4–20% SDS-polyacrylamide gradient gel. Membranes were incubated in blocking solution with the indicated primary antibodies at 4°C overnight. Immunoreactive proteins were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and then visualized by chemiluminescence (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Quantification of band intensities was performed using an RAS4000 system. The primary antibodies used were anti-NQO1 (1:1000, Abcam), anti-NRF2 (1:1000, Santa Cruz Biotechnology) and β-actin (1:5000, Cell Signaling).
CCCP and Baf A1 treatments
Neurons were cultured with 30 μM CCCP (Sigma-Aldrich) or DMSO, with or without 5 μM Baf A1 (Sigma-Aldrich), for 48 h. The cells were then fixed and stained for βIII-tubulin and C-III Core I, and counterstained with Hoechst. To quantify the IMM area of the neurons, the cytoplasmic area was extracted as shown in Figure 3C. The C-III Core I-positive signals within the extracted area were then converted to gray-scale and digitized. The IMM area was quantified from the digitized values using Image J software.
Tetramethylrhodamine ethyl ester (TMRE) staining
iPSC-derived neurons were incubated with 1nM TMRE (Invitrogen) for 15 min at 37°C and then observed under an Olympus IX81 microscope.
Cells were fixed with 2% glutaraldehyde/2% PFA in 0.1 M phosphate buffer (PB) (pH7.2), post-fixed with 1% OsO4 in 0.1 M PB (pH 7.2), blocked and stained with a 2% aqueous solution of uranyl acetate, dehydrated with a graded series of ethanol, and then embedded in Epon 812 (TAAB). Coverslips were detached and the embedded samples were placed under a stereomicroscope to identify the cells of interest. Ultrathin sections were cut with a Leica UC6 or UC7 ultramicrotome (Leica Microsystems) and then stained with uranyl acetate and lead citrate. Samples were observed with a Hitachi H7100 or HT7700 electron microscope.
Morphometric analysis was used to measure the volume density of mitochondria in the neuronal perikarya as previously described. Briefly, electron micrographs of neurons (n = 20, 23, 41, and 44 for control A (B7), control B (WD39), PA9 and PB2, respectively) were obtained at a magnification of ×7000. After enlarging to three times the original magnification, point-counting was carried out to determine the volume density using a double-lattice test system with 1.5 cm spacing. Mitochondria were classified as normal, abnormal, or undetermined. The abnormal mitochondria were defined as those with irregularly arranged cristae, or with a high electron-dense matrix. The volume density (Vv) of each type of mitochondrion was expressed as percent volume according to the following formula: Vv = (Pi/Pt) × 100 (%), where Pi is the number of points falling on each mitochondrial structure and Pt is the number of points falling on the neuronal perikarya.
Immunohistochemical analysis of autopsied brain tissue
The ethical committee of the Kitasato University School of Medicine and Juntendo University School of Medicine reviewed and approved the protocol for analysis of autopsied brain tissue. Patients and control subjects were informed of the study and gave written informed consent. Brain tissue from patient PA was obtained following her death at age 72; brain tissue from the father of patient PB was obtained when he died at age 70. Tissue was fixed with 10% formalin and then embedded in paraffin. Midbrain sections (6 μm thick) were cut, deparaffinized with xylene, and then rehydrated in ethanol. After being boiled and treated with H2O2, sections were subjected to immunofluorescence staining with antibodies to the following proteins: α-synuclein (1:500, Invitrogen), pα-synuclein (1:1000, Wako), and TH (1:1000, Calbiochem). After washing with PBS, sections were incubated with a biotinylated secondary antibody (1:500; Vector Laboratories Inc.) at RT for 1 hr followed by incubation with an avidin-biotin peroxidase complex (Vector Laboratories Inc.) for 1 hr. Immunoreactive proteins were visualized using 3,3-diaminobenzidine (DAB; Wako Pure Chemical Industries) and nuclear fast red staining. For immunofluorescence, FITC-conjugated and Cy3-conjugated secondary antibodies (1:500; Jackson Immunoresearch Laboratories) were used. Images were obtained using a BIOREVO (Keyence) and a confocal laser-scanning LSM710 (Zeiss) microscope.
Values represent the mean ± SEM. The Mann–Whitney U-test was used to evaluate differences between groups. A P value of < 0.05 was considered significant.
We would like to thank S. Yamanaka (CiRA) for the 201B7 iPSCs; N. Nakatsuji (Kyoto University) for the KhES cells; N. Izawa, S. Banno, Y. Matsuzaki, M. Fujiwara, Y. Nagahata, N. Hirose (Keio University), C. Kishi (Tokyo Medical and Dental University), M. Ogino, S. Miyakawa, (Kitasato University) and G. Takata (GE Healthcare Biosciences) for technical assistance and suggestions. This work was supported by the Project for the Realization of Regenerative Medicine and Support for the Core Institutes for iPS cell research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) to H.O., Exploratory Research for Advanced Technology (ERATO), Suematsu Gas Biology Project from Japan Science and Technology Agency (JST) to M.S., a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) to W.A. and Y.O., the Keio Kanrinmaru Project and a Grant-in-Aid for Scientific Research on Innovative Areas to Y.O., a Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the MEXT to Y. O. and Y. I., a Grant-in-Aid for Encouragement of Young Medical Scientists from Keio University and the Japan Society for the Promotion of Science Fellows to Y.I., and a Grant-in-Aid for the Global COE Program at Keio University.
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