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. 2012 Oct 6:5:35.
doi: 10.1186/1756-6606-5-35.

Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue

Yoichi Imaizumi  1 , Yohei Okada, Wado Akamatsu, Masato Koike, Naoko Kuzumaki, Hideki Hayakawa, Tomoko Nihira, Tetsuro Kobayashi, Manabu Ohyama, Shigeto Sato, Masashi Takanashi, Manabu Funayama, Akiyoshi Hirayama, Tomoyoshi Soga, Takako Hishiki, Makoto Suematsu, Takuya Yagi, Daisuke Ito, Arifumi Kosakai, Kozo Hayashi, Masanobu Shouji, Atsushi Nakanishi, Norihiro Suzuki, Yoshikuni Mizuno, Noboru Mizushima, Masayuki Amagai, Yasuo Uchiyama, Hideki Mochizuki, Nobutaka Hattori, Hideyuki Okano
Affiliations

Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue

Yoichi Imaizumi et al. Mol Brain. .

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

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Figures

Figure 1
Figure 1
Generation of PARK2 iPSCs. (A) iPSCs derived from control and PARK2 subjects, embryoid bodies (EBs), neurospheres (NSs), and neurons. Left three rows: iPSCs from Control A (YA9), Control B (WD39), PA (PA9), and PB (PB2) were immunopositive for the pluripotency markers Oct4 (green) and Nanog (red). Right three rows: Differentiation of iPSCs into tyrosine hydroxylase (TH)-positive (red) neurons via EB and NS formation. Scale bars: Phase contrast, 400 μm; Nanog and Oct4 immunostaining, 100 μm; EBs, 25 μm; NSs, 50 μm; neurons, 10 μm. (B) Deletion of exons 2–4 in clones PA1, 9 and 22; and of exons 6 and 7 in clones PB1, 2, 18, and 20 was confirmed. (C) Exons 2–4 were deleted in human dermal fibroblasts (HDFs) from PA and in PA1 iPSC lines. Exons 6 and 7 were deleted in HDFs from PB and PB1 iPSC lines. (D) Copy number profiles of whole chromosomes in PARK2 HDFs and iPSCs were assessed by comparative genomic hybridization (CGH) microarray analysis; there was no evidence that genomic aberrations were introduced during the process of establishing PARK2 iPSCs.
Figure 2
Figure 2
Increased oxidative stress accompanied by activation of the Nrf2 pathway in PARK2 iPSC-derived neurons. (A) GSH levels were significantly reduced in PARK2 (PA1, 9 and 22, and PB2, 18 and 20) iPSC-derived neurospheres compared with those in control A (YA9) and B (WD39) neurospheres. (B, C) DCF fluorescence intensity in PARK2 (PA1, 9 and 22, and PB2 and 20) iPSC-derived neurons was significantly higher than that in control A (B7) and B (WD39) neurons. (D, E) Immunoblot analysis of Nrf2 and NQO1 levels in iPSC-derived neurons from PA and PB. Expression of Nrf2 and NQO1 in PARK2 (PA9 and PB2) iPSC-derived neurons was significantly higher than that in control A (YA9) and B (WD39) neurons. Relative protein abundance was normalized to β-actin. ** indicates P < 0.01 (Mann–Whitney U-test). Data represent the mean and SEM of at least three experiments for each group.
Figure 3
Figure 3
Dysregulation of mitochondrial homeostasis in PARK2 iPSC-derived neurons. (A) Electron micrographs of control A (B7), control B (WD39) and PARK2 (PA9 and PB2) iPSC-derived neurons. Boxed areas are shown in the enlarged images to the right. Control mitochondria showed a characteristically long, cylindrical profile with well-organized cristae, and the electron density of the matrix was relatively low (white arrowheads). By contrast, increased electron density of the matrix was evident in PARK2 mitochondria (black arrowheads), and the cristae often appeared swollen. As shown in PB2, some of the neurons contained both morphologically intact (white arrowheads) and abnormal (black arrowheads) mitochondria. Furthermore, abnormal tubulovesicular structures (asterisks) were observed adjacent to the Golgi cisternae (G). (B) The relative perikaryal volume of the abnormal mitochondria was significantly increased, and that of the normal mitochondria was decreased, in PARK2 neurons compared with control neurons. (C) Double labeling for the IMM marker, ComplexIII coreI (CIII-Core I; magenta) and βIII-tubulin (green) of control A (B7), control B (WD39) and PARK2 (PA9 and PB2) iPSC-derived neurons. The volume of the IMM area was reduced in control neurons treated with CCCP, but not in PARK2 neurons treated with CCCP. Administration of Baf A1 rescued the CCCP-induced phenotype in control neurons. (D) The CCCP/DMSO ratio in control A (B7 and YA9) and B (WD39) neurons was reduced after CCCP treatment. This reduction was not observed in PARK2 (PA1, 9 and 22, and PB2 and 20) iPSC-derived neurons (black bars indicate CCCP/DMSO ratio; white bars indicate Baf A1+CCCP/Baf A1 ratio). ** indicates P < 0.01 compared with the control; ¶¶ indicates P < 0.01 when comparing the black and white bars (Mann–Whitney U-test). At least three experiments were performed for each group, with 5–36 cells quantified per experiment. Scale bars: a, 1 μm; c, 10 μm. Error bars represent the SEM. N.D., not detected.
Figure 4
Figure 4
Accumulation of LBs in the postmortem brain of patient PA. (A–C) Immunohistochemical staining of postmortem brain tissue from patient PA. (A) Low magnification image of a midbrain section stained with hematoxylin and eosin (H&E). (A’) High magnification image of the boxed area. Melanin levels were reduced in most of the substantia nigra (SN). (B) (Left) High magnification image of a midbrain section stained with H&E showing the presence of Lewy bodies (LBs) in the SN. (Middle and Right) α-synuclein-positive and pα-synuclein-positive cells in the SN. (C) Confocal microscopy image of a TH (green) and pα-synuclein (red) double-positive SN neuron and a projected merged image: pα-synuclein accumulated in the TH-positive neuron. (D) Melanin levels were reduced in most of the SN. No LBs or α-synuclein-positive neurons were observed in postmortem brain tissue from the father of patient PB. Scale bars: A, 1000 μm; A’, 350 μm; B, C, 50 μm; D, 100 μm.
Figure 5
Figure 5
α-synuclein accumulation in PARK2 iPSC-derived neurons. (A–C) Triple labeling for α-synuclein (red), tyrosine hydroxylase (TH; cyan), and βIII-tubulin (green) along with Hoechst (blue) staining of control A (B7), control B (WD39), Cent1-8, and PARK2 (PA9 and PB20) iPSC-derived neurons. (A) Arrows indicate α-synuclein+/TH+/βIII-tubulin+ neurons; arrowheads indicate α-synuclein+/TH-/βIII-tubulin+ neurons. Note the presence of α-synuclein+/TH-/βIII-tubulin- non-neural cells (asterisks). (B) High magnification confocal projection image of an α-synuclein (magenta)/βIII-tubulin (green) double-positive PA9 iPSC-derived neuron. (C) The proportion of α-synuclein+/βIII-tubulin+ neurons relative to βIII-tubulin-positive neurons was significantly higher in PA (PA1, 9 and 22) iPSC-derived neurons than in control A (B7 and YA9), control B (WD39) and Cent1-8 iPSC-derived neurons. Scale bars: A, 50 μm; C, 5 μm. ** indicates P < 0.01; * and ¶ indicate P < 0.05 (Mann–Whitney U-test). Data represent the mean and SEM of at least three experiments for each group.
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