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Differential effects of PINK1 nonsense and missense mutations on mitochondrial
function and morphology
A. Grünewald
a,b
, M.E. Gegg
c
, J.-W. Taanman
c
, R.H. King
c
, N. Kock
a,b
, C. Klein
a,b
, A.H.V. Schapira
c,
⁎
a
Department of Neurology, Lübeck University, Lübeck, Germany
b
Department of Human Genetics, Lübeck University, Lübeck, Germany
c
University Department of Clinical Neurosciences, Institute of Neurology, University College London, Rowland Hill Street, London NW3 2PF, UK
abstractarticle info
Article history:
Received 30 January 2009
Revised 21 May 2009
Accepted 22 May 2009
Available online 3 June 2009
Keywords:
PINK1
Parkinson's disease
Mitochondria
Respiratory chain
Oxidative stress
Complex I
Mutations of the PINK1 gene are a cause of autosomal recessive Parkinson's disease (PD). PINK1 encodes a
mitochondrial kinase of unknown function which is widely expressed in both neuronal and non-neuronal
cells. We have studied fibroblast cultures from four family members harbouring the homozygous p.Q456X
mutation in PINK1, three of their wild-type relatives, one individual with the homozygous p.V170G
mutation and five independent controls. Results showed bioenergetic abnormalities involving decreased
activities of complexes I and IV along with increased activities of complexes II and III in the missense p.
V170G mutant. There were increased basal levels of mitochondrial superoxide dismutase in these cells and
an exaggerated increase of reduced glutathione in response to paraquat-induced free radical formation.
Furthermore, swollen and enlarged mitochondria were observed in this sample. In the p.Q456X nonsense
mutants, the respiratory chain enzymes were unaffected, but ATP levels were significantly decreased. These
results confirm that mutations of PINK1 cause abnormal mitochondrial morphology, bioenergetic function
and oxidative metabolism in human tissues but suggest that the biochemical consequences may vary
between mutations.
© 2009 Elsevier Inc. All rights reserved.
Introduction
Parkinson's disease (PD) is characterised clinically by bradykinesia,
tremor, and rigidity. The aetiology of PD is varied (Warner and
Schapira, 2003), and an increasing proportion of patients, albeit still a
minority, have an identifiable gene mutation (Klein and Schlossma-
cher, 2006). The biochemical mechanisms by which these single gene
mutations cause cell dysfunction and death provide valuable insight
into the pathogenesis of PD.
Recessive mutations in PTEN-induced putative kinase 1 (PINK1,
GenBank accession no. AB053323) are responsible for a familial form
of early-onset parkinsonism, previously mapped to chromosome 1p36
(the PARK6 locus). The protein product of PINK1 is ubiquitously
transcribed and encodes a kinase predominantly localised to mito-
chondria, but also expressed in the cytosol (Haque et al., 2008;
Valente et al., 2004).
Mitochondrial dysfunction has long been implicated in the
pathogenesis of PD. Evidence first emerged when 1-methyl-4-
phenyl-1,2,3,4-tetrahydropyridine (MPTP), an environmental toxin,
was discovered to produce parkinsonian features in drug abusers
(Langston et al., 1983). This finding was further supported by the
detection of a complex I deficiency in the substantia nigra of PD
patients (Schapira et al., 1989). Subsequently, complex I dysfunction
has also been observed in platelets (Benecke et al., 1993; Krige et al.,
1992; Parker et al., 1989) as well as in fibroblasts of patients (Hoepken
et al., 2007; Winkler-Stuck et al., 2004).
The complex I deficiency is thought to result in increased free
radical production, and it contributes to the oxidative mediated
damage seen in the PD nigra (Schapira, 2008). This relationship is bi-
directional. The enhanced release of free radicals causes a decrease in
the activity of the respiratory chain enzymes, particularly of
complexes I and IV (Schapira, 2006). The mitochondrial deficiency
can contribute to the dysfunction of the energy-dependent ubiquitin
proteasomal system (UPS) and to enhanced dopaminergic cell
damage and death (Hoglinger et al., 2003).
Preliminary data suggest that PINK1 may play a role in
protecting cells against stress conditions that affect the mitochon-
drial membrane potential (Valente et al., 2004). Recent mitochon-
drial studies on patient cells support these findings but different
mutation types appear to induce different functional consequences.
Hoepken et al. observed elevated levels of oxidative stress and a
mild decrease in complex I activity in lymphoblasts from three
patients homozygous for the p.G309D PINK1 missense mutation
(Hoepken et al., 2007). In contrast, Piccoli et al. studied fibroblasts
Experimental Neurology 219 (2009) 266–273
⁎Corresponding author.
E-mail address: a.schapira@medsch.ucl.ac.uk (A.H.V. Schapira).
0014-4886/$ –see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2009.05.027
Contents lists available at ScienceDirect
Experimental Neurology
journal homepage: www.elsevier.com/locate/yexnr
from a PD patient with the p.W437X nonsense mutation and observed
unaltered levels of the antioxidant glutathione but a decrease in the
superoxide dismutase activity. The enzyme activities of complexes
I–IV were unaffected, however ATP-ase activity was reduced (Piccoli
et al., 2008).
Similar differences emerged when the mitochondrial morphology
was investigated in mutant PINK1 cell models and patient fibroblasts.
Swollen and ruptured mitochondria were observed in Drosophila
PINK1 null mutants (Clark et al., 2006; Park et al., 2006; Poole et al.,
2008; Yang et al., 2006) and in human missense mutant fibroblasts (p.
Q126P and p.G309D PINK1) (Exner et al., 2007). In contrast, no
structural changes were seen in PD patient cells with the p.W437X
PINK1 mutation (Piccoli et al., 2008).
Here, we investigated the consequences of the PINK1 p.Q456X
nonsense and the p.V170G missense mutations on mitochondrial
function and morphology, and on free radical metabolism in fibroblast
cultures from patients with PD.
Materials and methods
PD patients with mutations in PINK1 and controls
Family W consists of three generations. Among the members there
were four patients with a homozygous c.1366CNT, p.Q456X nonsense
mutation (three female, mean age: 66.8 years [±4.8], mean age at
onset: 50.0 years [±9.3]). They had an average Hoehn and Yahr score
of 2.6 [± 0.5] and an average motor Unified Parkinson's Disease Rating
Scale (UPDRS) III score of 30.8 [± 4.4]. Three healthy family members
were also included (three female, mean age: 44.6 [± 10.0]) (for details
see Hedrich et al., 2006).
In addition, one patient (female, age: 70 years, age at onset:
31 years) harbouring a homozygous missense mutation c.509TNG, p.
V170G was studied. For this case, a Hoehn and Yahr score of 3.0 and an
UPDRS III score of 35.5 was determined (see also Moro et al., 2008). No
relatives of this individual were available.
Five independent age-matched healthy individuals of British origin
(C3, 4, 5, 7 and D5) served as additional controls (one female, mean
age: 64.8 years [±13.1]).
The investigated wild-type members of Family W and the
independent controls did not show significant differences in any of
the performed experiments, therefore they were combined in the
following analyses. All control subjects were examined and found to
be free of any signs of PD.
Phenotypic features of all individuals, investigated in this study,
are summarised in Table 1.
Tissue culture
Primary fibroblasts cultures were established from skin biopsies.
Fibroblasts were cultured in high glucose (4.5 g/l) or 0.9 g/l galactose
Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen) supple-
mented with 10% foetal bovine serum (BioSera) and 1 mM sodium
pyruvate (Sigma) at 37 °C, 5% CO
2
. The medium was changed weekly.
To mimic conditions of oxidative stress, cells were treated with
0.5 mM paraquat (PQ; Aldrich) for 48 h. The medium containing PQ
was refreshed after 24 h.
Real-time PCR: mitochondrial DNA levels
Gene dosage analysis was performed in a quantitative SYBR green
PCR on the LightCycler according to manufacturer's protocol (Roche
Diagnostics). The mitochondrial DNA concentration was measured
using primers against the mitochondrial displacement loop (D-loop)
(for sequences see Bai and Wong, 2005). The expression of the nuclear
single copy gene thymidine kinase 2 (TK2) was quantified as an
external standard (forward primer: 5′-TCC TGC AGA TGC CAC TTT GA-
3′; reverse primer: 5′-CCC CAA GTC TGA AGA AAA CG-3′). PCR
conditions were as follows: 95 °C for 15 min, 95 °C for 15 s, 60 °C for
30 s, 72 °C for 30 s (40 cycles); measurement of fluorescence in each
cycle at 72 °C. The mitochondrial DNA level was measured at least
twice per fibroblast sample.
Protein determination
Sample protein concentration was determined by use of the
bicinchoninic acid (BCA) protein assay kit (Pierce) according to the
manufacturer's protocol. Sample protein concentration was detected
in duplicate and calculated from the bovine serum albumin standard
calibration curve (0–150 0 μg/ml).
Mitochondrial enzyme assays and citrate synthase activity measurement
Fibroblasts were trypsinised and washed in PBS supplemented
with 1 mM phenylmethanesulphonylfluoride (PMSF), 1 μg/ml of
leupeptin and 1 μg/ml of pepstatin A. Cells were then homogenised in
isolation medium (250 mM sucrose, 10 mM Tris, pH 7.4, 1 mM
ethylenediaminetetraacetic acid (EDTA)), nuclei and cell debris were
removed by centrifugation at 1500 ×g, and mitochondria were
isolated by centrifugation at 11,800 ×g. The mitochondrial pellet was
then resuspended in isolation medium. Mitochondrial respiratory
chain complex and citrate synthase (CS) activities were measured by
Table 1
Genotypic and phenotypic characterisation of investigated individuals.
Mutational status Code Sex Age (yr) Age of onset (yr) Mutation (homozygous) H-Y stage UPDRS III motor score (off)
Nonsense mutants (Family W) IP2123 M 69 39 p.Q456X 3.0 34.0
R2122 F 67 61 p.Q456X 2.0 27.0
R2124 F 60 53 p.Q456X 2.5 27.0
R2126 F 71 47 p.Q456X 3.0 35.0
Mean+/−STD 66.8+/−4.8 50.0 +/−9.3 2.6 +/−0.5 30.8 +/−4.4
Missense mutant IP1703 F 70 31 p.V170G 3.0 35.5
Controls (Family W) R2132 F 35 –None ––
R2133 F 46 –None ––
R2134 F 42 –None ––
Mean+/−STD 44.6+/−10.0
Controls (independent) C3 F 60 –None ––
C4 M 73 –None ––
C5 M 45 –None ––
C7 M 79 –None ––
D5 M 67 –None ––
Mean+/−STD 64.8+/−13.1
The index patients are denoted by IP.Relatives of IP2123 are marked by an R. Given are the patients' ages at the time of their first examination. Sex: F —female, M —male; other: H-Y —
Hoehn and Yahr, STD —standard deviation, UPDRS —Unified Parkinson Disease Rating Scale.
267A. Grünewald et al. / Experimental Neurology 219 (2009) 266–273
spectrophotometric methods in these mitochondrial preparations as
reported (Bolanos et al., 1995). Data were expressed as a ratio with
citrate synthase activity. The activities of each complex were
determined in 2–4 assays per individual.
Malate dehydrogenase assay
Fibroblasts were harvested 72 h after feeding with fresh culture
media and resuspended in isolation media. Malate dehydrogenase
(MDH) activity was determined spectrophotometrically according to
Lai and Clark (1976). Samples were measured in duplicate and activity
expressed as μmol/min/mg protein.
Blue native polyacrylamide gel electrophoresis (PAGE)
Preparation of blue native gels was performed on isolated
mitochondria according to Schagger and Pfeiffer (2000). All gels
were transferred to Hybond-P membranes (GE Healthcare). The
membranes were incubated with primary antibodies raised against
subunits of the respiratory chain enzyme complexes (mouse anti-
complex I NDUFA9, mouse anti-complex II SDHA, mouse anti-complex
III UQCRC2, mouse anti-complex IV MTCO2, mouse anti-complex V
ATP5A1; Mitosciences). This was followed by incubation with a
horseradish peroxidase (HRP)-conjugated secondary mouse antibody
(Dako) and enhanced chemiluminescence (ECL) detection (Pierce).
Mitochondrial membrane potential
The mitochondrial membrane potential (ψm) was analysed by
means of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcar-
bocyanine iodide (JC-1, Invitrogen). Fibroblasts (5× 10
4
cells per 12-
well) were treated with 1 μg/ml of JC-1 for 15 min at 37 °C. The cells
were washed with phosphate-buffered saline (PBS), and mitochon-
drial JC-1 aggregates were measured with a fluorescent plate reader
(excitation 530 nm, emission 590 nm). In a sister well, JC-1
fluorescence was measured in the presence of the ionophore
valinomycin (100 nM), which destroys the mitochondrial membrane
potential, and was subtracted from the data. JC-1 fluorescence was
expressed against protein.
Cellular ATP levels
ATP levels were determined with the ATP Bioluminescence
Assay Kit HS II (Roche). Prior to the measurement with a Jade
luminometer (Labtech International), the samples were corrected
for cell number. The experiment was performed with 1× 10
5
cells
per ml of PBS. Each measurement was performed at least three
times per sample.
ATP synthesis
Fibroblasts were trypsinised and washed three times with ice cold
PBS. Cells were resuspended at 2×10
5
cells/ml in incubation medium
(25 mM Tris, 150 mM KCl, 2 mM K
+
-EDTA, 10 mM K
2
HPO4, pH 7.4). An
aliquot of cells (2 × 10
4
) was mixed with an equal volume of
incubation buffer containing 1 mg/ml bovine serum albumin, 1 mM
ADP and substrates (complexes I, III, IV: glutamate+ malate (10 μM);
complexes II, III, IV: succinate (10 μM) + rotenone (40 μg/ml);
complex IV: ascorbate (2 mM)+ N,N,N′,N′-tetramethyl-p-phenylene-
diamine (TMPD; 50 μM)), permeabilised with digitonin (40 μg/ml),
and incubated at 37 °C for 20 min. The reaction was stopped with
perchloric acid, and samples neutralised with 3 M K
2
CO
3
dissolved in
0.5 M tri-ethanolamine. Debris was removed by centrifugation and
ATP measured with the ATP Bioluminesence Assay kit HSII (Roche).
Data were expressed as pmoles ATP synthesised/min/10
5
cells and
represent means of two analyses per sample.
Western blotting
Cell lysates were separated by 12% sodium dodecyl sulfate (SDS)-
PAGE and protein transferred to Hybond-P membranes. Blots were
incubated with rabbit polyclonal anti-superoxide dismutase 2 (SOD2;
sc-30080, Santa Cruz Biotechnology) or mouse monoclonal anti-porin
31HL (clone 89-173/016, Calbiochem). The blots were then incubated
with HRP-conjugated secondary antibodies and bands detected by ECL.
Equal loading was assessed using an antibody against succinate dehy-
drogenase subunit (SDHA) of the electron transport chain (Mitos-
ciences) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH;
Abcam). Band density was measured using Alpha DigiDoc software.
For quantitative studies three separate western blots were analysed.
Cellular reduced glutathione (GSH) levels
The levels of GSH in fibroblasts were determined by trypsinising the
cells, resuspending in isolation medium and extracting into 15 mM ortho-
phosphoric acid. The cellular GSH concentration was then measured by
reverse-phase HPLC and electrochemical detection (Riederer et al., 1989).
Oxyblot
Protein carbonyl levels were measured in fibroblasts using the
Oxyblot Protein Oxidation Detection kit (Millipore). Briefly, cells were
lysed with 0.25% (v/v) Triton X-100 in PBS supplemented with 50 mM
dithiotreitol and protease inhibitors. Following removal of insoluble
material, protein carbonyls were derivatised to 2,4-dinitrophenylhy-
drazone (DNP) by reaction with 2,4-dinitrophenylhydrazine. The
DNP-derivatised protein samples were then separated by SDS PAGE
followed by western blotting. Carbonyls were detected by incubation
with an antibody specific to DNP. Equal loading was determined by
reprobing the same blot with GAPDH antibody.
MitoTracker Green staining of fibroblasts
The mitochondrial network in live fibroblasts was assessed with
MitoTracker Green FM (Invitrogen). Cells were cultured on coverslips
for 72 h in media containing either glucose (4.5 g/l) or galactose
(0.9 g/l). Cells were then incubated with 5 μg/ml 4′-6-diamidino-2-
phenylindole (DAPI) for 2 h in the respective media. The cells were
then loaded with 250 nM MitoTracker Green FM in their respective
culture media for 45 min at 37 °C.
Electron microscopy
Cells of one 80% confluent T-175 flask were harvested and the pellet
was treated as described by Kinget al. (2002). Electron microscopy was
performed on ultrathin sections of four p.Q456X mutants, the p.V170G
mutant and five controls. Sections were contrasted with uranyl acetate
and lead citrate. Images were captured with a digital camera attached
to a Zeiss 902C electron microscope. Images were analysed blinded.
Statistical analysis
A Mann–Whitney-Utest (nonparametric) was applied to evaluate
the statistical significance of differences measured in the different
data-sets. Values outside of the 99% conference interval of the control
group were defined as outliers in the assays.
Results
Mitochondrial respiratory chain enzyme complexes
To test for the effects of PINK1 mutations on mitochondrial
function, the activities of respiratory chain enzyme complexes were
268 A. Grünewald et al. / Experimental Neurology 219 (2009) 266–273
investigated. No difference in complex I activity was found when
comparing four homozygous PINK1 nonsense mutants with six
controls. In the culture of the patient with a PINK1 missense mutation
the complex I activity was reduced, although not significantly, to 81%
of the median control level (Fig. 1A).
For the combined activity measurement of complexes II+III there
was no difference between the nonsense mutants and the control
samples. However, in the missense mutant, the complex II+III
activity was markedly higher (231%) than the median control level
(Fig. 1B).
The activity of complex IV in the sample with the missense
mutation was decreased to 54% of the median control activity. The
median activity in the nonsense mutants was indistinguishable from
the activity in the controls (Fig. 1C).
To investigate if the detected changes in the respiratory chain
complex activities were a result of changes in expression levels, the
assembly of the respiratory chain enzyme complexes was investigated
by blue native PAGE. The western blot analysis of complexes I to V
displayed bands of expected molecular weight. No additional bands
indicative of complex subassemblies were detected (Fig. 1D). When
comparing the expression of the complexes, no overt changes were
observed.
Mitochondrial content and mtDNA levels
The activities of the citric acid cycle (CAC) enzymes CS (data not
shown) and MDH (median MDH
controls
: 7.38 μg/min/mg, n=6;
median MDH
p.Q456X
: 5.81 μg/min/mg, n=4; MDH
p.V170G
: 6.91 μg/
min/mg, n=1) were similar in the cell lysates of control, nonsense
and missense cultures, indicating that the mitochondrial number was
comparable in all cultures. This finding was further supported by
similar levels of mitochondrial DNA in all fibroblast cultures (median
[mtDNA]
controls
: 72.71, n= 7; median [mtDNA]
p.Q456X
:70.10,n=4;
[mtDNA]
p.V170G
: 64.52, n= 1).
Since complex II is a component of both the electron transport
chain and the CAC, the observation that both CS and MDH activities
Fig. 1. Activities and protein expression of respiratorychain enzyme complexes I–IV. (A) Controls and patients with a nonsense mutation showed comparable complex I activities. The
activity in the missense mutation sample was at the lower limit of the control range. (B) In the p.V170G mutant elevated complex II + III activity was detected. The activity of the
missense mutant amounted to 231% of the median control activity. (C) The complex IV activity of the p.V170G mutant was 54% of the median control activity. The activities of
complexes I–IV were normalised to CS activity. (D) Protein expression of the respiratory chain enzyme complexes. Western blot showing complex I to V for two controls, two
homozygous PINK1 p.Q456X mutants and the p.V170G mutant. Horizontal bars represent medians of the investigated groups of individuals. CI–IV —complex I–IV activity, CS —
citrate synthase activity, ⁎⁎ — value outside of the 99% confidence interval of the control group.
269A. Grünewald et al. / Experimental Neurology 219 (2009) 266–273
were unaffected, suggests that the increased activities of complexes
II+III (Fig. 1B) in the missense culture are a result of increased
respiratory chain activity rather than the CAC.
Mitochondrial membrane potential
The influence of mutant PINK1 on ψm was investigated by means
of a cytofluorimetric assay. Neither the nonsense nor the missense
mutation had a significant effect on ψm (median ψm
controls
: 100%,
n=3; median ψm
p.Q456X
: 100% of the median control value, n=3;
ψm
p.V170G
: 79% of the median control value, n=1). PINK1 was
previously reported to function in the response to cellular stress
(Pridgeon et al., 2007; Wang et al., 2007). Therefore, the effect of
treatment with the free radical generator paraquat (0.5 mM) on ψm
was measured. Ψm was unaffected in control or mutant PINK1
fibroblasts following stress, when compared to basal conditions
(median ψm
controls
: 87% of the median basal control value, n=3;
median ψm
p.Q456X
: 106% of the median basal control value, n=3;
median ψm
p.V170G
: 74% of the median basal control value, n=1).
Cellular ATP content and synthesis
The total cellular ATP concentration was quantified in all availa-
ble patient fibroblast cultures and eight control cultures. The
experiment revealed a significant decrease of 21% (pb0.05) in the
nonsense mutants (median level) when compared with the median
of the control individuals (Fig. 2A). The ATP concentration in cells
of the missense mutant patient did not differ from the average
control level.
Furthermore, we examined ATP synthesis in digitonin-permeabi-
lised fibroblasts via complexes I, III, IV (substrates: glutamate and
malate), complexes II, III, IV (succinate and rotenone) or complex IV
(ascorbate and TMPD) only. Measuring ATP production via complexes
I, III and IV the median activity in the missense mutant fibroblasts was
Fig. 2. Cellular ATP levels (A) and ATP synthesis (B). (A) Controls and the missense mutation sample showed comparable cellular ATP concentrations under basal conditions. In
patients with a nonsense mutation a significantly reduced median ATP level was detected (pb0.05). Results are displayed relative to the median control level. (B) In the missense
mutant fibroblasts enhanced ATP synthesis was determined when measuring the activity by use of glutamate and malate (ATP generation via complexes I, III and IV; for details see
Materials and methods section), or succinate and rotenone (ATP generation via complexesII, III and IV) as substrates. Here, the PINK1 p.V170G sample was compared to four p.Q456X
cultures and five controls. Horizontal bars represent medians of the investigated groups of individuals. Vertical lines indicate the 95% confidence interval.⁎⁎ — value outside of the 99%
confidence interval of the control group.
Fig. 3. Indicators of oxidative stress. (A) SOD2 protein expression under basal conditions (normalised to porin and the median value in the control group). The missense
mutant showed a markedly higher SOD2 expression level (284%) than seen in controls (100%). The median expression in the nonsense mutants was comparable with the
median control level (85%). (B) GSH levels under basal conditions and following paraquat treatment. For controls (100%), patients with the p.Q456X mutation (95%) and the
sample with the p.V170G mutation (95%) comparable median GSH levels were detected when measured under basal conditions. After treatment with 0.5 mM paraquat, all
samples showed a significant increase in the cellular glutathione concentration. However, the missense mutant sample tended to show a greater increase (+134%) than the
other groups (controls: + 85%; p.Q456X mutants: + 60%). All data are expressed relative to the median of the control group under basal conditions. Horizontal bars represent
medians of the investigated groups of individuals. ⁎⁎ — value outside of the 99% confidence interval of the control group.
270 A. Grünewald et al. / Experimental Neurology 219 (2009) 266–273
71% higher than in controls and via complexes II, III and IV, the median
activity increased by 88%. However, no significant differences in ATP
synthesis were observed when using ascorbate and TMPD as
substrates (Fig. 2B).
Oxidative stress in PINK1 mutant fibroblasts
SOD2 and GSH are important antioxidants, which participate in the
oxidant stress defence system. Whereas SOD2 can only be found in
mitochondria, GSH is additionally detectable in the cytosol. The
median cellular SOD2 level was determined relative to porin
expression by densitometric analysis of three western blots prepared
with cell lysates of all available mutant samples and six controls. SOD2
expression in the missense cells was markedly increased (284%). The
median SOD2 expression in the lysates of the nonsense mutation cells
was normal (85%) (Fig. 3A).
No significant changes of the median GSH levels were found in cell
lysates of control, nonsense or missense PINK1 fibroblast cultures.
Fig. 4. Mitochondrial network in primary fibroblasts grown in glucose and galactose medium. MitoTracker Green FM was used to visualise the mitochondrial network in control,
nonsense mutant (p.Q456X) and missense (p.V170G) mutant fibroblasts. Cell nuclei are stained with DAPI.
Fig. 5. Mitochondrial morphology in fibroblasts. Transverse sections of control and PINK1 mutant fibroblast pellets were examined using electron microscopy. (A) The micrograph
shows mitochondria of normal size and structure (highlighted by arrow heads) in a control. (B) In the p.V170G sample mitochondria fusion was detected. Abnormal mitochondria are
marked by arrows. Scale bar 2 μm.
271A. Grünewald et al. / Experimental Neurology 219 (2009) 266–273
Following treatment with paraquat, the median GSH levels were
elevated in control and nonsense cultures by 85% and 60%,
respectively. The median GSH level was elevated to a greater extent
(134%) in the missense culture (Fig. 3B).
Finally, the Oxyblot Protein Oxidation Detection kit from Millipore
was used for the immunoblot detection of carbonyl groups introduced
into proteins by oxidative reactions. There were no differences
between the three groups (data not shown).
Mitochondrial network
The mitochondrial networks in live control, nonsense and
missense fibroblast cultures were stained with Mitotracker Green.
When grown in glucose, a typical reticular structure was seen in all
cultures. Culturing of the cells in galactose media for 72 h had no effect
on the mitochondrial network in control cells. However, a noticeable
minority of cells exhibited a more fragmented mitochondrial network
in both nonsense (22%) and missense cells (19%), when cultured in
galactose media (Fig. 4).
Electron microscopy studies
To study the mitochondrial morphology in the cells, pellets of
four controls, the four nonsense mutant samples and the missense
mutant sample were prepared for electron microscopy and
assessed blind. In the PINK1 p.V170G mutant fibroblasts a small
subset of mitochondria exhibited abnormal morphology when
compared with controls. The respective mitochondria in the missense
sample were swollen and noticeably enlarged with abnormal cristae
(Fig. 5).
Discussion
In this paper we have investigated the mitochondrial function in
fibroblasts containing homozygous nonsense (p.Q456X) or missense
(p.V170G) PINK1 mutations. The p.Q456X mutation, which is causing
nonsense-mediated mRNA decay (Grünewald et al., 2007), probably
leads to a loss of protein function in the four homozygous siblings
investigated here. However, this mutation had no effect on the
enzyme activities of the respiratory chain in these samples. This result
is in agreement with a previous report, which showed that another
PINK1 nonsense mutation (p.W347X) did not affect the activity of the
complexes of the electron transport chain when measured spectro-
photometrically. Although no decrease in complex IV activity was
detected spectrophotometrically in fibroblasts from this patient,
polarographic studies did indicate perturbed complex IV activity
(Piccoli et al., 2008).
In contrast, the missense mutant fibroblast culture exhibited
decreased activities of complexes I and IV, while the activity of
complexes II+ III was increased. These data lack statistical signifi-
cance because only one p.V170G mutant fibroblast culture was
available for study and because of the wide range of control values,
a phenomenon that has been observed earlier (Williams et al., 2001).
Nevertheless, our results are in agreement with recently reported
findings describing a link between reduced activity of complex IV and
loss of PINK1 activity (Gegg et al., 2009).
Despite the mitochondrial dysfunction observed in the missense
culture, mitochondrial membrane potential and steady-state ATP
levels were unaffected. In fact, the rate of ATP synthesis was increased
in the missense culture when electron donors feeding into the
respiratory chain at complexes I or II were used. This correlates with
the unexpected increase in the specific activity of complex II+III.
Since the activities of complexes I and IV appear decreased in these
cells, the respiratory chain activity via complexes II and/or III may be
increased to compensate, thereby maintaining the membrane poten-
tial, and subsequently ATP levels.
Steady-state ATP levels were significantly decreased in the
nonsense cultures, although no loss of respiratory chain function or
ATP synthesis was detected in these cells. The lower ATP levels may be
areflection of increased ATP consumption in the cell (e.g. main-
tenance of the mitochondrial membrane potential, increased activity
of Na
+
/K
+
transporters). Indeed, mitochondrial membrane potential
has been shown to be maintained in cells lacking PINK1 expression by
complex V hydrolysing ATP (Gandhi et al., 2009).
Recently, PINK1 deficiency has been reported to cause mitochon-
drial accumulation of calcium in mammalian neurons, resulting in a
mitochondrial calcium overload which then stimulates the production
of reactive oxygen species (ROS) via NADPH oxidase (Gandhi et al.,
2009). Increased SOD2 expression levels in mitochondria of the
missense culture are most likely indicative of a cellular defence
mechanism against elevated ROS production. Similar effects have also
been detected earlier in fibroblasts harbouring PINK1 mutations
(Hoepken et al., 2007; Piccoli et al., 2008). Baseline GSH levels were
unaffected in both the missense and nonsense cultures and this
correlated with no detectable difference in the levels of oxidised
proteins.
GSH levels can be elevated in cells following exogenous oxidative
stress (Gegg et al., 2003; Moellering et al., 1999). Following paraquat
treatment, GSH levels were increased in control and nonsense cultures
to a similar extent, while the increase was much greater in the
missense culture. This result strengthens the idea that antioxidant
pathways have been activated in the missense culture to deal with the
oxidative stress induced by defective mitochondria.
The reason for altered mitochondrial function in the missense
culture is unclear. Studies have indicated that PINK1 function is
required to maintain the correct mitochondrial structure (Deng et al.,
2008; Exner et al., 2007; Poole et al., 2008; Yang et al., 2008). The
decreased activities of complexes I and IV in the missense fibroblasts
may be a direct result of decreased PINK1 activity, or a consequence of
the abnormal mitochondrial morphology seen in these cells. Culturing
both the nonsense and missense fibroblasts in galactose appears to
exacerbate PINK1-mediated changes in the mitochondrial network. A
similar phenomenon has also been reported in fibroblasts with
different PINK1 mutations (Exner et al., 2007). The use of galactose as
the major carbon source means that the cell predominantly relies on
oxidative phosphorylation for ATP synthesis (Robinson et al., 1992). It
is therefore expected that respiratory chain dysfunction as a result of
mutated PINK1 will be amplified under these conditions. Indeed, ATP
levels have been shown to be lower in PINK1 fibroblasts containing a
nonsense mutation only when they are cultured in galactose media
(Piccoli et al., 2008).
In summary, fibroblasts containing mutated PINK1 resulted in
mitochondrial dysfunction, with the missense PINK1 mutation in
particular having an effect on the respiratory chain. In support of
previous studies we have also found alterations in mitochondrial
morphology as a result of loss of PINK1 activity. The observation that
the missense mutation appears to be more pathogenic than the
nonsense mutation is intriguing. It should be noted that the PD patient
with the missense mutation had both an earlier age of onset and a
more severe clinical condition than the four patients with the
nonsense mutation. However, it remains to be determined in a larger
sample whether there may be a correlation between the severity of
clinical findings and the degree of mitochondrial dysfunction seen in
the patients' cells. In addition, it is conceivable that the significantly
longer duration of disease in the patient with the missense mutation
may have a possible impact on the mitochondrial integrity and
bioenergetic properties. Although this study on mitochondrial func-
tion comprises the largest number of fibroblast cultures from PD
patients and control individuals investigated to date, the results
received from the single p.V170G PINK1 missense mutant sample will
need further confirmation. To obtain a complete picture of the
situation on PINK1 missense and nonsense mutant backgrounds,
272 A. Grünewald et al. / Experimental Neurology 219 (2009) 266–273
cellular samples from PD families with a large number of affected and
unaffected individuals will be required.
Acknowledgments
This study was supported by the Parkinson's Disease Society UK,
the Kattan Trust, the Fritz Thyssen Foundation, the DFG and the DAAD.
We are grateful to Prof. Simon Heales and Dr. Lee Stanyer (Institute of
Neurology, London) for allowing the use of the reverse phase HPLC to
measure GCH levels.
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