Science Supporting online material
Hereditary Early-Onset Parkinson’s Disease Is Caused by Mutations in PINK1
Enza Maria Valente, Patrick M. Abou-Sleiman, Viviana Caputo, Miratul M. K. Muqit,
Kirsten Harvey, Suzana Gispert, Zeeshan Ali, Domenico Del Turco, Anna Rita Bentivoglio,
Daniel G Healy, Alberto Albanese, Robert Nussbaum, Rafael González-Maldonado,
Thomas Deller, Sergio Salvi, Pietro Cortelli, William P. Gilks, David S. Latchman, Robert J.
Harvey, Bruno Dallapiccola, Georg Auburger, Nicholas W. Wood.
Contents
Materials and Methods
SOM text
Fig. S1-S5
Tables S1, S2.
References
1
Materials and Methods
Genetic analysis of PINK1. PCR reactions were performed in 25µl containing 1x PCR
buffer, 1.5mM MgCl2, 200µM of each dNTP, 0.3µM of both forward and reverse primers,
1.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Warrington, UK) and 50ng
of genomic DNA. Cycle conditions were: 11 min at 94°C; 30 cycles of 30s denaturation at
94°C, 30s annealing, and 90s extension at 72°C; final extension 7 min at 72°C (Primers
sequences are given in Table S1). Bidirectional sequencing was performed using Big Dye
Terminator v3.1 (Applied Biosystems, Warrington, UK) and electrophoresed on an ABI
3100 automated sequencer.
PINK1 cDNA was synthesized using SuperScript (Invitrogen, Paisley, UK) amplified using
PINK1 cDNA: fw-I 5’-CCA AGT TTG TTG TGA CCG GC-3’, rv-I 5’-CTT CAT AAC GAG
GAA CAG CGT CC-3’, fw-II 5’-ATC CAA GAG AGG TCC CAA GCA AC-3’, rv-II 5’-AGT
AAT TCA CCA GCT CCA TGC A-3’. PCR conditions were as above, with annealing
temperature of 63°C for 35 cycles.
Bioinformatics of PINK1 protein. Multiple sequence alignments were produced with
ClustalW software at the European Bioinformatics Institute
(http://www2.ebi.ac.uk/clustalw). The mitochondrial targeting motif was predicted by
using Mitoprot-2 (http://ihg.gsf.de/ihg/mitoprot.html) (mitochondrial score >99.8%).
The kinase domain was predicted using the following dedicated software: SMART
(http://smart.embl-heidelberg.de/), Prodom
(http://prodes.toulouse.inra.fr/prodom/current/html/home.php) and PFAM
(http://pfam.wustl.edu/).
Constructs for mammalian cell expression. Full-length PINK1 cDNAs were amplified
using the primers hPINK1 (5'-ggcggatccatggcggtgcgacaggcg-3') and hPINK2 (5'ctcgaattcgggacatcacagggctgc-3') and cloned into the BamHI and EcoRI sites of the vector
pRK5myc. The expressed PINK1 protein had a c-myc epitope tag attached to the Nterminus. The G309D mutation was introduced into pRK5-myc-PINK1 using the
Quikchange site directed mutagenesis kit (Stratagene, Amsterdam, Netherlands).
Cell cultures. Human dopaminergic neuroblastoma SH-SY5Y cells were maintained in
85cm2 flasks in Ham’s F12 and minimum essential medium (MEM) with Earles salts (1:1)
(Gibco-BRL, Paisley, UK) supplemented with 15% fetal calf serum (PAA Laboratories),
2
100U/mL penicillin, 100µg/mL streptomycin, 2mM L-glutamine, and 1X non-essential
amino acids (all from Gibco-BRL, Paisley, UK). COS-7 cells were maintained in 175cm2
flasks in Dulbecco’s modified Eagle’s medium containing sodium pyruvate and
pyridoxine (DMEM, Gibco-BRL, Paisley, UK), 10% fetal calf serum, 100U/mL penicillin,
100µg/mL streptomycin, and 2mM L-glutamine. All cells were incubated in a humidified
5% CO2 atmosphere at 37°C.
Immunofluorescence and Microscopy. COS-7 or SH-SY5Y cells were transiently
transfected with constructs encoding c-Myc-tagged wild type or mutant PINK1 using
Lipofectamine 2000 (Invitrogen, Paisley, UK). Cells were stained with mouse monoclonal
anti-c-myc (clone 9E10) antibody (1:100; Sigma, Paisley, UK) and Alexa Fluor 488 goat
anti-mouse IgG conjugates (1:1000; Molecular Probes, Leiden, Netherlands). For
mitochondrial staining, cells were incubated with Mitotracker red (1µg/ml; Molecular
Probes, Leiden, Netherlands) for 1 hour in a humidified 5% CO2 atmosphere at 37°C prior
to fixing. Standard immunofluorescence was performed using a Zeiss Axioskop 2 plus
microscope and digital images were captured using a Zeiss Axiocam camera and
processed in Adobe Photoshop (S1). Negative controls omitting primary antibody
separately or in combination with mitotracker was performed and no significant staining
was observed.
Western blot assays. COS-7 cells were transfected as described above. Cells were
harvested 48 hours after transfection and lysed in RIPA buffer (50mM Tris, 150mM NaCl,
0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) containing protease inhibitors (complete
mini protease inhibitor cocktail, Roche, Lewes, UK). Protein concentrations in all lysates
were determined using a bicinchoninic acid (BCA) assay (Pierce, Rockford, USA). Lysates
were run on a 10% SDS-PAGE gel; transferred to nitrocellulose membranes and probed
with an anti-c-myc (clone 9E10) antibody overnight at 4°C (1:100; Sigma, Gillingham, UK)
and HRP-conjugated anti-mouse IgGs (1:2000; Dako, Ely, UK) for 1 hour at room
temperature. Membranes were developed using ECL reagent (Bio-Rad, Hemel
Hempstead, UK). Equal loading of samples was confirmed by stripping membranes and
reprobing with either a mouse monoclonal anti-glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) antibody (1:1000; Chemicon, Temecula, CA) or a goat polyclonal
anti--actin antibody (1:2500; Santa Cruz Biotechnology, Santa Cruz, CA) and horseradish
peroxidase (HRP)-conjugated anti-mouse IgGs and anti-goat IgGs respectively (1:2000;
1:2500; Dako, Ely, UK).
3
Mitochondrial fractionation studies. COS-7 cells were grown in 175cm2 flasks and
transfected with wild-type myc-PINK1 cDNA using Fugene 6 (Roche, Lewes, UK). Cells
were harvested 48 hours post-transfection and washed twice in PBS and resuspended in
buffer containing 250mM sucrose, 20mM HEPES, 3mM EDTA, pH 7.5 and protease
inhibitors. Cells were then disrupted using a glass hand held homogeniser (10 passes) and
spun at 3000rpm for 10min. The supernatant was removed and retained. The pellet was
rehomogenised and spun as before. Supernatant was removed and both supernatants
were then centrifuged at 13,500rpm for 10min. The mitochondrial pellets were combined
and resuspended in the above buffer (S2). The cytoplasmic fractions were concentrated
using centricon YM-30 devices (Millipore, Watford, UK) as per the manufacturer’s
instructions. Protein concentrations in cytoplasmic and mitochondrial enriched fractions
were determined by BCA assay and equal amounts of cytoplasmic and mitochondrial
lysates were analyzed by Western blotting using c-myc antibody as described above. To
confirm relative purity of fractions, the membrane was stripped and reprobed with the
following primary antibodies: mouse monoclonal hsp 60 (1:1000; Stressgen, San Diego,
CA); GAPDH and mouse monoclonal complex I antibody (1:1000; Molecular Probes,
Leiden, Netherlands).
FACS mitochondrial membrane potential (m) assay. To measure the effect of wildtype and mutant PINK1 on mitochondrial membrane potential ( m) we used a
fluorescence-activated cell sorting (FACS)-based assay (S3). SH-SY5Y cells were cotransfected with a GFP reporter plasmid and vector, wild-type or G309D PINK1
constructs. GFP transfection efficiency was similar for all constructs. Cells were treated
with vehicle or 15µM MG-132 for 24 hours, then incubated with 10µM verapamil (Sigma,
Paisley, UK) and 100nM of the m-sensitive dye tetramethylrhodamine methyl ester
(Molecular Probes, Leiden, Netherlands) in a humidified 5% CO2 atmosphere at 37°C for
45 min. Cells were then harvested with trypsin, pelleted and resuspended in phosphate
buffered saline (PBS) on ice. For each sample, 20,000 cells (events) were analyzed on an
Epics XL flow cytometer with a 488-nm argon laser. The TMRM signal was analysed in the
FL2 channel gated for GFP positive events. The channel was equipped with a band-pass
filter at 580±30 nm; the photomultiplier value of the detector was 631V. Data were
acquired on a logarithmic scale. Arithmetic mean values of the median fluorescence
intensities (MFI) were generated for graphic representation. Experiments were performed
in duplicate or triplicate and results reported are an average of three independent
4
experiments. Similar results were obtained when depolarizing the plasma membrane with
high potassium prior to TMRM incubation confirming that MFI values corresponded to
m (data not shown). Statistical analysis was performed using one way ANOVA and
post hoc multiple comparisons with Bonferroni correction.
FACS Annexin V-PE assay. To determine the effect of wild-type and mutant PINK1 on
apoptosis, we used a FACS based assay. SH-SY5Y cells were transfected and stressed with
vehicle or MG-132 as described above. Cells were then harvested and washed in PBS and
pelleted. Pellets were resuspended in 1X Annexin V binding buffer (BD Biosciences,
Oxford, UK) and then incubated with Annexin V conjugated with phycoerythrin (Annexin
V-PE) for 15mins at room temperature before being analyzed immediately on an Epics XL
flow cytometer. For each sample, 20,000 cells were analyzed and Annexin V-PE
fluorescence was determined in the FL2 channel gated for GFP positive events.
Experiments were performed in triplicate and results reported are an average of four
independent experiments. Statistical analysis was performed using one way ANOVA.
SOM text
Clinical Phenotype
The clinical phenotype resembles sporadic or idiopathic PD (IPD), although the age at
onset was earlier, usually in the fourth-fifth decade (range 24-48 years) compared to IPD
where the average age of onset is 60-70 years (S4). Disease progression was slower than in
IPD and sustained response to L-dopa and occurrence of L-dopa associated dyskinesias of
variable severity were observed. Distinguishing clinical features, such as dystonia at onset,
sleep benefit and psychiatric disturbances, were rarely reported in PINK1 mutated
patients.
Clinical Samples
Mitochondrial dysfunction in idiopathic PD has previously been associated with Complex
I deficiency. We were unable to determine Complex I activity in the PARK6 families due to
a lack of suitable clinical material.
PINK1 variants
The following PINK1 polymorphisms were found in control individuals:
5
IVS1 -7 A>G = 24.4% (17.1% heterozygous A/G, 7.3% homozygous A/A)
G936A --> R312R = 1.2% heterozygous
IVS4 -5 A>G = 23% (20.6% heterozygous G/A, 2.4% homozygous G/G)
G1018A --> A340T = 9% heterozygous
IVS6 +43 C>T= 7% heterozygous C/T
A1562C --> N521T= 39.2% (35.5% heterozygous A/C, 3.9% homozygous C/C)
PINK1 expression
PINK1 is endogenously expressed in both COS7 and SH-SY5Y cells as determined by RTPCR.
6
A
7
B
Fig. S1. (A) Representative electropherograms of the exon 4 G>A (G309D) transition and
the exon 7 G>A (W437OPA) nonsense mutation. (B) Alignment of PINK1 orthologues
demonstrating the conservation of the G309D residue indicated by the arrow.
8
A
B
Fig. S2. PINK1 protein expression is not affected by G309D mutation. Cells were
transiently transfected with empty c-Myc vector (lane 1), wild-type (lane 2) and G309D
(lane 3) c-Myc-tagged PINK1 and lysates analysed by Western blotting using a c-Myc
antibody. Membranes from different experiments were re-probed with (A) GAPDH or (B)
-Actin
to
confirm
equal
loading.
9
Fig. S3. PINK1 is localized to mitochondria in neuronal cells. SH-SY5Y cells were
transfected with wild-type (A to C) or G309D (D to F), c-Myc-tagged PINK1 cDNA.
Immunofluorescence was carried out with c-Myc antibody and mitotracker. c-Myc-PINK1
[green (A, D)]; mitotracker [red (B, E)]; c-Myc-PINK1 and mitotracker merged (C, F).
10
Fig. S4. Representative flow cytometric histogram profiles of GFP-gated events after
treatment with MG-132 showing increased proportion of TMRM negative events in
cells transfected with G309D (B) compared with wild-type PINK1 (A).
11
Fig. S5. Representative flow cytometric Annexin V-PE plots of GFP-gated events after
treatment with MG-132 showing decreased percentage of Annexin V-PE positive
events in cells transfected with (B) wild-type PINK1 compared to (A) vector and (C)
G309D.
12
Marker
d1s378
d1s2647
d1s199
d1s2843
d1s2732
PINK1
d1s478
d1s2828
d1s2702
Kb (UCSC)
18984
19297
19426
19980
20106
20430-20450
21069
21412
22018
Family 1
1-1
1-1
1-1
3-3
3-3
Family 2
1-1
1-1
1-1
3-3
3-3
4-4
10-10
5-5
4-4
10-10
5-5
Table S1. Genotypes of the two Italian families around the PARK6 locus demonstrating a
common haplotype.
13
Exon
1A
Forward Primer 5’-3’
TCACTGCTAGAGGCGCCAG
Reverse Primer 5’-3’
anneal.
size
Temp.
(bp)
GCACCACGAACTGCCGCTG
64°
452
T
1B
TCGGGCTCGGGCTCCCTAA
CGGCCCTCGATCTGCTCAG
64°
330
2
ATTGATCTGGTCGACGTGG
CCTTTCCTGTGGATAATCTG
57°
522
AC
TC
CTCGAAGGTCAGAGCCAAT
CTGTCATATCAGACACTGTA 57°
323
TC
CC
GAATGTCAGTGCCAGTGTT
AGATATGTTCCCTTTGCATG
GG
GC
CGTATTGGGAGTCGTCGAT
GACCTGAAGAGTCAGTCCT
GT
AAA
GTCAGCTATGTCTTGCTGGT
ATCACAAGGCATCGAGTCT
3
4
5
6
7
8
60°
429
57°
300
59°
309
G
CC
TGGATCAGGTGATGTGCAG
AGGATCTGTCACTGTGGCTC 60°
416
GA
T
GAGAAGGGAAGACCCTCAC CAGACTGAACTCTCACTCA
TA
60°
520
AGT
Table S2. PCR-amplification conditions for PINK1 exons
14
References
S1.
M.M. Muqit et al., Hum. Mol. Genet. 13, 117 (2004).
S2.
R.S. Kler et al., J. Biol. Chem. 266, 22932 (1991).
S3.
M. Kalbacova, M., Vrbacky, Z. Drahota, Z. Melkova. Cytometry 52A, 110 (2003).
S4.
D. Twelves, K.S. Perkins, C. Counsell. Mov. Disord. 18, 19 (2003).
15