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. 2011 Apr 20;31(16):5970-6.
doi: 10.1523/JNEUROSCI.4441-10.2011.

Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells

Philip Seibler  1 , John Graziotto, Hyun Jeong, Filip Simunovic, Christine Klein, Dimitri Krainc
Affiliations

Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells

Philip Seibler et al. J Neurosci. .

Abstract

Genetic Parkinson disease (PD) has been associated with mutations in PINK1, a gene encoding a mitochondrial kinase implicated in the regulation of mitochondrial degradation. While the studies so far examined PINK1 function in non-neuronal systems or through PINK1 knockdown approaches, there is an imperative to examine the role of endogenous PINK1 in appropriate human-derived and biologically relevant cell models. Here we report the generation of induced pluripotent stem (iPS) cells from skin fibroblasts taken from three PD patients with nonsense (c.1366C>T; p.Q456X) or missense (c.509T>G; p.V170G) mutations in the PINK1 gene. These cells were differentiated into dopaminergic neurons that upon mitochondrial depolarization showed impaired recruitment of lentivirally expressed Parkin to mitochondria, increased mitochondrial copy number, and upregulation of PGC-1α, an important regulator of mitochondrial biogenesis. Importantly, these alterations were corrected by lentiviral expression of wild-type PINK1 in mutant iPS cell-derived PINK1 neurons. In conclusion, our studies suggest that fibroblasts from genetic PD can be reprogrammed and differentiated into neurons. These neurons exhibit distinct phenotypes that should be amenable to further mechanistic studies in this relevant biological context.

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Figures

Figure 1.
Figure 1.
Generation of iPS cells from a PD patient harboring a PINK1 mutation and a healthy control individual. IPS-PINK1 was established from a PD patient with mutant PINK1 (c.1366C>T) and iPS-WT from a healthy family member. A, Immunofluorescence analysis shows presence of pluripotency markers OCT4, Tra-1-60, NANOG, and SSEA-4. B, Expression levels of pluripotency markers NANOG, GDF3, OCT4, and SOX2 in fibroblasts and iPS cell lines relative to β-actin (a loading control) as assessed by quantitative RT-PCR. The values from parental fibroblasts were set to 1. C, Residual expression levels of transgenes OCT4, SOX2, cMYC, and KLF4 (relative to β-actin) were examined by quantitative RT-PCR. The values from the infected fibroblasts (isolated 7 d after infection) were set to 1. Uninfected fibroblasts were used as negative controls. The error bars indicate SD. D, RT-PCR analyses of various differentiation markers for the three germ layers (endoderm: GATA4, AFP; mesoderm: RUNX1, BRACHYURY; ectoderm: PAX6, NCAM) in iPS cells that were undifferentiated (U) and after 4 d in suspension culture followed by 7 d in adherent culture (D). E, Direct sequencing confirmed the PINK1 mutation c.1366C>T (p.Q456X; nonsense mutation) in iPS-PINK1.
Figure 2.
Figure 2.
Generation of human DA neurons from wild-type and mutant PINK1 iPS cells. A, Immunofluorescence staining of neuronal cultures derived from iPS cell lines iPS-WT and iPS-PINK1 for neuron-specific TUJ1 (red), the DA marker TH (green), and nuclear DAPI (blue). The two lower panels show high-magnification images. B, Quantification of TUJ1-positive neurons and TUJ1/TH-coexpressing DA neurons. The graph displays the percentage of cells that stain positive relative to nuclear DAPI staining (total cells). Error bars indicate the SD generated from two independent differentiation experiments. The total number of cells analyzed exceeded 12,000 cells. C, Western blot analysis of neuronal culture lysates from iPS-WT and iPS-PINK1. The Western blot was probed with TH and α-Tubulin (as loading control) antibodies. D, RT-PCR revealed 80% reduction in expression of PINK1 in mutant compared to control neurons (control normalized to 1). β-Actin was used as control gene. The results were analyzed with an unpaired t test (p < 0.001), with error bars representing SEM, n = 3.
Figure 3.
Figure 3.
Stress-induced mitochondrial translocation of Parkin is impaired in mutant PINK1 iPS cell-derived human DA neurons. A, Neuronal cultures were infected with WT Parkin and treated with 1 μm valinomycin or vehicle for 12 h. Cells were fixed and immunostained with antibodies against Parkin (red), mitochondrial marker TOM20 (blue), and the DA marker TH (green). Parkin colocalizes with mitochondria in control DA neurons (iPS-WT, left panel) but not in mutant PINK1 neurons (iPS-PINK1 mutants, middle panel). Lentiviral transduction of mutant PINK1 neurons with WT PINK1 restored Parkin translocation (right panel). B, Validation of lentiviral Parkin and PINK1–V5 infection of iPS cell-derived neurons. Neuronal culture was infected with lenti-Parkin lentivirus (left panel), or lenti-PINK-V5 (middle and right panels), coimmunostained with anti-Parkin antibody (green), anti-V5 antibody (red), and DAPI (blue). PINK1 is synthesized as a full-length form (∼66 kDa) that is proteolytically processed upon mitochondrial entry to its cleaved ∼55 kDa form. In untreated neuronal culture C-terminal V5-tag is cleaved and only a few cells are PINK1–V5-positive (middle panel). Treatment with 1 μm valinomycin for 12 h results in PINK1–V5 accumulation in its uncleaved full-length form (∼66 kDa; right panel). The infection efficiency was 70–90%. C, The quantitative colocalization analysis of Parkin and Tom20 signals was performed with ImageJ and JACoP plug-in (Bolte and Cordelières, 2006) to determine Manders' coefficient (Manders et al., 1992) from 0 to 1 (0 = non-overlapping images and 1 = colocalized images). The highest values were found for valinomycin-treated iPS-WT (sample 2) and PINK1-WT-infected iPS-PINK1 mutants (sample 6), suggesting a translocation of Parkin to mitochondria in the presence of a functional PINK1 protein. The results were analyzed with an unpaired t test with error bars indicating SD, n = 4 (samples 1, 2: p = 0.002; samples 5, 6: p = 0.0003).
Figure 4.
Figure 4.
Abnormalities in mtDNA copy number and PGC-1α expression in mutant PINK1 iPS neurons. Neurons were either untreated or treated with 1 μm valinomycin for 12 h. A, Upon treatment, there is significant reduction in mtDNA copy number in the iPS-WT and mutant iPS-PINK1 neurons transduced with wt-PINK1, but not in the PINK1 mutant line. B, PGC-1α gene expression is significantly upregulated after valinomycin treatment in the PINK1 mutant line but not in the WT or lenti-PINK1-expressing mutant line. Results in A and B are shown as fold change relative to untreated samples and normalized to β-actin. The results were analyzed with an unpaired t test, with error bars representing SEM (mtDNA untreated vs treated: p < 0.005; PGC-1α untreated vs treated: p = 0.02; n = 3).
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