PINK1 Defect Causes Mitochondrial Dysfunction, Proteasomal Deficit and ??-Synuclein Aggregation in Cell Culture Models of Parkinson's Disease

Article · February 2009with107 Reads
DOI: 10.1371/journal.pone.0004597 · Source: PubMed
Abstract
Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased alpha-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and alpha-synclein aggregation.
9 Figures
PINK1 Defect Causes Mitochondrial Dysfunction,
Proteasomal Deficit and a-Synuclein Aggregation in Cell
Culture Models of Parkinson’s Disease
Wencheng Liu
1.
, Cristofol Vives-Bauza
1.
, Rebeca Acı
´
n-Pere
´
z-
1
, Ai Yamamoto
4
, Yingcai Tan
3
, Yanping
Li
1
, Jordi Magrane
´
1
, Mihaela A. Stavarache
2
, Sebastian Shaffer
1
, Simon Chang
1
, Michael G. Kaplitt
2
, Xin-
Yun Huang
3
, M. Flint Beal
1
, Giovanni Manfredi
1
*, Chenjian Li
1
*
1 Department of Neurology and Neurosciences, Weill Medical College of Cornell University, New York, New York, United States of America, 2 Department of Neurological
Surgery, Weill Medical College of Cornell University, New York, New York, United States of America, 3 Department of Physiology, Weill Medical College of Cornell
University, New York, New York, United States of America, 4 Judith P. Sulzberger M.D. Columbia Genome Center and Department of Physiology and Cellular Biophysics,
Columbia University, New York, New York, United States of America
Abstract
Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson’s
disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with
respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated
complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in
mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly,
these deficits are accompanied by increased a-synclein aggregation. Our results indicate that it will be important to delineate
the relationship between mitochondrial functional deficits, proteasome dysfunction and a-synclein aggregation.
Citation: Liu W, Vives-Bauza C, Acı
´
n-Pere
´
z- R, Yamamoto A, Tan Y, et al. (2009) PINK1 Defect Causes Mitochondrial Dysfunction, Proteasomal Deficit and a-
Synuclein Aggregation in Cell Culture Models of Parkinson’s Disease. PLoS ONE 4(2): e4597. doi:10.1371/journal.pone.0004597
Editor: Mark R. Cookson, National Institutes of Health, United States of America
Received September 12, 2008; Accepted January 20, 2009; Published February 26, 2009
Copyright: ß 2009 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported partially by grants to CJL (NIH/NINDS NS054773), MFB (Department of Defense), and to MAS (Parkinson’s Disease Foundation).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: gim2004@mail.med.cornell.edu (GM); chl2011@med.cornell.edu (CL)
. These authors contributed equally to this work.
Introduction
Parkinson’s Disease (PD) is a neurodegenerative disorder with
pathological hallmarks of dopaminergic neuron degeneration in
the substantia nigra pars compacta, and cytoplasmic inclusion
Lewy bodies that contain mainly a-synuclein aggregates. The PD
pathogenic pathways involve defects in many cellular processes
such as protein degradation, oxidative stress, phosphorylation
signaling, and mitochondrial function.
Mitochondria are complex subcellular organelles that play
diverse and critical roles in energy production, pyrimidine
biosynthesis, fatty acid metabolism, calcium homeostasis, oxidative
stress response and apoptotic cell death. Mitochondrial dysfunction
is implicated in normal as well as pathological aging, especially in
many neurodegenerative diseases such as Alzheimer’s disease,
Huntington’s disease, Amiotrophic Lateral Sclerosis and PD [1,2,3].
In idiopathic PD patients, a 30–40% decrease of mitochondrial
electron transport chain (ETC) complex I activity was observed in
platelets, skeletal muscle and brain [4,5,6,7]. Moreover, complex I
inhibitors 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
and rotenone induce a clinical syndrome that replicates the
hallmarks of PD in human, non-human primates and rodents
[8,9,10,11]. Remarkably, mitochondria are also implicated in most
genetic forms of familial PD: ETC Complex IV activity is reduced
in an a-synuclein mouse model [12]; the mitochondrial respiratory
capacity is decreased and oxidative damage is increased in both
Parkin-knockout mouse and Drosophila [13,14]; a portion of DJ-1
and LRRK2, two PD-associated proteins, localize to mitochondria
[15,16]; overexpression of DJ-1 is protective against oxidative stress
and mitochondrial damage, while loss of DJ-1 is harmful [16,17].
Despite this wealth of evidence, it is still unclear whether
mitochondrial dysfunction is a primary causal event or a secondary
consequence of PD.
The most direct evidence indicating a primary role of
mitochondria in PD pathogenesis comes from the identification
of PINK1 as the causal gene for PARK6, an autosomal recessive
form of PD. PINK1 is a Ser/Thr kinase localized in mitochondria
[18,19,20]. Thus, the primary causal event in PARK6 has to be
associated with mitochondria. Recent studies have begun to shed
light on the mechanisms through which PINK1 causes PD, and
implicate cellular functions such as mitochondrial respiration
[21,22], mitochondrial morphology and dynamics
[23,24,25,26,27], and oxidative stress [21,28]. Our study directly
addresses the consequences of PINK1 defects on mitochondrial
function, and on cellular abnormalities that are important to PD.
We report here that in cell culture systems, mutant PINK1 or loss
of PINK1 induces mitochondrial dysfunction, defective protea-
some function and increases a-synuclein aggregation.
PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4597
Materials and Me thods
Plasmid and viral constructs
Full length PINK1 cDNA was cloned into a pshuttle-1-3XFlag-
IRES-GFP vector (Stratagene) to generate Flag-tagged PINK1.
PINK1 cDNA carrying Del 245, L347P or E417G mutation was
made by staggered PCR. To make adenoviruses, wild type-, Del
245-, L347P- or E417G-PINK1-3XFlag-IRES-EGFP in pshuttle-
1 vector were used to re-combined into pAdeasy (Stratagene).
These viral constructs were transfected into HEK293 cells to
obtain PINK1 recombinant viruses, followed by virus amplifica-
tion and purification based on the manufacture’s protocol. Viral
titers were determined by method previously described [29]. To
generate various V5 tagged PINK1, wild type and mutant PINK1
cDNAs were cloned into a pcDNA3.1 vector (Invitrogen).
Transfection and immuno-precipitation
DNA was transfected into cells using Lipofectamine 2000
according to the manufacturer’s protocol. 48 hours post transfection,
cells were harvested and re-suspended in lysis buffer (50 mM Tris,
pH 7.4, 150 mM NaCl,1 mM EDTA and 0.1% Triton-100)
supplemented with 26 protease inhibitor (Roche) and homogenized
with dounce homogenizer. The cell homogenates were then
centrifuged at 11,000 g for 10 min, and cell lysates were collected.
For immuno-precipitation, cell lysates were first pre-cleared with
protein A & G agarose beads for one hour at 4uC, and then incubated
with rabbit anti-Flag or rabbit anti-V5 Ab for at least 2 hours at 4uC
with constant agitation, followed by four washes of 20 min each with
the lysis buffer. The beads that captured PINK1 complexes were
mixed with equal amount of 26 SDS sample buffer and heated at
95uC for 10 min to elute the complex proteins. Eluents were used for
SDS-PAGE, followed by Western analyses with mouse anti-V5
(Invitrogen) or mouse anti-Flag antibody (Ab, Sigma). Human
PINK1 antibodies from Novus (BC100-494) were used for the
detection of endogenous PINK1.
Cell lines stably expressing shRNAs against PINK1
DNA oligos encoding for small hairpin RNAs (shRNA) targeting
rat PINK1 were annealed and cloned into a rAAV vector. The
following oligos were used: PINK1 59-ATCCCC
GCACACTCT-
TCCTCGTTATCTTCCTGTCAATAACGAGGAAGAGTGT-
GCTTTTTGGAA-39. The gene specific sequences are underlined
and the loop from the murine miR-23 is shown in bold. These rAAV
expression plasmids were subcloned into a vector containing the
neomycin resistance gene. The plasmids were transfected into PC12
cells. Neomycin resistant clones were isolated, expanded and ana-
lyzed for PINK1 expression following G418 selection (500 mg/ml).
Quantitative PCR (Q-PCR)
Real-time PCR was performed using SYBR Green Master Mix
(Applied Biosystems) on an ABI Prism 7000 Sequence Detection
System (Applied Biosystems). The level of PINK1 transcript was
normalized against GAPDH.
Infection of SH-SY5Y cells with recombinant adenovirus
Purified recombinant adenoviruses (50 particles per cell) were
used to infect SH-SY5Y cells cultured in DMEM/F12 (1:1)
medium containing 10% fetal bovine serum. 48 hours post
infection, cells were harvested for further experiment.
Measurements of O
2
consumption
Oxygen consumption in intact cells was measured in a 300-ml
reaction chamber equipped with a Clark-type polarographic
electrode (Hansatech Instrument, UK) as described previously
(Hofhaus, et al., 1996). Briefly, cells were trypsinized, counted in a
Z1 automated cell counter (Beckman-Coulter, Miami, FL), and re-
suspended at 1.5610
6
cells in DMEM containing no glucose and no
fetal bovine serum and supplemented with 1 mM sodium pyruvate.
After a baseline trace of coupled respiration was recorded with
adequate time for accurate slope measurement, the endogenous
respiration was completely inhibited by 1.5 mM KCN.
ATP synthesis measurement
ATP synthesis was measured in digitonin-permeabilized SH-
SY5Y with a luciferase-based kinetic assay as previously described
[30]. Briefly, cells were collected by trypsinization, pelleted by
centrifugation, counted, and resuspended at 1610
7
cells/ml in
buffer A containing 150 mM KCl, 25 mM Tris-HCl, 2 mM
EDTA, 0.1% bovine serum albumin, 10 mM potassium phos-
phate, 0.1 mM MgCl
2
, pH 7.4. 160 ml of the cell suspension was
incubated with 50 mg/ml digitonin for 1 min at room tempera-
ture, diluted with 1 ml of buffer A, and centrifuged at 6000 r.p.m.
for 1 min. The cell pellet was re-suspended in 160 ml of buffer A,
and 0.15 mM diadenosine-pentaphosphate Ap
5
A, 0.1 mM ADP,
1 mM malate, 1 mM pyruvate, and 10 ml of buffer B containing
0.8 mM luciferin and 20 mg/ml luciferase were added. The light
emission was recorded by an Optocomp I luminometer (MGM
Electronics) at 15-sec intervals for a total recording time of 4 min.
A standard ATP/luminescence curve was constructed by measur-
ing the luminescence of different ATP concentrations.
ATP content and proteasome activity
10 cm plates of HeLa cells were transiently transfected by CFP-
degron (CFPde) with Lipofectamine 2000. Cells were plated into a
96-well plate. After a 1 hr pre-incubation of cells in glucose-free
DMEM, cells were placed in glucose-containing media, but
treated with 0 to 6 mM of 2-deoxyglucose (2-DG) for 5 hrs.
Proteasome activity was indirectly assessed in half of the wells of
cells by the degradation of CFPde as previously described [31].
The other half of the cell wells of the 96-well plate were used to
measure ATP content using the luminescent ATP Assay Kit
(Calbiochem), for which the release of luminescence reflects the
breakdown of ATP.
Assays for protesome activity affected by PINK1
CFP-degron method: 24-well plate of HeLa cells were transfected with
a CFP-degron construct (CFPde) alone or co-transfected with 10 nM
siSCRAMBLE sequence (siSCR) or 10 nM siRNA directed against
PINK1 (Dharmacon), with Lipofectamine 2000. 24 hrs post
transfection, cells were plated into 96 well format. 48 hrs post
transfection, cells that were transfected with CFPde alone were split
and half of it were treated with 1 uM MG-132, a proteasome
inhibitor, for 24 hrs. All cells were fixed, stained with Hoechst 333342
for nuclei, and scanned on the automated confocal INCELL
Analyzer (INCA) 3000, as previously described [31]. The fluorescent
intensity of the cytoplasm of individual cells was assessed using the
object intensity module. SiRNA sequences: GAGAGGUCCAAG-
CAACUA TT and CCUGGUCGACUACCCUGAU TT.
Suc-LLVY-AMC method:5610
5
SH-SY5Y cells were infected with
adenovirus expressing wild type, L347P and E417G-PINK1.
Three days post infection, cells were collected, re-suspended with a
buffer containing 10 mM HEPES, pH 7.4 and 0.5 mM MgCl
2
,
and homogenized with dounce homogenizer. Chymotrypsin
activity was measured as described by Ehlers [32]. In control
experiments, the proteasome was inhibited by addition of 50 mM
MG 132 to cell lysates 10 min before adding the fluorogenic
substrate. The assay was performed in a 96 cell plate; and
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 2 February 2009 | Volume 4 | Issue 2 | e4597
fluorescence was measured 1.5 hours after incubation on an HTS
7000 plus fluorescent plate reader (PerkinElmer, Boston, MA) with
excitation and emission wavelengths of 360 and 465 nm,
respectively. The same method was also applied to PC12 cells
with reduced PINK1 expression.
Filter trap assay
SH-SY5Y cells or stable HeLa cell lines expressing wild type or
A53T a-synuclein were transfected with siRNA against PINK1.
Two days post transfection, cell pellets were collected and lysed
with a buffer containing 20 mM Tris, pH 7.5, 1% Triton X-100,
1 mM EDTA, 1 mM DTT and 20 U/ml RQ1 RNase Free
DNase (Promega). 100 mg proteins from lysates were mixed with
SDS to a final concentration of 1% SDS. SDS insoluble and
soluble fractions in the lysates were separated with a modified filter
trap assay by co-filtration through two filters, a cellulose acetate
membrane on top to capture SDS insoluble proteins and a PVDF
membrane underneath to capture SDS soluble proteins. The
cellulose acetate membrane was probed with anti-GFP Ab,
whereas the PVDF membrane was incubated with anti-actin Ab
as a control for protein extraction and loading.
Statistical analysis
Differences between mutant PINK-1 cell lines and KO PINK-1
cells and their respective controls were assessed by ANOVA and
by the nonparametric Kruskal-Wallis test. Paired genotype
differences were assessed by the post-hoc Fisher’s PLSD test. All
test and calculations were done with the statistical package
StatView 5.0 for PC (SAS Institute).
Results
The PINK1 constructs and expression
PINK1 has a mitochondrial targeting signal, followed by a
conserved Ser/Thr kinase domain, and a C-terminal domain. PD-
causing mutations have been found in both the kinase and the C-
terminal domains [18,19,20]. Among these mutations, G309D,
L347P and E417G are on amino acid residues that are well
conserved throughout evolution and thus are expected to be
important for PINK1 kinase function. Another human disease-
causing mutation is a large deletion of PINK1 after amino acid
245 (Del 245), which eliminates most of the kinase domain.
Therefore, we introduced the mutations L347P, E417G, and Del
245 into human PINK1 cDNAs, and then cloned into mammalian
expression plasmid vectors. In order to identify the recombinant
proteins, V5, Flag and GFP tags were added to the C-termini
(Fig 1A). These constructs allowed efficient expression of PINK1
in mammalian cells (Fig 1B). It is worth noting that L347P-
PINK1, although prone to degradation by some purification
methods, was stable in our experimental systems and conditions
(Figure 1B, lane 3 and 8), in contrast to a previously reported
instability of L347-PINK1 [33].
PINK1 proteins form dimers via the kinase domain
We confirmed that PINK1 is indeed a kinase, that it is auto-
phosphorylated, and that familial mutations impair the kinase
activity (Figure 2), as others have shown [33]. One of the
mechanisms to regulate kinase activity is by homo-dimerization of
the kinase [34,35]. We therefore investigated whether PINK1 also
dimerizes, and if disease-causing mutations disrupt this dimerization
and impair kinase activity. V5, Flag, and GFP tag were each fused
to a wild type PINK1, L347P-PINK1, E417G-PINK1 and Del-245-
PINK1. All combinations of the above constructs were transfected
in HEK293 cells for co-immunoprecipitation (co-IP) experiments.
Co-transfection of PINK1-V5 with PINK1-Flag followed by pull-
down of the protein with the anti-Flag antibody and detection by
WB with the anti-V5 antibody showed that wild type PINK1
protein exist as a dimer (Figure 3A, left panel). The same results
were obtained in a reverse direction when the protein was pulled-
down with anti-V5 antibody and detected with anti-Flag
(Figure 3A, middle panel). Co-transfection of PINK1-GFP with
PINK1-V5, followed by IP with anti-GFP antibody and detection of
PINK1 with anti-V5 antibody also proved the dimerization of
PINK1 (Figure 3A, right panel). These results were confirmed in
COS and HeLa cells (data not shown). To map the domain required
for dimerization, we used Del 245, Del 509 and Del 525-PINK1
(Fig 1A) for co-IP with the wild type PINK1. Whereas Del 509 and
Del 525 dimerized with wild type PINK1 (Fig 3B, lane 2 and 3 of
the top panel), Del 245 lost such ability (Fig 3B, lane1 of the top
panel). Therefore, the interaction for PINK1 dimerization requires
amino acid 246–509 within the kinase domain. A further co-IP
experiment with L347P-PINK1 and E417G-PINK1 demonstrated
that these mutations do not affect the homo-dimerization of PINK1
kinase (Figure 3B, lane 5 and 6, top panel). This implies that
the kinase deficit in L347P-PINK1 and E417G-PINK1 is not due to
a failure of dimerization. We also showed that L347P-PINK1 and
E417G-PINK1 can form hetero-dimers with the wild type PINK1
(Fig 3C).
Recombinant and endogenous PINK1 are associated with
protein complexes in mitochondria
PINK1 has a mitochondrial targeting signal at its N terminal,
and was localized to mitochondria in our experimental systems
(Figure 4A) as in other systems [19,33]. Since many proteins in
mitochondria are compartmentalized and tightly organized in
functional multi-protein complexes such as the ETC complexes,
we investigated if this is also true for PINK1, and if mutations in
PINK1 could impair this association. Using sucrose gradients to
sub-fractionate mitochondrial extracts, we demonstrated that
indeed wild type PINK1 exists in protein complexes that
fractionate with respiratory chain complexes I, II, III, IV,
(Figure 4B, top panel) and mutations in PINK1 do not alter
the localization of the protein within the multi-protein complexes
(Figure 4B,2
nd
and 3
rd
panels). PINK1 was not detected in lanes
1 and 2 that contain proteins of molecular mass close to that of
PINK1 monomers, further suggesting that PINK1 protein does
not exist as a monomer in these cells. Furthermore, the
endogenous PINK1 migrates within the same fractions as the
recombinant proteins along the sucrose gradient (Figure 4B,
bottom panel), indicating that endogenous PINK1 is present as
a dimer and associates with larger protein complexes.
Mutant PINK1 impairs mitochondrial respiration and ATP
synthesis in cultured SH-SY5Y cells
A cardinal mitochondrial function is to produce energy through
oxidative phosphorylation (OXPHOS). Recent studies suggest that
the OXPHOS system is regulated by phosphorylation
[36,37,38,39,40]. To determine whether mutant PINK1 affects
mitochondrial function, we generated Flag-tagged wild type,
L347P, E417G or Del 245 PINK1-adenovirus to infect human
neuroblastoma SH-SY5Y cells with high efficiency. We measured
cellular respiration 48 hours post-infection. A statistically signifi-
cant defect in O
2
consumption rate was observed in intact SH-
SY5Y cells expressing L347P and E417G PINK1 using pyruvate
as a respiratory substrate, compared to the wild type PINK1
controls (Figure 5A). Since SH-SY5Y cells express endogenous
PINK1, our data suggest that over-expressed L347P- and E417G-
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4597
PINK1 exert a dominant negative effect, probably through the
formation of hetero-dimers of mutants L347P and E417G with
wild type PINK1 as shown in Fig 3C. In fact, Del 245-PINK,
which lacks the kinase and C-ternimal domains, and consequently
cannot form heterodimers with wild type PINK1 (Fig 3C), acts as
a null control for the dominant negative effect. And as expected,
Del 245 PINK1 did not cause a respiratory defect (Figure 5A).
One of the major deleterious consequences of a defective ETC
is an ATP synthesis deficit that has a wide spread impact on most
cellular functions. Therefore, we measured mitochondrial ATP
synthesis using the Complex I substrates malate and pyruvate, and
demonstrated that cells expressing L347P- and E417G-PINK1
have statistically significant reductions of mitochondrial ATP
synthesis, compared to non-infected cells or cells infected with wild
type PINK1 (Figure 5B). Consistent with the respiration
measurements, Del 245-PINK1, as a null control, did not show
a defect in ATP synthesis. By controlling and normalizing for
various recombinant PINK1 (Figure 5C, top panel), and a
mitochondrial protein, Tim23 (Figure 5C, bottom panel), we
conclude that the respiration and ATP synthesis deficit is a
functional defect of mitochondria, rather than a depletion of
mitochondrial mass, or a variation of transgene expression.
Figure 1. PINK1 constructs and their expressions. A) A schematic depiction of PINK1 constructs. Full length wild type, L347P-, or E417G- PINK1
tagged with Flag, V5, or GFP are indicated. Several truncated PINK1 tagged with Flag or V5 are also depicted. M stands for mitochondrial targeting
sequence. B) Confirmation of the expression of the above constructs in HEK 293 cells. HEK293 cells were transfected by various PINK1 constructs, and
their lysates were analyzed by Western blots with Flag antibody (Left panel), V5 antibody (the middle panel) or GFP antibody (the right Panel). Lane
1–10 are lysates from cells transfected by plasmids with the same numbering as shown in A). The lysates from the cells transfected with the empty
cloning vector without PINK1 insert were used as controls (labeled as C). The results demonstrated that the expression of all constructs yielded
recombinant PINK1 proteins with expected molecular weights.
doi:10.1371/journal.pone.0004597.g001
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 4 February 2009 | Volume 4 | Issue 2 | e4597
Loss of PINK1 impairs mitochondrial respiration and ATP
synthesis
In order to elucidate whether the OXPHOS deficit observed
above was the result of a PINK1 defect, rather than an artifact of
protein over-expression, we knocked-down PINK1 expression by
80% in PC12 cells stably transfected with siRNA against PINK1
(Figure 6A). PINK1 knockdown led to a statistically significant
reduction in oxygen consumption (Figure. 6B), similar to
expression of mutant PINK1 (Figure 5A). Importantly, this
respiration deficit could be partially rescued by wild type PINK1
but not by mutant PINK1, further demonstrating that the
respiration deficit is specifically due to mutant PINK1
(Figure 6C).
Consistent with the observation described in Figure 5B,a
deficit in ATP synthesis rate was detected in mitochondria isolated
from PINK1 knock-down (KD) cells (Figure 6D). Since
mitochondrial quantity was normalized between control and
PINK1-KD cells as demonstrated by Western analysis with Tim
23 Ab (Figure 6E and 6F), we conclude that the defect in
respiration and ATP synthesis seen in the cells with reduced
PINK1 is a functional deficit and not due to a sheer reduction of
mitochondria. This result also suggests that the decline in steady-
state ATP levels previously reported in flies [41] is likely due to
defective mitochondrial ATP synthesis.
Thus, taken together, OXPHOS function can be impaired by
either mutant PINK1 or loss of PINK1.
Proteasome deficits caused by mutant PINK1 or loss of
PINK1
Among the many cellular functions that require ATP,
proteasomal activity is particularly important for PD. PD
pathology is generally characterized by the presence of SDS-
insoluble protein inclusions of a-synuclein. These inclusions are
often positive for polyubiquitin, implicating a possible impairment
of the ubiquitin proteasome system (UPS). The UPS comprises a
tightly regulated succession of steps that first tag proteins with a
Figure 2. PINK1 auto-phosphorylation is impaired by L347P and E417G mutations. PINK1 protein is auto-phosphorylated in the cells
expressing wild type PINK1 and the auto-phosphorylation was decreased in cells expressing L347P- or E417G-PINK1. Lysates from the SH-SY5Y cells
expressing wild type-PINK1-Flag (1
st
and 2
nd
panels), L347P-PINK1-Flag (3
rd
and 4
th
panels) or E417G- PINK1-Flag (5
th
and 6
th
panels) were subjected
to 2-dimensional gel electrophoresis, followed by Western analyses using anti-Flag Ab. Of the same protein, the more phosphorylated ones migrated
to the more acidic end (left hand) of the isoelectric focusing (IEF) gel, while less or non-phosphorylated ones migrated to the more basic end (right
hand) of IEF. 1
st
panel: Wild type PINK1 along the pH gradient of the IEF. Spot 1, 2 (64 kD), and 3 (50 kD) migrated to the acidic end, and several
others (open arrows) to the basic end. 2
nd
panel: A control of wild type PINK1 proteins treated with alkaline phosphotase (AP). While the spots on the
basic end still remained in the same region of the gel as in the 1
st
panel, spot 1, 2, and 3 were missing. This indicated that these 3 spots were
phospho-PINK1 in 1
st
panel, and were de-phosphorylated in the 2
nd
panel. The difference between spot 1 and 2 reflected the degree of
phosphorylation on PINK1. In cells expressing L347P or E417G-PINK1, spot 1, 2 and 3 were greatly reduced, indicating reduced auto-phosphorylation
on L347P- or E417G-PINK1 (3
rd
and 5
th
panels). The 4
th
and 6
th
panels are controls (treated with AP) for the 3
rd
and 5
th
panels respectively, similar to
the 2
nd
panel.
doi:10.1371/journal.pone.0004597.g002
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 5 February 2009 | Volume 4 | Issue 2 | e4597
polyubiquitin chain, then target the proteins for degradation by
the 26S proteasome [42]. Covalent attachment of the polyubiqui-
tin chain is dependent upon a series of ubiquitin ligases E1, E2 and
E3 that systematically bind ubiquitin to its substrate in an ATP-
dependent manner. The tagged protein is then rapidly degraded
by the 26S proteasome, which requires ATP to assemble from the
19S and 20S subunits. The importance of the UPS in PD is
exemplified by the identification of mutations in Parkin, an E3
ligase, as the cause of autosomal recessive PARK2 form of PD.
Since mutant and PINK1 KD cells evidenced a significant ATP
production deficit (Figure 5B and 6D), we decided to study
whether this ATP deficit which is induced by loss of PINK1 or
mutant PINK1 could also influence the proteasome function. To
pursue this line of investigation, we first examined whether ATP
affects proteasome activity in our experimental systems. We
treated SH-SY5Y and HeLa cells with 2-deoxyglucose (2DG) to
decrease ATP levels via inhibition of glucose utilization. In these
cells, proteasome degradation of a degron fused monomeric CFP
(mCFP-degron) [43] was diminished in a 2DG dose-dependent
manner (Figure 7A, 7B). Therefore, in our systems, progressive
ATP depletion decreases proteasome activity, consistent with the
observation in other cell culture systems [44].
Figure 3. Dimerization of PINK1 via the kinase domain. In all experiments, PINK1 with different tags were co-transfected in pairs into HEK293
cells. A tag was used for immunoprecipitation (IP), and Ab against another tag was used for Western blots (WB). Results were confirmed in COS and
HeLa cells (data not shown). A) Wild type PINK1 form dimers. Left lanes in all three panels: lysate from cells without transfection as negative controls;
right lanes in all panels: lysate from cells co-transfected with a pair of PINK1 constructs. In the left and middle panels, PINK1-Flag and PINK1-V5 were
co-transfected. Anti-Flag Ab could co-IP PINK1-V5 (left panel) and vice versa (middle panel), indicating that PINK1-Flag and PINK1-V5 form a dimer.
PINK1 dimerization is confirmed when PINK1-GFP and PINK1-V5 were co-transfected, anti-GFP Ab could co-IP PINK1-V5 (right panel). B) Wild type and
mutant PINK1 form homo-dimers via the kinase domain. Lysates from cells expressing PINK1-V5 and PINK1
1-245
-Flag (lane 1), PINK1-Flag and PINK1
1-
509
-V5 (lane 2), or PINK1-Flag and PINK1
1-525
-V5 were isolated (lane 3), and subject to Western analysis with V5 antibody (middle panel, input control)
or Flag antibody (bottom panel, input control) to confirm the expression of expected tagged recombinant protein. These lysates were then
immunoprecipitated with mouse anti-V5 Ab, and subjected to Western analyses with rabbit anti-Flag Ab (upper panel; Co-IP). PINK1
1-245
-Flag
abolished dimerization (lane 1), whereas PINK1
1-525
-V5 and PINK1
1-509
-V5 could dimerize normally (lanes 2 and 3). Thus amino acid residues 246–509
are necessary for dimerization. L347P and E417G mutations did not disrupt the PINK1-PINK1 interaction (lane 5 and 6). C) Mutant PINK1 can also form
hetero-dimers with wild type PINK. Lysates from cells expressing 1) PINK1-V5, 2) PINK1-V5 and PINK1-Flag, 3) PINK1-V5 and PINK1-L347P-Flag, 4)
PINK1-V5 and PINK1-E417G-Flag were isolated and immunoprecipitated with rabbit anti-Flag Ab. The IP and Co-IP fractions were then subjected to
Western analyses with mouse anti-V5 Ab (upper panel, Co-IP) or mouse anti-Flag Ab (lower panel, control IP). Wild type PINK1 form dimers (lane 2),
and the disease-causing PINK1 mutations did not affect the dimerization (lanes 3, 4).
doi:10.1371/journal.pone.0004597.g003
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 6 February 2009 | Volume 4 | Issue 2 | e4597
Next, cells over-expressing L347P-PINK1 and E417G-PINK1
were assessed for proteasome function. Consistent with their effect
on ATP production, L347P-PINK1 and E417G-PINK1 led to
decreased degradation of the proteasome substrate Suc-Leu-Leu-
Val-Tyr-AMC, which was reflected by decreased fluorescence
(Figure 8A). The wild type PINK1 overexpression did not lead to
a deficit in proteasome function, indicating that the effect of
L347P-PINK1 and E417G-PINK1 on proteasome was not due to
protein overexpression per se. By controlling and normalizing for
the proteasomal 20S a subunit, we conclude that the proteasome
deficit is a functional deficit rather than a decrease of proteasome
(Figure 8A, bottom panel).
To further prove that the proteasome dysfunction is the result of
a PINK1 defect, we examined the proteasome function in PINK1
depleted cells. PINK1 KD led to a statistically significant reduction
in proteasome function (Figure. 8B and 8C), similar to
Figure 4. Recombinant and endogenous PINK1 are associated with protein complexes in mitochondria. In all experiments,
adenoviruses of PINK1-Flag, L347P-PINK1-Flag, E417G-PINK1-Flag or Del 245 PINK1-Flag were infected into SH-SY5Y cells. A) Western blot analysis of
PINK1 sub-cellular distributions with anti-Flag Ab. The two forms of PINK1 (a 64 kD full length protein and a truncated form of 50 kD, presumably a
proteolytic product) are present in mitochondria and cytosol. The L347P, E417G or Del 245 mutant PINK1 did not affect this distribution. S: cytosolic
fraction; M: mitochondrial fraction. B) PINK1 is associated with protein complexes. Mitochondrial proteins were sub-fractionated by 15% to 35%
discontinuous sucrose gradient, from which fractions 1–10 were collected from top (lighter proteins) to bottom (heavier proteins or complexes). They
were subjected to SDS-PAGE, and Western analyses with anti-Flag Ab for PINK1 (the top three panels); anti-39 kD protein Ab for complex I (4
th
panel);
anti-70 kD protein Ab for complex II (5
th
panel); anti-core 2 Ab for complex III (6
th
panel); and anti-cox I Ab for complex IV (7
th
panel). No PINK1 was
observed in lane 1 and 2, the fractions that contained proteins of the sizes for monomeric PINK1. Instead, PINK1 was associated with protein
complexes ranging from 130–900 kD, which co-migrated with ETC complexes. The L347P and E417G mutations did not affect the PINK1 association
and distribution of these complexes. More importantly, anti-human PINK1antibodies (Novus) detected endogenous PINK1 in SH-SY5Y cells with
similar distribution along the sucrose gradient (bottom panel).
doi:10.1371/journal.pone.0004597.g004
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 7 February 2009 | Volume 4 | Issue 2 | e4597
expression of mutant PINK1 (Figure 8A). Proteasome amounts
were comparable between control and PINK1 depleted cells, as
demonstrated by Western analysis with proteasomal 20S a subunit
Ab (Figure 8B, bottom panel), suggesting that the defects
observed in proteasome activity upon PINK1 depletion may be
due to a functional deficit rather than a decrease in the amount of
proteasome. To further prove the deleterious effects of knocking
down PINK1 on the proteasome function, we measured protea-
some activity by proteasome-mediated degradation of mCFP-
degron, corroborating that PINK1 KD significantly impairs
proteasome function (Figure 8D).
a-synuclein aggregation
Abnormal protein accumulation and aggregation are common
features in neurodegenerative diseases. In PD, a-synuclein-
containing Lewy body pathology is present in most cases, and
PINK1 protein is found in Lewy bodies [45,46]. An important
factor for a-synuclein accumulation and aggregation is whether
the protein degradation pathway is functioning properly. In fact,
transgenic mouse models for a-synuclein have defective protea-
some function [47].
Since the loss of PINK1 function impairs proteasome-mediated
degradation (Figure 8), we next examined if it would promote the
formation of SDS-insoluble inclusions of a-synuclein. Co-trans-
fection of a-synuclein-mCFP with either of the two different
siRNA against PINK1 (siPINK1) in SH-SY5Y cells led to the
formation of mCFP-positive inclusions as revealed by confocal
microscopy. A control scramble sequence siRNA (siSCR) led to no
visible aggregates (Figure 9A). The same results were also
obtained in stable cell lines that express a-synuclein-mCFP at low
levels (data not shown).
We next used a modified filter trap method to better quantify
the amount of SDS-insoluble aggregates (Figure 9B and 9C)
[48]. Loss of PINK1 led to increased SDS-insoluble aggregates in
both the wild type a-synuclein and the human disease form A53T-
a-synuclein. Taken together, these results show that loss of PINK1
is sufficient to lead to a decrease of proteasome function and
accumulation of insoluble a-synuclein aggregates.
Discussion
In an effort to dissect the molecular pathway for PINK1
mediated pathogenesis, we have obtained new insights into the
nature of mitochondrial dysfunction and its deleterious cellular
consequences.
Figure 5. Mutant PINK1 impairs mitochondrial respiration and
ATP synthesis in cultured SH-SY5Y cells. A) Oxygraphic measure-
ment of SH-SY5Y cells infected with adenovirus of wild type (WT) PINK1,
L347P-PINK1, and Del 245 PINK1. Normal respiration was measured with
pyruvate as a substrate in non-infected SH-SY5Y control (C), cells
infected with WT, L347P, E417G or Del 245 PINK1. A statistically
significant deficit in oxygen consumption was detected in cells
expressing L347P-PINK1 compared to wild type PINK1 (37.7% reduction;
n = 3, p,0.01, ANOVA) or to non-infected control cells (39.3% reduction;
n = 3, p,0.01, ANOVA). In cells expressing E417G-PINK1, oxygen
consumption was significantly reduced compared to cells expressing
wild type PINK1 (23.1% reduction, n = 3, p,0.05, ANOVA) or to non-
infected cells (25.2% reduction, n = 3, p,0.05, ANOVA). There is no
significant difference in respiration changes between cells expressing
WT-PINK1 and non-infected control cells or between cells expressing
WT-PINK1 and Del 245-PINK1. B) Measurements of ATP synthesis with
malate/pyruvate as substrates in cultured SH-SY5Y cells. A statistically
significant deficit in ATP synthesis was detected in cells expressing
L347P-PINK1 compared to wild type PINK1 (23% reduction; n = 15,
p = 0.001, Student t test) or to non-infected control cells (30% reduction;
n = 5, p = 0.012, student t test). In cells expressing E417G-PINK1, ATP
synthesis was significantly reduced compared to cells expressing wild
type PINK1 (19.7% reduction, n = 12, p = 0.001, student t test) or to non-
infected cells (27.4% reduction, n = 12, p = 0.011, student t test). There is
no significant difference in ATP synthesis between cells expressing WT-
PINK1 and non-infected control cells (n = 5, p = 0.646, student t test) or
between cells expressing WT-PINK1 and Del 245-PINK1 (n = 5, p = 0.849,
student t test). C) As controls, lysates used for respiration and ATP
synthesis were subsequently subjected to Western analysis with Flag Ab
(top panel) and Tim 23 Ab (bottom panel). Results indicate that equal
expression of various recombinant PINK1 and equal amount of
mitochondria were used for all the experiments.
doi:10.1371/journal.pone.0004597.g005
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 8 February 2009 | Volume 4 | Issue 2 | e4597
Figure 6. Loss of PINK1 impairs OXPHOS function in PC12 cells with reduced PINK1 expression. Normal respiration is impaired in the
PC12 cell lines with PINK1 knocked-down by RNAi. A) PINK1 mRNA is significantly reduced in two stable cell lines expressing PINK1 siRNA. Compared
to the wild type control, there is an 81.7% and 91% reduction in PINK1 mRNA in SiPINK1-2 and SiPINK1-4 cell line. PINK1 mRNA is significantly
reduced in two stable cell lines expressing PINK1 siRNA. The level of PINK1 mRNA is normalized to GAPDH. B) Oxygen consumption is significantly
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 9 February 2009 | Volume 4 | Issue 2 | e4597
The mitochondrial OXPHOS system is known to be regulated
by kinases. We have demonstrated that mutant PINK1 or loss of
PINK1 cause deficits in respiration and ATP synthesis. Similar
and yet different observations were made in other systems. In
Drosophila, mitochondrial structural defect accompanied by
reduced ATP content were reported [23,49]; Additionally,
complex I and II driven respiration deficits were also detected in
mice, though surprisingly, this was in the absence of any ATP
deficit [21]. While the molecular details need to be elucidated, it is
likely that PINK1 mediated defects are caused by changes in the
phosphorylation states of ETC Complexes. In support of this
notion, it is known that several subunits of mitochondrial ETC
Complexes are phospho-proteins, and their activity can be
regulated via PINK1 mechanism [50,51].
It is important to note that PINK1-induced deficit in ETC and
subsequent cellular dysfunctions are similar to those induced by
rotenone toxicity in rats [10]. Pharmacological inhibition of the
ETC, particularly Complex I, result in PD-like phenotypes, but
how these models reflect actual PD pathogenesis remains to be
elucidated. Our results offer a possible link between specific
mitochondrial dysfunctions and cellular abnormalities that are
highly relevant to PD.
We found that proteasome function is impaired by mutant or
reduced amounts of PINK1. The proteasome is one of the major
pathways for protein degradation. Parkin, the disease gene for
PARK2 type of PD, encodes an E3 ubiquitin ligase. One of
Parkin’s proposed roles is the proteasomal degradation of its
protein substrates. In Drosophila, it was shown that Parkin and
PINK1 have a genetic interaction [49,52]. It will be interesting to
investigate if the interaction between Parkin and PINK1 occurs in
mitochondria, and whether it affects mitochondrial function.
Our results identified concomitant deficits in mitochondrial
bioenergetics, proteasomal activity, and a-synuclein aggregation.
Are they consequential to each other or independent events? We
postulate that since PINK1 is predominantly localized in
mitochondria, the primary pathogenic event is likely to be in the
same place. The ATP deficit is a potential link between
mitochondrial abnormality and proteasome deficit, although
proteasome deficit could also be caused by other mechanisms
such as abnormal post-translational modification including
phosphorylation, assembly and targeting, etc. a-synuclein is known
to be a target of proteasome degradation in the cytosol [53,54].
Therefore, a-synuclein aggregation could be the consequence of
proteasome dysfunction. In addition, since a-synuclein aggrega-
tion has been shown to affect proteasome function directly [47], it
is tempting to speculate a vicious cycle of proteasomal dysfunction
and a-synuclein aggregation, although further experimental data
are needed to support this hypothesis.
A common theme in neurodegeneration is that for any given
disease-causing mutant protein, a large number of interwoven
cellular dysfunctions have been discovered. Our observations start
to unravel a subset of PINK1 pathogenic processes, and will
certainly lead to other highly relevant pathways. For example,
deficits in respiratory complexes identified in our experiments, in
addition to bioenergetic impairment, may also lead to increased
oxidative stress, as was shown in rotenone models and other
studies [10,55]. Furthermore, the ATP deficit is likely to have a
much wider negative impact on many cellular functions in
addition to proteasome activity. Clearly, it will be important to
further assess as many mitochondrial and other cellular functions
as possible to dissect the full spectrum of PINK1 pathogenesis. As
this manuscript was reviewed, Gautier et al reported respiration
deficit in the PINK1 knockout mice [21]. Our data, not only show
Figure 7. Proteasome function is impaired by reduction of ATP.
Proteasome function is ATP dependent. Fluorescent CFP was fused to
degron, a signaling peptide that directs its protein to proteasome for
degradation. An increase of fluorescence (open circle) indicates a
reduction of proteasome function. ATP production was inhibited by 2-
deoxyglucose (2DG), and ATP content was measured with the ATP
Assay Kit (Calbiochem) for luminescence (filled circle). A) Increasing
dosages of 2DG caused a decrease in ATP production (filled circle) and
enhanced proteasome inhibition (open circle). B) Compared to non-
treated cells in DMEM, there is a significant proteasome inhibition by
6 mM 2DG (p = 0.0004, ANOVA) and the proteasome inhibitor MG132
(p = 0.0001, ANOVA).
doi:10.1371/journal.pone.0004597.g007
reduced in both SiPINK1-2 (22.4% reduction, n = 10, p,0.05, ANOVA) and SiPINK1-4 (33.1% reduction, n = 11, p,0.01, ANOVA) cell lines compared to
that of control cells. SiPINK1-2: 46.3863.5SE; SiPINK1-4: 39.9661.93SE; control cell: 59.7062.1SE. C) The respiratory deficit in SiPINK1-4 cells can be
partially rescued by wild type (n = 7, p = 0.008, student T test) but not E417G-PINK1 (n = 4, p = 0.76, student T test) or Del 245 PINK1 (n = 3, p = 0.1,
student T test). Control: 60 63.38SE; SiPINK1-4: 3762.32SE; SiPINK1-4/wt-PINK1: 47.962.39SE; SiPINK1/E417G: 38.5363.35SE; SiPINK1/Del 245:
28.6364.75SE. D) With glutamate/malate as substrates, ATP synthesis rate was significantly reduced in both SiPINK1-2 (41.3% reduction, n = 7,
p,0.01, ANOVA) and SiPINK1-4 (29.8% reduction, n = 8, p,0.01, ANOVA) cell lines compared to that of control cell. SiPINK1-2: 8.7360.99SE; SiPINK1-
4:10.5960.69SE; control cells: 14.88360.78SE. E) Western analysis of the samples used for the rescued experiment shown in (C) with Tim 23 Ab. The
result demonstrates that equal amount of mitochondria is present in all the samples subject to respiration experiment. F) Equal amount of
mitochondria were used for the experiments shown in B and D as demonstrated by identical Tim 23 in all the samples.
doi:10.1371/journal.pone.0004597.g006
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 10 February 2009 | Volume 4 | Issue 2 | e4597
similar decrease in respiration, but also pointed out the
downstream deleterious consequence of this deficit, such as
decrease in ATP synthesis rate, proteasomal deficit, and a-
synuclein accumulation. Therefore, our results provide a frame-
work for PINK1 mediated pathogenesis, upon which future studies
can be designed and pursued.
Figure 8. Proteasome function is impaired by mutant or loss of PINK1. Proteasome activity was measured from SH-SY5Y cells expressing
mutant PINK1 (A), and PC12 cells expressing siRNA against PINK1 (B, C, D). A) Fluorescence of fluorogenic proteasome substrate Suc-LLVY-AMC
(Calbiochem) is positively correlated with proteasome function. No statistically significant changes were detected in proteasome activity between
control SH-SY5Y cells and the cells expressing wild type PINK1 (n = 8, p = 0.484, paired student t test). There was a statistically significant decrease of
proteasome activity in the SH-SY5Y cells expressing L347P-PINK1 (23% reduction, n = 7, p = 0.018, paired student t test) or in SH-SY5Y cells expressing
E417G-PINK1 (19.4% reduction, n = 8, p = 0.012, paired student t test) compared to cells expressing wild type PINK1. MG132, a proteasome inhibitor,
was used as a negative control. The bottom panel is a Western analysis of the above samples with the 20S a subunit Ab for normalization. B)
Proteasome activity was measured in 20 mg of cell lysate isolated from wild type control PC12 cells (open diamond) or SiPINK1-4 PC12 cell line (filled
circle) for 60 min after 30 min incubation. Wild type PC12 cells lysate treated with MG132 (filled triangle) was used as a negative control. The result
revealed that the kinetic of proteasome activity monitored over 60 min was markedly decreased in the cells with reduced PINK1. The bottom panel is
a Western analysis of the above samples with the 20S a subunit antibody for normalization. C) Histographic presentation for Figure 7B. The reduction
of PINK1 by siRNA impairs the proteasome activity (31.8% reduction, n = 8, p = 0.01, ANOVA). Experiments were repeated with SiPINK1-2 PC12 cell
line, and consistent results were obtained (data not shown). D) PINK1 mediated proteasome activity deficit confirmed by another independent
method in the HeLa cells. Compared to control (CFP-de transfection), siRNA against PINK1 (siPINK1) knocked down PINK1 and led to a sigfinicant
inhibition of CFP degradation (p = 0.0001, ANOVA) to an extent similar to direct proteasome inhibition by MG132 (p = 0.0014, ANOVA). A scrambled
siRNA (siSCR) had no effect (p = 0.876, ANOVA). The RNAi sequences are: GAGAGGUCCAAGCAACUA TT and CCUGGUCGACUACCCUGAU TT.
doi:10.1371/journal.pone.0004597.g008
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 11 February 2009 | Volume 4 | Issue 2 | e4597
Figure 9. Loss of PINK1 function leads to a-synuclein aggregation. Knockdown of PINK1 by siRNAs in cell lines expressing wild type a-
synuclein-mCFP or A53T a-synuclein-mCFP leads to the accumulation and aggregation of a-synuclein as measured by confocal microscopy or filter
trap assay. A) Representative confocal images of SH-SY5Y cells co-transfected with a -synuclein-mCFP and siRNA. The results indicate that loss of
PINK1 leads to the formation of mCFP-positive inclusions. B) Dot blot filter trap assay captured SDS-insoluble inclusions formed by loss of PINK1
function. Two different siRNA with sequences against PINK1 (siPINK1-1 and siPINK1-2) were transfected into stable HeLa cell lines expressing wt a-
synuclein or A53T a-synclein. The lysate was collected and filtered through two filters, with the cellulose acetate (ca) membrane on the top and the
PVDF membrane at the bottom. The ca membrane trapped the insoluble aggregates, whereas PVDF membrane caught the soluble protein. The
results indicate that reduction of PINK1 by either siRNA leads to increased accumulation of SDS insoluble a-synuclein compared with the scrambled
siSCR control when filtered through ca membrane. PVDF membrane was blotted with actin as loading control. C) Quantification of filter trap assay
using densitometry analysis via NIH Image. Each bar represents 4 samples. siRNA-mediated knockdown of PINK1 leads to a significant increase of
SDS-insoluble a-synuclein (*: p,0.05). ANOVA analysis reveals a significant effect of a-synuclein (F
(1,18)
= 56.482; p,0.001) and siRNA (F
(2,18)
= 15.559;
p,0.001), but no significant interaction between the two variables (F
(2,18)
= 55.413; p = 0.949), thus indicating that PINK1 knockdown has a similar
effect on both forms of a-synuclein.
doi:10.1371/journal.pone.0004597.g009
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 12 February 2009 | Volume 4 | Issue 2 | e4597
Author Contributions
Conceived and designed the experiments: WL CVB RAP AY XYH FB
GM CL. Performed the experiments: WL CVB RAP AY YT YL MAS SS
SC CL. Analyzed the data: WL CVB RAP AY YT XYH FB GM CL.
Contributed reagents/materials/analysis tools: WL CVB AY JM MAS
MK XYH FB GM CL. Wrote the paper: WL CVB AY GM CL.
References
1. Melov S (2004) Modeling mitochondrial function in aging neurons. Trends in
Neurosciences 27: 601–606.
2. Li C, Beal MF (2005) Leucine-rich repeat kinase 2: a new player wit h a familiar
theme for Parkinson’s disease pathogenesis. Proc Natl Acad Sci U S A 102:
16535–16536.
3. Kwong JQ, Beal MF, Manfredi G (2006) The role of mitochondria in inherited
neurodegenerative diseases. J Neurochem 97: 1659–1675.
4. Parker WD Jr, Boyson SJ, Parks JK (1989) Abnormalities of the electron
transport chain in idiopathic Parkinson’s disease. Ann Neurol 26: 719–723.
5. Schapira AH, Cooper JM, Dexter D, Clark JB, Jenner P, et al. (1990)
Mitochondrial complex I deficiency in Parkinson’s disease. Journal of
Neurochemistry 54: 823–827.
6. Schapira AH, Mann VM, Cooper JM, Dexter D, Daniel SE, et al. (1990)
Anatomic and disease specificity of NADH CoQ1 reductase (complex I)
deficiency in Parkinson’s disease. J Neurochem 55: 2142–2145.
7. Shoffner JM, Wa tts RL, Jun cos JL, To rroni A, Wa llace DC ( 1991)
Mitochondrial oxidative phosphorylation defects in Parkinson’s disease. Ann
Neurol 30: 332–339.
8. Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, et al.
(2000) Chronic systemic pesticide exposure reproduces features of Parkinson’s
disease. Nature Neuroscience 3: 1301–1306.
9. Dauer W, Przedborski S (2003) Parkinson’s disease: mechanisms and models.
Neuron 39: 889–909.
10. Betarbet R, Canet-Aviles RM, Sherer TB, Mastroberardino PG, McLendon C,
et al. (2006) Intersecting pathways to neurodegeneration in Parkinson’s disease:
effects of the pesticide rotenone on DJ-1, alpha-synuclein, and the ubiquitin-
proteasome system. Neurobiol Dis 22: 404–420.
11. Richardson JR, Caudle WM, Guillot TS, Watson JL, Nakamaru-Ogiso E, et al.
(2007) Obligatory role for complex I inhibition in the dopaminergic
neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Toxicol
Sci 95: 196–204.
12. Martin LJ, Pan Y, Price AC, Sterling W, Copeland NG, et al. (2006) Parkinson’s
disease alpha-synuclein transgeni c mice develop neuronal mitochondrial
degeneration and cell death. J Neurosci 26: 41–50.
13. Palacino JJ, Sagi D, Goldberg MS, Krauss S, Motz C, et al. (2004)
Mitochondrial dysfunction and oxidative damage in parkin-deficient mice.
Journal of Biological Chemistry 279: 18614–18622.
14. Greene JC, Whitworth AJ, Kuo I, Andrews LA, Feany MB, et al. (2003)
Mitochondrial pathology and apoptotic muscle degeneration in Drosophila
parkin mutants. Proceedings of the National Academy of Sciences of the United
States of America 100: 4078–4083.
15. West AB, Moore DJ, Biskup S, Bugayenko A, Smith WW, et al. (2005)
Parkinson’s disease-associated mutations in leucine-rich repeat kinase 2 augment
kinase activity. Proc Natl Acad Sci U S A 102: 16842–16847.
16. Canet-Aviles RM, Wilson MA, Miller DW, Ahmad R, McLendon C, et al.
(2004) The Parkinson’s disease protein DJ-1 is neuroprotective due to cysteine-
sulfinic acid-driven mitochondrial localization. Proceedings of the National
Academy of Sciences of the United States of America 101: 9103–9108.
17. Yokota T, Sugawara K, Ito K, Takahashi R, Ariga H, et al. (2003) Down
regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and
proteasome inhibition. Biochemical & Biophysical Research Communications
312: 1342–1348.
18. Valente EM, Salvi S, Ialongo T, Marongiu R, Elia AE, et al. (2004) PINK1
mutations are associated with sporadic early-onset parkinsonism. Annals of
Neurology 56: 336–341.
19. Valente EM, Abou-Sleiman PM, Caputo V, Muqit MM, Harvey K, et al. (2004)
Hereditary early-onset Parkinson’s disease caused by mutations in PINK1.[see
comment]. Science 304: 1158–1160.
20. Hatano Y, Li Y, Sato K, Asa kawa S, Yamamura Y, et al. (2004) Novel PINK1
mutations in early-onset parkinsonism.[erratum appears in Ann Neurol. 2004
Oct;56(4):603]. Annals of Neurology 56: 424–427.
21. Gautier CA, Kitada T, Shen J (2008) Loss of PINK1 causes mitochondrial
functional defects and increased sensitivity to oxidative stress. Proc Natl Acad
Sci U S A 105: 11364–11369.
22. Piccoli C, Sardanelli A, Scrima R, Ripoli M, Quarato G, et al. (2008)
Mitochondrial respiratory dysfunction in familiar parkinsonism associated with
PINK1 mutation. Neurochem Res 33: 2565–2574.
23. Clark IE, Dodson MW, Jiang C, Cao JH, Huh JR, et al. (2006) Drosophila pink1
is required for mitochondrial function and interacts genetically with parkin.
Nature 441: 1162–1166.
24. Deng H, Dodson MW, Huang H, Guo M (2008) The Parkinson’s disease genes
pink1 and parkin promote mitochondrial fission and/or inhibit fusion in
Drosophila. Proc Natl Acad Sci U S A 105: 14503–14508.
25. Yang Y, Ouyang Y, Yang L, Beal MF, McQuibban A, et al. (2008) Pink1
regulates mitochondrial dynamics through interaction with the fission/fusion
machinery. Proc Natl Acad Sci U S A 105: 7070–7075.
26. Poole AC, Thomas RE, Andrews LA, McBride HM, Whitworth AJ, et al. (2008)
The PINK1/Parkin pathway regulates mitochondrial morphology. Proc Natl
Acad Sci U S A 105: 1638–1643.
27. Exner N, Treske B, Paquet D, Holmstrom K, Schiesling C, et al. (2007) Loss-of-
function of human PINK1 results in mitochondrial pathology and can be
rescued by parkin. J Neur osci 27: 12413–12418.
28. Pridgeon JW, Olzmann JA, Chin LS, Li L (2007) PINK1 Protects against
Oxidative Stress by Phosphorylating Mitochondrial Chaperone TRAP1. PLoS
Biol 5: e172.
29. O’Carroll SJ, Hall AR, Myers CJ, Braithwaite AW, Dix BR (2000) Quantifying
adenoviral titers by spectrophotometry. Biotechniques 28: 408–410, 412.
30. Manfredi G, Yang L, Gajewski CD, Mattiazzi M (2002) Measurements of ATP
in mammalian cells. Methods 26: 317–326.
31. Yamamoto A, Cremo na ML, Rothman JE (2006) Autophagy-m ediated
clearance of huntingtin aggregates triggered by the insulin-signaling pathway.
J Cell Biol 172: 719–731.
32. Ehlers MD (2003) Activity level controls postsynaptic composition and signaling
via the ubiquitin-proteasome system. Nat Neurosci 6: 231–242.
33. Beilina A, Van Der Brug M, Ahmad R, Kes avapany S, Miller DW, 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.
34. Hao W, Takano T, Guillemette J, Papillon J, Ren G, et al. (2006) Induction of
apoptosis by the Ste20-like kinase SLK, a germinal center kinase that activates
apoptosis signal-regulating kinase and p38. J Biol Chem 281: 3075–3084.
35. Liu CY, Schroder M, Kaufman RJ (2000) Ligand-independent dimerization
activates the stress response kinases IRE1 and PERK in the lumen of the
endoplasmic reticulum. J Biol Chem 275: 24881–24885.
36. Hansford RG (1991) Dehydrogenase activation by Ca2+
in cells and tissues.
Journal of Bioenergetics & Biomembranes 23: 823–854.
37. Thomson M (2002) Evidence of undiscovered cell regulatory mechanisms:
phosphoproteins and protein kinases in mitochondria. Cellular & Molecular Life
Sciences 59: 213–219.
38. van den Heuvel L, Ruitenbeek W, Smeets R, Gelman-Kohan Z, Elpeleg O, et
al. (1998) Demonstration of a new pathogenic mutation in human complex I
deficiency: a 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ)
subunit. American Journal of Human Genetics 62: 262–268.
39. Petruzzella V, Vergari R, Puzziferri I, Boffoli D, Lamantea E, et al. (2001) A
nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit
of complex I abolishes assembly and activity of the complex in a patient with
Leigh-like syndrome. Human Molecular Genetics 10: 529–535.
40. Sardanelli AM, Technikova-Dobrova Z, Scacco SC, Speranza F, Papa S (1995)
Characterization of proteins phosphorylated by the cAMP-dependent protein
kinase of bovine heart mitochondria. FEBS Letters 377: 470–474.
41. Park J, Lee SB, Lee S, Kim Y, Song S, et al. (2006) Mitochondrial dysfunction in
Drosophila PINK1 mutants is complemented by parkin. Nature 441:
1157–1161.
42. Goldberg AL (2003) Protein degradation and protection against misfolded or
damaged proteins. Nature 426: 895–899.
43. Bence NF, Sampat RM, Kopito RR (2001) Impairment of the ubiquitin-
proteasome system by protein aggregation. Science 292: 1552–1555.
44. Hoglinger GU, Carrard G, Michel PP, Medja F, Lombes A, et al. (2003)
Dysfunction of mitochondrial complex I and the proteasome: interactions
between two biochemical deficits in a cellular model of Parkinson’s disease.
J Neurochem 86: 1297–1307.
45. Muqit MM, Abou-Sleiman PM, Saurin AT, Harvey K, Gandhi S, et al. (2006)
Altered cleavage and localization of PINK1 to aggresomes in the presence of
proteasomal stress. J Neurochem 98: 156–169.
46. Murakami T, Moriwaki Y, Kawarabayashi T, Nagai M, Ohta Y, et al. (2007)
PINK1, a gene product of PARK6, accumulates in {alpha}-synucleinopathy
brains. J Neurol Neurosurg Psychiatry.
47. Chen L, Thiruchelvam MJ, Madura K, Richfield EK (2006) Proteasome
dysfunction in aged human alpha-synuclein transgenic mice. Neurobiol Dis 23:
120–126.
48. Bailey CK, Andriola IF, Kampinga HH, Merry DE (2002) Molecular
chaperones enhance the degradation of expanded polyglutamine repeat
androgen receptor in a cellular model of spinal and bulbar muscular atrophy.
Hum Mol Genet 11: 515–523.
49. Park J LS, Lee S, Kim Y, Song S, Kim S, Bae E, Kim J, Shong M, Kim JM,
Chung J (2006) Mitochondrial dysfunction in Drosophila PINK1 mutants is
complemented by parkin. Nature 441: 1157–1161.
50. Pagliarini DJ, Dixon JE (2006) Mitochondrial modulation: reversible phosphor-
ylation takes center stage? Trends Biochem Sci 31: 26–34.
51. Reinders J, Wagner K, Zahedi RP, Stojanovski D, Eyrich B, et al. (2007)
Profiling phosphoproteins of yeast mitochondria reveals a role of phosphory-
lation in assembly of the ATP synthase. Mol Cell Proteomics 6: 1896–1906.
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 13 February 2009 | Volume 4 | Issue 2 | e4597
52. Clark IE, Dodson MW, Jiang C, Cao JH, Huh JR, et al. (2006) Drosophila pink1
is required for mitochondrial function and interacts genetically with parkin.
nature 7097: 1162–1166.
53. Bennett MC, Bishop JF, Leng Y, Chock PB, Chase TN, et al. (1999)
Degradation of alpha-synuclein by proteasome. J Biol Chem 274: 33855–33858.
54. Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC (2003) Alpha-
Synuclein is degraded by both autophagy and the proteasome. J Biol Chem 278:
25009–25013.
55. Hoepken HH, Gispert S, Morales B, Wingerter O, Del Turco D, et al. (2007)
Mitochondrial dysfunction, peroxidation damage and changes in glutathione
metabolism in PARK6. Neurobiol Dis 25: 401–411.
PINK1 and Mitochondrial Defect
PLoS ONE | www.plosone.org 14 February 2009 | Volume 4 | Issue 2 | e4597
    • In this regard it is important to mention that PINK1 WT interacted with the mitochondrial import channel TOM40 specifically upon CCCP treatment, but both of the mutations that had reduced HSP90/CDC37 binding, failed. It is known that upon mitochondrial depolarization PINK1 auto-phosphorylates[73,80]and dimerizes into a higher molecular weight protein complex together with the TOM machinery in the OMM[10,12,81]. However, it is unclear whether PINK1 then remains in the TOM complex or integrates laterally into the lipid phase of the OMM [9, 10, 76] once it has acquired its orientation to phosphorylate Ub and PARKIN.
    [Show abstract] [Hide abstract] ABSTRACT: Background Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson’s disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future. Methods Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients’ fibroblasts and in cell-based, biochemical assays. ResultsUnder endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity. Conclusions Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon mitochondrial stress and due to conformational changes in the active site does not exert kinase activity towards Ub. In patients’ fibroblasts, biochemical assays and by structural analyses, we unraveled two pathomechanisms that lead to loss of function upon mutation of p.I368N and highlight potential strategies for future drug development.
    Full-text · Article · Dec 2017
    • Other evidence suggests that mutations in Parkin result in oxidative stress, impaired biogenesis, reduced mitochondrial dynamics and dysregulation of neuronal calcium (Parganlija et al., 2014;Scarffe et al., 2014). In contrast to the role of PINK1 deficit in mitophagy dysfunction, loss or mutations in this gene have been shown to cause proteasome dysfunction and an increase in a-synuclein aggregation (Liu et al., 2009), suggesting that mitochondrial quality control and protein aggregation are molecularly connected. Here, ATP-dependent autophagosomal transport may be implicated, contributing to dysfunctional mitochondrial quality control and protein clearance.
    [Show abstract] [Hide abstract] ABSTRACT: Neurodegenerative diseases are characterised by the presence of cytoplasmic and nuclear protein aggregates that result in toxicity and neuronal cell death. Autophagy is a physiological cellular process that engulfs primarily long-lived proteins as well as protein aggregates with subsequent cargo delivery for lysosomal degradation. The rate at which the material is degraded through autophagy is referred to as autophagic flux. Although we have progressed substantially in unravelling the role and regulation of the autophagy machinery, its dysfunction in pathology as well as its dynamic changes in the disease progression remains largely unclear. Furthermore, the magnitude of autophagic flux in neuronal subtypes is largely unknown and it is unclear to what extent the flux may be affected in distinct neurodegenerative disease states. In this review, we provide an introduction to autophagy in neuronal homeostasis and indicate how autophagy is currently measured and modulated for therapeutic purposes. We highlight the need not only to develop enhanced methodologies that target and assess autophagic flux precisely, but also to discern the dynamics of autophagy in different neuronal types and brain regions associated with the disease-specific pathology. Finally, we describe how existing and novel techniques for assessing autophagic flux could be implemented in order to distinguish between molecular defects associated with autophagic cargo and the machinery. In doing so, this review may provide novel insights in the assessment and control of autophagic flux that is aligned with the protein clearance dysfunction in neurodegenerative disorders.
    Full-text · Article · Apr 2017
    • All three genes have been demonstrated to have an effect on mitochondrial function. Deficiencies in these genes make mitochondria more vulnerable to oxidative stress [127][128][129][130]. A recent study demonstrated that MCU is involved in PD.
    [Show abstract] [Hide abstract] ABSTRACT: The mitochondrial calcium uniporter (MCU)—a calcium uniporter on the inner membrane of mitochondria—controls the mitochondrial calcium uptake in normal and abnormal situations. Mitochondrial calcium is essential for the production of adenosine triphosphate (ATP); however, excessive calcium will induce mitochondrial dysfunction. Calcium homeostasis disruption and mitochondrial dysfunction is observed in many neurodegenerative disorders. However, the role and regulatory mechanism of the MCU in the development of these diseases are obscure. In this review, we summarize the role of the MCU in controlling oxidative stress-elevated mitochondrial calcium and its function in neurodegenerative disorders. Inhibition of the MCU signaling pathway might be a new target for the treatment of neurodegenerative disorders.
    Full-text · Article · Feb 2017
    • For comparison of wild-type PINK1 and the p.G411S mutant, we focused mainly on the prediction of the catalytic kinase domain of PINK1. We modelled autophosphorylation at Ser228 and Ser402 as well as PINK1 dimerization, both of which correlate well with its enzymatic activity (Liu et al., 2009; Okatsu et al., 2012 Okatsu et al., , 2013 Aerts et al., 2015). Although mutant PINK1 p.G411S showed some differences to wild-type in the monomers, effects of the mutation were more pronounced in a dimer.
    [Show abstract] [Hide abstract] ABSTRACT: See Gandhi and Plun-Favreau (doi:10.1093/aww320) for a scientific commentary on this article. It has been postulated that heterozygous mutations in recessive Parkinson’s genes may increase the risk of developing the disease. In particular, the PTEN-induced putative kinase 1 (PINK1) p.G411S (c.1231G>A, rs45478900) mutation has been reported in families with dominant inheritance patterns of Parkinson’s disease, suggesting that it might confer a sizeable disease risk when present on only one allele. We examined families with PINK1 p.G411S and conducted a genetic association study with 2560 patients with Parkinson’s disease and 2145 control subjects. Heterozygous PINK1 p.G411S mutations markedly increased Parkinson’s disease risk (odds ratio = 2.92, P = 0.032); significance remained when supplementing with results from previous studies on 4437 additional subjects (odds ratio = 2.89, P = 0.027). We analysed primary human skin fibroblasts and induced neurons from heterozygous PINK1 p.G411S carriers compared to PINK1 p.Q456X heterozygotes and PINK1 wild-type controls under endogenous conditions. While cells from PINK1 p.Q456X heterozygotes showed reduced levels of PINK1 protein and decreased initial kinase activity upon mitochondrial damage, stress-response was largely unaffected over time, as expected for a recessive loss-of-function mutation. By contrast, PINK1 p.G411S heterozygotes showed no decrease of PINK1 protein levels but a sustained, significant reduction in kinase activity. Molecular modelling and dynamics simulations as well as multiple functional assays revealed that the p.G411S mutation interferes with ubiquitin phosphorylation by wild-type PINK1 in a heterodimeric complex. This impairs the protective functions of the PINK1/parkin-mediated mitochondrial quality control. Based on genetic and clinical evaluation as well as functional and structural characterization, we established p.G411S as a rare genetic risk factor with a relatively large effect size conferred by a partial dominant-negative function phenotype.
    Full-text · Article · Nov 2016
    • Recent studies demonstrated that PINK1 and Parkin are involved in the maintenance of mitochondrial morphology and membrane potential. Reduced transmembrane potential in PINK1 deficient cells has been reported in a wide variety of cells [43][44][45][46]. Parkin also plays a role in mitochondrial dynamics, presumably related to its E3 ubiquitin ligase activity.
    [Show abstract] [Hide abstract] ABSTRACT: Damage to mitochondria often results in the activation of both mitophagy and mitochondrial apoptosis. The elimination of dysfunctional mitochondria is necessary for mitochondrial quality maintenance and efficient energy supply. Here we report that miR-181a is a novel inhibitor of mitophagy. miR-181a is downregulated by mitochondrial uncouplers in human neuroblastoma SH-SY5Y cells. Overexpression of miR-181a inhibits mitochondrial uncoupling agents-induced mitophagy by inhibiting the degradation of mitochondrial proteins without affecting global autophagy. Knock down of endogenous miR-181a accelerates the autophagic degradation of damaged mitochondria. miR-181a directly targets Parkin E3 ubiquitin ligase and partially blocks the colocalization of mitochondria and autophagosomes/lysosomes. Re-expression of exogenous Parkin restores the inhibitory effect of miR-181a on mitophagy. Furthermore, miR-181a increases the sensitivity of neuroblastoma cells to mitochondrial uncoupler-induced apoptosis, whereas miR-181a antagomir prevents cell death. Because mitophagy defects are associated with a variety of human disorders, these findings indicate an important link between microRNA and Parkin-mediated mitophagy and highlights a potential therapeutic strategy for human diseases.
    Full-text · Article · Jun 2016
    • Our results support these previous observations as we showed that PINK1 deficiency disturbs the localization and activation of IGF-1R leading to alteration in Akt phosphorylation which ends up in cell survival decreasing. Several studies have demonstrated the association of PINK1 with mitochondrial homeostasis; PINK1-deficient neurons show higher levels of free radicals associated with decreased levels of glutathione (Wood-Kaczmar et al. 2008), increase in mitochondrial calcium (Marongiu et al. 2009), and reduced mitochondrial membrane potential (ΔΨm) (Wang et al. 2011), which are associated with a decrease in oxygen consumption leading to a deficiency in ATP synthesis, proteasome impairment, and α-synuclein aggregation (Liu et al. 2009). PINK1 deficiency also causes alterations in mitochondrial morphology characterized by increased mitochondrial fragmentation (Dagda et al. 2009), as described also in the present paper.
    [Show abstract] [Hide abstract] ABSTRACT: The etiology of Parkinson's disease remains unknown. Mutations in PINK1 have provided an understanding of the molecular mechanisms of this pathology. PINK1 and Parkin are important in the dismissal of dysfunctional mitochondria. However, the role of PINK1 in the control of neuronal survival pathways is not clear. To determine the role of PINK1 in the control of the phosphatidyl inositol 3-kinase (PI3K)/Akt pathway mediated by insulin-like grow factor type 1 (IGF-1), we use a model of mesencephalic neurons (CAD cells), which were transfected with lentiviral PINK1 shRNA or control shRNA constructs. Silencing of PINK1 was determined by RT-PCR and immunoblotting; cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays; proteins of the PI3K/Akt signaling pathway were tested by immunoblotting and IGF-1 receptor, and mitochondria were examined using fluorescence microscopy. PINK1 shRNA-transfected cells showed a reduction in cell survival compared to control shRNA cells. Exposure to IGF-1 induced a rapid and high increase in the phosphorylation level of IGF-1 receptor in control shRNA-transfected cells; however, silencing of PINK1 decreases phosphorylation level of IGF-1 receptor and downstream target proteins such as Akt, GSK3-beta, IRS-1, and hexokinase. Our results further suggest that PINK1 may be regulating the PI3K/Akt neuronal survival pathway through tyrosine kinase receptors such as IGF-1 receptor.
    Full-text · Article · Dec 2014
Show more
Article
February 2011 · Parkinsonism & Related Disorders · Impact Factor: 3.97
    Parkinson's disease (PD) is a complex neurodegenerative disorder contributed by both environmental and genetic factors. The inconsistent findings in genetic association studies may be due to unrecognized interactions with other genetic or environmental factors. Therefore, we assessed the combined effects of genetic variants of three candidate genes of familial PD and environmental exposure on... [Show full abstract]
    Article
    January 2011 · Neurobiology of Disease · Impact Factor: 5.08
      Mutations in PTEN-induced putative kinase 1 (PINK1) cause a recessive form of Parkinson's disease (PD). PINK1 is associated with mitochondrial quality control and its partial knock-down induces mitochondrial dysfunction including decreased membrane potential and increased vulnerability against mitochondrial toxins, but the exact function of PINK1 in mitochondria has not been investigated using... [Show full abstract]
      Article
      May 2009 · EMBO Molecular Medicine · Impact Factor: 8.67
        For many years research in Parkinson's disease (PD) has linked mitochondrial dysfunction with the characteristic loss of dopaminergic neurons of the substantia nigra, accumulation of cytoplasmic inclusions termed Lewy bodies, and motor dysfunction (Henchcliffe & Beal, 2008). The most compelling connection is that Parkinsonism can be observed in both humans and animals following exposure to... [Show full abstract]
        Discover more