Abstract
Mutations in PINK1 (PARK6), a serine/threonine kinase involved in mitochondrial homeostasis, are associated with early onset Parkinson’s disease. Fibroblasts from Parkinson’s disease patients with compound heterozygous mutations in exon 7 (c.1488 + 1G > A; c.1252_1488del) showed no apparent signs of mitochondrial impairment. To elucidate changes primarily caused by lack of functional PINK1, we over-expressed wild-type PINK1, which induced a significant downregulation of LRRK2 (PARK8). Indeed, we found that LRRK2 protein basal levels were significantly higher in the mutant PINK1 fibroblasts. To examine the interaction between the two PARK genes in a disease-relevant cell context, we generated induced pluripotent stem cell (iPSC) lines from mutant, carrier and control fibroblasts by lentiviral-mediated re-programming. Efficiency of neural induction and dopamine differentiation using a floor-plate induction protocol was similar in all genotypes. As observed in fibroblasts, PINK1 mutant neurons showed increased LRRK2 expression both at the RNA and protein level and transient over-expression of wild-type PINK1 efficiently downregulated LRRK2 levels. Additionally, we confirmed a dysregulation of LRRK2 expression in fibroblasts from patients with a different homozygous mutation in PINK1 exon 4, c.926G > A (G309D). Thus, our results identify a novel role of PINK1 modulating the levels of LRRK2 in Parkinson’s disease fibroblasts and neurons, suggest a convergent pathway for these PARK genes, and broaden the role of LRRK2 in the pathogenesis of Parkinson’s disease.
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Introduction
Rare monogenic forms of Parkinson’s disease (PD) have been correlated to specific gene mutations [1], providing the opportunity to identify novel pathogenic pathways or molecular mechanisms that may contribute to more frequent forms of the disease [2] and to examine interactions between PARK genes. Alternatively, early-onset genetic forms could represent a distinct entity at the molecular level.
PD-related mutations in PINK1 (PTEN-induced kinase 1, PARK6) compromise kinase function or protein stability [3]; thus, the phenotype is thought to result from a loss of function. PINK1 mitochondrial localization supports its involvement in the mitochondrial dysfunction extensively described in PD [4], and PINK1 protects against stress-induced mitochondrial dysfunction [3, 5]. In addition, the PINK1/Parkin signaling pathway controls mitochondrial quality and mitophagy [6], even if it appears that this pathway is not modified by endogenous human PINK1 mutations [7, 8]. Likewise, PINK1 regulates mitochondrial fusion and fission dynamics [9], although the net effect of PINK1 mutations is currently a matter of controversy because both increased fusion and increased fission have been reported in different species [10].
LRRK2 (PARK8) is one of the genes most frequently mutated in PD. LRRK2 has been implicated in a very broad range of cellular pathways so the precise mechanisms leading to neuronal degeneration remain to be defined [11]. An effect of LRRK2 on mitochondrial function was described in Caenorhabditis elegans [12]. Interestingly, in this model organism, the absence of lrk-1 rescues all phenotypic aspects of pink-1 loss-of-function mutants. Conversely, the hypersensitivity of lrk-1 mutant animals to stressors is reduced in a pink-1 mutant background, suggesting antagonistic effects of lrk-1 and pink-1 [13].
With the aim to define a PINK1-related phenotype for in vitro studies, we investigated expression pattern of genes involved in mitochondrial dynamics and PARK genes in fibroblasts and induced pluripotent stem cell (iPSC)-derived dopamine neurons from a PINK1-PD Spanish kindred [14]. This led us to unveil an upregulation of LRRK2 in PINK1 mutants and an interaction between these two PARK gene products in human cells which were ratified in a different pedigree.
Methods
Human Samples
Skin samples were obtained from subjects expressing mutated forms of PINK1 diagnosed at the Hospital Universitario Insular de Gran Canaria (La Palma de Gran Canaria, Spain) and from age-matched healthy individuals at the Hospital Donostia and Onkologikoa (San Sebastian, Spain). PD patients presented an early-onset, typical parkinsonian syndrome, characteristic of PINK1-associated PD [14]. Demographic data are provided in Table 1. Three samples from individuals from another Spanish family harboring the G309D (c.926G > A) mutation in exon 4 [3] were also analyzed to avoid possible confounding effects caused by consanguinity in the first family. Dermal fibroblasts were cultivated as described previously [15].
Protocol Approvals, Registrations, and Patient Consents
The study was approved by the Ethical Committee on the Use of Human Subjects in Research in Euskadi, Spain. All subjects gave informed consent for the study using forms approved by the ethical committees on the Use of Human Subjects in Research at Hospital Universitario Insular de Gran Canaria, La Palmas de Gran Canaria; Hospital Donostia and Onkologikoa, San Sebastián; and Hospital San Cecilio, Granada, respectively. Generation of iPSC lines was approved by the Advisory Committee for Human Tissue and Cell Donation and Use, Instituto Carlos III, Ministry of Health, Spain.
Genetic Analysis
PINK1 variants were analyzed by conventional PCR using a primer pair designed to amplify a region expanding exons 6 and 8 [14]. Total RNA and cDNA were obtained as described previously [15] for quantitative RT-PCR. Primer sequences [14] are provided in Supplementary Table S1. Comparative analysis of gene expression levels (∆∆Ct) was carried out using GAPDH as reference. Standard G-band karyotypes of the iPSC clones used in the study were performed at the Policlinica Gipuzkoa (San Sebastian, Spain).
ATP Content
Cellular ATP was measured using the Luminiscent ATP Detection kit (Abcam, Cambridge, UK). Cells were harvested, pelleted, and washed once in PBS. An aliquot was used for protein quantification. The rest was re-suspended in 50 μl growth medium, and cells were lysed by adding 50 μl of the detergent and mixing. After a 5-min incubation, 50 μl of substrate was added and luminescence was quantified in a GloMax® luminometer (Promega, Madison, WI, USA). An ATP standard curve was prepared, and values were calculated in picomole per microgram of protein in the cell extract.
Glycolytic Rate
The rate of glycolysis was determined in fibroblasts, seeded in flasks, by the conversion of [3-3H] glucose into 3H2O, as described previously [16].
Western Blotting
Whole-cell lysates were prepared in RIPA buffer with a protease inhibitor cocktail (Roche, Mannheim, Germany). SDS-PAGE and protein transfer and blotting were carried out according to standard procedures [15]. Primary and secondary antibodies are listed in Supplementary Table S2. Visualization of HRP-labeled proteins was performed using enzyme-linked chemifluorescence (ThermoFisher Scientific, Waltham, MA, USA) and quantified using ImageJ software. Data were normalized to control in order to compare different experiments.
Immunofluorescence
Cells plated onto glass coverslips were incubated with MitoTracker® Deep Red FM (M22426, Molecular Probes®, Life Technologies, Carlsbad, CA, USA) for 45 min and fixed for 10 min with 4% paraformaldehyde (15710-S, Electron Microscopy Sciences). Immunofluorescence staining was performed as previously reported [17]. Antibodies are listed in Supplementary Table S2. Images were acquired in a Zeiss LSM510 confocal microscope using the exact same settings for control and experimental samples and analyzed with ImageJ 1.42q software (NIH, http://rsb.info.nih.gov/ij). Automatic color level correction was used when required to enhance the contrast. Mitochondrial morphology was classified as tubular, mixed, or round (fragmented) according to published criteria [18]. Images were acquired at ×63 magnification, and 1000–5000 cells were counted by two blinded investigators on 16 randomly selected visual fields from at least two independent experiments, using ImageJ. Tyrosine hydroxylase positive neurons were counted over total ßIII-tubulin positive neurons at day 50–70 as previously described [17]. Live images were acquired using the Zoe™ Fluorescent Cell Imager (BioRad, Hercules, CA, USA) at ×20.
PINK1 Over-expression
Fibroblasts at 70–90% confluence were electroporated with the Neon® Transfection System (Invitrogen™, Waltham, MA, USA), using two pulses of 1500 V for 20 ms, with wild-type PINK1 (pcDNA-DEST47 PINK1 C-green fluorescent protein (GFP)) [19], from MR Cookson, Addgene no. 13316) or a control GFP plasmid, at 0.5 μg/106 cells. Additional controls in each group received only the pulses. Cells were collected for analysis at 24 and 48 h post-transfection given that expression declined rapidly to baseline levels after 72 h (data not shown). Electroporation in neural cells was done following the same procedure using two pulses of 1000 V. Neurons were collected for analysis at 4 days to minimize the effect of the electroporation on transcriptional changes.
iPSC and Neuronal Differentiation
Human-iPSC lines from the two Parkinson patients and one carrier and from age-matched control individuals were derived in our laboratory using lentiviral vectors and differentiated as previously described [17] (see Supplementary Fig. S1). The cell lines have been deposited in the Spanish National Cell Bank and are available at http://www.isciii.es/ISCIII/es.
Data Analysis and Statistics
Data analysis was carried out using GraphPad Prism software (v. 4.0c, La Jolla, CA, USA). One-way or two-way ANOVA with Bonferroni post-hoc tests were used to compare groups. Student’s t test was used to detect changes in fold expression whenever data were normalized to control levels. In all experiments using human fibroblast samples, four to five control individuals were assayed together with the two PINK1 -exon7/del patients and the two PINK1 -exon7 carriers or with the two G309D homozygous and one heterozygous carrier in at least two independent determinations. For neuronal experiments, data from three independent differentiations of the three mutant iPSC lines were included with two to three control pluripotent lines. In addition to the iPSC lines reprogrammed for this study, control cell lines [17] were differentiated and analyzed in parallel. Data in the figures represent the mean ± SEM of two to four independent experiments. The threshold for significance was set at p < 0.05.
Results
Characterization of Parkinson’s Disease PINK1 Mutant Fibroblasts
We established primary cultures of fibroblasts obtained from dermal biopsies of healthy subjects and individuals carrying modifications in the PINK1 gene that result in the inactivation of the normal kinase function [14]. Sample verification was confirmed by conventional PCR analysis (Fig. 1a) that identifies the shorter transcripts corresponding to the deletion (del) and the exon 7 skipping (-exon7) in the compound heterozygous patients (PINK1-exon7/del, PDP1 and PDP3) and heterozygous carriers (PINK1-exon7, PDP2 and PDP4). In spite of the mutations, PINK1 RNA expression was similar in mutant, carriers, and control fibroblasts (Fig. 1b). Baseline ATP levels were not different between groups (Fig. 1c). Nevertheless, using a sensitive method for glycolytic flux assessment, we detected an increase in the glycolytic rate in PINK1 mutants and carriers (Fig. 1d), as recently described in mouse Pink1 KO cells [16]. We next examined the morphology of the mitochondrial network using MitoTracker®; semi-quantitative analysis of tubularity (Fig. 1e, f) showed no differences between genotypes. This does not exclude subtle alterations in mitochondrial morphology or function, but it is in agreement with previous studies in fibroblasts carrying other PINK1 mutations [20]. We analyzed the expression levels of proteins involved in mitochondrial fusion (MNF2) and fission (DNM1L and MFF) to explore whether the lack of morphological changes in mitochondrial tubularity was due to compensatory adjustments in their levels. The results are shown in Supplementary Fig. S1 and revealed minor changes in fusion/fission dynamics that can reflect compensatory adaptations to the lack of PINK1 kinase function in fibroblasts. These results are consistent with previous studies that have used human fibroblasts to investigate disease and compensatory mechanisms in genetic PD [21].
Effect of PINK1 Over-expression in Fibroblasts
To identify changes causally related to PINK1 deficiency, we evaluated the capacity of wild-type PINK1 over-expression to modify the gene expression profile in PINK1 -exon7/del mutant fibroblasts. Following electroporation, average PINK1 levels determined by qPCR were elevated, 31 ± 4.5-fold over control cells, with no differences across genotypes in two independent experiments (Fig. 2a) and a clear expression of a 499 band corresponding to the wild-type PINK1 exons 6–8 in transfected mutant fibroblasts (boxed in Fig. 2b). Transient transfection of wild-type PINK1 did not modify the levels of genes involved in mitochondrial dynamics such as mitofusin 2 (MFN2), and the pro-fission DNM1L or MFF genes (Fig. 2c). Likewise, there were no significant changes in DJ1 (PARK7) or Parkin (PARK2, not shown), whereas, interestingly, we found a significant decrease in LRRK2 (PARK8). In view of these results, we analyzed the expression of UHRF2, an E3 ligase reported to be repressed in LRRK2G2019S mutant neurons [22] and found that it was upregulated (Fig. 2d), suggesting that the changes in LRRK2 expression in this paradigm are relevant.
LRRK2 Expression in PINK1 Mutant Fibroblasts
Since we identified a significant decrease in LRRK2 in PINK1 over-expression experiments, we went on to study the baseline expression of LRRK2 in mutant fibroblasts. LRRK2 RNA was elevated in PINK1-exon7/del fibroblasts although not significantly different from the control (Fig. 3a). Although some transcripts have been shown to increase with aging in fibroblasts and other tissues, we have not found any correlation between age and LRRK2 mRNA levels in fibroblasts from control individuals studied in our laboratory (age range 17–63 years; R 2 = 0.082, p = 0.42, data not shown). LRRK2 protein was remarkably increased in PINK1-exon7/del samples, with no change in PINK1-exon7 fibroblasts (Fig. 3b). LRRK2 sub-cellular distribution was similar in all groups (Fig. 3c).
LRRK2 Expression in PINK1 Mutant Neurons
Our findings in fibroblasts suggest that PINK1 and LRRK2 may act in a convergent pathway, with PINK1 regulating LRRK2 expression. Thus, to identify whether LRRK2 may be a factor contributing to the degeneration of dopamine neurons in PINK1-PD, we made iPSC lines from the two PINK1-exon7/del patients (PDP1 and PDP3 lines) and one carrier (PDP2) (Supplementary Fig. S2 and Fig. 4a, b). Unfortunately, the other carrier line (PDP4) was lost due to technical problems. Pluripotent cells were differentiated towards dopaminergic neurons using an inductive protocol combining developmental signals as described [17] (Fig. 4c). Because significant changes in the expression of genes and levels of proteins occur during neuronal maturation, we analyzed cultures at two time points, corresponding to neural progenitors (2–6 weeks) and neurons (6–12 weeks) (Fig. 4c). There were no apparent defects either in neural induction in the mutant cells, in agreement with published results for iPSC lines carrying homozygous point mutations in the PINK1 gene [7, 23, 24], or in the generation of dopamine neurons, with >30% of TH positive neurons in all genotypes (Fig. 4d, e). Analysis of the mitochondrial morphology in mature neurons showed more cells with a fragmented mitochondrial network in the PINK1-exon7/del cultures (Fig. 4f). However, RNA expression level of MNF2, DNM1L, and MFF were not significantly different between genotypes (Supplementary Fig. S3).
It is known that LRRK2 expression is rather low in the developing brain [25]. Indeed, in neural progenitors, LRRK2 RNA levels were barely detectable and there were no differences between groups. The expression increased at the neuronal stage, but only mutant neurons had significantly higher LRRK2 levels than progenitors, which were also higher than control neurons (Fig. 5a). At the protein level, the PINK1 -exon7/del mutant neurons showed increased protein levels of LRRK2 at the neuronal stage (Fig. 5b), corroborating our findings in fibroblasts in a disease-relevant cell context. Over-expression of PINK1 wild-type in PINK1 -exon7/del mutant cells was performed at the end of neural progenitor stage (5–6 weeks) with good survival and robust expression of the GFP reporter 1 week after transfection (Fig. 5c) that was confirmed by the presence of a strong band corresponding to the full length PCR product of exons 6–8 (Fig. 5d). Like in the fibroblasts, over-expression of wild-type PINK1 induced a marked decrease in LRRK2 expression, with no effect on the expression of neuronal βIII-tubulin, TUBB3, other PARK genes or mitochondrial gene expression (Fig. 5e).
To establish the relevance of LRRK2 deregulation in PINK1 mutants, we next examined LRRK2 levels in human fibroblasts harboring another mutation in PINK1, G309D (see Table 1), located in exon 4, that causes a modest decrease in kinase function with no change in protein stability [3, 19, 20]. The glycolytic rate was higher in the homozygous samples (105.7 ± 1, N = 2) than in controls (75.4 ± 6.9, N = 3, p < 0.05, data not shown). Fibroblasts from both PD patients had higher LRRK2 RNA levels (Fig. 6a), although in these samples, the protein levels were not significantly different from control (Fig. 6b). The mitochondrial network appeared to be normal (Fig. 6c).
Discussion
In this study, we sought to define a disease-related in vitro phenotype in human cells with PD-associated PINK1 mutations. We did not find consistent mitochondrial alterations as hypothesized but found instead a remarkable deregulation of LRRK2, revealing a previously under-recognized connection between these two PARK genes in human cells. We corroborated this association in fibroblasts from a second family with a different mutation in PINK1.
Our data suggest that PINK1 exerts, directly or indirectly, an inhibitory effect on LRRK2 at the transcriptional level. Indeed, over-expression of wild-type PINK1 decreased LRRK2 levels in control and mutant cells. In mutant fibroblasts and neurons, LRRK2 deregulation resulted in increased LRRK2 protein level.
Abnormal protein synthesis, cytoskeletal dynamics, and mitochondrial transport have all been implicated in LRRK2 pathogenesis [26]. Studies in C. elegans [12] and Drosophila [27] suggest an antagonistic effect of lrk-1 and pink-1 [13] with clear, if complex, interactions between these two genes, not unlike in our study.
In agreement with previous studies [8, 20, 28], PINK1-patient fibroblasts showed only subtle signs of mitochondrial dysfunction with normal levels of ATP—at the expense of an increased glycolytic rate—and no conspicuous alterations in the mitochondrial network. Similar findings have been documented for other PARK genes, like Parkin [29] and LRRK2 [30]. The net effect of PINK1 kinase deficiency on fusion/fission dynamics is a matter of controversy as different model organisms show opposite effects (increased tubularity or increased fission) [10]. It is worth noting that there are remarkable differences between vertebrate (mouse and zebrafish) and invertebrate (fly and worm) models regarding the effects of PARK genes on mitochondrial dynamics. Analysis of proteins involved in fusion and fission mitochondrial dynamics in PD fibroblasts showed alterations that are consistent with activation of compensatory mechanisms in the mutants but could also reflect a defective inhibition of fission through TRAP1 [31, 32].
Recent studies have proposed patient fibroblasts as an adequate system to investigate disease mechanisms and compensatory pathways in genetic PD [21], but we took advantage of reprogramming technology to generate iPSC and neurons from these patients and validate our findings in a disease-relevant cell type. iPSC-derived neurons offer the benefit of preserving cell-type specific endogenous expression and transcriptional regulation of the mutated gene. While modeling a complex, age-related and mostly sporadic disorder such as PD is challenging, iPSC harboring monogenic, early-onset variants like PINK1 may provide valuable insights into disease mechanisms [33]. Also critical is the capacity to generate a significant proportion of target cells from the iPSC. In this study, we obtained about 30% of TH+ neurons from all genotypes.
PINK1 -exon7/del mutant neurons had a significant increase in the percentage of cells with fragmented mitochondria suggesting a shift in mitochondrial dynamics towards fission. However, this should be interpreted with caution, because in neurons, many different pathways can result in this phenotype. Indeed, we did not find any significant changes in fusion or fission genes, besides a uniform developmental increase in their expression.
Interestingly enough, we observed a remarkable upregulation of LRRK2 in the PINK1 -exon7/del mutant neurons. Furthermore, like in fibroblasts, transient over-expression of wild-type PINK1 effectively downregulated LRRK2 expression without having any effect on neuronal markers, mitochondrial dynamics, or other PARK genes.
LRRK2 has been implicated in a very broad range of cellular pathways, and the precise mechanisms leading to neuronal degeneration in PD-associated LRRK2 mutations remain to be defined. Nonetheless, increased levels of LRRK2 appear to be directly related to the pathogenicity/toxicity of PD-related mutations, at least for the LRRK2 G2019S mutation [34], which is one of the mutations most frequently associated with PD. Importantly, not only mutations but also several polymorphisms in LRRK2 are associated to an increased risk of PD. Our data suggest that PINK1 and LRRK2 act on a common pathogenic pathway in an antagonistic manner.
Both LRRK2 and PINK1 have been found to modulate the phosphorylation state of several Rab GTP-ases [35, 36], and PD-related mutations could therefore affect vesicle trafficking. In this regard, it is also interesting that over-expression of Rab1 could rescue the SNCA mutant phenotype in invertebrate and mammalian models and in human neurons carrying PD-associated SNCA triplication [37, 38], indicating that several PARK genes with quite distinct clinical manifestations (early onset, typical PD, or dementia with Lewy body) may share common molecular mechanisms [39].
Further downstream, LRRK2 has recently been found to directly phosphorylate p53 (TP53), thus acting in a pro-apoptotic role—phosphorylation of p53 leads to transcriptional activation of pro-apoptotic genes such as BAX, PUMA (BBC3), NOXA (PMAIP1), and others, as well as activation of transcriptional independent pro-apoptotic mechanisms—in a tissue-specific manner [40]. It has been proposed that either LRRK2 or p53 could initiate cell death in dopamine neurons [41]. PINK1 negatively regulates p53 activity through activation (phosphorylation) of histone deacetylases, which could account for its pro-survival and anti-apoptotic role [42]. Therefore, in PINK1 mutant neurons, the lack of functional PINK1 could perhaps lead to activation of pro-apoptotic mechanisms through upregulation of LRRK2, although this remains to be proven. A better understanding of LRRK2 function is required to identify novel ways to re-establish this balance in susceptible neurons in PINK1-associated and, perhaps, more broadly in PD.
In conclusion, we report a novel role of PINK1 modulating the levels of LRRK2 in patient fibroblasts and neurons. Although LRRK2 is one of the genes that is most frequently associated with PD, its role in early-onset recessive forms of the disease had not been previously determined. Our results suggest a convergent pathway for these PARK genes, acting in an antagonistic manner, and broaden the involvement of LRRK2 in the pathogenesis of PD.
References
Farrer MJ (2006) Genetics of Parkinson disease: paradigm shifts and future prospects. Nat Rev 7:306–318
Berg D, Lang AE, Postuma RB et al (2013) Changing the research criteria for the diagnosis of Parkinson’s disease: obstacles and opportunities. Lancet Neurol 12:514–524
Valente EM, Abou-Sleiman PM, Caputo V et al (2004) Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 304:1158–1160
Pilsl A, Winklhofer KF (2012) Parkin, PINK1 and mitochondrial integrity: emerging concepts of mitochondrial dysfunction in Parkinson’s disease. Acta Neuropathol 123:173–188
Haque ME, Thomas KJ, D’Souza C et al (2008) Cytoplasmic Pink1 activity protects neurons from dopaminergic neurotoxin MPTP. Proc Natl Acad Sci U S A 105:1716–1721
Yu W, Sun Y, Guo S et al (2011) The PINK1/Parkin pathway regulates mitochondrial dynamics and function in mammalian hippocampal and dopaminergic neurons. Hum Mol Genet 20:3227–3240
Rakovic A, Shurkewitsch K, Seibler P et al (2013) Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)-dependent ubiquitination of endogenous Parkin attenuates mitophagy: study in human primary fibroblasts and induced pluripotent stem cell-derived neurons. J Biol Chem 288:2223–2237
Morais VA, Haddad D, Craessaerts K et al (2014) PINK1 loss-of-function mutations affect mitochondrial complex I activity via NdufA10 ubiquinone uncoupling. Science 344:203–207
Van Laar VS, Berman SB (2013) The interplay of neuronal mitochondrial dynamics and bioenergetics: implications for Parkinson’s disease. Neurobiol Dis 51:43–55
Scarffe LA, Stevens DA, Dawson VL et al (2014) Parkin and PINK1: much more than mitophagy. Trends Neurosci 37:315–324
Paisan-Ruiz C, Lewis PA, Singleton AB (2013) LRRK2: cause, risk, and mechanism. J Parkinsons Dis 3:85–103
Saha S, Guillily MD, Ferree A et al (2009) LRRK2 modulates vulnerability to mitochondrial dysfunction in Caenorhabditis elegans. J Neurosci 29:9210–9218
Samann J, Hegermann J, von Gromoff E et al (2009) Caenorhabditits elegans LRK-1 and PINK-1 act antagonistically in stress response and neurite outgrowth. J Biol Chem 284:16482–16491
Samaranch L, Lorenzo-Betancor O, Arbelo JM et al (2010) PINK1-linked parkinsonism is associated with Lewy body pathology. Brain 133:1128–1142
Lopez de Maturana R, Aguila JC, Sousa A et al (2014) Leucine-rich repeat kinase 2 modulates cyclooxygenase 2 and the inflammatory response in idiopathic and genetic Parkinson’s disease. Neurobiol Aging 35:1116–1124
Requejo-Aguilar R, Lopez-Fabuel I, Fernandez E et al (2014) PINK1 deficiency sustains cell proliferation by reprogramming glucose metabolism through HIF1. Nat Commun 5:4514
Aguila JC, Blak A, van Arensbergen J et al (2014) Selection based on FOXA2 expression is not sufficient to enrich for dopamine neurons from human pluripotent stem cells. Stem Cells Transl Med 3:1032–1042
Zhao J, Liu T, Jin S et al (2011) Human MIEF1 recruits Drp1 to mitochondrial outer membranes and promotes mitochondrial fusion rather than fission. EMBO J 30:2762–2778
Beilina A, Van Der Brug M, Ahmad R et al (2005) Mutations in PTEN-induced putative kinase 1 associated with recessive parkinsonism have differential effects on protein stability. Proc Natl Acad Sci U S A 102:5703–5708
Hoepken HH, Gispert S, Morales B et al (2007) Mitochondrial dysfunction, peroxidation damage and changes in glutathione metabolism in PARK6. Neurobiol Dis 25:401–411
Auburger G, Klinkenberg M, Drost J et al (2012) Primary skin fibroblasts as a model of Parkinson’s disease. Mol Neurobiol 46:20–27
Reinhardt P, Schmid B, Burbulla LF et al (2013) Genetic correction of a LRRK2 mutation in human iPSCs links parkinsonian neurodegeneration to ERK-dependent changes in gene expression. Cell Stem Cell 12:354–367
Seibler P, Graziotto J, Jeong H et al (2011) Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells. J Neurosci 31:5970–5976
Miller JD, Ganat YM, Kishinevsky S et al (2013) Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell 13:691–705
Giesert F, Hofmann A, Burger A et al (2013) Expression analysis of Lrrk1, Lrrk2 and Lrrk2 splice variants in mice. PLoS One 8:e63778
Esteves AR, Swerdlow RH, Cardoso SM (2014) LRRK2, a puzzling protein: insights into Parkinson’s disease pathogenesis. Exp Neurol 261:206–216
Venderova K, Kabbach G, Abdel-Messih E et al (2009) Leucine-rich repeat kinase 2 interacts with Parkin, DJ-1 and PINK-1 in a Drosophila melanogaster model of Parkinson’s disease. Hum Mol Genet 18:4390–4404
Exner N, Treske B, Paquet D et al (2007) Loss-of-function of human PINK1 results in mitochondrial pathology and can be rescued by parkin. J Neurosci 27:12413–12418
Zanellati MC, Monti V, Barzaghi C et al (2015) Mitochondrial dysfunction in Parkinson disease: evidence in mutant PARK2 fibroblasts. Front Genet 6:78
Mortiboys H, Furmston R, Bronstad G et al (2015) UDCA exerts beneficial effect on mitochondrial dysfunction in LRRK2G2019S carriers and in vivo. Neurology 85:846–852
Pridgeon JW, Olzmann JA, Chin LS et al (2007) PINK1 protects against oxidative stress by phosphorylating mitochondrial chaperone TRAP1. PLoS Biol 5:e172
Takamura H, Koyama Y, Matsuzaki S et al (2012) TRAP1 controls mitochondrial fusion/fission balance through Drp1 and Mff expression. PLoS One 7:e51912
Badger JL, Cordero-Llana O, Hartfield EM et al (2014) Parkinson’s disease in a dish—using stem cells as a molecular tool. Neuropharmacology 76 Pt A:88–96
Skibinski G, Nakamura K, Cookson MR et al (2014) Mutant LRRK2 toxicity in neurons depends on LRRK2 levels and synuclein but not kinase activity or inclusion bodies. J Neurosci 34:418–433
Lai YC, Kondapalli C, Lehneck R et al (2015) Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. EMBO J 34:2840–2861
Steger M, Tonelli F, Ito G et al (2016) Phosphoproteomics reveals that Parkinson’s disease kinase LRRK2 regulates a subset of Rab GTPases. eLife 5:e12813
Cooper AA, Gitler AD, Cashikar A et al (2006) Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science 313:324–328
Mazzulli JR, Zunke F, Isacson O et al (2016) Alpha-synuclein-induced lysosomal dysfunction occurs through disruptions in protein trafficking in human midbrain synucleinopathy models. Proc Natl Acad Sci U S A 113:1931–1936
Clague MJ, Rochin L (2016) Parkinson’s disease: a traffic jam? Curr Biol 26:R332–R334
Ho DH, Kim H, Kim J et al (2015) Leucine-rich repeat kinase 2 (LRRK2) phosphorylates p53 and induces p21(WAF1/CIP1) expression. Mol Brain 8:54
Levine AJ, Harris CR, Puzio-Kuter AM (2012) The interfaces between signal transduction pathways: IGF-1/mTor, p53 and the Parkinson disease pathway. Oncotarget 3:1301–1307
Choi HK, Choi Y, Kang H et al (2014) PINK1 positively regulates HDAC3 to suppress dopaminergic neuronal cell death. Hum Mol Genet 24:1127–1141
Acknowledgements
We are grateful to the patients, relatives, and healthy individuals that participated in the study and to Dr. Angel García Martin for control samples and technical advice.
Authors’ Contributions
GAz: Acquisition, analysis, and interpretation of data and drafting and revision of manuscript. RLM: Acquisition and analysis of data and drafting and revision of manuscript. PR: Acquisition and analysis of data and drafting and revision of manuscript. AS: Acquisition and analysis of data. NV: Acquisition and analysis of data. AZ: Acquisition and analysis of data. DJB: Acquisition and analysis of data. JPB: Supervision and data analysis, interpretation, and critical revision of manuscript. BM: Critical revision of manuscript. GAu: Data interpretation and critical revision of manuscript. JMA: Study conception and critical revision of manuscript. RSP: Study design, data acquisition, analysis and interpretation, and drafting and critical revision of manuscript. All: Revision and approval of manuscript.
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Ethics declarations
The study was approved by the Ethical Committee on the Use of Human Subjects in Research in Euskadi, Spain. All subjects gave informed consent for the study using forms approved by the ethical committees on the Use of Human Subjects in Research at Hospital Universitario Insular de Gran Canaria, La Palmas de Gran Canaria; Hospital Donostia and Onkologikoa, San Sebastián; and Hospital San Cecilio, Granada, respectively.
Conflict of Interest
The authors declare that they have no competing interests.
Funding
This study is funded by grants from the European Commission, CORDIS FP7, HEALTH-2, 278871 (DDPDGENES), and the Joint Program in Neurodegenerative Diseases (DAMNDPATHS) to RSP. JPB is the recipient of grants from MINECO (SAF2013-41177-R) and the NIH/NIDA (1R21DA037678-01). GAu is the recipient of grants from DFG (AU96/10-1) and the German National Genome Research Network NGFNplus (Neuron-Parkinson-subproject 7).
Additional information
Garikoitz Azkona, Rakel López de Maturana, and Patricia del Rio contributed equally to this work.
Electronic supplementary material
Fig. S1.
Analysis of mitochondrial dynamics related genes in fibroblasts. a. qPCR of MFN2, DNM1L and MFF showed a decrease in mitofusin2 and an increase in fission factor in the PINK1 mutants. b. At the protein level WB analysis of MFN2 showed a small increase, suggesting that the transcriptional decrease might be compensatory to a decreased degradation, while (C) MFF protein expression was elevated. Columns represent the mean ± SEM of four independent experiments in the same samples. One-way ANOVA; * p < 0.05 and ** < p 0.001. d-e. Representative confocal images, showing typical distribution and partial co-localization of D. MFN2 and E. MFF (green) with MitoTracker® (magenta) in all fibroblasts. Scale bar: 20 μm (GIF 627 kb)
Fig. S2.
Characterization of pluripotency of PINK1 mutant iPSC lines. a. Fingerprinting analyses of the clones used in the study and their corresponding fibroblasts; BJ: unrelated control. b. Karyotypes. c. iPSC colonies showed robust expression of pluripotent markers such as OCT4, SSEA-4 and Nanog. d. Pluripotency genes were expressed at similar levels than in control human embryonic stem cells (H9). e-f. Pluripotency was confirmed in vivo (E) by formation of teratomas in NOD-SCID mice and in vitro (F) by embryoid body formation and tri-lineage differentiation (GIF 214 kb)
Fig. S3.
Mitochondrial genes expression in PINK1 iPSC-derived neurons. Quantitative PCR analysis of RNA levels of mitochondrial fusion (MFN2) and fission (DNM1L and MFF) genes in neural progenitors (NP) and neurons (N) showed a significant effect of time (developmental stage) and no differences across genotypes. Two-way ANOVA; * p < 0.05 and ** < p 0.001 (GIF 27 kb)
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Azkona, G., López de Maturana, R., del Rio, P. et al. LRRK2 Expression Is Deregulated in Fibroblasts and Neurons from Parkinson Patients with Mutations in PINK1. Mol Neurobiol 55, 506–516 (2018). https://doi.org/10.1007/s12035-016-0303-7
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DOI: https://doi.org/10.1007/s12035-016-0303-7