Activation of FoxO by LRRK2 induces expression
of proapoptotic proteins and alters survival of
postmitotic dopaminergic neuron in Drosophila
Tomoko Kanao1, Katerina Venderova2, David S. Park2, Terry Unterman3, Bingwei Lu4,5
and Yuzuru Imai1,∗
1Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-Machi, Aoba-ku, Sendai 980-8575,
Japan,2Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road,
Ottawa, ON, Canada K1H 8M5,3University of Illinois College of Medicine/Chicago Area Veterans Health Care
System, Chicago, IL 60612, USA,4Department of Pathology, Stanford University School of Medicine, Stanford, CA
94305, USA and5Geriatric Research, Education and Clinical Center/Veterans Affairs Palo Alto Health Care System,
Palo Alto, CA 94304, USA
Received June 10, 2010; Revised and Accepted July 7, 2010
Missense mutations in leucine-rich repeat kinase 2 (LRRK2)/Dardarin gene, the product of which encodes a
kinase with multiple domains, are known to cause autosomal dominant late onset Parkinson’s disease (PD).
In the current study, we report that the gene product LRRK2 directly phosphorylates the forkhead box tran-
scription factor FoxO1 and enhances its transcriptional activity. This pathway was found to be conserved in
Drosophila, as the Drosophila LRRK2 homolog (dLRRK) enhanced the neuronal toxicity of FoxO. Importantly,
FoxO mutants that were resistant to LRRK2/dLRRK-induced phosphorylation suppressed this neurotoxicity.
Moreover, we have determined that FoxO targets hid and bim in Drosophila and human, respectively, are
responsible for the LRRK2/dLRRK-mediated cell death. These data suggest that the cell death molecules
regulated by FoxO are key factors during the neurodegeneration in LRRK2-linked PD.
Missense mutations in the Leucine-rich repeat kinase 2
(LRRK2) gene cause autosomal dominant late onset familial
Parkinson’s disease (PD). This form of PD demonstrates a
diverse pathology and includes the progressive loss of nigros-
triatal dopaminergic (DA) neurons, a-synucleinopathy and
tauopathy (1,2). The clinical symptoms and pathology
caused by LRRK2 mutations resemble those of the sporadic
form of PD, suggesting that the LRRK2 pathogenic pathway
may underlie general PD etiology. This is further supported
by a recent finding that several LRRK2 variants increase sus-
ceptibility to sporadic PD. The LRRK2 gene encodes a multi-
domain protein belonging to the ROCO family proteins. The
LRRK2 protein contains a GTPase domain called Ras of
complex proteins (Roc) domain, a conserved C-terminal of
Roc (COR) domain and a kinase domain. Furthermore,
LRRK2 harbors leucine-rich repeats (LRR) at its N-terminus
and WD40 repeats at the C-terminus (1,2).
Several amino acid substitutions have been identified
throughout the multiple domains, which include R1441G,
Y1699C, G2019S and I2020T mutations (3). Numerous patho-
logical substitutions in the kinase domain of full-length
LRRK2 such as G2019S and I2020T have been reported to
show moderately enhanced kinase activity in vitro (4–6).
However, how these mutations cause the progressive loss of
DA neurons and the other associated pathologies is currently
We have previously reported that transgenic expression of
the Drosophila LRRK2 orthologue (dLRRK) that contains
PD-associated mutations leads to DA neuronal loss in the Dro-
sophila brain (6). LRRK2 has been shown to phosphorylate
eukaryotic translation initiation factor 4E (eIF4E)-binding
protein (4E-BP), which modulates stress sensitivity and DA
∗To whom correspondence should be addressed. Tel/Fax: +81 227178490; Email: email@example.com
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Human Molecular Genetics, 2010, Vol. 19, No. 19
Advance Access published on July 12, 2010
at University of Ottawa on June 13, 2011
neuron survival in Drosophila. Forkhead transcription factor
FoxO, which controls various cellular processes involved in
cell cycle, cell death, metabolism and oxidative stress, regu-
lates 4E-BP transcription (7,8). The effects of FoxO are depen-
dent on the cellular context and cell type, as well as the
cofactors involved. FoxO controls redox metabolism by the
expression of anti-oxidative stress molecules, such as manga-
nese superoxide dismutase and catalase, which may affect the
lifespan of animals. On the other hand, cell death-related mol-
ecules, such as Hid, Bim, PUMA and Fas ligand, are induced
by FoxO in the cells destined to die.
In the current study, we report that FoxO is also phosphory-
lated by LRRK2, which in turn upregulates cell death regula-
tors, Hid and Bim in Drosophila and in human cells,
respectively. Moreover, our data suggest that the activation
of these molecules contribute to DA neurodegeneration fol-
lowing LRRK2 mutation.
LRRK2/dLRRK genetically interacts with FoxO
We have previously reported that LRRK2 phosphorylates
4E-BP, which in turn promotes the translation of numerous pro-
teins (6). While investigating the levels of Drosophila (d4E-BP)
expressed indLRRK mutants,wefound that the levelofd4E-BP
transcript was affected by dLRRK protein expression (Sup-
plementary Material, Fig. S1), even though differences in
d4E-BP protein level were not apparent (data not shown). It
has been reported that 4E-BP transcription is partly regulated
by FoxO (7,9). We then tested whether LRRK2 modulates
FoxO activity using Drosophila genetics (Fig. 1). Overexpres-
sion of Drosophila FoxO (dFoxO) in the fly eye imaginal disc
resulted in mild developmental defects in the eye (Fig. 1B).
This result was consistent with that reported previously (7,8).
Transgenic expression of either dLRRK or human LRRK2
(hLRRK2), even pathogenic mutants such as dLRRK I1915T
and hLRRK2 I2020T, in the eyes resulted in no apparent
degeneration (Fig. 1F and G, and Supplementary Material,
Fig. S2A and E), a result that has also been reported previously
(6). However, the dFoxO-mediated eye defect was significantly
worsened by the expression of either a wild-type (WT) dLRRK
or a PD-related mutant (I1915T corresponding to I2020T in
human) (Fig. 1C and D). Similar results were obtained in
dFoxO crosses combined with dLRRK harboring R1069G
mutation in the GTPase domain (corresponding to a pathogenic
G). In contrast, the effects of the kinase-dead form of dLRRK
(3KD) (6) were not as dramatic (Fig. 1E and Supplementary
Material, Fig. S2D). Similar results were also obtained when
hLRRK2 WT or PD-related mutant I2020T were co-expressed
with dFoxO (Fig. 1H and I). We found that dLRRK exhibited
no effect on eye degeneration induced by transgenic expression
of another forkhead box protein FoxA (Supplementary Material,
maintenance of the midbrain DA neurons in the mammalian
central nervous system (10). These results suggest that
hLRRK2/dLRRK specifically enhances the toxicity of FoxO
through its kinase activity.
Figure 1. LRRK2 enhances FoxO-mediated developmental defects in the Drosophila eye. The genotypes are: GMR-Gal4/UAS-EGFP (A), GMR-Gal4,
UAS-dFoxO/UAS-EGFP (B), GMR-Gal4, UAS-dFoxO/+; UAS-dLRRK WT/+ (C), GMR-Gal4, UAS-dFoxO/+; UAS-dLRRK I1915T/+ (D), GMR-Gal4,
UAS-dFoxO/+; UAS-dLRRK 3KD/+ (E), GMR-Gal4/+; UAS-dLRRK I1915T/+ (F), GMR-Gal4/+; UAS-hLRRK2 I2020T/+ (G), GMR-Gal4, UAS-dFoxO/+;
UAS-hLRRK2 WT/+ (H), GMR-Gal4, UAS-dFoxO; UAS-hLRRK2 I2020T/+ (I), GMR-Gal4, UAS-dFoxO/UAS-AKT1 (J).
3748 Human Molecular Genetics, 2010, Vol. 19, No. 19
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LRRK2/dLRRK activates FoxO through
FoxO activity has been shown to be regulated by acetylation,
ubiquitination and phosphorylation (11). We did not observe
any effects on the development of the eye phenotype caused
by dFoxO when overexpressed the FoxO deacetylase Sir2 or
removed one copy of the ubiquitin gene (Supplementary
Material, Fig. S4). The results may suggest that the acetylation
and ubiquitination pathways are not involved in the modu-
lation of dFoxO activity at least in the eye development.
Therefore, hLRRK2/dLRRK appeared likely to modulate
one of the phosphorylation pathways associated with FoxO
activity. We next investigated whether LRRK2 directly phos-
phorylated mammalian FoxO1. In vitro kinase assays
suggested that hLRRK2, but not the kinase-dead form 3KD
(6), may act as a FoxO kinase (Fig. 2A). Screening with
several mutant FoxO1 proteins revealed that the replacement
of the serine 319 residue with alanine (S319A) reduced the
phosphorylation signals generated by hLRRK2 (Fig. 2B and
Supplementary Material, Fig. S5). The S319 residue was
shown to be highly conserved among the mammalian FoxO
family and dFoxO (Fig. 2C). As expected, dLRRK also phos-
ophorylated dFoxO in in vitro kinase assay, and the introduc-
tion of dFoxO S259A mutation, which corresponds to S319A
in human FoxO1, reduced the phosphorylation signals gener-
ated by dLRRK (Fig. 2D). Overexpression of hLRRK2 (WT
or pathogenic G2019S), but not 3KD, was found to specifi-
cally stimulate phosphorylation of FoxO1 at S319 (pS319)
in cultured HEK 293T cells (Fig. 2E). We generated anti-
dFoxO antibody, which specifically recognized endogenous
dFoxO (Supplementary Material, Fig. S6A). In Drosophila,
the level of phosphorylation of endogenous dFoxO (FoxO-P)
was increased by dLRRK WT or I1915T overexpression
while abolished by 3KD or the loss of the dLRRK gene
(Fig. 2F). Reporter assays for FoxO activity suggested that
hLRRK2 WT, but not 3KD, stimulates transcriptional activity
of FoxO1 (Fig. 3A), whereas the S319A mutation in FoxO1
impairs hLRRK2-mediated activation of FoxO1 (Fig. 3B).
The expression of dFoxO S259A has exhibited milder toxicity
and has significantly attenuated dFoxO/dLRRK-mediated eye
degeneration (Supplementary Material, Fig. S6). In contrast,
a phospho-mimic mutant dFoxO S259E enhanced the eye phe-
notype (Supplementary Material, Fig. S6E). These results indi-
cate that hLRRK2/dLRRK activates FoxO through its
phosphorylation. We next examined whether the expression
of dLRRK 3KD does not affect the eye phenotype produced
by dFoxO S259A. Unexpectedly, the phenotype was dramati-
cally exacerbated, so that the flies came out only under a
milder gene expression condition, suggesting the existence
of a negative regulation pathway(s) mediated by LRRK2 in
FoxO activation (Supplementary Material, Fig. S6H).
Co-expression of pathogenic dLRRK and FoxO is toxic
to post-mitotic DA neurons
Endogenous dFoxO is widely expressed in the Drosophila
central nervous system, and is observed in the tyrosine
hydroxylase (TH)-positive neurons (Supplementary Material,
Fig. S7) while the expression of transgenic dFoxO during
the development of tissues is apparently toxic, as shown in
Figure 1. To examine the effects of FoxO in post-mitotic
neurons, we employed the mifepristone-inducible GAL4
system (GeneSwitch-GAL4) that drives the tissue-specific
expression of upstream activating sequence (UAS)-constructs
(Fig. 4A). To reduce the probability of non-specific neurotoxi-
city by conventional overexpression and to modulate the
signal pathway more specifically, Drosophila crosses was
treated with 25 mg/ml of RU486 throughout the lifespan
(refer to lanes 2 in Fig. 4A). Pan-neuronal expression of
dFoxO in WT (Fig. 4B) or dLRRK null (Fig. 4C) adult Droso-
phila had no effect on survival. The effect of dFoxO on neur-
onal cells was consistent with that reported previously (12).
The combined expression of dLRRK WT and dFoxO had
little effecton thelifespan
co-expression of pathogenic dLRRK and dFoxO significantly
shortened lifespan (Fig. 4E and F). The number of some clus-
ters of TH-positive neurons observed in adult Drosophila
co-expressing dFoxO and dLRRK was found to decrease in
an age-dependent manner (Fig. 5A and Supplementary
Material, Fig. S8). Consistent with the results of the lifespan
assay and the viability of TH-positive neurons, the motor
activity of the flies pan-neuronally expressing dFoxO/
dLRRK I1915T by the elav-GeneSwitch-GAL4 (elav-GS)
driver was more impaired with age than that of flies expressing
elav-GS, dFoxO or dLRRK I1915T alone (Fig. 5B). Treatment
with 1 mM L-3,4-dihydroxyphenylalanine (L-DOPA) signifi-
cantly improved the locomotor activity of dFoxO/dLRRK
I1915T-expressing flies (Fig. 5B). In this context, the introduc-
tion of the S259A mutation in dFoxO attenuated the toxic
interaction of pathogenic dLRRK and dFoxO in both lifespan
and TH-neuronal loss (Figs 4G and 5C). These results suggest
that dLRRK affects DA neuron survival through phosphoryl-
ation of dFoxO at the S259 residue.
(Fig.4D), whereas the
FoxO targets hid and bim affect viability of post-mitotic
We next examined which target of FoxO contributes to the
FoxO/LRRK2-mediated neurotoxicity. A lot of transcriptional
targets of FoxO have been characterized, which includes the
molecules involved in cell cycle arrest, oxidative stress resist-
ance, programmed cell death and metabolism (11). We tested
the reported FoxO targets, which may be involved in the neur-
onal maintenance by a combined screening of the Drosophila
eye assay and quantitative RT–PCR (qRT–PCR), and deter-
mined the proapoptotic hid gene as a responsible target. Over-
expression of hid caused dramatic eye degeneration as
reported (Fig. 6A) (13), whereas the removal of one copy of
hid genes significantly improved the eye phenotype of
dFoxO/dLRRK I1915T co-expression (Fig. 6B compared
with Fig. 1D). Drosophila inhibitor of apoptosis (DIAP) inhi-
bits cell death program by promoting the degradation of an
initiator caspase Dronc (13). Hid inactivates DIAP through
binding, which in turn activates the caspase activity and exe-
cutes cell death (13). Heterozygosity for DIAP promoted
loss of the ommatidia, while reduction in the gene dose of
Dronc partially rescued the eye phenotype of dFoxO/dLRRK
I1915T co-expression (Fig. 6C and D). dLRRK stimulated
expression of hid transcripts in Drosophila and a pathogenic
Human Molecular Genetics, 2010, Vol. 19, No. 193749
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Figure 2. LRRK2/dLRRK phosphorylates FoxO. (A and B) In vitro kinase assay of hLRRK2 using recombinant GST-FoxO1 as a substrate. hLRRK2 WT or
3KD was immunoprecipitated from hLRRK2-transfected HEK293T cells for kinase sources. Mock immunoprecipitate (Mock) served as a control. Autoradiog-
raphy (P32) and Coomassie brilliant blue (CBB) staining of the gels are shown. (B) FoxO1 S319 represents a major phosphorylation site for hLRRK2 in vitro. (C)
Alignment of putative hLRRK2/dLRRK target sequences in the FoxO family. The arrow indicates the potential phosphorylation sites. (D) In vitro kinase assay of
dLRRK using recombinant GST-dFoxO and its mutant S259A as substrates. The assay was performed as in (B). (E) Lysate from 293T cells transfected with
hLRRK2 WT, PD mutant (G2019S) and 3KD or b-galactosidase (control) was treated with or without dephosphorylation reaction (CIP) and analyzed by western
blot. Overexpression of hLRRK2 with kinase activity stimulated phosphorylation of the S319 residue in endogenous FoxO1. The graph shows relative levels of
phospho-S319 (pS319) after normalization with total FoxO1 levels. Data are presented as mean+SE of three experiments (∗∗P , 0.01, Tukey–Kramer test).
(F) The levels of dFoxO phosphorylation are decreased in the dLRRK null fly. Fly brain extracts treated with (+) or without (2) dephosphorylation reaction with
alkaline phosphatase (CIP) were subjected to western blot analysis using dFoxO antibody (left panel). Endogenous dFoxO expression was analyzed in the flies
harboring the UAS genes (EGFP, dLRRK, dLRRK I1915T and dLRRK 3KD) crossed with the ubiquitous daughterless (Da)-Gal4 driver (right), dLRRK(+/+)
and dLRRK(2/2) (middle). Bands corresponding to phosphorylated (dFoxO-P) and non-phosphorylated forms (dFoxO) of dFoxO are indicated. Actin signals
indicate that equivalent amounts of lysates were loaded. The genotypes of dLRRK(+/+) and dLRRK(2/2) are w2and dLRRKe03680/dLRRKe03680, respectively.
3750Human Molecular Genetics, 2010, Vol. 19, No. 19
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mutant I1915T had a higher activity (Fig. 6E). Clusters of
FoxO response elements were reported in the first intron of
the hid gene (14). We confirmed that dFoxO binds to the
sites in the first hid intron by chromatin-immunoprecipitation
(ChIP) for endogenous dFoxO in Drosophila S2 cells
(Fig. 6F). Furthermore, a combined expression of dFoxO
S259A along with dLRRK I1915T mutant impaired the
binding of FoxO to the hid gene in S2 cells and subsequent
hid expression in flies, compared with dFoxO WT/dLRRK
I1915T co-expression (Fig. 6G and H). We next examined
whether Hid and its mammalian homologue are involved in
the DA neurodegeneration by FoxO/LRRK2 signaling. DA
neurodegeneration caused by the inducible expression of
dFoxO/dLRRK I1915T in the adult fly brain tissues was sig-
nificantly suppressed on the hid heterozygous genetic back-
ground (Fig. 6I). In mammals, FoxO family regulates the
expression of a proapoptotic Bcl-2 family protein Bim in
various contexts of neuronal death, which is a cellular regu-
lation similar to Hid in Drosophila (15–17). Bim, especially
a shorter isoform Bim-S, stimulates caspase activation by pro-
moting the release of cytochrome c from mitochondria
(18,19). A clear increase of alternative splicing isofoms of
Bim (Bim-L and Bim-S) expression was observed in cells
transfected with hLRRK2 G2019S mutant as well as a consti-
tutive active form of FoxO (Fig. 7A), suggesting that hLRRK2
G2019S activates endogenous FoxO through phosphorylation.
A similar tendency was also seen in hLRRK2 WT-transfected
cells although the difference was not statistically significant
(Fig. 7A). The FoxO/LRRK2-mediated cell death was also
observed in SH-SY5Y cells. Co-transfection of hLRRK2
along with FoxO1 resulted in around 30% of cell death,
which was partially suppressed by one of the mammalian
IAP family proteins XIAP (Fig. 7B). We next examined to
what extent endogenous hLRRK2 and its downstream Bim
contribute to FoxO/LRRK2-mediated cell death in SH-SY5Y
cells. RNA interference (RNAi) against hLRRK2 or Bim
significantly inhibited cell death by FoxO1 (Fig. 7C and D).
Furthermore, a FoxO1 mutant that is resistant to phosphoryl-
ation by LRRK2 (FoxO1 SA) attenuated the cell death
(Fig. 7E). These results suggested that Hid/Bim is required
downstream of FoxO/LRRK2 signaling for neurodegeneration.
Mutations in the LRRK2 gene have been reported to be the
most common cause of familial PD (20). In addition, recent
genome-wide association studies have identified LRRK2 as
well as SNCA/a-synuclein as major risk loci for general PD,
strongly suggesting that LRRK2 signal pathway has a
central pathogenic role across the spectrum of PD (21,22).
However, the physiological and pathogenic functions of
LRRK2 are poorly understood. Here we have found that
LRRK2 phosphorylates and stimulates transcriptional activity
in both human and Drosophila FoxO proteins through phos-
phorylation. Numerous transcriptional targets of FoxO have
been characterized, and include molecules involved in cell
cycle arrest, oxidative stress, programmed cell death and
metabolism (11). We tested by a combination of fly genetics
and qRT–PCR the reported key target molecules that might
have roles in neurodegeneration, which included 4E-BP
(7,8), Polo (23), Cyclin B (23) and Hid (14) (Fig. 6 and Sup-
plementary Material, Fig. S1 and S9). Abnormal activation of
Cyclins and its regulators has been reported to be one of the
causes for DA neuron death (24,25). However, there was no
evidence that Polo and Cyclin B contribute to the neurodegen-
eration by FoxO/dLRRK in Drosophila (Supplementary
Material, Fig. S9). In contrast, hid expression showed the
best correlation with the neuronal loss in this context.
Although hid is not conserved in the mammalian system,
known programmed cell death target molecules such as Bim,
PUMA and Fas ligand are possible candidates for effectors
of the LRRK2/FoxO pathway in humans (11). Indeed, Bim
expression and subsequent cell death were promoted by a
combined expression of FoxO and LRRK2 in the human cul-
tured cells, whereas the knockdown of Bim or overexpression
of XIAP suppressed cell death in this context. Thus, Bim
seems to be a functional homologue of Hid downstream of
LRRK2/FoxO signaling in mammalian system.
We have previously reported that LRRK2 phosphorylates
one of the transcriptional targets of FoxO, 4E-BP, and attenu-
ates 4E-BP function (6). The relationship between LRRK2,
FoxO and 4E-BP appears to be complex. Although LRRK2/
dLRRK stimulates the expression of 4E-BP in parallel with
Bim/Hid through FoxO phosphorylation, our previous study
suggested that LRRK2 promotes the inactivation of a neuro-
protective function of 4E-BP through its phosphorylation.
The idea that LRRK2 has a dual effect on 4E-BP is supported
by a result of the genetic interaction analysis, where the
removal of d4E-BP gene exacerbated the FoxO/dLRRK-
mediated DA neurodegeneration (Supplementary Material,
We have demonstrated that the co-expression of FoxO and
LRRK2/dLRRK causes synergistic effect of neurotoxicity.
However, single expression of PD-related LRRK2/dLRRK
mutants by the GMR-GAL4 driver was not toxic in the eyes,
Figure 3. LRRK2 stimulates FoxO transcriptional activity through the phos-
phorylation of the FoxO S319 site. (A) FoxO transcriptional activity was
measured in extracts prepared from 293T cells transfected with the indicated
plasmids and a plasmid for FoxO1, a FoxO reporter plasmid containing Firefly
luciferase and a plasmid for Renilla luciferase to monitor the transfection effi-
ciency. The relative FoxO transcriptional activity (Firefly luciferase activity)
normalized to Renilla luciferase activity is presented. Data are presented as
the mean+SE from three independent experiments. b-galactosidase (Mock)
served as a transfection control.∗P , 0.05 versus Mock. RT–PCR was per-
formed for the estimation of mRNA levels of Firefly luciferase (luciferase)
and b-actin in the extracts. (B) Introduction of the S319A mutation in
FoxO1 reduced FoxO activity.∗∗P , 0.01.
Human Molecular Genetics, 2010, Vol. 19, No. 193751
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suggesting that the phosphorylation of endogenous FoxO by
pathogenic LRRK2/dLRRK is not sufficient for the retinal
degeneration in developing eyes. Cell death signaling path-
ways are known to have neutralization mechanisms, in
which the balance between death activators and repressors
determines the threshold for cell death. Hid induction by
ectopic expression of LRRK2/dLRRK will lower the threshold
for cell death, neutralizing the cell protective activity of DIAP,
but it might not be sufficient to undergo neurodegeneration in
this context. In fact, we observed age-dependent DA neurode-
generation by pathogenic LRRK2/dLRRK alone (6,26),
suggesting that ageing and cell type are key factors to modu-
late the cell death threshold.
The S319 residue in FoxO1 has been reported to constitute
one of the major phosphorylation sites for the serine–
threonine kinases AKT/protein kinase B and serum- and
glucocorticoid-inducible kinase (SGK) (11). Phosphorylation
of S319 along with T24 and S256 by AKT and SGK has
been shown to stimulate sequential phosphorylation of S322
and S325, which in turn accelerates nuclear export of FoxO1
and suppresses its activity in mammalian cells (27). In
addition, the co-expression of AKT with dFoxO has been
Figure 4. Neuronal activation of dFoxO by dLRRK affects the Drosophila lifespan. Neuron-specific expression of dLRRK and dFoxO was induced following the
administration of the activator RU486 in the elav-GeneSwitch-GAL4 (elav-GS) crosses. (A) Expression levels of the indicated proteins were determined by
western blot analysis. The endogenous protein levels are shown in the non-inducible samples (RU486, 0 mg/ml). LE, longer exposure;∗non-specific bands.
(B–F) Flies from each genotype were subjected to survival assays at 298C. Female adults (n ¼ 85–91) were fed yeast paste containing 25 mg/ml RU486.
dFoxO expression in normal (B: elav-GS. dFoxO versus elav-GS), dLRRK null [C: dLRRK(2/2), dFoxO versus dLRRK(2/2)] or ectopically dLRRK
WT-expressing flies (D: dLRRK WT, dFoxO versus dLRRK WT) had no effect on survival (P . 0.05 by log-rank test). Co-expression of dFoxO with pathogenic
dLRRK mutants (I1915T and Y1383C corresponding to Y1699C in human) shortened the lifespan compared with the expression of the dLRRK mutant alone (E:
I1915T/dFoxO versus I1915T, P , 0.001; F: Y1383C/dFoxO versus Y1383C, P , 0.05), while single expression of dLRRK WT, I1915T or Y1383C had little
effect (elav-GS versus dLRRK WT, I1915T or Y1383C, P . 0.05 by log-rank test). (G) Flies from each genotype (n ¼ 88–90) were subjected to survival assay as
in (B–F). Pan-neuronal expression of dFoxO S259A (SA) alone had no significant effect on the lifespan when compared with that of dFoxO WT. Co-expression
of dFoxO SA with dLRRK I1915T attenuated the effect of dFoxO WT/dLRRK I1915T co-expression on the lifespan. Statistical comparison by log-rank test:
elav-GS versus dFoxO WT, dFoxO WT versus dFoxO SA and dFoxO SA versus dFoxO SA/I1915T, P . 0.05 (not significant). dFoxO SA/I1915T versus dFoxO
WT/I1915T, P ¼ 0.04. The genotypes used were: elav-GS/+ versus UAS-dFoxO/+; elav-GS/+ (B); elav-GS, dLRRKe03680/dLRRKe03680versus UAS-dFoxO/+;
elav-GS, dLRRKe03680/dLRRKe03680(C); UAS-dLRRK WT/+; elav-GS/+ versus UAS-dFoxO/UAS-dLRRK WT; elav-GS/+ (D); UAS-dLRRK I1915T/+;
elav-GS/+ versus UAS-dFoxO/UAS-dLRRK I1915T; elav-GS/+ (E); UAS-dLRRK Y1383C/+; elav-GS/+ versus UAS-dFoxO/UAS-dLRRK Y1383C;
elav-GS/+ (F); UAS-dFoxO/+; elav-GS/+ (dFoxO WT), UAS-dFoxO S259A/+; elav-GS/+ (dFoxO SA), UAS-dFoxO/UAS-dLRRK I1915T; elav-GS/+
(dFoxO WT, I1915T), UAS-dFoxO S259A/UAS-dLRRK I1915T; elav-GS/+ (dFoxO SA, I1915T) (G).
3752 Human Molecular Genetics, 2010, Vol. 19, No. 19
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reported to partially rescue dFoxO-mediated eye degeneration
in Drosophila (8). In our study, we also observed inhibitory
effects of AKT on the eye phenotype in Drosophila
(Fig. 1J), and in a FoxO reporter assay in mammalian cells
(data not shown). In contrast to AKT, LRRK2 appears to
stimulate FoxO activity by selectively targeting S319, and
the phosphorylation status at S322 and S325 was relatively
unchanged (Supplementary Material, Fig. S11). Thus, selec-
tive phosphorylation of FoxO S319 site by LRRK2 may
have a novel effect on FoxO function. For example, specific
phosphorylation of S319 might stimulate the recruitment of
a co-factor(s) of dFoxO, which may activate cell death
targets of FoxO preferentially.
In contrast to the results reported here, Tain et al. (28) have
demonstrated that FoxO protects DA neurons in a parkin-linked
PD model fly. We speculate that the inconsistency comes from
differences in a primary cause of neurodegeneration. Loss of
parkin causes mitochondrial degeneration in tissues with high-
energy demands in Drosophila. Since it has been reported that
the genes for mitochondrial biogenesis are regulated by FoxO in
Drosophila (29), FoxO might compensate for the mitochondrial
loss by parkin inactivation via the expression of those genes as
well as 4E-BP. Thus the effects of FoxO appear to be context-
We demonstrated that pan-neuronal expression of dFoxO
and PD-associated mutant dLRRK leads to motor dysfunction
and degeneration of DA neurons in Drosophila, suggesting
that our findings might be relevant to clinical and pathological
features of PD. However, we also observed similar degener-
ation phenotypes in retinal neurons of developing eye tissues
by higher expression of dFoxO and LRRK2/dLRRK (Fig. 1).
Considering relatively ubiquitous distribution of LRRK2/
dLRRK and FoxO in human and Drosophila brain tissues
(Supplementary Material, Fig. S7) (6,30,31), we speculate
Figure 5. dFoxO activated by dLRRK affects the maintenance of DA neurons in Drosophila. (A) Graph presents the number of PAL, PPM 1 and 2, PPM3, PPL1,
PPL2 and VUM clusters of TH-positive neurons in 24-day-old adult flies treated as in Figure 4B–F. PPM1 and PPM2 cluster neurons were counted together.
Data are presented as the mean+SE from three repeated experiments (∗P , 0.05;∗∗P , 0.01). The total number of flies examined is shown in parentheses.
PAL, protocerebral anterior lateral; PPM, protocerebral posterior medial; PPL, the protocerebral posterior lateral; VUM, ventral unpaired medial. (B) Adult
aged flies expressing dFoxO/dLRRK I1915T under the elav-GS control showed motor defect, whereas single expression of dFoxO or dLRRK I1915T had
little effect. Treatment with 1 mM L-DOPA in phosphate-buffered saline (PBS), but not with PBS only, for 4 days rescued the loss of climbing ability in
dFoxO/dLRRK I1915T-expressing flies. The values represent means+SE from 20 trials in six independent experiments (∗P , 0.05;∗∗P , 0.01). (C) Graph
shows the number of TH-positive neurons in 24-day-old adult flies as treated in Figure 4G. Data are presented as the mean+SE from three repeated experi-
ments. The differences between I1915T/dFoxO SA and I1915T in each cluster of DA neurons were not significant. The total number of flies examined is shown in
Human Molecular Genetics, 2010, Vol. 19, No. 193753
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that a sensitivity threshold for cell death could determine
selective degeneration of DA neurons as well as other types
of neurons. It is widely accepted that early non-motor signs,
which include neuropsychiatric complications, autonomic dis-
orders, sleep disturbances and sensory symptoms, precede the
onset of motor symptoms in PD. Although the pathophysiol-
ogy of non-motor symptoms is still poorly understood, the
degeneration of both DA and non-DA systems is thought to
contribute to their pathogenesis. Thus the FoxO-LRRK2
pathway could be a common cell death pathway in various
Figure 6. A FoxO target Hid is responsible for dLRRK-mediated DA neurodegeneration in Drosophila. (A–D) SEM images of the fly eyes. (A) Ectopic expression
of hid resulted in eye degeneration. Reduction of the gene dose of hid (B) and Dronc (D) improved the eye phenotype of dFoxO/dLRRK I1915T shown in
Figure 1D, whereas the phenotype was exacerbated on a DIAP heterozygous mutant background (C). (E) dLRRK regulates the level of hid transcript. The
amounts of hid and rp49 transcripts were measured by real-time RT–PCR using total RNA from the transgenic flies indicated genotypes crossed with the
Da-Gal4 driver. The amounts of hid transcripts normalized with those of rp49 are graphed. The values are presented as the mean+SE from three independent
experiments. EGFP served as a control.∗P , 0.05;∗∗P , 0.01. (F) FoxO binds to the hid locus. ChIP assay was performed using S2 cells incubated in serum-free
medium for 16 h. FoxO/DNA complex was immunoprecipitated using anti-FoxO antibody or control antibody. The region of the first hid intron containing the
FoxO-binding sites was amplified by PCR. (G) FoxO SA mutation impairs the binding of FoxO to the hid gene. S2 cells transfected with dLRRK I1915T
along with dFoxO WT or SA were subjected to ChIP assay as in (F). (H) FoxO SA mutation impairs FoxO/dLRRK-mediated hid expression. Pan-neuronal
expression of the indicated genes was induced by elav-GS crosses after 100 mg/ml RU486 administration for 4 days. Flies of the indicated genotypes were subjected
to real-time RT–PCR for hid as in (E). The values are presented as the mean+SE from three independent experiments.∗∗P , 0.01. (I) Hid is required for dLRRK/
dFoxO-mediated DA neurodegeneration in Drosophila. Graph shows the number of PPM1/2 and PPL1 TH-positive neurons in 24-day-old adult flies (n ¼ 10) as
treated in Figure 4E. Data are presented as the mean+SE from three repeated experiments.∗P , 0.05;∗∗P , 0.01; N.S., not significant. Fly genotypes in Figure 6
are: GMR-Gal4, UAS-hid/CyO (A), GMR-Gal4, UAS-dFoxO/+, UAS-dLRRK I1915T/hid1(B), GMR-Gal4, UAS-dFoxO/+; UAS-dLRRK I1915T/DIAP11(C),
GMR-Gal4, UAS-dFoxO/+; UAS-dLRRK I1915T/DroncG02994(D), UAS-dFoxO/+; elav-GS/+ [dFoxO], UAS-dFoxO/+; elav-GS/hid1[dFoxO, hid(+/2)],
UAS-dFoxO/UAS-dLRRK I1915T; elav-GS/+ [dFoxO, I1915T], UAS-dFoxO/UAS-dLRRK I1915T; elav-GS/hid1[dFoxO, I1915T, hid(+/2)] (I).
3754Human Molecular Genetics, 2010, Vol. 19, No. 19
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cell types including DA neuron, but the sensitivity to this
signal might be cell-type dependent.
Although the neurotoxic effect of LRRK2/dLRRK appears
to require kinase activity, the expression of a kinase-dead
form of dLRRK 3KD in the eye also had a mild toxic effect
(Fig. 1E compared with B). Previous biochemical studies indi-
cated that a dimeric form of LRRK2 possesses kinase activity
in vitro and that kinase-dead forms of LRRK2 lack the dimer-
ization activity (32,33). Therefore it is unlikely that hLRRK2/
dLRRK 3KD recruits and activates the endogenous hLRRK2/
dLRRK. We recently found that a LRRK2-associated kinase
also targets the same residue of FoxO (manuscript in prep-
aration). Such associated kinases might be recruited and acti-
vated even by 3KD. Indeed the expression of hLRRK2 3KD
mildly increased the pS319 signal of FoxO1 in cultured cells
(Fig. 2E), and slightly activated the FoxO reporter construct
(Fig. 3A) though the difference is statically not significant.
Another possibility is that the overexpression of LRRK2/
dLRRK proteins in itself is neurotoxic unlike EGFP since
we did not see a clear difference of the eye phenotype
between WT and pathogenic mutants (Fig. 1C and D, and
Supplementary Material, Fig. S1F and G). However, milder
expressions of dLRRK WT and mutants using the GeneSwitch
system revealed a pathogenic mutant-specific effect in the life-
span assay (Fig. 4). Thus, these findings warrant further study
in the neurodegeneration of LRRK2-linked PD.
It is particularly worth noting that the co-expression of
dLRRK 3KD and dFoxO S259A dramatically worsened the
eye phenotype (Supplementary Material, Fig. S6H). The unex-
pected effect suggests that there remains a negative regulation
mechanism to be clarified, and might explain the aforemen-
tioned effect of LRRK2/dLRRK 3KD. One of plausible
explanations for this observation is that dLRRK stimulates
AKT or its activators, and that dLRRK 3KD acts as a
Figure 7. Bim is required for LRRK2-mediated neuronal death in human cells. (A) LRRK2 induces Bim expression. SH-SY5Y cells transfected with plasmids
for the indicated cDNA were subjected to western blot for Bim or Actin. b-Galactosidase (Control) served as a negative control. The graph shows relative levels
of Bim-L and Bim-S after normalization with actin levels. Data are presented as mean+SE of three independent experiments (∗P , 0.05). (B) Cell death
induced by FoxO/LRRK2 was suppressed by XIAP in SH-SY5Y cells. XIAP lacking a RING-finger domain (XIAPDRING), which fails to ubiquitinate caspases,
did not fully protect the cells (44). Graph represents the percentage of cells with apoptotic nuclei 48 h after transfection. Data are presented as the mean+SE
from three independent experiments. (C and D) Endogenous LRRK2 and Bim are required for FoxO-mediated neuronal death. (D) SH-SY5Y cells treated with
short interfering RNA (siRNA) against LRRK2, Bim or a control siRNA were subsequently transfected with plasmids for FoxO1 or b-galactosidase (Mock).
Graph represents the percentage of cells with apoptotic nuclei 48 h after FoxO1 transfection. Data are presented as the mean+SE from three independent experi-
ments.∗P , 0.05. (C) The efficiencies of knockdown with the siRNA were estimated by western blot with the indicated antibodies. (E) The S319A mutation of
FoxO1 suppresses FoxO/LRRK2-mediated neuronal death. Cell death assay was performed as in (B). Data are presented as the mean+SE from three indepen-
dent experiments. FoxO1 SA; FoxO1 S319A.∗∗P , 0.01.
Human Molecular Genetics, 2010, Vol. 19, No. 193755
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dominant-negative mutant. However, there is no evidence that
LRRK2/dLRRK modulates AKT activity (data not shown).
Thus, the elucidation of the entire LRRK2-FoxO pathway
must await further studies.
In conclusion, our results identify FoxO proteins as novel
substrates in the LRRK2 pathogenic pathway and suggest a
role for dFoxO in the neurodegeneration caused by mutant
LRRK2/dLRRK expression in Drosophila. That is, LRRK2/
dLRRK confers a neurotoxic activity to FoxO through a
novel phosphorylation mechanism in Drosophila. Since the
LRRK2-FoxO pathway appears to be partially conserved
between human and Drosophila, downstream molecules of
Bim/Hid such as IAP family proteins and caspases could be
therapeutic targets for LRRK2-linked PD. Further studies in
the mammalian system may also uncover other roles for
LRRK2 and FoxO in the underlying pathogenic mechanisms
mediating sporadic as well as familial PD.
MATERIALS AND METHODS
Drosophila culture and crosses were performed on standard fly
food containing yeast, cornmeal and molasses, and flies were
raised at 258C unless otherwise stated. We used UAS-dLRRK
WT and mutant lines that exhibit similar levels of protein
expression (6). For the construction of UAS-dFoxO S259A
and UAS-dFoxO S259E transgenic lines, dFoxO cDNA
obtained by RT–PCR from adult Drosophila total RNA was
cloned into the pBluescript vector. The mutant forms of
dFoxO generated by site-directed mutagenesis were sub-cloned
into the pUAST vector. The introduction of transgenes into
Drosophila germ lines and the establishment of transgenic
lines were performed in the w2background using the embryo
transformation service of BestGene, Inc. (Chino Hills, CA,
USA). All additional general fly stocks and GAL4 lines were
obtained from the Bloomington Drosophila stock center. These
flies have been described previously: UAS-dLRRK WT,
R1069G, Y1383C, I1915T, 3KD and e03680 (dLRRK null) (6);
UAS-hLRRK2 WT and I2020T (26); elav-GeneSwitch (34);
dFoxO21and dFoxO25(7); dSir2EP2300(35); UAS-dFoxA (36).
Rabbit anti-dFoxO and anti-hLRRK2 polyclonal antibodies
were raised against recombinant GST-N-terminal dFoxO (1–
214 amino acids) and GST-hLRRK2 (823–1004 amino
acids), respectively, and were affinity-purified with the anti-
gens. Anti-a-tubulin (DM1A), anti-b-tubulin (Tub2.1) and
anti-FLAG (M2) antibodies were purchased from Sigma.
Anti-FoxO1 (no. 9454), anti-phospho-FoxO1 (Ser319, no.
2487; Thr24, no. 9464; Ser256, no. 9461) and anti-Bim
(no. 2819) were obtained from Cell Signaling Technology.
Anti-Actin (MAB1501) and anti-phospho-FoxO1 (Ser319,
51136-1) antibodies were purchased from Chemicon and
Signalway, respectively. Mouse anti-TH monoclonal antibody
was purchased from ImmunoStar. The rabbit anti-Drosophila
TH polyclonal antibody has been described previously (37).
Anti-phospho-FoxO1 (Ser322 and Ser325) antibodies were
kindly provided by Drs G. Rena and C.J. Hastie (38).
RT–PCR and plasmids
For real-time RT–PCR analysis, RT and PCR reactions with
total RNA extracted from adult flies were performed using
Superscript VILO cDNA Synthesis Kit (Invitrogen) and
SYBR GreenER qPCR SuperMix (Invitrogen), respectively.
To generate GST-FoxO1 for bacterial expression, we amplified
the corresponding coding sequences from pcDNA3-mouse
FoxO1-Myc-His (a kind gift from Dr T. Furuyama) and
cloned it into the pGEX6P-1 vector. The introduction of
mutations was performed using the QuikChange II XL Site-
directed Mutagenesis Kit (Stratagene). Although the S316
residue in mouse FoxO1 corresponds to S319 in human
FoxO1, we describe both residues as S319 to avoid confusion.
Plasmids for FLAG-hLRRK2, FLAG-dLRRK, FLAG-XIAP,
FLAG-XIAPDRING and myr-AKT have been reported else-
where (6,39,40). The luciferase reporter plasmid for FoxO
(TK.IRS3) were generated by the insertion of three copies of
an insulin response sequence derived from the IGFBP-1 pro-
moter to 81 bp upstream of the thymidine kinase promoter
TK.IRS3 has been reported elsewhere (41).
ChIP assay was performed using ChIP-IT Express Kit (Active
Motif). S2 cells treated with 1% formaldehyde were subjected
to homoginization, sonication and subsequent fractionation.
Immunoprecipitation for dFoxO was carried out using anti-
dFoxO in the sheared chromatin fractions. PCR was performed
with ExTaq (TAKARA bio) to estimate bound genomic DNA
in the precipitates. The hid first intron region was amplified
with the same primer pairs as reported (14).
In vitro phosphorylation assay
FLAG-hLRRK2, FLAG-dLRRK or mock fractions immuno-
purified from transfected and mock-transfected 293T cells
were used as kinase sources. Five micrograms of GST-
FoxO1 was incubated with FLAG-hLRRK2 or FLAG-dLRRK
in kinase reaction buffer containing 20 mM HEPES (pH 7.4),
15 mM MgCl2, 5 mM EGTA, 0.1% Triton X-100, 0.5 mM
DTT, 1 mM b-glycerolphosphate and 2.5 mCi [g-32P]-ATP
for 30 min at 308C. The reaction mixture was then suspended
in SDS sample buffer and subjected to SDS–PAGE and auto-
Cell culture, immunopurification and western blot analysis
Transfection of 293T and SH-SY5Y cells, immunopurification
of FLAG protein from the transfected cell lysate and western
blot analysis were performed as described previously (42,43).
For the preparation of fly samples for western blot analysis, fly
heads were directly homogenized in 20 ml/head of SDS
sample buffer using a motor-driven pestle. After centrifugation
at 16 000g for 10 min, the supernatant was subjected to SDS–
PAGE and subsequent western blot analysis. Densitometric
analysis was performed using Image J software from the US
National Institute of Health (http://rsb.info.nih.gov/ij/).
3756Human Molecular Genetics, 2010, Vol. 19, No. 19
at University of Ottawa on June 13, 2011
Scanning electron microscopy analysis
Adult flies directly soaked in 70% ethanol were subjected to
stepwise dehydration, and the samples were processed as
described previously (6). Scanning electron microscopy
(SEM) images were obtained at The Biomedical Research
Core of Tohoku University Graduate School of Medicine.
Lifespan and climbing assays
Twenty female adult flies per vial were maintained at 298C,
transferred to fresh fly food vials containing 250 ml of yeast
paste and 25 mg/ml of RU486 and scored for survival every
4 days. To control for isogeny, the dLRRK(2/2) mutant
and the UAS-dFoxO WT flies were backcrossed to w2wild-
type background for six generations. The dLRRK and dFoxO
S259A transgenics were generated on w2background and
thus have matched genetic backgrounds. Climbing assay was
performed as described previously using 24-day-old female
adult flies as treated in the lifespan assay (6).
Total TH-positive neuron numbers were calculated following
whole-mount immunostaining of brain samples as described
previously (37). Brain tissues were isolated and stained by
T.K. and TH-positive neurons were counted by Y.I. under
blinded conditions. All immunohistochemical analyses were
performed using a Carl Zeiss laser scanning microscope
Cell death assay
SH-SY5Y cells transiently co-transfected with Venus (an
improved version of YFP) and various combinations of plas-
mids were fixed and stained with DAPI 48 h after transfection.
At least 260 Venus-positive cells with healthy or apoptotic
nuclei from randomly chosen fields were counted. For a com-
bination of RNAi and plasmid transfection experiments in
Figure 7C, Stealth RNAi duplexes (Invitrogen) were intro-
duced with LipofectAmine RNAiMAX reagent (Invitrogen)
24 h before plasmid transfection for maximum gene knock-
down efficiencies. Total amounts of DNA for transfection
were adjusted to equal amounts with a plasmid for
b-galactosidase upon combined transfection.
One-way repeated measures ANOVA was performed to deter-
mine significant differences between multiple groups unless
otherwise indicated. If a significant result was achieved
(P , 0.05), the mean of the control and the specific test
groups was analyzed using the Tukey–Kramer test. For life-
span assays, the Kaplan–Meier analysis with log-rank test
Supplementary Material is available at HMG online.
We thank A. Yasui, S. Nakajima, S. Kanno, S. Imai and
M. Kaji for their excellent technical support and equipment
use, and R. Takahashi, T. Furuyama, T. Kimura, R. Reuter,
S.L. Helfand, E. Hafen, T. Osterwalder, G. Rena and
C.J. Hastie for their generous supply of materials.
Conflict of Interest statement. None declared.
This study was supported by the Uehara Memorial Foun-
dation, the Inamori Foundation Research Grant, the Takeda
Science Foundation Research Grant and the Program for
Young Researchers from Special Coordination Funds for
Promoting Science and Technology commissioned by MEXT
in Japan (Y.I.).
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