Azkona Et Al. 2015 Increased LRRK2 in PINK1 Mutants

Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

Lack of functional PINK1 is accompanied by increased LRRK2 levels in Parkinson disease fibroblasts and iPSC-neurons

Garikoitz Azkona1,2#, Rakel López de Maturana2, Patricia Del Rio2, Amaya Sousa2,

Nerea Vazquez2, Amaia Zubiarrain2, Dani...3,4, Juan Pedro Bolaños3,4, José Matias

Arbelo5, Rosario Sánchez-Pernaute2*

1. Animal Model Unit, Inbiomed, San Sebastian (Spain) 2. Laboratory of Stem Cells and Neural Repair, Inbiomed, San Sebastian (Spain) 3. Institute of Functional Biology and Genomics (IBFG), University of Salamanca- CSIC, Salamanca (Spain) 4. Institute of Biomedical Research of Salamanca (IBSAL), University Hospital of Salamanca, Salamanca (Spain) 5. Parkinson’s and Movement Disorders Unit. Department of Neurology. Hospital Universitario Insular de Gran Canaria. Las Palmas de Gran Canaria (Spain) # Present address: Animal Research Facility. Scientific and Technological Centers. University of Barcelona, Barcelona (Spain).

* Corresponding author

Rosario Sánchez-Pernaute Laboratory of Stem Cells and Neural Repair, Inbiomed P. Mikeletegi, 81; 2009 San Sebastian (Spain) Tlf: +34 943 309 064 Ext. 225 Fax: +34 943 308 022 E-mail: [email protected]

RunningTitle: Increased LRRK2 in PINK1 mutant cells

1 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

Abstract

Mutations in PINK1 (PARK6), a serine/threonine kinase involved in mitochondrial homeostasis, are associated with early onset Parkinson disease. Analysis of fibroblasts from two PINK1 Parkinson’s disease patients (c.1488+1G >A; c.1252_1488del) showed subtle signs of mitochondrial impairment and compensatory changes in fusion and fission proteins to maintain mitochondrial network morphology. To elucidate changes primarily caused by lack of functional PINK1 we first over-expressed wild-type PINK1, which induced minor adjustments in the expression of fusion and fission genes but a significant down-regulation of LRRK2 (PARK8). Furthermore, we found that, under basal conditions, LRRK2 protein levels were significantly higher in the mutant PINK1 fibroblasts. Lentiviral-mediated silencing of LRRK2 modified the expression of genes involved in mitochondrial dynamics in control and carrier cells (c.1488+1G >A) but was ineffective in cells from the PINK1 Parkinson’s disease patients, suggesting that

LRRK2 effect on mitochondrial homeostasis is mediated by functional PINK1 kinase activity. To further examine this interaction we generated induced pluripotent stem cell lines (iPSC) from mutant, carrier and control fibroblasts by lentiviral-mediated reprogramming. Neural induction and dopamine differentiation were similar in all genotypes. As observed in fibroblast, PINK1 mutant neurons showed increased LRRK2 expression both at the RNA and protein level. Finally, we investigated the expression of alpha-synuclein in iPSC-derived neural progenitors and neurons but these were not increased in the PINK1 mutant neurons. Our results identify a novel role of PINK1 modulating the levels of LRRK2 in Parkinson’s disease patient fibroblasts and iPSC- derived neurons and suggest a convergent pathway for these two PARK genes in the pathogenesis of Parkinson’s disease.

2 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

Keywords: Parkinson disease, dopamine, neurodegeneration, mitochondria, iPSC,

PARK genes, PINK1, LRRK2, alpha synuclein

Abbreviations:

3 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

Introduction

Parkinson’s disease is the second most common neurodegenerative disease (de Rijk et al., 1995), and results from the progressive loss of dopamine neurons in the substantia nigra pars compacta (Lees et al., 2009). Although Parkinson’s disease is a complex, multifactorial and largely sporadic disease, rare familiar monogenic forms have been correlated to specific genetic mutations (Farrer, 2006), providing the opportunity to identify causes of parkinsonism and study new pathways in cell biology.

An early-onset recessive Parkinson’s disease gene was found on chromosome 1 at the

PARK6 locus in a large Italian pedigree (Valente et al., 2002). The 8-exon PARK6 gene encodes a 581 amino acid protein, phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) with a C-terminal serine/threonine kinase domain and a mitochondrial targeting sequence at the N terminus (Valente et al., 2004). Parkinson’s disease related mutations cluster around the kinase domain and compromise kinase function or protein stability, so the phenotype is thought to result from a loss of function. Besides, the mitochondrial localization supported its involvement in mitochondrial dysfunction observed in Parkinson’s disease. ADD a sentence about this family ?.

Dopamine neurons are especially dependent on PINK1, which protects againts stress- induced mitochondrial dysfunction (Valente et al. 2004). PINK1 plays an important role in mitochondrial fidelity in Parkinson’s disease (for review see, (Pickrell and

Youle, 2015). Upon mitochondrial damage PINK1 is stabilized on the outer membrane of the mitochondria and recruits the E3 ubiquitin (Ub) ligase Parkin (PARK2) from the cytosol to mediate mitophagy (Matsuda et al., 2010; Vives-Bauza et al., 2010) thus,

PINK1/Parkin signaling pathway controls mitochondrial quality. The proper function of the PINK1/Parkin pathway requires PINK1’s kinase activity (Yu et al., 2011). However, despite the interest in the role of PINK1 in mitophagy, it appears that this pathway is not

4 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants modified by PINK1 mutations under physiological conditions, i.e. in systems with endogenous expression (Morais et al., 2014; Rakovic et al., 2013).

Besides its role in mitophagy, PINK1 signaling regulates mitochondrial fusion and fission (Van Laar and Berman, 2013). Mitofusin 2 (MFN2) is a GTPase involved in mitochondrial fusion that is phosphorylated by PINK1 and ubiquitinated by Parkin for degradation (Youle and van der Bliek, 2012). Mitochondrial fission factor (MFF) is required for mitochondrial recruitment of Dynamin 1-Like (DNM1L) during fission in mammalian cells (Otera et al., 2010). It has been shown that PINK1 phosphorylates

TNF-receptor associated protein 1 (TRAP1) (Pridgeon et al., 2007), that in turn downregulates the expression of fission proteins MFF and DNM1L (Takamura et al.,

2012). Therefore a defective PINK1 function may favor fission, although the effect of

PINK1 kinase deficiency on fusion/fission dynamics is a matter of controversy (Scarffe et al., 2014).

In this study, we first investigated the mitochondrial dynamics and the expression pattern of other PARK genes in fibroblast and differentiated dopamine neurons from patients and asyntomatic carriers from a Spanish kindred (Samaranch et al., 2010) and age-matched controls. This led us to unveil an up-regulation of LRRK2 in the PINK1 mutants and a bidirectional interaction between these two PARK gene products.

5 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

Material and Methods

Human samples

Skin samples were obtained from four 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). Subjects with PINK1 gene alterations included two individuals with a c.1488+1G >A mutation (asymptomatic carriers, denoted as

PINK1-exon7) and two individuals with an additional deletion c.1252_1488 (affected

Parkinson’s disease patients, denoted as PINK1-exon7/del). Demographic data are provided in Supplementary Table 1. The two Parkinson’s disease patients presented an early- onset, typical parkinsonian syndrome, characteristic of PINK1-associated Parkinson’s disease (Samaranch et al., 2010). All subjects gave informed consent for the study using forms approved by the Ethical Committee on the Use of Human Subjects in Research at the Hospital Universitario Insular de Gran Canaria and the Hospital Donostia. Dermal fibroblasts were cultivated in DMEM (High Glucose modification, Sigma Aldrich®) with 10% of fetal calf serum (Invitrogen), 2 mM L-glutamine (Sigma) and 1 %

Penicillin/Streptomycin (Sigma), as described previously (Lopez de Maturana et al.,

2014).

Genetic analysis

PINK1 variants were analyzed by conventional PCR using a primer pair designed to amplify a region expanding exons 6 and 8 (Samaranch et al., 2010). Total RNA and cDNA were obtained as detailed below. Cycling conditions were standard. Primer sequences (Samaranch et al., 2010) are provided in Supplementary Table 2. GAPDH was used as the reference gene.

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Standard G-band karyotypes of the iPSC clones used in the study were performed at the

Clinical Genetics Unit, Policlinica Gipuzkoa, San Sebastian (Spain).

Quantitative RT-PCR

Total RNA was extracted using TRIzol® total RNA isolation reagent (Gibco®, Life

Technologies) and the RNeasy Qiaprep (Qiagen). cDNA was synthesized from total

RNA using random hexamers according to the GeneAmp® RNA PCR Core Kit (Life

Technologies) and the High-Capacity cDNA RT kit (Applied Biosystems®). Real- time

PCR was performed using SYBR® Green PCR Master Mix (Applied Biosystems®) and the StepOneTM Detection System (Applied Biosystems®). Primer sequences are provided in Supplementary Table 2. Comparative analysis of gene expression levels

(∆∆Ct) was carried out using GAPDH as reference.

ATP content

Cellular ATP was measured using the Luminiscent ATP Detection kit (Abcam). Briefly, cells were harvested with Accutase, pelleted and washed once in PBS. An aliquot was set aside for protein quantification. The rest of the cells was pelleted again and resuspended in 50 µl growth medium. Cells were lysed by adding 50 µl of the detergent and mixing. After a 5-min incubation, 50 µl of Substrate was added and 10 min later luminescence was quantified in a GloMax® luminometer (Promega). An ATP standard curve was prepared as indicated by the manufacturer. ATP values are calculated in pmol per µg of protein in the cell extract. Only samples that yielded >15 µg protein in the

ATP assay extract were included.

Glycolytic rate

Rates of glycolysis in fibroblasts were calculated as previously described (Requejo-

Aguilar et al., 2014) by measuring the production of tritiated water from labeled [6-

14C]glucose in sealed flasks.

7 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

Western blotting

Whole-cell lysates were prepared in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1%

NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM NaF) with a protease inhibitor cocktail (Roche). SDS-PAGE and protein transfer and blotting were carried out according to standard procedures (Lopez de Maturana et al., 2014). Primary and secondary antibodies are listed in Supplementary

Table 3. Visualization of HRP-labeled proteins was performed using enzyme-linked chemifluorescence (ThermoFisher Scientific) 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) for 45 minutes and fixed for 10 minutes with 4% paraformaldehyde (15710-S, Electron Microscopy Sciences).

Immunofluorescence staining was performed as previously reported (Aguila et al.,

2014). Primary and secondary antibodies are listed in Supplementary Table 3. The cells were counterstained with Hoechst 33342 (Sigma-Aldrich) and in some cases with phalloidin A12380 (Alexa Fluor® 568 conjugated). Images were acquired in a Zeiss

LSM510 confocal microscope using the same settings for control and experimental samples and analyzed with Image J 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 the criteria included in (Zhao et al., 2011). Cell counts were performed on randomly selected visual fields from at least two independent experiments. In each field, images were acquired at 63X magnification and cells were counted using Image J

1.42q Cell Counter plugin. On average, 16 visual fields were acquired per coverslip, for

8 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants a total of 1,000-5,000 Hoechst labeled cells counted per condition, by 2 investigators

(PdelR and RLdeM) in a blinded manner. For an estimate of dopamine differentiation from iPSC, tyrosine hydroxylase positive neurons were counted over total ßIII-tubulin positive neurons at day 50-70 as previously described (Aguila et al., 2014).

PINK1 over-expression

Fibroblasts at 70-90% confluence from the two patients, two carriers and three to five control subjects were electroporated with the Neon® Transfection System

(InvitrogenTM), using 2 pulses of 1000V for 20 ms, with wild-type PINK1 (pcDNA-

DEST47 PINK1 C-GFP (Beilina et al., 2005) a gift from Marc Cookson, Addgene

#13316) or a control GFP plasmid at 0.5µg/106 cells. Additional controls in each group received the pulses only with no plasmid. Cells were collected for analysis at 24 and 48 hours post-transfection given that expression declined rapidly to baseline levels after

72h in preliminary experiments (data not shown).

LRRK2 gene knockdown

LRRK2 expression was stably silenced in fibroblasts using commercially available shRNA sequences cloned into the pLKO.1 vector (TRCN0000368513 and

TRCN0000358257 MISSION® shRNAs, Sigma-Aldrich®) as previously reported

(Lopez de Maturana et al., 2014). Lentiviral particles were produced and titrated at the

Viral Vector Platform in Inbiomed according to standard protocols. Transduction was performed in adhesion, overnight (16h) with a multiplicity of infection of 10. Cells were collected for analysis 5 days after transduction. iPSC and neuronal differentiation

Human induced pluripotent stem cell (iPSC) lines from the two patients and one carrier and from age-matched control individuals were derived in our laboratory using lentiviral vectors with the 4 Yamanaka factors fused to fluorescent reporters

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(Papapetrou et al., 2009). iPSC colonies expressed alkaline phosphatase and pluripotency markers such as NANOG, SSEA4 and REX1, and showed tri-lineage differentiation capacity in vitro upon formation of embryoid bodies, and in vivo by teratoma assay in SCID mice (data not shown). iPSC were maintained on irradiated human foreskin fibroblasts (HFF1, SCRC-1041, ATCC) in stem cell media composed of Knockout-DMEM (Invitrogen), supplemented with 2 mM L-glutamax (Invitrogen),

50 µM β-mercaptoethanol (Invitrogen), non-essential amino acids (Sigma), 20 % knockout serum replacement (Invitrogen), 50 U/ml penicillin, 50 μg/ml streptomycin and 10 ng/ml of FGF2 (PeproTech) as previously described (Aguila et al., 2014).

Dopamine differentiation was induced using a modified dual-SMAD inhibition protocol

(Chambers et al., 2009) on matrigel with addition of the sonic hedgehog agonist, N-

Methyl-N′-(3-pyridinylbenzyl)-N′-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4- diaminocyclohexane (SAG) (0.5µM, Milipore) and GSK3 inhibitor, CHIR 99021 (0.5

µM, Cayman Chemical) from day 2 to 12. At this time media was gradually shifted to

N2 media (DMEM/F-12 with 1X N2 supplement (Invitrogen) and the SAG concentration was reduced to 100nM as described (Aguila et al., 2014). On day 14 neural rosettes were manually passaged to 0.1% gelatine (GL, Sigma) / 15 μg/ml laminin/ 1 μg/ml fibronectin coated plates and expanded at high cell densities

(>300,000/cm2) in N2 media supplemented with 20 ng/ml BDNF, 200 μM ascorbic acid (AA), 100 nM SAG, 100 ng/ml FGF8 and 10 ng/ml FGF2. For terminal differentiation, media was replaced with neurobasal media containing 1X B27 supplement (Invitrogen), 2mM L-glutamine (Sigma-Aldrich), 20 ng/mL BDNF, 20 ng/ml GDNF, 200 uM AA, 0.5mM dibutyryl cAMP and 1ng/ml TGF-b III and laminin.

Data analysis and statistics

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Data analysis was carried out using GraphPad Prism software (v. 4.0c, La Jolla, CA).

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 when data were normalized to control levels (Western blotting). In experiments using human fibroblast samples, 2 to 3 individuals were assayed in each group in at least 2 independent determinations. For neuronal data we included data from 3 independent differentiations of the iPSC lines. In addition to the lines reprogrammed for this study other control lines (Aguila et al., 2014) were differentiated and analyzed in parallel. Data in the figures are represented as the mean ± SEM of two to four independent experiments. The threshold for significance was set at p < 0.05.

11 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

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 (Samaranch et al., 2010). Sample verification was confirmed by conventional PCR analysis using primers that result in the amplification of a 499-bp region expanding along exons 6 and 8 (Fig. 1A). This allows the identification of asymptomatic carriers (PINK1-exon7) and Parkinson’s disease patients (PINK1-exon7/del). In spite of the mutations, PINK1 expression was similar in mutant, carriers and control fibroblasts, both at the RNA (Fig. 1B) and protein (Fig 1C) levels and also in its distribution in the cytoplasm, partially co-localized with mitochondria (Fig. 1D). We examined the morphology of the mitochondrial network using MitoTracker®; semi-quantitative analysis of tubularity (Fig. 1E) showed no differences between genotypes (Fig. 1F). Baseline ATP levels were no different between groups (Fig. 1G). A sensitive method of glycolytic flux measurement demonstrated an increase in the glycolytic rate in the PINK1 mutants and carriers (Fig.

1H), as recently described in PINK1 KO cells (Requejo-Aguilar et al., 2014). These results are in agreement with previous studies that have validated human fibroblasts as a suitable model to investigate disease and compensatory mechanisms in genetic

Parkinson’s disease (Auburger et al., 2012).

Mitochondrial dynamics in PINK1 mutant fibroblasts

We next analyzed the expression levels of several proteins involved in mitochondrial fusion (MNF2) and fission (DNM1L and MFF) to determine whether the lack of morphological changes in tubularity was due to compensatory adjustments to PINK1 kinase defect. At the RNA level we found that MNF2 expression was significantly

12 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants reduced in Parkinson’s disease patients fibroblast, whereas MFF expression was significantly elevated (Fig. 2A). At the protein level, MFN2 levels were significantly increased in Parkinson’s disease patient fibroblasts (Fig. 2B), suggesting that the reduced RNA levels represent a transcriptional adjustment to compensate decrease degradation. On the other hand, MFF protein levels were significantly elevated in the patients (Fig. 2C), corresponding to the increased transcription. By immunofluorescence we observed a normal pattern of distribution of these two proteins, partially co-localized with the mitochondrial network visualized with MitoTracker® staining (Fig. 2D and E, respectively). These results reveal a series of changes in fusion/fission dynamics that can compensate the lack of PINK1 kinase function in fibroblast, as quantification of mitochondrial morphology showed no differences between genotypes (Fig. 1). This does not exclude subtle alterations in mitochondrial morphology or function, but it is in agreement with previous studies in fibroblasts with PINK1G309D mutations (Hoepken et al., 2007).

Effect of PINK1 over-expression in fibroblasts

To identify the changes causally related to PINK1 deficiency, we next evaluated the capacity of wild-type PINK1 over-expression to modify the mitochondrial gene expression profile observed in PINK1 mutant fibroblasts. 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. 3A) and clear expression of a

499 band corresponding to the wild-type PINK1 (exon 6-8) in transfected mutant fibroblasts (boxed in Fig. 3B). Transient transfection of wild-type PINK1, did not significantly modify the levels of either MNF2, DNM1L or MFF genes (Fig. 3C). We examined the effect of PINK1 over-expression on the expression pattern of other genes

13 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants related to Parkinson’s disease. 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 that has been described to be repressed in LRRK2G2019S mutant neurons

(Reinhardt et al., 2013), and observed and up-regulation in the PINK1-exon7/del fibroblasts

(Fig. 3C), suggesting that the changes in LRRK2 level in this paradigm are meaningful.

By immunofluorescence we observed that a few cells that had very high levels of GFP-

PINK1 showed low signal for LRRK2 (Fig. 3D).

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 PINK1 mutant fibroblasts. LRRK2 RNA was elevated in PINK1-exon7/del fibroblasts (Fig. 4A) but not significantly different from the control. Remarkably, LRRK2 protein was significantly increased (2.49 ± 0.24) in PINK1-exon7/del samples, with no apparent change in PINK1-exon7 fibroblasts (Fig. 4B). By immunofluorescence LRRK2 subcellular distribution was similar in all groups (Fig. 4C). To further characterize the relationship between LRRK2 and the mitochondrial proteins in PINK1 mutant fibroblasts, we knocked down the expression of LRRK2 by lentiviral silencing. Five days after transduction LRRK2 RNA expression was reduced by more than 50%, with no differences across groups (average decrease of 67.34 ± 19.07%; Fig. 4D, inset). PINK1 expression was slightly reduced in all the samples (Fig. 4D). In control fibroblasts, LRRK2 silencing resulted in a trend to increase MNF2, DNM1L and MFF RNA levels (118.07 ± 27,4, 159.29 ± 21.33 and

137.33 ± 12.67 %, respectively). We found a similar trend in PINK1-exon7 fibroblasts. In contrast, in fibroblasts from PINK1-exon7/del subjects, LRRK2 knockdown did not modify

14 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants the expression of any of these mitochondrial genes (Fig. 4D). This is better observed by plotting the variances for the combined genes (Fig. 4E) and suggests that LRRK2 effect on mitochondrial dynamics requires functional PINK1.

LRRK2 expression in PINK1 mutant in Parkinson’s disease patients’ 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-

Parkinson’s disease, we made iPSC lines from the two patients (PDP1 and PDP3 lines) and one of the carriers (PDP2) (Fig. 5A-B). Pluripotent cells were differentiated towards dopamine neurons using an inductive protocol combining developmental signals as described (Aguila et al., 2014)(Fig. 5C). There were no apparent defects in neural induction in the mutant cells, in agreement with results for iPSC lines carrying the G309D mutation in the PINK1 gene (Cooper et al., 2012; Rakovic et al., 2013;

Seibler et al., 2011), nor in the generation of dopamine neurons, with around 35% of tyrosine hydroxylase (TH) positive neurons at 2 months in all genotypes (Fig. 5D-E).

Because there are significant changes in the expression of genes and levels of proteins during neuronal maturation, we analyzed our cultures at two time points, corresponding to neural progenitors (2-6 weeks) and neurons (6-12 weeks). We first analyzed the mitochondrial morphology in mature neurons and observed that mutant neurons presented more cells with a fragmented mitochondrial network (Fig. 6A). Expression of mitochondrial fusion/fission genes, MFN2, DNM1L and MFF, showed a significant age- related effect. However, neither at the neural progenitor, nor at the neuronal stages, were there any significant differences between genotypes (Fig. 6B).

15 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

It is known that LRRK2 expression is rather low in the developing brain (Giesert et al.,

2013). Indeed, in neural progenitors LRRK2 RNA levels were barely detectable and there were no differences between groups. The expression increased slightly at the neuronal stage, but only in the mutant neurons were LRRK2 levels significantly higher than in the progenitors (p < 0.05). At the protein level, LRRK2 expression in neural progenitors showed no differences between groups. However, at the neuronal stage the

PINK1 mutant neurons (PDP1/PDP3) showed increased levels of LRRK2 (Fig. 6C), thus corroborating our findings in fibroblasts in a disease relevant context.

Finally we examined the expression of alpha-synuclein in these cells, because LRRK2 has been reported to modulate them (Skibinski et al., 2014). In addition, the postmortem analysis of a member of this family revealed alpha-synuclein in neurites and Lewy bodies in the midbrain (Fiesel et al., 2015; Samaranch et al., 2010). As expected, there was a robust increase in alpha-synuclein/SNCA RNA levels in neurons with respect to neural progenitors, corresponding to normal neuronal maturation. At the protein level, both the carrier and the mutant neural progenitors showed lower alpha-synuclein expression compared with controls, whereas no differences were observed at the neuronal stage (Fig. 6D). Taken together these results confirm an up-regulation of

LRRK2 in PINK1 mutant neurons but its putative pathogenic effect does not appear to involve an increase in alpha-synuclein.

16 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

Discussion

In this study we found a remarkable up-regulation of LRRK2 in human fibroblasts and iPSC-derived neurons from Parkinson’s disease-PINK1 patients, revealing a previously unknown connection between these two genes involved in familial Parkinson’s disease pathogenesis.

Our novel data suggests that PINK1 exerts an inhibitory effect on LRRK2 at the transcriptional level. Indeed, over-expression of wild-type PINK1 decreased LRRK2 levels even in control cells. In mutant fibroblast this deregulation resulted in increased

LRRK2 protein level. Interestingly, silencing LRRK2 modified the expression of PINK1 and genes involved in mitochondrial dynamics, in control and carrier fibroblasts but was ineffective in PINK1 mutant cells (Fig 4F), suggesting that the interaction is bidirectional and that LRRK2 operates upstream, or requires functional PINK1 on this pathway.

Deregulations of protein synthesis, cytoskeletal dynamics and mitochondrial transport have been implicated in LRRK2 pathogenesis. An effect of LRRK2 on mitochondrial function was described in C. elegans (Saha et al., 2009). In this model organism another study proposed an antagonistic effect of lrk-1 (the C. elegans homologue of human

LRRK2) and pink1 because in the absence of lrk-1 all phenotypic aspects of pink-1 loss-of-function mutants were suppressed. Conversely, the hypersensitivity of lrk-1 mutant animals to the endoplasmic reticulum stressor tunicamycin was reduced in a pink-1 mutant background (Samann et al., 2009). In drosophila, hPINK1 can rescue some of the eye defects caused by hLRRK2 over-expression while PINK1-RNAi severe phenotype was made worse by hLRRK2 over-expression suggesting clear, if complex, bidirectional interactions between these genes (Venderova et al., 2009) like in our study.

In agreement with previous studies (Exner et al., 2007; Hoepken et al., 2007; Morais et

17 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants al., 2014), PINK1-patient fibroblasts showed subtle signs of mitochondrial dysfunction but no conspicuous alterations in the mitochondrial network. Similar findings have been documented for other PARK genes, like Parkin (Zanellati et al., 2015) and LRRK2

(Mortiboys et al., 2015). Importantly, and in contrast with knockout models, expression and localization of PINK1 in our mutant fibroblasts was not different from control cells, and showed partial co-localization with mitochondria. 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)(Scarffe et al., 2014). Analysis of proteins involved in fusion and fission mitochondrial dynamics in our fibroblasts showed complex alterations that are consistent with activation of compensatory mechanisms in the mutant fibroblasts. MFN2 protein levels were only marginally (but significantly) increased, probably reflecting an adjustment at the transcriptional level, given that the RNA level was decreased. Therefore, in the mutant fibroblasts a lower degradation rate of MFN2 is partially compensated by decreased transcription. In contrast, MFF, was upregulated both at transcriptional and protein levels (and DNM1L show a trend to higher levels). While this could be compensatory suggesting that PINK1 promotes mitochondrial fission in human fibroblasts, it is also possible that the increase in MFF is a direct consequence of defective PINK1 function inhibiting fission through TRAP1 (Pridgeon et al., 2007) (Takamura et al., 2012).

Several recent studies have proposed and validated patient fibroblasts as an adequate system to investigate disease mechanisms and compensatory pathways in genetic Parkinson’s disease (Auburger et al., 2012) although the model has limitations. Therefore, we took advantage of reprogramming technology to generate iPSC and neurons from these patients. We used a well-established differentiation protocol towards dopamine neurons. Nevertheless, this results in a heterogeneous

18 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants neuronal population, with 30 to 40% of dopamine neurons, which has to be taken into consideration when interpreting the results. Another question to take into account is the developmental dynamic nature of this system. In order to control for different maturation rates we analyzed the cells at two different time points that were set with very wide intervals, 2 to 6 weeks for neural progenitors and 6 to 12 weeks for neurons to avoid confusing effects of different maturation rates between cell lines and experiments.

A first difference we observed in the PINK1 mutant neurons was a significant increase in the percentage of cells with fragmented mitochondria. This points to a shift in mitochondrial dynamics towards fission but it should be interpreted with caution as in neurons many different pathways can result in this phenotype. Indeed, we did not find significant changes in fusion or fission genes besides a consistent developmental increase at the neuronal stage.

Interestingly enough, we observed a remarkable up-regulation of LRRK2 also in the

PINK1 mutant neurons. Low expression of LRRK2 in neurons makes difficult to study its physiological role. LRRK2 has been implicated in a very broad range of cellular pathways so the precise mechanisms leading to neuronal degeneration in LRRK2 mutations remain to be defined. Nonetheless, increased levels of LRRK2 appear to be directly related to the pathogeneicity/toxicity, at least for the LRRK2G2019S mutation

(Skibinski et al., 2014), which is the most frequent mutation associated with Parkinson’s disease. Importantly, and in contrast with other PARK genes (including PINK1), LRRK2 has been found to increase the risk of Parkinson’s disease at several levels, i.e. several point mutations cause familial Parkinson’s disease but also GWAS analyses have identified rare polymorphisms that are a strong risk factor and also more frequent variants that confer a small increase in the risk (Paisan Ruiz 2013; van der Brug 2015).

19 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

Modulation of alpha-synuclein levels by LRRK2 has been proposed as a pathogenic mechanism in Parkinson’s disease with LRRK2G2019S mutation (Skibinski et al., 2014).

However, in our system we could not confirm this correlation that may be limited to the specific mutation G2019S in the kinase domain of LRRK2 that increases kinase activity. Likewise, although unlikely, we cannot exclude that our observation is limited to this specific PINK1 mutation.

Recent work has identified other possible pathways in which PINK1 and LRRK2 effects may converge. A recently identified role of PINK1 modulating mitochondrial import and translation (de-repression) of nuclear encoded mRNAs for mitochondrial respiratory chain complexes is disrupted by the PINK1G309D mutation in primary

Parkinson’s disease fibroblasts and induced neurons (Gehrke et al., 2015). Such mechanism could also provide an explanation for the positive rescue effect of phosphomimetic NDUFA10 previously described (Morais et al., 2014), in the absence of direct phosphorylation of this complex I subunit by PINK1. The role of PINK1 in transcriptional de-repression (probably by phosphorylating repressors, like histone deacetylases, HDACs) is underscored by a recent quantitative phosphoproteomic analysis of PINK1 targets (Qin et al., 2014). Indeed, PINK1 negatively regulates p53

(TP53) activity through activation (phosphorylation) of HDACs and this could account for its pro-survival anti-apoptotic role (Choi et al., 2014). Interestingly, LRRK2 has recently been found to directly phosphorylate p53 and induce p21, thus acting in an antagonistic pro-apoptotic role (Ho et al., 2015), in a tissue specific manner. Moreover

LRRK2 promoter has RE-1 binding site and is de-repressed by inhibitors of the repressor complex that contain HDAC/KDM (RSP, unpublished data). Therefore in

PINK1 mutant neurons the lack of functional PINK1 would lead to an up-regulation of

LRRK2 and activation of pro-apoptotic mechanisms. Such antagonistic effect of

20 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

LRRK2 and PINK1 on p53 pathway fits within the general hypothesis that portrays cancer and neurodegeneration as flipsides of the same process. A better understanding of the intricate relations between PINK1 and LRRK2 is required to identify novel ways to re-establish balance in susceptible neurons in PINK1-Parkinson’s disease.

21 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

Figure Legends

Fig. 1 Characterization of fibroblasts. A. The mutation was confirmed by a PCR analysis and agarose gel electrophoresis, which revealed the exon 7 loss in Parkinson’s disease patient (PDP1 and PDP3) and carriers (PDP2 and PDP4) and a 33-bp deletion in

PINK1-exon7/del samples (PDP1 and PDP3). GAPDH was used as the reference gene. B.

RNA levels were assessed by quantitative RT-PCR and C. protein levels by western blotting analysis; a representative blot is shown above the quantification. Columns represent the mean ± SEM of at least two independent experiments. D. PINK1 subcellular distribution was analyzed by immunofluorescence. Confocal images show partial co-localization (yellow) of PINK1 (green) and MitoTracker® (red), which was used to define mitochondrial morphology. E. Examples of mitochondrial network tubularity visualized with MitoTracker® staining - a tubular and round network in the left panel and a tubular in the right. F. Quantification of tubularity in fibroblast samples showed no differences between genotypes. Mitochondrial morphology was assessed in randomly selected fields and > 150 cells were analyzed. Columns represent the mean ±

SEM of three independent experiments. Scale bar: 50 µm. G. ATP levels were not different in control, carrier and mutant fibroblasts. Scatter plot graphic of two to three independent experiments. H. Glycolytic rate measured by incorporation of tritiated water showed a significant increase in PINK1 fibroblasts. One-way ANOVA and post- hoc analysis * p < 0.05 and ** p < 0.01.

Fig. 2 Mitochondrial protein expression. A. RNA levels analyzed by quantitative RT-

PCR of MFN2, DNM1L and MFF in fibroblasts. B. Western blotting analysis of MFN2 and C. MFF protein expression; representative blots are shown above the respective quantifications. Columns represent the mean ± SEM of four independent experiments.

22 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

One-way ANOVA; * p < 0.05 and ** < p 0.001. d. Representative confocal images of d.

MFN2 and e. MFF (green) and MitoTracker® (magenta), showing partial co- localization of these proteins (white) in all fibroblasts. Scale bar: 20 µm.

Fig. 3 PINK1 overexpression in fibroblasts. A. PINK1 RNA levels analyzed by quantitative RT-PCR and B. a representative image of an agarose gel electrophoresis showing the expression of the 499 bp band corresponding to the exons 6 to 8 of PINK1 in PINK1-exon7/del samples (PDP1 and PDP3; boxed). Average increase by qPCR was not different across groups (31±4.5 fold increase over mock GFP transfection). C. RNA levels analyzed by quantitative RT-PCR of MFN2, DNM1L, MFF, DJ1, LRRK2 and

UHRF2 in fibroblasts after PINK1 over-expression. All data are expressed as fold change over mock (GFP) transfected samples. Scatter plot graphics of two to three independent experiments. One-way ANOVA. * p < 0.05 and ** p < 0.01. d. By confocal analysis a few cells showing high levels of GFP (PINK1) appeared to have less

LRRK2 immunoreactivity. Scale bar 10µm.

Fig. 4. LRRK2 baseline expression in fibroblasts. A. LRRK2 RNA levels analyzed by quantitative RT-PCR and, B. LRRK2 protein levels in fibroblasts; a representative blot is shown above the quantification. Columns represent the mean ± SEM of four independent experiments. *** p < 0.001. C. LRRK2 subcellular distribution was visualized by confocal microscopy showing a similar pattern in all genotypes. Cells were counterstained with phalloidin (actin) to visualize the cytoskeleton. Scale bar: 50

µm. D. Transcriptional changes after LRRK2 knocked down (inset) in PINK1, MNF2,

DNM1L and MFF RNA levels. E. Analysis of the variance for the combined genes in the different genotypes caused by LRRK2 silencing.

23 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

Fig. 5. Mutant PINK1 iPSC-derived neurons. A. Representative brightfield images of iPSC colonies from selected clones from the 2 Parkinson’s disease patients (PDP1 and

PDP3) and one carrier (PDP2) and control used for differentiation. Scale bar 0.5 mm. B.

PCR analysis showed the same splicing pattern in the cell lines as in the original fibroblasts. C. Schematic representation of the differentiation protocol and the stages defined for analysis. D. Confocal images of immunofluorescence characterization at neural progenitor, SOX2 (red) and Nestin (green) positive cells and neuronal differentiation, TuJ1 (red) and TH (green) positive, stages. Scale bar 50µm e.

Quantification of TH positive neurons (over TuJ1) at the neuronal stage showed no differences across different cell lines in 2 independent experiments.

Fig. 6. PINK1 related changes in iPSC-derived neurons. A. Representative images of

Mitotracker labeling in 8-week-old neurons from control, carrier and mutant lines. Scale bar 10µm. Quantification of cells presenting predominantly tubular, mixed or fragmented mitochondrial morphology showed that there were more PINK1 mutant neurons with fragmented network (one-way ANOVA and post-hoc analysis p < 0.05).

B. 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 but no differences across genotypes. C. LRRK2 RNA and protein levels in NP and N. RNA levels significantly increased in PINK1 mutants from

NP to N stage (p < 0.05); Quantification of Western blots showed a significant increase in the mutant neurons (p < 0.05). D. SNCA RNA and protein levels in NP and N showed a slight decrease of protein levels in the PINK1 carrier and mutant NPs with respect to the control. Graphs represent the results of two to three independent

24 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants experiments. Columns represent the mean ± SEM. Two-way ANOVA and post-hoc analysis; * p < 0.05.

25 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

Acknowledgements

We are grateful to the member of this family that participated in the study. Technical help from Alfredo Santana, Adriana Aranguren and Jon Ondarro is greatly appreciated.

Funding

This work was funded by grants of the European Commission FP7, HEALTH-2 278871

(DDPDGENES) and the Joint Program in Neurodegenerative Diseases JPND

(DAMNDAPATHS) to RSP.

Supplementary material. 3 tables

Table 1. Dermal fibroblast samples Table 2. Primers used in the study Table 3. Antibodies used in the study

26 Azkona et al. 2015 Increased LRRK2 in PINK1 mutants

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