1 Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030,
USA
2 Department of Pathology, Baylor College of Medicine, Houston, TX 77030,
USA
3 Program in Developmental Biology, Baylor College of Medicine, Houston, TX
77030, USA
4 Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA
5 Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX
77030, USA
* Author for correspondence (e-mail: gmardon{at}bcm.tmc.edu)
Accepted 20 January 2004
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SUMMARY |
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Key words: parkin, Drosophila, Parkinson disease, Paraquat, Oxygen radicals, Cell size, Infertility, Neurodegeneration, Dopaminergic, Brain, Apoptosis
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Introduction |
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Recent identification of a number of familial PD cases and the genes
involved therein has provided new insights into the pathogenesis of PD.
Missense mutations in -Synuclein, identified in a small number of
families, result in an autosomal dominant familial PD
(Kruger et al., 1998
;
Polymeropoulos et al., 1997
).
-Synuclein is a presynaptic protein and is a major component of LBs
(Clayton and George, 1998
).
Misexpression of
-Synuclein in cultured cells results in aggregate
formation, abnormal mitochondrial morphology, and increased levels of free
radicals (Hsu et al., 2000
). A
number of transgenic models of PD have been generated using overexpression of
-Synuclein in both mice and flies
(Feany and Bender, 2000
;
Giasson et al., 2002
;
Lee et al., 2002
;
Masliah et al., 2000
).
Mutations in PARK2 have been linked to Autosomal
Recessive-Juvenile Parkinson's (AR-JP)
(Kitada et al., 1998). The
dopaminergic loss in AR-JP is generally not associated with LB formation
(Mizuno et al., 1998
).
PARK2 is highly expressed throughout the brain, is present in both
the cytoplasm and the nucleus, and is also associated with mitochondrial
membranes (Darios et al.,
2003
; Kubo et al.,
2001
; Shimura et al.,
1999
; Stichel et al.,
2000
). The human PARK2 protein consists of four domains: an
N-terminal ubiquitin-homology domain and two RING finger domains flanking a
cysteine-rich in between RING fingers (IBR) domain
(Fig. 1A)
(Morett and Bork, 1999
).
Proteins containing RING finger domains frequently act as ubiquitin E3 ligases
(Joazeiro and Weissman, 2000
).
E3 proteins function as part of the ubiquitin-proteasome system required to
degrade proteins that are misfolded, damaged by oxidative stress, nitrated or
ubiquitinated (Grune et al.,
1998
; Halliwell,
2001
; Sitte et al.,
2000
; Stadtman and Berlett,
1998
). Vertebrate parkin also acts as an E3 ligase, helping to
ubiquitinate and degrade several proteins, including Pael receptor (Pael-R),
-Synuclein, CDCrel-1 and Synphilin 1. Familial mutations in PARK2
abolish its E3 activity (Chung et al.,
2001
; Imai et al.,
2000
; Shimura et al.,
2000
; Zhang et al.,
2000
). This and other data (see below) suggest that loss of
PARK2 may result in increased amounts of damaged and misfolded
proteins, leading to cellular dysfunction and apoptosis of DA neurons
(Bence et al., 2001
;
Mezey et al., 1998
). A
familial mutation in another ubiquitin/proteasome system member, ubiquitin
carboxy-terminal hydrolase I, also results in an early onset PD, underscoring
the importance of this system in PD (Leroy
et al., 1998
).
|
Previous work has shown that Drosophila parkin is required for
longevity, male fertility and survival of flight muscle fibers
(Greene et al., 2003). In this
report, we confirm these observations and extend the requirement for
parkin to several other processes. Specifically, we show that
parkin mutant flies have reduced mass, cell size and number; female
infertility; and increased sensitivity to chemical and environmental stress.
We also confirm that loss of parkin does not result in any
significant DA neuron loss.
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Materials and methods |
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Molecular characterization of parkin
Using a BLAST search of the Drosophila genome database, we
identified an EST clone (SD01679) that encodes Drosophila parkin. We
used this EST to probe a Drosophila genomic library (kindly provided
by Ron Davis) and identified lambda phage clones that span the parkin
locus. Phage integrity was verified by restriction digests as well as sequence
analysis of the parkin locus. These phage were used to generate the
dpkR (Rescue) genomic rescue construct that encompasses the following
genomic coordinates: chromosome 3L, 21098849-21124696
(www.ensembl.org)
(Fig. 2A). This rescue fragment
was cloned into the pCasper 4.0 vector to generate transgenic flies. The
dpkSR (Stop-Rescue) construct was created by introducing stop codons
in all three reading frames of parkin using PCR mutagenesis but is
otherwise identical to dpkR. These stop codons are located 66 base
pairs 3' of the parkin AUG. Two and five independent transgenic
lines of dpkSR and dpkSR, respectively, were obtained. In
addition to the parkin mutant chromosome,
dpk21, we also used an
independent chromosome that had a deficiency uncovering the entire
parkin gene, Df(3L) pc-MK (Bloomington, stock BL-3068).
UAS-HA::parkin transgene was built using the pUAST vector
with an engineered HA-tag sequence (courtesy or Beril Tavsanli) fused
to the parkin-coding sequence from SD01679 EST clone. This EST clone
contained an A
T difference at base pair 760 (of 1446) of the coding
sequence compared to the genome database and our genomic phage clones.
Therefore, we replaced a NarI fragment of SD01679 with the same
fragment from EST clone AT25577 to correct for this change.
|
Immunohistochemistry
Whole-mount immunohistochemistry was performed as previously described
(Davis et al., 2003). Brains
from parkin mutant and heterozygous adults, aged 2 and 21 days post
eclosion, were fixed in PLP, washed in PAXDG and stained with rabbit
polyclonal anti-Drosophila-tyrosine hydroxylase antibody (courtesy of
Wendi Neckameyer) (Mardon et al.,
1994
; Neckameyer et al.,
2001
). Subsequently, brains were stained with Alexa goat
anti-rabbit antibodies in PAXDG. Brains were mounted in Vectashield and
dorsomedial clusters identified and the number of cells counted using confocal
microscopy (Zeiss LSM 510 microscope).
Light microscopy and TEM muscle fiber analysis
Two-day old adult flies were dissected by removing the head and abdomen in
ice cold fixative (2% paraformaldehyde/3% glutaraldehyde/0.1 M sodium
cacodylate/0.05% CaCl2). Whole thoraces were fixed overnight at
4°C and then washed with 0.1 M cacodylate. Postfixation was performed with
2% OsO4 for 2 hours at room temperature and the sample was washed
with water before going through a series of progressive dehydration steps in
ethanol:water mixtures. The sample was then embedded in Spurr's resin
(Spurr, 1969). Thoraces were
then trimmed to retain only the dorsal longitudinal muscles. Ultrathin
sections of 50 nm were then made with a diamond knife and stained with
Toluidine Blue dye, which labels nucleic acids, thereby marking both nuclei
and cytoplasm (Balabanova et al.,
2003
). For TEM, sections were stained on the grid first with 4%
uranyl acetate, then with 2.5% lead nitrate, five minutes each at room
temperature. The sections were observed with a JEOL 1010 transmission electron
microscope. Late pupae were removed from their pupal cases in the same
fixative as above and processed as described above.
Courtship behavior and fertility assays
The courtship behavior assay was carried out on parkin mutant and
heterozygous males that were aged individually for one week as previously
described (Hall, 1994).
Individual virgin w- females, comparable in age to the
males, were placed in a food vial with individual males, and the elements of
the courtship behavioral sequence were noted for a total of 1 hour. Mating
pairs were considered to be successful when the courtship behavior ended with
copulation. Fertility was assayed by placing single parkin males with
3-4 virgin w- females and by placing single virgin parkin
females with two w- males. Vials were then checked 3-7 days later
for the presence of larvae.
Mass and cell size analysis
Mass analysis was performed as described
(Bohni et al., 1999). Twenty
individual males and females were weighed on a precision scale (range 0.01 mg
to 50 g; Mettler Toledo AG245). In addition, groups of 20 and 30-100 flies of
each genotype were weighed to confirm the accuracy of the individual fly
weighing. Flies were reared under the same conditions and were age matched
(48-72 hours post eclosion). Cell counts to determine the differences in cell
size in the wing were assessed by counting the number of wing hairs on the
dorsal wing surface in the same arbitrary area just posterior to the posterior
cross vein for all genotypes (n=20 wings/genotype,
P<0.001 one-way ANOVA test). Wing area was determined using NIH
image 1.60 software
(http://rsb.info.nih.gov/nih-image/Default.html)
(n=20 wings/genotype, P<0.001 one-way ANOVA test).
Jump/flight analysis
Flies were anesthetized with CO2 and individually placed in
vials 24 hours before the assays were conducted to allow time for a full
recovery from effects of CO2. Each fly was dispelled into a plastic
petri dish by gently tapping the vial. The dish was covered with a lid to
prevent flies from escaping. The petri dish was then tapped lightly on the
bench to initiate a jumping reaction. Flight tests were performed by removing
the lid of the petri dish and inverting the dish at a height of 29 inches. The
lid was lightly tapped to loosen the fly. Flies either fell in a straight
line, fell by veering or flew. The number of flies exhibiting each behavior
was recorded. Twenty flies of each genotype were assayed.
Oxidative stress toxicity
Paraquat (methyl viologen) toxicity was performed as previously described
(Clancy et al., 2001). Two-day
old animals were placed in vials (10 per vial) with food media made of 0.8%
agar containing 5% sucrose and 2 mM paraquat (Sigma). Control flies for each
genotype were placed under the same conditions minus paraquat. All experiments
were performed in the dark. Animals were scored daily and the media was
replaced every 3-4 days.
Cold stress
Resistance to cold stress of 2-day old flies was performed as previously
described (Mockett et al.,
2003). Flies were placed (n=10) in a vial and kept at
4°C, except for a 1 hour recovery period (25°C) daily prior to scoring
mortality.
Longevity
Flies were maintained on standard media at 25°C, ten per vial, and
transferred to new food medium daily. Mortality was scored daily.
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Results |
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parkin is expressed at multiple stages of development
To determine the developmental expression of parkin we performed
northern blot analysis of embryonic, larval, pupal and adult RNAs. This
reveals a 1.6 kb transcript, which is similar in length to the longest
parkin EST clone identified (SD01679)
(Fig. 1C). parkin
transcript is first detected early (0- to 2-hour-old embryos) and is probably
due to a maternal contribution. Although parkin transcript is not
detected at high levels during later embryonic stages, it is present in third
instar larvae, as well as throughout pupal development and into adulthood
(Fig. 1C).
parkin mutants have reduced body mass, cell size, and cell number
We used P-element mutagenesis to generate null mutations in parkin
(Fig. 2A,B)
(Kaiser and Goodwin, 1990). A
P-element located 26 kb 3' of parkin [EP(3)3515] was
used to conduct a local hop screen for new insertions in or near the
parkin locus (see Materials and methods)
(Fig. 2A). We obtained a new
insertion 1 kb 3' of parkin, termed dpkP30. After
precisely excising the starting element [EP(3)3515], the new element
(dpkP30) was mobilized to generate new insertions in and
flanking parkin (dpkP21, dpkP23, and
dpkP24) (Fig.
2B and data not shown). Subsequent simultaneous mobilization of
dpkP30 and dpkP21 resulted in deletion
of the entire parkin-coding region. PCR and Southern analyses were
used to confirm deletion of the gene (data not shown). We also obtained two
insertions directly in the parkin-coding region,
dpkP23 and dpkP24, which behave
genetically as null alleles (data not shown). To confirm that observed
phenotypes are specific to parkin we generated two rescue transgenes.
The first transgene contains parkin and 10-12 kb flanking it on
either side. The second transgene is identical to the first with the exception
that stop codons were introduced in the coding sequence of parkin
shortly after the translational start site. Rescue by the first transgene,
dpkR (Rescue), but not the second, dpkSR (Stop
Rescue), indicates that observed phenotypes are specific to loss of
parkin function.
parkin mutant animals are homozygous viable but eclose one day later than heterozygous controls. In addition, loss of parkin results in a significant pupal lethality (Fig. 2C). Although there is little or no lethality prior to pupation, 68% of parkin mutant animals fail to eclose from their pupal cases, suggesting that parkin plays an important role during pupal development (Fig. 2C). parkin null animals that do eclose exhibit a significant reduction in body size (Fig. 3A). Mutant flies present a mass a reduction of 34% in females and 30% in males when compared with controls of the same age and sex. This phenotype is partially but significantly rescued with dpkR but not dpkSR transgenes, confirming that the mass reduction is due to parkin and not any of the surrounding genes that could have been affected by the parkin deletion (Fig. 3B).
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Loss of parkin results in infertility, apoptotic indirect flight muscle degeneration, and mitochondrial dysfunction
Previous work has shown that parkin is required for male fertility
and in particular for proper sperm maturation
(Greene et al., 2003). We
confirmed that parkin mutant males are sterile and have found that
females are infertile as well. Neither sex yields progeny when crossed with
wild-type animals of the opposite sex. Furthermore, only three out of 20
mutant males exhibited one of seven tested courtship behaviors (data not
shown, see Materials and methods) (Hall,
1994
). This suggests that parkin is important in either
sexual behavior, function or both.
We also confirmed that parkin null animals are unable to fly
(Table 2) and exhibit a drooped
wing phenotype: mutant wings are positioned at an approximately 45° angle
to the ground, while wild-type flies hold their wings parallel to the ground
(Greene et al., 2003). To
investigate the flight and drooped wing phenotypes, we analyzed thoracic
indirect flight muscles (IFMs) using light microscopic analysis of thin resin
sections and transmission electron microscopy (TEM) at both pupal and adult
stages (Figs 5,
6). At a late pupal stage (94
hours after pupation) all IFMs appear normal
(Fig. 5A-D). By contrast, 2
days post eclosion parkin mutant animals have significant IFM
degeneration (Fig. 5F,H, asterisks). By 11 days, most of the IFMs show signs of degeneration
(Fig, 5J,L, asterisks).
Heterozygous controls and mutant animals containing the rescue transgene,
dpkR, did not show any signs of a muscle loss, while parkin
mutants carrying the dpkSR transgene were not rescued
(Fig. 5E,G,I,K).
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parkin mutants do not show an age dependent dopaminergic neuronal loss
The pathological presentation of familial cases of Parkinson disease is an
age-dependent degeneration of dopaminergic neurons in the substantia nigra of
the midbrain (Cotran, 1999). Therefore, we were interested to determine if
there was a corresponding age-dependent loss of dopaminergic neurons in
parkin mutant animals. We specifically focused on the dorsomedial
clusters (DMC) of the Drosophila brain dopaminergic system. DMC
neurons were previously reported to be affected in transgenic
Drosophila misexpressing -Synuclein and Pael
receptor. These clusters contain 14-18 tyrosine hydroxylase (TH)-positive
cells that were shown to degenerate in an age-dependent manner in the
previously reported transgenic models
(Auluck and Bonini, 2002
;
Auluck et al., 2002
;
Feany and Bender, 2000
;
Yang et al., 2003
). To assay
DA neurons in these clusters, we used whole-mount brain staining and confocal
microscopy (Fig. 7A,B). We
analyzed 2- to 3-day-old and 21-day-old animals, and did not observe any
significant difference in the number of TH-positive cells
(Fig. 7C). Furthermore, we did
not observe any defects in DA cell body appearance. These findings are
consistent with previously published results in flies and mice, and suggest
that parkin is not essential for DA neuron survival at least during
the 21 days post eclosion (Greene et al.,
2003
; Itier et al.,
2003
). In addition, parkin mutants do not have an
abnormal electroretinogram (ERG) shortly after eclosion or at 2 weeks of age
(data not shown). This suggests that photoreceptor neurons are also
intact.
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Discussion |
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parkin mutant animals have reduced body and cell size at eclosion,
suggesting possible defects in cell growth, proliferation and/or cell
survival. This phenotype is particularly interesting given the interaction of
human Parkin with cyclin E, an important regulator of cell cycle progression
(Staropoli et al., 2003). The
reduced body size of parkin mutants is similar to the phenotypes of
insulin growth factor (IGF) receptor mutant flies and mice
(Bruning et al., 2000
).
Physiological effects of insulin in the brain are not limited to regulation of
food intake and control of glucose uptake, but are also important in trophic
actions on neurons and glial cells. Administration of the N-terminal
tripeptide of IGF1 prevents loss of DA neurons after chemically
(6-hydroxydopamine)-induced DA cell lesion in rats
(Guan et al., 2000
). In
addition, IGF1 also protects against DA-induced neurotoxicity in vitro
(Offen et al., 2001
). This,
together with our data, suggests parkin may play a role in the
insulin signaling pathway during development or in adults.
Our results show that parkin mutant flies have increased
sensitivity to paraquat toxicity. Paraquat, with its two N-methyl pyridinium
moieties, is structurally similar to MPP(+), a toxic metabolite of the MPTP.
In humans, toxins such as MPTP cause DA neuron-specific death in the
substantia nigra because production of oxygen radicals, resulting in
Parkinsonian symptoms. Glutathione and superoxide dismutase (SOD) inactivate
H2O2 and superoxide radicals, respectively, thereby
reducing MPTP neurotoxicity in mice
(Przedborski et al., 2001).
Paraquat also causes high oxygen radical production and interferes with
mitochondrial respiration, resulting in cell death. The effects of paraquat
are not specific to DA cells and sensitivity to its toxicity reflects a
general impairment of oxygen radical defense
(Ding and Keller, 2001
;
Phillips et al., 1989
). Loss
of Drosophila SOD also results in sensitivity to low doses of
paraquat, reflecting a similar defect in oxygen radical defense
(Kirby et al., 2002
;
Phillips et al., 1989
). Our
results suggest that antioxidant defenses in parkin mutant flies are
also impaired and that parkin may play a role in the oxidative stress
response. One likely mechanism of Parkin function is to help rid the cell of
specific proteins that are misfolded because of oxygen radical damage. This is
consistent with the identification of human Parkin as an E3 ligase
(Imai et al., 2000
;
Shimura et al., 2000
;
Staropoli et al., 2003
;
Zhang et al., 2000
).
parkin mutant flies show a progressive apoptotic degeneration of
indirect flight muscles (IFMs). Furthermore, this degeneration is accompanied
by mitochondrial disintegration and loss of christae
(Greene et al., 2003).
Drosophila IFMs are groups of specialized muscle that are in a
constant state of vibration. They require a high oxygen supply to sustain
their respiratory activity, making this tissue especially susceptible to
mitochondrial dysfunction. It is possible that the mitochondrial degeneration
we observe in parkin mutants results in increased susceptibility to
oxygen radical damage because of the impairment of antioxidant defenses by
mitochondria, culminating in cell death. The data from animal and tissue
culture models of PD outlined above suggest that mitochondrial dysfunction and
oxygen radical damage are two crucial factors in the development of PD
pathology. In addition, they also underscore the importance of the apoptotic
pathway in DA cell loss. Oxidative stress such as that produced by MPTP can
trigger apoptosis (Kruman et al.,
1998
). Transgenic mice overexpressing the anti-apoptotic gene
Bcl2 and mice null for the proapoptotic gene Bax are
resistant to MPTP (Offen et al.,
1998
; Vila et al.,
2001
). Loss of parkin results in similar phenotypes in
flies: increased sensitivity to oxygen radical stress and IFM apoptosis,
suggesting that underlying mechanisms of cellular dysfunction maybe be similar
between flies and humans.
Individuals with AR-JP exhibit a loss of DA neurons in the substantia nigra
similar to that of idiopathic PD. Therefore, we expected to see a similar
phenotype in parkin mutant animals. We focused on the dorsomedial
cluster (DMC) neurons because they were shown to be affected in other
Drosophila models of PD (Auluck et
al., 2002; Feany and Bender,
2000
). However, we do not observe any significant loss of DA
neurons at three weeks of age in parkin mutant animals compared with
controls. In addition, in contrast to a previously published report, we do not
see any changes in DA cell morphology in parkin mutants
(Greene et al., 2003
). Loss of
mouse PARK2 also does not result in DA cell loss. However, increased
extracellular DA concentration, abnormal neurophysiology and motor and
cognitive behavioral deficits are observed in PARK2 mutant mice
(Goldberg et al., 2003
;
Itier et al., 2003
). By
contrast, only motor deficits have been observed thus far in
Drosophila (Greene et al.,
2003
). In another model of PD, overexpression of
-synuclein
in mice does not result in DA cell loss but does cause DA neuron dysfunction
(Giasson et al., 2002
;
Masliah et al., 2000
;
van Der Putten et al., 2000
).
Similarly, in Alzheimer's disease mouse models employing overexpression of
amyloid precursor protein, cognitive defects are present in the absence of
cortical neuronal loss (Hock and Lamb,
2001
). The absence of cell loss in multiple models of
neurodegenerative disease may reflect the shorter life span of flies and mice
compared with humans. Although the mean onset of PD symptoms in AR-JP is the
earliest of all genetically defined forms of Parkinson's disease, it is not
clear how to compare fly, mouse and human life spans in this regard
(Gasser, 1998
).
Even though we do not observe DA loss in parkin mutant animals, the mechanism of cell loss may be similar between humans and Drosophila. Neurons and muscle are two of the most energy-dependent tissues, and therefore have high numbers of mitochondria and are highly sensitive to mitochondrial insults. Although the mechanisms of cell death in IFMs and in DA neurons might be similar, it is possible that Drosophila IFMs are more sensitive than DA neurons to mitochondrial defects. The nature of this defect is still unclear; however, three mechanisms are likely. First, it is possible that parkin functions in a trophic factor pathway that promotes cell survival, and in its absence cells become more susceptible to insults such as oxygen radicals. Second, parkin might be important in the stress response pathway, and in its absence the cell becomes more susceptible to various stimuli such as oxygen radical damage that trigger apoptosis. Finally, it is possible that parkin is part of the cell death pathway, and its absence results in susceptibility to proapoptotic insults. Drosophila parkin mutants will serve as an invaluable model for understanding the biological role of parkin and may provide important clues concerning the molecular mechanisms of PD.
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ACKNOWLEDGMENTS |
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