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Apoptosis-inducing factor deficiency sensitizes dopaminergic neurons to parkinsonian neurotoxins.

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ORIGINAL ARTICLE
Apoptosis-Inducing Factor Deficiency
Sensitizes Dopaminergic Neurons to
Parkinsonian Neurotoxins
Celine Perier, PhD,1 Jordi Bové, PhD,1 Benjamin Dehay, PhD,1
Vernice Jackson-Lewis, PhD,2 Peter S. Rabinovitch, MD, PhD,3
Serge Przedborski, MD, PhD,2,4,5,6 and Miquel Vila, MD, PhD1,7
Objective: Mitochondrial complex I deficits have long been associated with Parkinson disease (PD). However, it
remains unknown whether such defects represent a primary event in dopaminergic neurodegeneration.
Methods: Apoptosis-inducing factor (AIF) is a mitochondrial protein that, independently of its proapoptotic properties, plays an essential physiologic role in maintaining a fully functional complex I. We used AIF-deficient
harlequin (Hq) mice, which exhibit structural deficits in assembled complex I, to determine whether primary
complex I defects linked to AIF depletion may cause dopaminergic neurodegeneration.
Results: Despite marked reductions in mitochondrial complex I protein levels, Hq mice did not display apparent
alterations in the dopaminergic nigrostriatal system. However, these animals were much more susceptible to
exogenous parkinsonian complex I inhibitors, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Subtoxic
doses of MPTP, unable to cause damage to wild-type animals, produced marked nigrostriatal dopaminergic
degeneration in Hq mice. This effect was associated with exacerbated complex I inhibition and increased production of mitochondrial-derived reactive oxygen species (ROS) in Hq brain mitochondria. The antioxidant superoxide dismutase-mimetic compound tempol was able to reverse the increased susceptibility of Hq mice to MPTP.
Supporting an instrumental role for mitochondrial-derived ROS in PD-related neurodegeneration, transgenic mice
overexpressing mitochondrially targeted catalase exhibited an attenuation of MPTP-induced mitochondrial ROS and
dopaminergic cell death.
Interpretation: Structural complex I alterations linked to AIF deficiency do not cause dopaminergic neurodegeneration but increase the susceptibility of dopaminergic neurons to exogenous parkinsonian neurotoxins, reinforcing
the concept that genetic and environmental factors may interact in a common molecular pathway to trigger PD.
ANN NEUROL 2010;68:184 –192
C
omplex I dysfunction has long been associated with
Parkinson disease (PD). Reduced activity of mitochondrial complex I has been widely demonstrated in experimental models of PD and in postmortem PD samples.1
Impaired mitochondrial respiration linked to complex I
blockade in experimental PD leads to (1) increased production of reactive oxygen species (ROS); (2) oxidative damage
to proteins, lipids, and DNA; and (3) activation of
mitochondria-dependent programmed cell death pathways,
all of which have been shown to play an instrumental role
in PD-related dopaminergic neurodegeneration.1–3 However, whether complex I defects represent a primary event
in PD-related dopaminergic neurodegeneration remains
currently unknown. A major obstacle to addressing this
question has been the absence of viable in vivo genetic
models with specific primary deficits in complex I.4
In this context, it has been recently reported that
the inner mitochondrial membrane protein apoptosis-
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.22034
Received Nov 13, 2009, and in revised form Mar 5, 2010. Accepted for publication Mar 12, 2010.
Address correspondence to Dr Vila, Neurodegenerative Diseases Research Group, Research Institute of the University Hospital Vall d’Hebron,
Pg. Vall d’Hebron 119-129, 08035 Barcelona, Spain. E-mail: mvila@ir.vhebron.net
From the 1Vall d’Hebron Research Institute and Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED),
Barcelona, Spain; 2Department of Neurology, Columbia University, New York, NY; 3Department of Pathology, University of Washington, Seattle,
WA; 4Department of Pathology, 5Department of Cell Biology, and 6Center for Motor Neuron Biology and Disease, Columbia University,
New York, NY; and 7Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain.
Additional Supporting Information can be found in the online version of this article.
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© 2010 American Neurological Association
Perier et al: AIF and PD-Linked Toxins
inducing factor (AIF) is essential for the maintenance of a
fully functional complex I.5–10 AIF was originally identified
as a mitochondrial proapoptotic protein that on mitochondrial outer membrane permeabilization occurring during
apoptosis is abnormally released from the mitochondria to
the cytosol along with other mitochondrial proteins.10,11
Once into the cytosol, AIF is translocated to the nucleus,
where it can directly induce chromatin condensation and
chromatolysis in a caspase-independent manner.10,11 In
healthy cells, the physiologic role of AIF in sustaining complex I-driven oxidative phosphorylation appears related to
the local redox activity of AIF and is independent of its
proapoptotic properties.6 –10 AIF-depleted cells exhibit reduced levels of complex I subunits, decreased complex I
activity, and impaired complex I-driven mitochondrial respiration.6 –9 Complete ablation of AIF in mice results in
embryonic lethality because of reduced mitochondrial respiratory function.8,12–14 In contrast, mice with a partial AIF
deficiency, named harlequin (Hq), are fertile and viable.15
In these animals, the levels of AIF are reduced by 80 to
90% due to a fortuitous retroviral insertion in the first intron of the AIF gene, which is located on the chromosome
X.15 Hq-derived brain mitochondria display reduced levels
of complex I, impaired assembly of complex I subunits,
and variable deficits in complex I-driven mitochondrial respiration.6,16,17 These animals exhibit a phenotype typically
associated with mitochondrial respiratory chain diseases,
such as cerebellar neurodegeneration with ataxia and progressive retinal degeneration,15,18 and have been considered
as genetic models of human complex I disorders.6
Here, we used the Hq mice to determine whether
primary complex I deficits linked to AIF depletion may
cause dopaminergic neurodegeneration. We found that, despite marked reductions in complex I levels, the dopaminergic nigrostriatal system of Hq mice was fully intact.
However, these animals were much more sensitive to exogenous parkinsonian complex I inhibitors, such as 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The latter effect was associated with an exacerbation of complex I
blockade and mitochondria-derived ROS production in
Hq brain mitochondria. The increased sensitivity of Hq
mice to MPTP was reversed by the antioxidant superoxide
dismutase-mimetic compound tempol. Further supporting
an instrumental role for mitochondrial-derived ROS in
PD-related dopaminergic neurodegeneration, transgenic
mice overexpressing catalase specifically targeted to mitochondria exhibited an attenuation of MPTP-induced mitochondrial ROS production and dopaminergic neuron cell
death. Overall, our results indicate that primary complex I
defects linked to mitochondrial AIF deficiency do not trigger dopaminergic neurodegeneration but increase the susAugust, 2010
ceptibility of dopaminergic neurons to exogenous parkinsonian mitochondrial toxins. In this context, mitochondrialderived ROS appear as pivotal in setting the threshold for
dopaminergic mitochondrial-dependent toxicity.
Materials and Methods
Animals
Mice hemizygous for the Hq mutation in the pcd8 gene encoding
AIF and age-matched male wild-type (WT) controls of the same
background were obtained from Jackson Laboratory (Bar Harbor,
ME). Transgenic mice overexpressing human catalase targeted to
the mitochondria (MCAT mice) were obtained as previously described.19 For MPTP experiments, (1) 6- to 9-month-old male
Hq mice and WT age-matched controls (n ⫽ 3– 6 animals per
group) received 1 intraperitoneal (i.p.) injection of MPTP-HCl
(10mg/kg/day of free base; Sigma-Aldrich, St. Louis, MO) or saline for 5 consecutive days, and (2) 10- to 12-week old WT or
MCAT mice (n ⫽ 10 –13 animals per group) received one i.p.
injection of MPTP-HCl per day (30mg/kg/day of free base) for 5
consecutive days. In an additional group of MPTP-intoxicated
Hq mice, tempol was given once daily at a dose of 200mg/kg at
45 minutes to 1 hour before each MPTP injection and up to 5
days after the last MPTP injection. All animals were sacrificed 21
days after the last MPTP or saline injection.20
Quantitative Morphology
Twenty-one days after the last MPTP injection, mice were
killed, and their brains were removed and processed for immunohistochemistry using a polyclonal anti–tyrosine hydroxylase
(TH) (1:1,000; Calbiochem, San Diego, CA). The total number
of TH-positive substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) neurons was estimated by stereology
using the optical fractionator method.21 Optical densitometry of
striatal TH-positive terminals was determined using Scion Image
(Frederick, MD) software. The number of substantia nigra apoptotic profiles was determined as described in Vila et al.22
Isolated Brain Mitochondria
Nonsynaptosomal brain mitochondria was isolated from 2- to
3-month-old Hq (n ⫽ 3 animals per group), MCAT (n ⫽ 6
animals per group), and WT (n ⫽ 3– 6 animals per group) mice
as previously described.23 Oxygen consumption was monitored
using a Clark-type electrode (Hansatech Instruments, PP Systems, Haverhill, MA), as in Perier et al.23 Mitochondrial H2O2
production was measured using 5␮M Amplex Red (Molecular
Probes, Eugene, OR) and 5U/ml horseradish peroxidase, as in
Perier et al.23 The activities of mitochondrial complexes I, II,
and III/IV and citrate synthase were measured as in Tieu et al.24
Statistical Analysis
All values are expressed as mean ⫾ standard error of the mean.
One- or 2-way analysis of variance was used (unless indicated
otherwise), followed by Student Newman-Keuls post hoc testing
for pairwise comparison. The null hypothesis was rejected at the
0.05 level.
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Results
Primary Complex I Defects in AIF-Deficient
Hq Mice Sensitize Dopaminergic Neurons to
Parkinsonian Mitochondrial Toxins
AIF is abundantly expressed in brain mitochondria from
WT mice (Fig 1). In contrast, AIF levels are reduced by
90% in brain mitochondria derived from Hq mice (see Fig
1 and Klein et al15). It has been previously reported that
AIF-deficient Hq brain mitochondria exhibit partial defects
in protein levels of different complex I subunits,6,16,17 as
exemplified here by a ⬃40% decrease in complex I
NDUFA9 subunit, compared to WT mitochondria. No
such defects were observed in Hq brain mitochondria for
other respiratory chain complexes, such as complex IV (see
Fig 1 and Vahsen et al6). To determine whether complex I
defects linked to AIF depletion in Hq mice influence the
viability of ventral midbrain dopaminergic neurons, we examined the nigrostriatal dopaminergic system in these animals. Six- to 9-month-old Hq mice, in which cerebellar
and retinal degeneration is already established,15 exhibited a
fully intact nigrostriatal dopaminergic system, as determined by stereological cell counts of TH-positive dopaminergic neurons in the SNpc and the assessment of striatal
TH-positive dopaminergic fibers in these animals (Fig 2
and Fig S1). However, SNpc dopaminergic neurons from
Hq mice were much more susceptible to mitochondrial
FIGURE 2:
Harlequin (Hq) mice are more sensitive to
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced
dopaminergic neurodegeneration. Stereological cell counts of
substantia nigra pars compacta (SNpc) tyrosine hydroxylase
(TH)-immunoreactive neurons (left panel) and optical densitometry of striatal TH immunoreactivity (right panel) from
saline- and MPTP-intoxicated 6-month-old wild-type (wt) or
Hq mice. Top panels display representative micrographs of
TH-immunostained (brown) thionin-counterstained (blue) SNpc
from the different groups of animals. Insets display representative images of TH-immunostained striata in these animals.
*p < 0.05, compared to saline-injected wild-type and Hq
mice and to MPTP-injected wild-type mice. Fgenotype(1, 11) ⴝ
10, 36, Ftreatment(1, 11) ⴝ 12, 15, and Fgenotype x treatment(1,
11) ⴝ 7, 37. Scale barⴝ500␮m.
parkinsonian toxin MPTP than their WT counterparts. Indeed, whereas a subtoxic regimen of MPTP intoxication
(10mg/kg/day for 5 consecutive days) did not cause any
damage to the nigrostriatal dopaminergic system of WT
mice, it produced a marked reduction of dopaminergic
SNpc neurons and striatal dopaminergic terminals in Hq
mice (see Fig 2). Similarly, VTA dopaminergic neurons
from Hq mice were also more susceptible to MPTPinduced cell death than those of WT animals (Fig S2).
MPTP-induced SNpc dopaminergic cell loss in Hq mice
was associated with an increased number of apoptotic profiles in the substantia nigra of these animals (Fig 4C).
Overall, our data indicate that primary complex I defects
linked to AIF depletion in Hq mice increase the susceptibility of dopaminergic neurons to exogenous parkinsonian
mitochondrial toxins.
FIGURE 1:
Decreased complex I levels in apoptosisinducing factor (AIF)-deficient brain mitochondria from harlequin (Hq) mutant mice. Immunoblot analyses of AIF, complex I NDUF9 subunit, and cytochrome oxidase subunit IV
(COX IV) protein levels in isolated whole brain mitochondria from Hq and wild-type (wt) mice. *p < 0.05, compared to wild-type brain mitochondria.
186
Enhanced Complex I Inhibition by
Parkinsonian Neurotoxins in AIF-Deficient Hq
Brain Mitochondria
To determine the molecular substrate for the increased
susceptibility of Hq mice to MPTP, selected experiments
were performed in nonsynaptosomal whole brain mitoVolume 68, No. 2
Perier et al: AIF and PD-Linked Toxins
in complex I protein levels, and in contrast to some previous reports,6 the enzymatic activity of complex I was
not decreased in Hq brain mitochondria (Table). Also,
the rates of adenosine diphosphate (ADP)-stimulated state
3 respiration or resting state 4 respiration driven by complex I-linked substrates did not significantly differ between Hq and WT brain mitochondria (Fig 3A, B). Similarly, no differences in the activity of other mitochondrial
respiratory complexes (Table) or in mitochondrial respiration driven by complex II- or complex III/IV-linked substrates (see Fig 3C, D) were detected between WT and
Hq brain mitochondria. However, when challenged with
parkinsonian complex I inhibitors, such as 1-methyl-4phenylpyridinium (MPP⫹), MPTP’s active metabolite, or
FIGURE 3: Enhanced complex I inhibition by parkinsonian
neurotoxins in Hq brain mitochondria. Rates of adenosine
diphosphate-stimulated state 3 respiration in harlequin (Hq)and wild-type (wt)-derived whole brain mitochondria oxidizing either the complex I-linked substrates malate and glutamate (A, B), the complex II-linked substrate succinate (C), or
the complex III/IV-linked substrates ascorbate/trimethyl pentanediol (D). Mitochondria were incubated with parkinsonian
complex I inhibitors 1-methyl-4-phenylpyridinium (MPPⴙ) (A)
or rotenone (B), complex II inhibitor malonate (C), or complex
IV inhibitor potassium cyanide (KCN) (D). *p < 0.05, compared to nontreated mitochondria; #p < 0.05, compared to
MPPⴙ- or rotenone-treated wild-type mitochondria. For
MPPⴙ, Fgenotype(1, 17) ⴝ 4, 42, Ftreatment(2, 17) ⴝ 47, 851,
and Fgenotype ⴛ treatment(2, 17) ⴝ 2, 74; for rotenone,
Fgenotype(1, 29) ⴝ 14, 43, Ftreatment(2, 29) ⴝ 79, 77, and
Fgenotype ⴛ treatment(2, 29) ⴝ 5, 9.
chondria isolated from Hq and WT animals. Citrate synthase activity did not differ between Hq and WT brain
mitochondria, indicating that mitochondrial mass was not
altered in Hq animals (Table). Despite marked reductions
TABLE: Activity of Mitochondrial
Chain Complexes in Hq Mice
Respiratory
Complex
Wild Type
Hq
I
II
III/IV
Citrate synthase
27.39 ⫾ 2.23
29.71 ⫾ 4.08
99.32 ⫾ 26.45
379.68 ⫾ 88.04
28.80 ⫾ 4.73
33.29 ⫾ 13.17
108.24 ⫾ 15.42
316.44 ⫾ 3.5
Enzymatic activities of individual respiratory chain
complexes (nmol/min/mg protein) were assessed in isolated
brain mitochondria from Hq mice and wild-type controls.
Citrate synthase was determined as an index of
mitochondrial mass. Data represent mean ⫾ standard error
of the mean of 3 to 6 mice per group.
Hq ⫽ harlequin.
August, 2010
FIGURE 4: Enhanced reactive oxygen species (ROS) production contributes to the increased susceptibility of harlequin
(Hq) mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP)-induced dopaminergic neurodegeneration. (A, B) Mitochondrial ROS production quantified in Hq and wild-type
(wt) brain mitochondria using the dye Amplex Red, in presence or absence of 1-methyl-4-phenylpyridinium (MPPⴙ)
(100␮m, A) or rotenone (100nM, B). (C) Quantification of apoptotic profiles in the substantia nigra of MPTP-injected wildtype or Hq mice, treated or not with tempol. (D) Stereological cell counts of substantia nigra pars compacta (SNpc)
tyrosine hydroxylase (TH)-immunoreactive neurons in salineor MPTP-injected wild-type or Hq mice, treated or not with
tempol. In A and B, *p < 0.05, compared to nontreated
mitochondria; #p < 0.05, compared to MPPⴙ- or rotenonetreated WT mitochondria. For MPPⴙ, Fgenotype(1, 11) ⴝ 4, 42,
Ftreatment(1, 11) ⴝ 229, 249, Fgenotype ⴛ treatment(1, 11) ⴝ 1,
256; for rotenone, Fgenotype(1, 11) ⴝ 9, 150, Ftreatment(1,
11) ⴝ 195, 763, Fgenotype ⴛ treatment(1, 11) ⴝ 0, 694. In C,
*p < 0.05, compared to MPTP-injected WT mice; #p < 0.05,
compared to MPTP-injected Hq mice; F2,15 ⴝ 11, 93, p <
0.001. In D, *p < 0.05, compared to saline-injected wild-type
mice; #p < 0.05, compared to MPTP-injected Hq mice;
F2,16 ⴝ 6, 82.
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rotenone, Hq brain mitochondria exhibited exacerbated
impairment of complex I-driven mitochondrial respiration, compared to WT mitochondria. The increased susceptibility of Hq brain mitochondria to respiratory chain
inhibitors was specific to complex I, as no differential effects in the extent of mitochondrial respiration impairment were observed between WT and Hq mitochondria
when challenged with complex II inhibitor malonate or
complex IV inhibitor potassium cyanide. Taken together,
our results indicate that, despite structural complex I deficits, AIF-deficient Hq brain mitochondria do not display
any apparent functional defect in mitochondrial respiration, which may account for the lack of dopaminergic nigrostriatal damage in Hq mice at basal conditions. However, Hq brain mitochondria display enhanced impairment
of complex I-driven respiration, compared to WT mitochondria, when challenged with parkinsonian mitochondrial toxins, which concurs with the increased susceptibility
of Hq mice to MPTP-induced dopaminergic neurodegeneration.
Mitochondrial-derived ROS Set the Threshold
for the Increased Susceptibility of Hq Mice to
MPTP-induced Dopaminergic Neurodegeneration
AIF deficiency has been previously associated with increased mitochondrial ROS production, linked either to
the presumed antioxidant properties of AIF or to its role
in maintaining a fully functional complex I.9,13 We thus
hypothesized that excessive mitochondrial ROS production in Hq mice may drive the enhanced susceptibility of
these animals to parkinsonian complex I inhibition. Using
the dye Amplex Red to quantify superoxide or H2O2 generation in isolated brain mitochondria, we did not observe
any statistically significant difference in net ROS production between Hq and WT brain mitochondria at basal
conditions (see Fig 4A, B). In agreement with the known
effects of complex I inhibition on mitochondrial ROS
production,23 complex I blockade with MPP⫹ or rotenone exacerbated ROS production in both Hq and WT
brain mitochondria. However, the extent of MPP⫹- or
rotenone-induced mitochondrial ROS production was
significantly greater (⬃18%) in Hq than in WT brain
mitochondria. Supporting an instrumental deleterious role
for enhanced ROS production in Hq brain mitochondria,
the membrane-permeable superoxide dismutase-mimetic
compound tempol was able to reverse the increased susceptibility of Hq mice to parkinsonian complex I inhibition, as it significantly attenuated MPTP-induced apoptotic morphology and dopaminergic neurodegeneration in
these animals (see Fig 4C, D). The neuroprotective effects
of tempol on SNpc dopaminergic neuron cell bodies were
not extended, however, to striatal dopaminergic terminals
188
FIGURE 5: Overexpression of catalase targeted to mitochondria attenuates mitochondrial reactive oxygen species (ROS)
production induced by complex I inhibition. (A) Immunoblot
levels of human catalase (hCAT) in brain mitochondria isolated from MCAT transgenic mice. The mitochondrial marker
Hsp60 was used as a loading control. (B, C) Mitochondrial
ROS production quantified in MCAT and wild-type (wt) brain
mitochondria using the dye Amplex Red, in presence or absence of 1-methyl-4-phenylpyridinium (MPPⴙ) (B) or rotenone
(C). *p < 0.05, compared to nontreated wild-type mitochondria; $p < 0.05, compared to nontreated MCAT mitochondria; #p < 0.05, compared to MPPⴙ- or rotenone-treated
wild-type mitochondria. For MPPⴙ, Fgenotype(1, 35) ⴝ 5, 94,
Ftreatment(2, 35) ⴝ 23, 22, and Fgenotype ⴛ treatment(2, 35) ⴝ 4,
12; for rotenone, Fgenotype(1, 31) ⴝ 15, 3, Ftreatment(2, 31) ⴝ
62, 82, and Fgenotype ⴛ treatment(2, 31) ⴝ 1, 75.
(Fig S3). Overall, our results indicate that increased ROS
production plays an instrumental role in the enhanced
susceptibility of Hq mice to MPTP-induced dopaminergic neurodegeneration.
Overexpression of Catalase Targeted to
Mitochondria in Transgenic Mice Attenuates
MPTP-induced Mitochondrial ROS Production
and Dopaminergic Cell Death
Because increased ROS production can emanate from
sources other than mitochondria following MPTP intoxication,21,25 we assessed the specific contribution of
mitochondria-derived ROS to MPTP-induced neurodegeneration by using transgenic mice overexpressing human catalase (an antioxidant enzyme normally localized in
the peroxisome) specifically targeted to the mitochondria.19 These animals, called MCAT, exhibit decreased
aging-related oxidative stress and extended life span, indicating that mitochondrial ROS are an important limiting
factor in determining mammalian longevity.19 Although
the original biochemical characterization of MCAT mice
was mainly performed in heart-derived mitochondria,
these animals also express human catalase in their brains,
although to a lesser extent than in cardiac tissue.19 Immunoblot analyses confirmed the expression of human
catalase in MCAT brain mitochondria (Fig 5A). Using
the dye Amplex Red to measure mitochondrial ROS proVolume 68, No. 2
Perier et al: AIF and PD-Linked Toxins
FIGURE 6: Overexpression of catalase targeted to mitochondria attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP)-induced dopaminergic neurodegeneration in a gene dosage-dependent fashion. (Left panel) Stereological cell counts
of substantia nigra pars compacta (SNpc) tyrosine hydroxylase (TH)-immunorective neurons in saline- or MPTP-injected
wild-type (wt) or MCAT mice. (Right panel) Double immunofluorescence of human catalase (hCAT, green) and TH (red) in
ventral midbrain sections from MPTP-intoxicated MCAT mice. Scale barsⴝ250␮m (low magnification images) and 25␮m (high
magnification images).
duction, MCAT and WT brain mitochondria did not display significant differences in net ROS production at
basal levels (see Fig 5B, C). However, MCAT brain mitochondria exhibited a marked attenuation of MPP⫹- or
rotenone-induced ROS production, compared to WT mitochondria. In ventral midbrain sections from MCAT
mice, immunohistochemical examination revealed a punctuate expression of human catalase in SNpc dopaminergic
neurons, in agreement with its mitochondrial localization
(Fig 6). The levels of human catalase expression in these
neurons, however, markedly differed from one transgenic
animal to another. Remarkably, MCAT mice displaying
high levels of mitochondrial human catalase in SNpc dopaminergic neurons (see Fig 6, top panels) exhibited an
attenuation of dopaminergic cell death induced by toxic
doses of MPTP (30mg/kg/day for 5 consecutive days),
compared to WT animals. In contrast, MCAT mice exhibiting low levels of mitochondrial human catalase in
SNpc dopaminergic neurons (see Fig 6, low panels) were
not protected against MPTP-induced dopaminergic neurodegeneration. These results indicate that ectopic expression of catalase into mitochondria is able to protect SNpc
dopaminergic neurons, in a gene dosage-dependent manner, against PD-related complex I inhibition, thus supporting a pivotal role for mitochondria-derived ROS in
PD-related dopaminergic neurodegeneration.
Discussion
Here we show that complex I structural alterations linked
to mitochondrial AIF deficiency do not trigger dopaminergic neurodegeneration but sensitize dopaminergic neuAugust, 2010
rons to parkinsonian mitochondrial neurotoxins through
ROS-mediated toxicity. Mitochondrially-derived ROS, in
particular, appear as pivotal in setting the threshold for
mitochondria-dependent dopaminergic neuron cell death.
It is now well established that AIF deficiency results
in reduced levels of mitochondrial complex I, either in
Hq mice with partial AIF defects,6,16,17,26 mice with a
complete knockout of AIF,8,13,14,27 or AIF-silenced
cells.7,9 In mutant mice with a complete ablation of AIF,
complex I structural alterations are accompanied with impaired complex I activity and impaired complex I-driven
mitochondrial respiration,8,13,14,27 which accounts for the
embryonic lethality of these animals.8,12,14 In contrast,
Hq mice with partial AIF deficiencies do not display consistent defects in mitochondrial function, despite exhibiting decreased complex I levels, and are fertile and viable.15 Initial reports indicated that brain and retinal tissue
from Hq mice displayed functional defects in complex I,
whereas unaffected tissues, such as muscle and kidney, did
not exhibit such metabolic changes.6 However, we (see
Fig 3 and Table) and others16 did not observe any basal
defect in complex I activity or complex I-driven respiration in Hq-derived brain mitochondria, which may account for the lack of dopaminergic nigrostriatal damage
in these animals at basal conditions. These observations
suggest that only when below a critical threshold, which
may be organ specific,6 AIF deficiency may cause alterations in oxidative phosphorylation. It is not known,
however, how AIF influences complex I structure and activity. Although AIF itself is not a component of complex
I,6 it may be required for the maintenance of a functional
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complex I either by reducing the oxidation of complex I
proteins9 or by promoting the optimal assembly of complex I subunits.6 In addition, some reports indicate that
AIF deficiency results in aberrant mitochondrial morphology, such as abnormally dilated cristae, which may result
in impaired mitochondrial respiration.8,17
Although Hq mice did not display apparent defects
in the nigrostriatal dopaminergic system, these animals exhibited an exacerbation of complex I inhibition, mitochondrial ROS production, and dopaminergic neuron cell
death following MPTP intoxication, compared to their
WT counterparts. Enhanced ROS production appeared to
be pivotal to the increased susceptibility of Hq mice to
MPTP, as the latter was reversed by the superoxidedismutase mimetic tempol. However, although Hq brain
mitochondria exhibited a significantly greater exacerbation
of ROS production following complex I inhibition, compared to WT mitochondria, the extent of this difference
(⬃18%) seems small compared to the striking differential
susceptibility of Hq mice to MPTP-induced neurodegeneration. These results suggest that, in addition to an exacerbation of mitochondrial ROS production in Hq mice,
the threshold for ROS-induced toxicity may be lowered
in these animals. Previous studies have associated AIF deficiency with increased sensitivity to oxidative stress, both
in vitro and in vivo. For instance, AIF-deficient cells are
more susceptible to peroxide-induced apoptosis,6,7,15,28
and tissue-specific AIF-deficient mice display increased
markers of oxidative stress in mutant tissues.13 It remains
to be determined, however, whether increased oxidative
stress linked to AIF deficiency is mostly related to the
presumed antioxidant properties of AIF, which have been
questioned,7 or to its role in maintaining a functional
complex I.10,29
Although MPTP-induced ROS production can emanate from sources other than mitochondria, such as microglial cells,21,25 here we showed a specific role for
mitochondria-derived ROS in MPTP-induced dopaminergic cell death by the observation that overexpression of mitochondrially targeted catalase in MCAT transgenic mice
was able to protect dopaminergic neurons against MPTPinduced neurotoxicity in a gene dosage-dependent manner.
In light of these results, mitochondrial ROS appear to be
pivotal in setting the threshold for mitochondria-dependent
dopaminergic neuronal toxicity. In agreement with this, we
have previously shown that mitochondrial ROS production
following complex I inhibition sensitizes dopaminergic
neurons to proapoptotic proteins, such as Bax, by increasing the releasable soluble pool of cytochrome c in the mitochondria intermembrane space through oxidation of the
inner mitochondrial lipid cardiolipin.23 From a therapeutic
190
point of view, our results indicate that targeting ROS production specifically at the level of the mitochondria may be
envisaged to potentially attenuate neurodegeneration in
PD. Furthermore, because MCAT mice exhibit an extended life span,19 mitochondria-derived ROS production
may represent a molecular link between aging and increased risk of developing PD.
It is now well established that dopaminergic neurodegeneration linked to complex I inhibition in experimental PD occurs, at least in part, through mitochondrial
outer membrane permeabilization and subsequent release
of proapoptotic proteins, such as cytochrome c.3,22,23,30
In vitro experimental reports have shown that AIF can
also be released from the mitochondria, along with cytochrome c, following complex I inhibition with MPP⫹ in
dopaminergic cell lines or primary ventral midbrain cultures.31 Furthermore, small interfering RNA-mediated
knockdown of AIF in dopaminergic cell lines is able to
delay MPP⫹-induced cell death.31 However, whether AIF
plays an actual pro–cell death role in experimental in vivo
models of PD remains to be determined. The latter possibility is plausible, given the fact that activation of
poly(ADP-ribose) polymerase-1, which has been identified as a cellular signal for the mitochondria-to-nucleus
translocation of AIF in various cellular settings,32 contributes to MPTP-induced dopaminergic neurodegeneration.30,33,34 Our results using the Hq mice, however,
failed to demonstrate a proapoptotic role of AIF in
MPTP-induced dopaminergic neurodegeneration, as these
animals not only were not protected against MPTP intoxication but were much more sensitive to this parkinsonian
neurotoxin. Although caution has to be taken in inferring
the potential proapoptotic role of AIF based on the use of
the Hq mice, various studies have used these animals for
this purpose. For instance, it has been reported that the
Hq mice exhibit a markedly reduced infarct volume in
experimental models of cerebral ischemia, compared to
WT animals.35,36 Moreover, excitotoxic studies using
kainic acid-induced seizures demonstrated less hippocampal damage in Hq mice than in WT mice.37 The unequivocal demonstration that AIF may contribute or not
to PD-related neurodegeneration as a proapoptotic factor
would require, however, the generation of genetically
modified cell lines and mouse models in which the vital
(mitochondrial) and lethal (nuclear) functions of AIF
could be dissociated.8,10
Overall, our results indicate that complex I structural defects linked to AIF deficiency do not trigger
dopaminergic neurodegeneration per se, but increase the
susceptibility of dopaminergic neurons to exogenous parkinsonian neurotoxins. These results support the notion
Volume 68, No. 2
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Acknowledgment
This work was supported by the European Commission’s
Marie Curie Excellence Grant (MEXT-CT-2005-24929,
M.V.), European Commission’s Marie Curie International Reintegration Grant (MIRG-CT-2004-6505,
M.V.), Fundació la Caixa, Spain (BM06-153, M.V.),
Fondo de Investigación Sanitaria-Instituto de Salud Carlos III, Spain (PI071019, M.V.), National Institutes of
Health/National Institute on Aging (AG021617, M.V.
and S.P.), US Department of Defense (DAMD 17-03-10482, M.V.), and Ramón y Cajal program from Ministerio de Ciencia e Innovación (MICINN, C.P.).
We thank A. Parent, E. Pérez, and M. Humà for
their technical assistance.
Authorship
C.P. and J.B. contributed equally to this work.
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192
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