LRRK2 functions as a Wnt signaling scaffold, bridging cytosolic proteins and membrane-localized LRP6.
ABSTRACT Mutations in PARK8, encoding leucine-rich repeat kinase 2 (LRRK2), are a frequent cause of Parkinson's disease (PD). Nonetheless, the physiological role of LRRK2 remains unclear. Here, we demonstrate that LRRK2 participates in canonical Wnt signaling as a scaffold. LRRK2 interacts with key Wnt signaling proteins of the β-catenin destruction complex and dishevelled proteins in vivo and is recruited to membranes following Wnt stimulation, where it binds to the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) in cellular models. LRRK2, therefore, bridges membrane and cytosolic components of Wnt signaling. Changes in LRRK2 expression affects pathway activity, while pathogenic LRRK2 mutants reduce both signal strength and the LRRK2-LRP6 interaction. Thus, decreased LRRK2-mediated Wnt signaling caused by reduced binding to LRP6 may underlie the neurodegeneration observed in PD. Finally, a newly developed LRRK2 kinase inhibitor disrupted Wnt signaling to a similar extent as pathogenic LRRK2 mutations. The use of LRRK2 kinase inhibition to treat PD may therefore need reconsideration.
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ABSTRACT: Mutations in LRRK2 (leucine-rich repeat kinase 2) are found associated with both sporadic and familial Parkinson´s disease (PD). Pathogenic mutations are localized to the catalytic domains of LRRK2, including kinase and GTPase domains. Altered catalytic activity correlates with neurotoxicity, indicating that targeting those activities may provide clues as to novel therapeutic strategies for LRRK2-linked PD. However, the cellular readout of such altered catalytic activities remains largely unknown. Recent cell biological studies have started to highlight possible early cellular events which are altered in the presence of pathogenic LRRK2 and may ultimately lead to neuronal demise, and these studies link altered LRRK2 function to various abnormal endolysosomal vesicular trafficking events. This review examines our current knowledge of LRRK2 neurobiology and how pathogenic mutations may lead to neurodegeneration in PD.Neuropharmacology 05/2014; · 4.82 Impact Factor
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ABSTRACT: Wnt/β-catenin signaling is required for specification and neurogenesis of midbrain dopaminergic (mDA) neurons, the pivotal neuronal population that degenerates in Parkinson's disease (PD) and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Wnt/β-catenin signaling plays a vital role in adult neurogenesis but whether it might engage DA neurogenesis/neurorepair in the affected PD brain is yet unresolved. Recently, the adult midbrain aqueduct periventricular regions (Aq-PVRs) were shown to harbor neural stem/progenitor cells (mNPCs) with DA potential in vitro, but restrictive mechanisms in vivo are believed to limit their DA regenerative capacity. Using in vitro mNPC culture systems we herein demonstrate that aging is one most critical factor restricting mNPC neurogenic potential via dysregulation of Wnt/β-catenin signaling. Co-coculture paradigms between young/aged (Y/A) mNPCs and Y/A astrocytes identified glial age and a decline of glial-derived factors including Wnts as key determinants of impaired neurogenic potential, whereas Wnt activation regimens efficiently reversed the diminished proliferative, neuronal and DA differentiation potential of A-mNPCs. Next, in vivo studies in wild (Wt) and transgenic β-catenin reporter mice uncovered Wnt/β-catenin signaling activation and remarkable astrocyte remodeling of Aq-PVR in response to MPTP-induced DA neuron death. Spatio-temporal analyses unveiled β-catenin signaling in predopaminergic (Nurr1+/TH-) and imperiled or rescuing DAT+ neurons during MPTP-induced DA neuron injury and self-repair. Aging inhibited Wnt signaling, whereas β-catenin activation in situ with a specific GSK-3β antagonist promoted a significant degree of DA neurorestoration associated with reversal of motor deficit, with implications for neurorestorative approaches in PD. Stem Cells 2014Stem Cells 03/2014; · 7.70 Impact Factor
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ABSTRACT: Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset autosomal dominant Parkinson's disease (PD), and sequence variations at the LRRK2 locus are associated with increased risk for sporadic PD. LRRK2 contains both GTPase and kinase domains flanked by protein interaction motifs, and mutations associated with familial PD have been described for both catalytic domains. LRRK2 has been implicated in diverse cellular processes, and recent evidence pinpoints to an important role for LRRK2 in modulating a variety of intracellular membrane trafficking pathways. However, the underlying mechanisms are poorly understood. Here, by studying the classical, well-understood, degradative trafficking pathway of the epidermal growth factor receptor (EGFR), we show that LRRK2 regulates endocytic membrane trafficking in a Rab7-dependent manner. Mutant LRRK2 expression causes a slight delay in early-to-late endosomal trafficking, and a pronounced delay in trafficking out of late endosomes, which become aberrantly elongated into tubules. This is accompanied by a delay in EGFR degradation. The LRRK2-mediated deficits in EGFR trafficking and degradation can be reverted upon coexpression of active Rab7 and of a series of proteins involved in bridging the EGFR to Rab7 on late endosomes. Effector pull-down assays indicate that pathogenic LRRK2 decreases Rab7 activity both in cells overexpressing LRRK2, as well as in fibroblasts from pathogenic mutant LRRK2 PD patients as compared to healthy controls. Together, these findings provide novel insights into a previously unknown regulation of Rab7 activity by mutant LRRK2 which impairs membrane trafficking at very late stages of the endocytic pathway.Human Molecular Genetics 07/2014; · 6.68 Impact Factor
LRRK2 functions as a Wnt signaling scaffold,
bridging cytosolic proteins and membrane-
Daniel C. Berwick and Kirsten Harvey∗
Department of Pharmacology, UCL School of Pharmacy, University College London, 29-39 Brunswick Square,
London WC1N 1AX, UK
Received August 12, 2012; Revised and Accepted August 13, 2012
Mutations in PARK8, encoding leucine-rich repeat kinase 2 (LRRK2), are a frequent cause of Parkinson’s dis-
ease (PD). Nonetheless, the physiological role of LRRK2 remains unclear. Here, we demonstrate that LRRK2
participates in canonical Wnt signaling as a scaffold. LRRK2 interacts with key Wnt signaling proteins of the
b-catenin destruction complex and dishevelled proteins in vivo and is recruited to membranes following Wnt
stimulation, where it binds to the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) in
cellular models. LRRK2, therefore, bridges membrane and cytosolic components of Wnt signaling. Changes
in LRRK2 expression affects pathway activity, while pathogenic LRRK2 mutants reduce both signal strength
and the LRRK2–LRP6 interaction. Thus, decreased LRRK2-mediated Wnt signaling caused by reduced bind-
ing to LRP6 may underlie the neurodegeneration observed in PD. Finally, a newly developed LRRK2 kinase
inhibitor disrupted Wnt signaling to a similar extent as pathogenic LRRK2 mutations. The use of LRRK2
kinase inhibition to treat PD may therefore need reconsideration.
Parkinson’s disease (PD) is a progressive movement disorder
characterized by the degeneration of dopaminergic neurons of
the ‘substantia nigra pars compacta’ and is the second most
common neurodegenerative disease worldwide (1–3). Al-
though typically idiopathic, genetic linkage has led to the iden-
tification of PD-causing mutations in PARK genes. Interest in
PARK8 is particularly strong, since PARK8 mutations account
for up to40%of PDcases insomepopulations,and elicitsymp-
toms and brain pathologies resembling idiopathic PD (1–3).
PARK8 encodes leucine-rich repeat kinase 2 (LRRK2), a 2527
amino acid protein with two distinct enzymatic activities,
namely serine/threonine kinase activity and GTPase activity,
the latter conferred by a RocCOR (Ras in complex; C-terminal
of Roc) tandem domain. The combination of these enzymatic
activities has inevitably suggested a possible function for
LRRK2 in signal transduction (1,2,4,5). However, despite a
growing body of data linking LRRK2 to various cellular func-
tions including autophagy and endocytosis, much remains
unknown about the role of this protein. In particular, the
precise cellular mechanisms by which LRRK2 mutations
elicit neurodegeneration are still a mystery.
Wnt (Wingless/Int) pathways are evolutionarily conserved
signaling cascades (6–8). Activation of the well-defined ca-
nonical Wnt pathway leads to nuclear accumulation of the
transcriptional co-factor b-catenin and resultant changes in
transcription (6–8). Under basal conditions, b-catenin is
retained in a cytoplasmic multi-protein complex known as
the b-catenin destruction complex (BDC). Here, b-catenin is
phosphorylated by glycogen synthase kinase-3b (GSK3b)
triggering b-catenin ubiquitination and degradation (6–8).
However, when cells are stimulated by the binding of an extra-
cellular Wnt ligand to frizzled (Fz) receptors, the BDC is
recruited to membranes via interaction with a second cytosolic
complex, dishevelled (DVL) polymers. DVL proteins are
believed to interact with the intracellular face of Fz receptors,
while the BDC associates with a second transmembrane
protein, the Wnt signaling co-receptor low-density lipoprotein
receptor-related protein 6 (LRP6). These aggregates of
∗To whom correspondence should be addressed. Tel: +44 2077535888; Fax: +44 2077535902; Email: email@example.com
# The Author 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is prop-
Human Molecular Genetics, 2012, Vol. 21, No. 22
Advance Access published on August 16, 2012
at OUP site access on July 12, 2013
cytosolic protein complexes and membrane-localized recep-
tors have been described as ‘signalsomes’ and their formation
as a crucial step in the transduction of canonical Wnt signals.
Within the signalsome, b-catenin phosphorylation is inhibited
by mechanisms involving the subsequent internalization of the
signalsome complex, resulting in the sequestration of GSK3b
into multi-vesicular bodies (6–9). No longer phosphorylated
and targeted for degradation, b-catenin is free to enter the
nucleus and modulate downstream transcription (Fig. 1A).
The importance of canonical Wnt signaling in embryonic
development and carcinogenesis is well described, but
growing evidence suggests a role for this pathway in the func-
tion of mature neurons. For example, Wnt ligands are now
well established as modulators of synaptic plasticity (10–
17), while N-methyl-D-aspartate receptor stimulation has
been reported to cause b-catenin activation (18). Wnt signal-
ing has also been linked to neurodegenerative disease—
indeed, dysregulated Wnt signaling has been suggested as a
unifying hypothesis underlying Alzheimer’s disease (19).
Despite this, few connections between Wnt signaling and
PD exist, although there are some clues that this might be a
fruitful area of study. For example, Parkin, encoded by
PARK2, has been described as a repressor of b-catenin activa-
tion (20), while we have reported a protein–protein interaction
between overexpressed LRRK2 and the DVL proteins DVL1–
3 (21). LRRK2 has also been described as a GSK3b interactor
in Drosophila (22), although it was not reported if this repre-
sents the small fraction of total cellular GSK3b that is
involved in Wnt signaling.
Here, we demonstrate a functional role for LRRK2 in the
canonical Wnt pathway. Through interactions spanning
DVL, BDC and LRP6 proteins, we present data indicating a
scaffolding role for LRRK2 at the heart of canonical Wnt sig-
naling. Our investigation has allowed LRRK2 to be placed
physically and functionally into a well-defined signaling
cascade for the first time. The following results suggest an in-
timate relationship between the canonical Wnt pathway and
LRRK2 interacts with the BDC in vivo
We investigated the association of endogenous LRRK2 with Wnt
signaling proteins by immunoprecipitation with anti-LRRK2
and anti-DVL antibodies from the cytosolic fraction of mouse
brain. Since these experiments used tissue from animals that
had not been treated to stimulate the canonical Wnt pathway,
it can be assumed that these samples are representative of
basal Wnt activity. These immunoprecipitations confirmed
interaction between LRRK2 and DVL proteins in vivo
(Fig. 1B; Supplementary Material, Fig. S1A). Importantly,
LRRK2 was also found to exist in complex with multiple com-
ponents of the BDC, including b-catenin, GSK3b and Axin1
(Fig. 1B). In addition, LRRK2 associated with b-arrestin—
another protein implicated in canonical Wnt signaling (23)
(Supplementary Material, Fig. S1B). The relative enrichment
ofGSK3binLRRK2 murinebrain immunoprecipitatesincom-
parison with GSK3a, a protein not considered to play a major
role in Wnt signaling, and the failure to co-immunoprecipitate
cytosolic Rab5b, confirmed the specificity of these assays.
Thus, these assays implicated endogenous LRRK2 in two dis-
tinct Wnt signaling complexes in the murine brain: DVL
protein complexes and the BDC.
In some of the following experiments, the LRRK2 homolog
LRRK1 was used to establish similarities and differences
between these proteins in Wnt signaling. However, it is im-
portant to note that sequence variations in the LRRK1 gene
have not been found to segregate with PD (24,25). In addition,
the introduction of pathogenic LRRK2 missense mutations
into equivalent positions in LRRK1 demonstrated that
LRRK2 mutants are more prone to form inclusion bodies in
transfected cells and are more toxic than the equivalent, artifi-
cial changes in LRRK1 (26).
The effect of LRRK2 knockdown on canonical Wnt activity
was investigated to probe the functional role of LRRK2 in the
BDC. These experiments were performed in dopaminergic
which has previously been shown to respond to treatment with
the Wnt ligand, Wnt3a (27). Using the TOPflash reporter (28)
to measure b-catenin activity, the siRNA-mediated knockdown
of LRRK2 reproducibly increased basal and Wnt3a-stimulated
canonical Wnt activity, although to a lesser extent than the
knockdown of the core BDC component Axin1 (Fig. 1C). The
specificity of these siRNAs for LRRK2 and Axin1 was demon-
strated in HEK293 cells, which unlike SH-SY5Y cells express
with the TOPflash data, siRNAs targeting LRRK2 or Axin1
induced an increase in lower-molecular-weight b-catenin
species, likely caused by decreased b-catenin ubiquitination
(Supplementary Material, Fig. S3). In addition, we observed
an increase in LRRK2 protein levels upon Axin1 knockdown.
This is consistent with a destabilization of the BDC upon
Axin1 knockdown, allowing LRRK2 to leave the BDC and to
bind to DVL proteins. The co-expression of LRRK2 and DVL
was shown previously to increase LRRK2 protein levels (21).
LRRK2 knockdown also increased TOPflash activity in a
second in vitro model of canonical Wnt pathway activation,
tary Material, Fig. S1C). Moreover, similar data were obtained
using siRNA probes to LRRK1, suggesting some redundancy
of function between LRRK proteins (Fig. 1D). Thus, our data
support the idea that LRRK2 is a component of the BDC and
that LRRK2 knockdown compromises this complex, leading
to b-catenin stabilization and Wnt signaling activation.
LRRK2 enhances canonical Wnt signaling at membranes
The role of LRRK2 in canonical Wnt signaling was also inves-
tigated in overexpression studies. When averaged across all
experiments, LRRK2 overexpression had no effect on basal
TOPflash activity relative to the control FOPflash reporter
(Supplementary Material, Fig. S2A). Furthermore, ectopic
LRRK2 had no reproducible effect on Wnt3a-stimulated TOP-
flash activity (Supplementary Material, Fig. S2B). Thus, the
overexpression of LRRK2 alone was insufficient to signifi-
cantly affect the canonical Wnt pathway. However, when
this signal transduction cascade was activated by transient
transfection of any of the three human DVL proteins,
Human Molecular Genetics, 2012, Vol. 21, No. 22 4967
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Figure 1. LRRK2 associates with the BDC and DVL proteins. (A) Overview of canonical Wnt signaling and potential interactions with LRRK2. (B) LRRK2
co-immunoprecipitates from mouse brain cytoplasm with components of the BDC and DVL proteins. Immunoprecipitations with anti-LRRK2 antibody (MJFF2).
Anti-LRRK2 IP confirmed by western blotting with a second anti-LRRK2 antibody (NeuroMab N138/6). LRRK2 and co-complexed proteins are present in
MJFF2 eluates and cell lysate, but not IgG control. (C) siRNA-mediated knockdown of LRRK2 increased basal and Wnt3a-induced TOPflash activity in
SH-SY5Y cells. For each treatment condition, values are normalized to control siRNA to show the effect of LRRK2 knockdown. siRNA to Axin1 used as a
positive control. P-values relative to siRNA control are shown. (D) Knockdown of LRRK1 and/or LRRK2 enhances DVL1-mediated Wnt signaling in
HEK293 cells. P-values relative to siRNA control are shown.
4968 Human Molecular Genetics, 2012, Vol. 21, No. 22
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enhancement of reporter activity (Fig. 2A). Increased canonic-
al Wnt activity was also observed when LRRK2 was
co-transfected with LRP6 or the combination of LRP6 and
Fz5 (Fig. 2B). Analogous results could be obtained using
LRRK1 (Fig. 2C; Supplementary Material, Fig. S4). Since
both LRRK proteins contain a kinase domain with homology
to the MAPKKK superfamily (4), these observations raised
the concern that the enhanced activation of canonical Wnt sig-
naling might be a common and likely non-specific observation
upon the overexpression of other kinases with MAPKKK
homology. Importantly, the MAPKKK MEKK3 had the op-
posite effect to LRRK protein overexpression on Wnt signaling,
repressing rather than activating DVL1-mediated TOPflash ac-
tivation (Fig. 2D). This indicates that the effects of LRRK1
and LRRK2 are specific and unlikely to be an artifact of over-
expressing proteins with MAPKKK homology.
The observation that either LRRK protein could significant-
ly enhance canonical Wnt signaling upon co-expression with
additional Wnt pathway components suggested that the
co-transfected proteins modify the behavior of LRRK1 and
LRRK2. Consistent with this suggestion, overexpressed DVL
proteins form polymeric structures (29) and we have previous-
ly reported that LRRK2 is efficiently recruited to these bodies,
most likely via the direct association of the DVL DEP domain
with the LRRK2-RocCOR domain (21). Intriguingly, DVL
polymers have been suggested to at least partially localize to
membranous compartments (9,29), while LRP6 is a transmem-
brane protein and present at cell membranes. We therefore
hypothesized that overexpressed DVL and LRP6 could
recruit LRRK2 to membranes and that membrane-associated
LRRK2 exerted a positive effect on Wnt signaling. To test
this idea, two LRRK2 expression constructs were generated
with membrane-targeting mutations: the first containing
amino acids 1–12 of c-Src to confer N-terminal myristoyla-
tion (30), the second encoding a T2524C amino acid substitu-
tion to create a CAAX motif conferring C-terminal prenylation
(31). Supporting a role for LRRK2 in Wnt signaling at mem-
branous compartments, both membrane-targeted LRRK2
mutants enhanced DVL1-stimulated TOPflash reporter activ-
ity to a greater extent than wild-type LRRK2 (Fig. 3A). In
contrast, no increased activation was observed using the com-
bination of Fz5 and LRP6 to activate canonical Wnt signaling
(Fig. 3B), suggesting that either Fz5 or LRP6 is sufficient to
confer the maximal membrane relocalization of wild-type
LRRK2. Confocal microscopy
co-transfection of LRRK2 with any of three Fz receptors
caused a limited redistribution of LRRK2 to membranes and
some co-localization with Fz receptors (Supplementary Mater-
ial, Fig. S5). However, the co-expression with LRP6 elicited a
co-localization between the two proteins (Fig. 4A).
LRRK2 interacts with LRP6 and is recruited
to membranes by Wnt ligands
Since the high degree of co-localization between LRRK2 and
LRP6 suggested a potential physical interaction, we confirmed
that overexpressed LRRK2 was able to co-immunoprecipitate
LRP6 (Fig. 4B) but not Fz1, Fz4 or Fz5 receptors. To deter-
mine whether the interaction was direct, yeast two-hybrid
(YTH) assays were performed using the intracellular domain
of LRP6 as bait and various portions of LRRK2 as prey.
This experiment revealed a strong direct interaction between
LRP6 and the LRRK2-RocCOR tandem domain (Fig. 4C),
but not between LRP6 and additional LRRK2 domains (Roc,
COR, kinase and WD40; data not shown). In agreement
with this observation,the
co-localized with LRP6 in a similar manner to full-length
LRRK2 (Fig. 4D). Taken together, these data indicate that
LRRK2 can interact with the intracellular domain of LRP6
in cells and that this binding is likely to be direct, via the
A number of Wnt signaling proteins, including DVL pro-
teins, have been reported to translocate to membranous com-
partments in response to acute stimulation with Wnt ligands
(29). Indeed, the recruitment of the BDC to juxtamembrane
protein aggregates is considered a key step in the stabilization
of b-catenin (6–9). Thus, the interaction between LRRK2 and
BDC in mouse brain cytoplasm and the recruitment of LRRK2
to intracellular membranes following LRP6 overexpression
raised the possibility that the cellular distribution of LRRK2
might be regulated by Wnt stimulation. To this end,
HEK293 cells, which unlike SH-SY5Y cells express LRRK2
at readily detectible levels, were treated with recombinant
Wnt3a for 0, 30 and 60 min and the localization of endogen-
ous LRRK2 to membrane fractions was determined. These
experiments revealed a strong increase in the level of
LRRK2 present at membranes at both time points following
Wnt3a treatment (Fig. 5, Supplementary Material, Fig. S6).
The enhancement of DVL1-driven canonical Wnt signaling
is weakened by PARK8 mutations and LRRK2 kinase
An increase in the catalytic activity of LRRK2, in particular
LRRK2 kinase activity, has been suggested as the underlying
cause of the neurodegeneration observed in PARK8 patients.
Therefore, we investigated the importance of LRRK2 kinase
and GTPase activities in canonical Wnt signaling. Kinase ac-
tivity was first investigated using a pharmacological inhibitor
of LRRK2, LRRK2-IN-1 (32). This compound markedly
reduced the TOPflash activation elicited by DVL1 or a com-
bination of LRRK2 and DVL1 (Fig. 6B). In agreement with
a requirement for LRRK2 kinase activity for canonical Wnt
signal activation, the effect of LRRK2 on DVL1-mediated
Wnt signaling was also decreased by two artificial kinase-dead
mutations (Fig. 6C). The importance of GTP binding was
investigated in parallel using a K1347A substitution to block
guanyl nucleotide binding. This mutant showed a similar defi-
ciency in the ability to increase Wnt signaling to that of the
kinase-dead constructs (Fig. 6C). Interestingly, introduction
of the analogous mutation into LRRK1 did not appear to
affect the function of this protein in identical experiments
(Fig. 6D), while enzymatic inhibition did not appear to com-
promise the effect of LRRK proteins on Fz5 and LRP6-
Fig. S7A and B). Thus, our data suggest the importance of
LRRK2, but not LRRK1, catalytic activity for Wnt signaling
Human Molecular Genetics, 2012, Vol. 21, No. 22 4969
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Figure 2. LRRK2 enhances DVL and LRP6-driven Wnt signaling. Overexpressed LRRK2 enhances the TOPflash activation elicited by overexpressed (A) DVL
proteins or (B) LRP6 and LRP6 plus Fz5 in SH-SY5Y cells. In both figures, the fold effect of LRRK2 is included as inset graphs. (C) An analogous effect on
DVL1-mediated Wnt signaling is seen with LRRK1. (D) In contrast, MEKK3 suppresses DVL1-mediated TOPflash activation, indicating that the effect of
LRRK1 and LRRK2 is not a generic effect caused by the overexpression of proteins with MAPKKK domains. In all figures, P-values for the effect of
LRRK1, LRRK2 or MEKK3 overexpression relative to the appropriate vector control are shown.
4970 Human Molecular Genetics, 2012, Vol. 21, No. 22
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Numerous papers have investigated the influence of familial
PARK8 mutations on the interaction of LRRK2 with accessory
Forexample, the G2019Skinase domain mutationincreasesthe
binding of LRRK2 to GSK3b (22). Furthermore, Roc domain
mutations at R1441 increase the binding of LRRK2 to DVL1,
while the Y1699C COR domain mutation weakens interaction
with all three DVL proteins (21). Intriguingly, the binding of
the LRRK2-RocCOR tandem domain to LRP6 is weakened
by R1441C, R1441G and Y1699C mutations (Fig. 6E). In
light of these observations and the requirements for LRRK2
kinase and GTPase activity (Fig. 6B and C), we determined
used to activate canonical Wnt signaling, no statistically signi-
ficant differences of any of the mutations in comparison
with wild-type LRRK2 were found (Supplementary Material,
Fig. S7A). However, in co-transfection experiments with
DVL1 performed in SH-SY5Y or HEK293 cells, all mutations
tested (R1441C, Y1699C and G2019S) decreased TOPflash ac-
tivity relative to wild-type LRRK2 (Fig. 6F).
Taken together, our data support a central role for LRRK2 in
canonical Wnt signaling (summarized as a schematic in
Fig. 7). In particular, our data are consistent with a model
where under basal cell conditions without measurable canon-
ical Wnt signaling activity, LRRK2 is associated with the
BDC. Following Wnt stimulation, LRRK2 is recruited to the
plasma membrane and directly associates with the intracellular
domain of LRP6. LRRK2 also interacts with DVL proteins
and proteins of the BDC, which are also recruited to the cell
membrane after Wnt signal stimulation. Therefore, LRRK2
is able to assist in the formation of LRP6 signalosomes at
the cell membrane. As such, despite the observed importance
of GTP-binding and kinase activity, the role of LRRK2 is
likely to be primarily as a scaffold for Wnt signaling com-
plexes. This scaffold function takes place under basal condi-
tions in the cytoplasmic BDC and after signal activation in
association with LRP6 at membranes. LRP6 signalosomes
are then internalized into the endosomal system, leading to
the sequestration of GSK3b and other Wnt signaling compo-
nents into multi-vesicular bodies. While it remains to be deter-
mined whether efficient intracellular trafficking of LRP6
signalosomes requires LRRK2, it is intriguing to note that
both LRRK proteins are implicated in endocytosis (33–36).
The interaction of LRRK2 with DVL proteins, b-arrestin
and GSK3b also suggests the possibility that LRRK2 partici-
pates in non-canonical Wnt signaling, although this possibility
requires further investigation.
Importantly, PARK8 mutations to the LRRK2 Roc, COR
and kinase domains all weaken the activation of canonical
Wnt signaling elicited by the overexpression of DVL1 and
LRRK2 (Fig. 6F), although whether these mutations disrupt
Wnt signaling in vivo remains to be determined. However,
given that LRRK2 interactions with LRP6 (Fig. 6E), DVLs
(21) and GSK3b (22) are all altered by pathogenic PARK8
mutations, it seems unlikely that b-catenin activation will
remain unaffected. Such in vivo studies might be difficult
however, since PARK8 mutations are not fully penetrant—in
fact, the most prevalent G2019S mutation is thought to have
a penetrance of around 30% at the age of seventy (2)—sug-
gesting that observed effects on Wnt signaling might only be
obvious in aged animals or following additional insults, e.g.
challenge with agents causing oxidative stress or co-existing
Wnt ligands, Wnt antagonists and b-catenin modulators are
expressed in overlapping patterns throughout the developing
central nervous system (37,38), demonstrating the redundancy
of function for some as well as specific function for other Wnt
Figure 3. LRRK2 facilitates DVL1-mediated Wnt signaling at membranes. (A) Membrane-targeting of LRRK2 by myristoylation or prenylation potentiates the
enhancement of DVL1-driven TOPflash activation elicited by wild-type LRRK2. (B) In contrast, membrane-targeting of LRRK2 has no significant effect on the
ability of this protein to enhance LRP6 and Fz5-driven Wnt activation. In both figures, P-values for the effect of membrane targeting are shown.
Human Molecular Genetics, 2012, Vol. 21, No. 224971
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signaling components. The pattern of Wnt b-catenin activity
in the adult brain is also complex mirroring suggested Wnt sig-
naling functions in neuronal maintenance, including synapse
formation, synaptic plasticity and cell proliferation, and is
further complicated by the observation that the expression of
some Wnt components is age- and sex-dependent (39–41).
The most evident Wnt-responsive tissues in the adult brain
include the dentate gyrus, hippocampus, sensory telencephalic
cortex, several thalamic nuclei, collicula and cerebellar cortex
(40). Importantly, Wnt signaling pathways have also been
demonstrated to be of fundamental importance for the
biology of dopaminergic neurons in the ventral midbrain
(42–45). For example, impaired dopaminergic development
and altered midbrain morphology is seen upon the disruption
Figure 4. LRRK2 binds directly to the intracellular domain of LRP6. (A) Myc-tagged LRRK2 (red) and HA-tagged LRP6 (green) show almost complete
co-localization in HEK293 cells (a–g). Almost no co-localization was seen between FLAG-tagged DVL1 (red) and HA-tagged LRP6 (green) (h–n). (B) FLAG-
tagged LRRK2 and HA-tagged LRP6 co-immunoprecipitate from HEK293 cells. (C) The intracellular domain of LRP6 binds the LRRK2-RocCOR domain but
not DVL2 in yeast. X-gal freeze-fracture assays indicate protein–protein interactions in blue. All negative controls show no color change. (D) Consistent with a
requirement for the LRRK2-RocCOR domain for the LRP6–LRRK2 interaction the myc-tagged LRRK2-RocCOR domain (green) co-localizes with HA-tagged
LRP6 similar to full-length protein in HEK293 cells (A, a–g). In (A) and (D), DNA staining with 4’,6-diamidino-2-phenylindole (blue) is shown, scale bar:
4972 Human Molecular Genetics, 2012, Vol. 21, No. 22
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of Wnt1 (46) and Lrp6 (47) genes encoding two proteins that
function specifically within the canonical Wnt pathway. A
recent study also suggested an interaction between dopamine
D2 receptors and b-catenin in the adult brain (48).
LRRK2 is also a ubiquitously expressed protein, found in
relative abundance in most brain regions, including the sub-
stantia nigra, thalamus, striatum, cortex, olfactory tubercle,
nucleus accumbens, hippocampus and cerebellum (49–53).
Interestingly, LRRK2 was also shown to be present in the sub-
ventricular zone (SVZ), suggesting a role for LRRK2 in adult
neurogenesis (53,54). Deregulated Wnt signaling has also
been shown to play an important role in impaired adult neuro-
genesis seen in PD. In particular, reactive astrocytes and
microglia were shown to protect dopaminergic neurons in
animal models of PD by activating canonical Wnt signaling
and promoting neurogenesis from adult SVZ neuroprogenitor
cells, by a mechanism based on the interplay between inflam-
mation and canonical Wnt signaling (55–57). Investigation of
an interaction between LRRK2 and Wnt signaling contributing
to adult neurogenesis in the SVZ would be of great interest to
further research into the pathogenesis of PD and the identifica-
tion of therapeutic targets.
by PARK8 mutations in our study (Fig. 6F) is in good accord
with decreased Wnt signaling observed in neurodegeneration
causing Alzheimer’s disease and frontotemporal dementia (58).
Moreover, a general requirement for Wnt signaling for the de-
velopment of dopaminergic neurons and negative effects of
signaling might also lead to neurodegeneration, as observed in
PARK2 early onset PD and JNPL3 frontotemporal dementia
ing is subject to regulation within defined boundaries to permit
normal neuronal and synaptic function without eliciting cell
cycle reentry leading to cell death in postmitotic neurons. Thus,
Dysregulated canonical Wnt signaling represents an intri-
guing candidate mechanism for PD pathogenesis. The import-
ance of Wnt signaling for basic neuronal functions is well
established, not least synaptic plasticity (10,12–18,63) and
the control of microtubule stability (64–67). It is important
to note that not all of these events require b-catenin-dependent
Figure 5. LRRK2 is recruited to membranes by Wnt3a. Acute treatment of HEK293 cells with recombinant Wnt3a increases the amount of endogenous LRRK2
present in crude membrane fractions. (A) A representative experiment. (B and C) Quantifications of the relative levels of membrane-associated LRRK2 at each
time point from four independent experiments, normalized to (B) calnexin and (C) Rab5b. (D) Confocal images showing HEK293 cells expressing LRRK2 under
basal conditions (A–D) and after activation with Wnt3a (E–H), scale bar: 10 mm.
Human Molecular Genetics, 2012, Vol. 21, No. 224973
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Figure 6. Pathogenic PARK8 mutations and loss of kinase or GTPase activity impair LRRK2 function in Wnt signaling. (A) Schematic of the pathological
PARK8 mutations and strategies employed to inhibit kinase and GTPase function. (B) The LRRK2 kinase inhibitor, LRRK2-IN-1 inhibits TOPflash activation
elicited by DVL1 or DVL1 and LRRK2. (C) The LRRK2-mediated enhancement of DVL1-driven TOPflash activation is inhibited by kinase-dead (D1994A and
D2017A) or GTP/GDP-binding site mutations (K1347A). (D) An analogous GTP/GDP-binding mutation to LRRK1 has no effect on DVL1-driven Wnt signal-
ing. (E) Pathogenic PARK8 mutations weaken the binding of the LRRK2-RocCOR tandem domain to LRP6. (F) PARK8 mutations weaken LRRK2-mediated
enhancement of DVL1-driven TOPflash activation in two cell lines.
4974 Human Molecular Genetics, 2012, Vol. 21, No. 22
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transcriptional regulation, indicating both genomic and non-
genomic actions. Moreover, the development of ventral mid-
brain dopaminergic neurons appears particularly dependent on
canonical Wnt signaling (42,47,59–61). Thus, dopaminergic
neurons could be particularly sensitive to mild perturbations
in canonical Wnt signaling that would elicit a slow but progres-
sive loss of neuronal function ultimately ending in cell death.
Furthermore, dysregulated Wnt signaling has the potential to
explain the link between PD and variations in MAPT (encoding
the microtubule-binding protein, tau) linked to PD in numerous
substrate, and this phosphorylation event can be modulated by
Wnt ligands (19,69). Moreover, tau phosphorylation is a patho-
logical hallmark of Alzheimer’s disease (19,69) and frontotem-
poral dementia and has been strongly linked to PARK8
and animal models (22,68,70,71). Thus, in addition to suggest-
ing an intriguing mechanism for PD pathogenesis, our data also
suggest tantalizing links to Alzheimer’s disease and frontotem-
Our observations are also likely to have implications beyond
neurodegeneration. For example, constitutive activation of
b-catenin is well described in numerous types of cancer and we
note that an increased risk of non-skin cancer has been observed
for individuals with PARK8 mutations (72,73). Moreover, the
capacity of the LRRK2-IN-1 compound to inhibit canonical
Wnt signaling (Fig. 6B) suggests the possibility of targeting
LRRK2 kinase activity in the treatment of tumors linked to
elevated b-catenin activation. Unfortunately, LRRK2 kinase
inhibitors showed an inhibitory effect on Wnt signaling that
was similar to that seen with pathogenic LRRK2 mutations.
This suggests that the usefulness of LRRK2 inhibitors for the
treatment of PD might be limited. In fact, LRRK2 inhibitors
could have detrimental effects on disease progression and cogni-
tive ability, since decreased Wnt signaling was observed in Alz-
heimer’s disease (19).
In conclusion, our data show LRRK2 to be a central compo-
nent of canonical Wnt signaling. We propose that dysregulated
Wnt signaling is a new potential pathomechanism leading to
PARK8 PD and suggest that alterations in Wnt signaling
Figure 7. Schematic summary of the main study findings. (A1) In the basal state, LRRK2 is associated with the BDC. The distribution of BDCs between mem-
brane and cytosol is in dynamic equilibrium dependent on interactions between Fz receptors (FzR) and DVL proteins, between DVL proteins and LRRK2 and
between LRP5/LRP6 co-receptors and LRRK2. (A2) Following stimulation with Wnt ligand, DVL proteins are polymerized, thereby recruiting BDCs to the
membrane via interaction with LRRK2 and additional BDC proteins. LRRK2 also interacts with the intracellular domain of LRP5/LRP6, thus bridging DVL
proteins, the BDC and LRP5/LRP6, and facilitating LRP5/LRP6 phosphorylation by GSK3b, and the formation of LRP5/LRP6 signalosomes. (A3) Signalo-
somes containing phosphorylated LRP5/LRP6 and GSK3b are internalized into the endosomal system and ultimately sequestered into multi-vesicular
bodies. Sequestration of GSK3b prevents phosphorylation of newly synthesized b-catenin allowing the protein to accumulate in the cytoplasm and activate
b-catenin-dependent transcription in the nucleus. Whether LRRK2 is sequestered into MVBs with LRP6 and GSK3b or recycled to the cytosol remains to
be determined. Note that LRRK2 is involved in multiple steps and interacts with multiple Wnt components. By altering strength of interaction with these pro-
teins, PARK8 mutations are expected to compromise canonical Wnt activity stimulated by Wnt ligands, and perhaps also to alter basal b-catenin activity. (B) The
primary effect of loss of LRRK2 is disruption of the BDC, leading to b-catenin stabilization. (C) Overexpressed DVL protein polymerizes and is sufficient to
recruit BDCs to the membrane via increased interactions with FzRs and LRRK2. Presumably, the overexpression of LRRK2 further increases the strength of the
LRRK2-DVL interaction. (D) Overexpressed LRP6 also increases the membrane recruitment of BDC via binding to LRRK2. (E) Membrane-targeting of LRRK2
ensures the DVL-LRRK2 interaction takes place at membranes, thereby maximizing the membrane recruitment of BDCs.
Human Molecular Genetics, 2012, Vol. 21, No. 22 4975
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pathways might also be a common cause of idiopathic PD. Our
data also suggest that the targeting of LRRK2 kinase activity
might not be the most suitable approach for the treatment of
neurodegeneration. In contrast, controlled Wnt signaling acti-
vation—e.g. with small molecules targeting LRP6—appears a
more promising therapeutic approach for PD.
MATERIALS AND METHODS
Plasmids, cloning and siRNA
previously (21). pACT2-RocCOR, pACT2-Roc and pACT2-
COR were made by inserting polymerase chain reaction (PCR)
fragments encoding amino acids 1288–1844, 1288–1516 and
1516–1844 into the BamHI and XhoI sites in pACT2. All
mutagenesis. pRK5-mycLRRK2CAAXwas also made by site-
directed mutagenesis to introduce a T2524C mutation, thereby
creating a C-terminal CAAX prenylation motif. pRK5-myr-
mycLRRK2 was generated from pRK5-mycLRRK2 by PCR
using a forward primer encoding the first 12 amino acids of
erated by the PCR amplification of the triple-FLAG-tagged
LRRK2 insert from pCHMWS-3flagLRRK2 (a kind gift from
Jean-Marc Taymans, Katholieke Universiteit Leuven, Belgium)
and insertion into pRK5. pEGFP-N1-Fzd1, -Fzd4 and -Fzd5
(74) were kindly provided by Yosuke Funato and Hiroaki Miki
(Osaka University, Osaka, Japan). TOPflash and FOPflash were
obtained from Addgene; pTK-RL is from Promega. To make
pRK5-LRP6-2xHA, human LRP6 was amplified from cDNA
using primers designed to introduce two HA epitopes at the
LRP6 C terminus and cloned into the pRK5 vector. pYTH16-
LRP6-ICD was made by PCR cloning the intracellular domain
of LRP6 (amino acids 1416–1613) and insertion into the
pYTH16 vector in frame with the Gal4 DNA-binding domain.
All constructs were verified by DNA sequencing.
siRNAs were synthesized by Eurofins and designed to rec-
ognize published sequences. siRNAs used were as follows:
axin CGAGAGCCAUCUACCGAAA (75); GFP GCUAC
GUCCAGGAGCGCAC (Eurofins); LRRK1#A GGAAUCA
CUCACUGACUAC (76); LRRK1#B CAGAGAUUCUUCC
UUUAUA (76); LRRK2#1 CGUCGACUUAUACGUGUAA
(77); LRRK2#2 GAAUUUCAUCAUAAGCUAA (77); LRR
K2#3 AUUAUUCUCUCCUCUUGUA∗; Fz5 UUGUAAUC
CAUGCAGAGGA (75); luciferase GL3 CUUACGCUGA
GUACUUCGA (Eurofins); non-specific control AGGUAGU
GUAAUCGCCUUG (Eurofins). The asterisk denotes the pre-
viously undescribed siRNA targeting the human LRRK2
Cell culture and transfection
All mammalian cells were grown at 378C in 5% CO2. Cells for
immunofluorescence experiments were seeded onto poly-
D-lysine-coated coverslips. HEK293 cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) containing
10% fetal bovine serum (FBS) plus 100 U/ml penicillin G
and 100 mg/ml streptomycin and were transfected using
Lipofectamine LTX (Invitrogen) at a 2:1 ml transfection
reagent to mg DNA ratio. SH-SY5Y cells were cultured in a
1:1 mixture of DMEM and F12 Ham’s media supplemented
with 10% FBS plus penicillin and streptomycin and were
transfected using FuGENE 6 (Roche) at a 2.5:1 ml transfection
reagent to mg DNA ratio.
Immunofluorescence staining and imaging
Cells were transfected with 500 ng of the relevant plasmid per
coverslip. Forty-eight hours post-transfection, cells were fixed
with 4% (w/v) paraformaldehyde and stained with antibodies
to myc and FLAG (Sigma) or HA (Covance) as described pre-
viously (21). Alexa-488, -546 and -633 conjugated secondary
antibodies were from Invitrogen. 4’,6-diamidino-2-phenylin-
dole stain (Invitrogen) was performed at a 300 nM concentra-
tion for 5 min. Confocal microscopy was performed using a
Zeiss LSM 710, with an AxioObserver.Z1 microscope. Fluor-
escence excited by a 30 mW 405 nm diode laser, a 25 mW
Argon ion laser (488 nm line), a 25 mW 565 nm diode laser
and a 5 mW 633 nm HeNe laser was detected separately. All
images were taken at room temperature with a 63× objective
with immersion oil, using Zen software.
Experiments were performed as described previously (21),
except that the Y190 strain (Clontech) was used, and cells
were grown in the presence of 10 mM 3-amino-1,2,4-triazole.
For quantitative experiments, sample means were calculated
from at least three replicates per experiment.
Unless indicated otherwise, TOPflash assays were performed
in human dopaminergic SH-SY5Y cells. Except for siRNA-
mediated knockdown experiments, assays were performed in
a 6-well plate format, using three wells per experimental con-
dition. Five hundred nanograms of pRK5-FLAG-DVL plas-
mids was used per well in Figure 2A; in subsequent
experiments, a mixture of 200 ng of DVL and 300 ng of
pRK5-FLAG was used. All other plasmid DNAs were used
at 500 ng/well, except pRL-TK (50 ng). siRNA experiments
were performed in 24-well plates using 100 ng of TOPflash
or FOPflash and 10 ng of pRL-TK, plus 10 pmol of each
siRNA per well. Cells were extracted 24 h post-transfection
using Passive Lysis Buffer (Promega). Luciferase assays
were performed using a Dual Luciferase Reporter Assay kit
(Promega) and Turner Instruments 20/20 luminometer.
Values obtained (relative luciferase units) are the ratio of luci-
ferase and Renilla from each well, to adjust for variations in
Biochemistry of mouse brains
Wild-type male CD1 mice for co-immunoprecipitation experi-
ments were obtained in house. Brains were snap-frozen in
liquid nitrogen immediately after removal and stored at
2808C prior to extraction. To isolate mouse brain cytosol,
brains were first homogenized into Tris-buffered saline
4976 Human Molecular Genetics, 2012, Vol. 21, No. 22
at OUP site access on July 12, 2013
[50 mM Tris, pH 7.5, 150 mM NaCl supplemented with 1×
complete protease inhibitor cocktail (Roche) and 1× Halt
phosphatase inhibitor cocktail (Pierce)] using a dounce hom-
ogenizer and then clarified by centrifugation at 14 000g for
10 min. All steps were performed at 48C. Anti-LRRK2 immu-
noprecipitation was performed on cytosolic extracts, using a
Dynabeads Co-immunoprecipitation kit (Invitrogen) coupled
with rabbit anti-LRRK2 antibody (MJFF2; Epitomics) or non-
specific rabbit IgG (Sigma).
Biochemistry of in vitro cell lines
In vitro biochemistry was performed in HEK293 cells due to
easeoftransfection andbecause thesecells express endogenous
LRRK2 to a much greater degree than SH-SY5Y cells. Cells
were grown in 10 cm dishes and, where indicated, transfected
with 4 mg of each plasmid 36 h prior to lysis. Wnt3a treatment
taining 20 mM Tris, pH 7.5, 50 mM NaCl, 2 mM ethylenediami-
netetraacetic acid, 1% Triton X-100 and 1× complete protease
tionat14 000gfor10 minat48C,andsupernatantsretained.For
Co-IP, 65 ml was retained as whole cell lysate, the remainder
wasadjustedto250 mMNaCl,and40 mlanti-FLAG-M2affinity
gel added, prior to incubation at 48C with rotation. After 4 h
beads were washed four times in extraction buffer containing
250 mM NaCl and bound protein eluted by incubation with
15 ng/ml3xFLAGpeptide(Sigma)for30 minatroomtempera-
ture. For membrane translocation studies, cells were extracted
according to the published protocols (78). The relative localiza-
tion of LRRK2 to membrane fractions was determined as the
ratio of LRRK2 to Calnexin or to membrane-bound Rab5b in
each sample, with images acquired from the same gel.
Eluates and cell lysate samples were denatured by the addition
of 4× LDS sample loading buffer and 10× sample reducing
agent (both Invitrogen) followed by heating to 998C for
10 min. Samples were resolved by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis using 4–12% Bis–Tris
pre-cast gels (Invitrogen) and transferred to polyvinylidene
difluoride membrane (Millipore). Membranes were rinsed in
PBS containing 0.1% Tween-20 (PBS-T), and then blocked
by incubation for 30 min in PBS-T containing 5% non-fat
dry milk powder. Primary antibody incubations were per-
formed overnight at 48C with gentle shaking, with all anti-
concentrations: anti-myc, anti-FLAG (both Sigma) 1:2000;
anti-GFP (AbCam) 1:3000; anti-HA (Covance) 1:5000; anti-
actin (Sigma) 1:6000. Horseradish peroxidase-conjugated sec-
ondary antibodies (1:2000 in blocking buffer; Santa Cruz)
were incubated with membranes for 1 h at room temperature.
Protein bands were visualized using SuperSignal West Pico
Chemiluminescent Substrate (Pierce) and images acquired
using a GeneGnome imager (Syngene). Quantification was
performed using GeneTools analysis software (Syngene).
LRRK2 membrane localization (Figs. 5B and C; n ¼ 4) and
quantitative YTH assays (Fig. 6E) were analyzed by paired
Student’s t-test with two-tailed distribution. Statistical signifi-
cance of luciferase assays was performed by unpaired t-test
with two-tailed distribution. In all cases, combined data
from a minimum of three independent experiments was
used. In all cases, P-values are indicated, with statistically sig-
nificant values (P , 0.05) highlighted in bold.
Supplementary Material is available at HMG online.
K.H. and D.C.B. thank Marian Blanca Ramirez for technical
assistance; Professor Robert Harvey for useful discussions
and critical reading of this manuscript and Dr Ana Antunes-
Martins for critical reading.
Conflict of Interest statement. None declared.
(WT088145AIA, WT095010MA to K.H.), The Michael
J. Fox Foundation (to D.C.B. and K.H.), a Vera Down
British Medical Association Research Grant (to K.H.) and an
Ibercaja Obra Social award (to Marian Blanca Ramirez).
Funding to pay the Open Access publication charges for this
article was provided by the Wellcome Trust.
work wassupported byTheWellcomeTrust
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