High LRRK2 Levels Fail to Induce or Exacerbate Neuronal
Alpha-Synucleinopathy in Mouse Brain
Martin C. Herzig, Michael Bidinosti, Tatjana Schweizer, Thomas Hafner, Christine Stemmelen,
Andreas Weiss, Simone Danner, Nella Vidotto, Daniela Stauffer, Carmen Barske, Franziska Mayer,
Peter Schmid, Giorgio Rovelli, P. Herman van der Putten, Derya R. Shimshek*
Department of Neuroscience, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland
The G2019S mutation in the multidomain protein leucine-rich repeat kinase 2 (LRRK2) is one of the most frequently
identified genetic causes of Parkinson’s disease (PD). Clinically, LRRK2(G2019S) carriers with PD and idiopathic PD patients
have a very similar disease with brainstem and cortical Lewy pathology (a-synucleinopathy) as histopathological
hallmarks. Some patients have Tau pathology. Enhanced kinase function of the LRRK2(G2019S) mutant protein is a prime
suspect mechanism for carriers to develop PD but observations in LRRK2 knock-out, G2019S knock-in and kinase-dead
mutant mice suggest that LRRK2 steady-state abundance of the protein also plays a determining role. One critical
question concerning the molecular pathogenesis in LRRK2(G2019S) PD patients is whether a-synuclein (aSN) has a
contributory role. To this end we generated mice with high expression of either wildtype or G2019S mutant LRRK2 in
brainstem and cortical neurons. High levels of these LRRK2 variants left endogenous aSN and Tau levels unaltered and
did not exacerbate or otherwise modify a-synucleinopathy in mice that co-expressed high levels of LRRK2 and aSN in
brain neurons. On the contrary, in some lines high LRRK2 levels improved motor skills in the presence and absence of
aSN-transgene-induced disease. Therefore, in many neurons high LRRK2 levels are well tolerated and not sufficient to
drive or exacerbate neuronal a-synucleinopathy.
Citation: Herzig MC, Bidinosti M, Schweizer T, Hafner T, Stemmelen C, et al. (2012) High LRRK2 Levels Fail to Induce or Exacerbate Neuronal Alpha-
Synucleinopathy in Mouse Brain. PLoS ONE 7(5): e36581. doi:10.1371/journal.pone.0036581
Editor: Patrick Lewis, UCL Institute of Neurology, United Kingdom
Received January 30, 2012; Accepted April 10, 2012; Published May 15, 2012
Copyright: ? 2012 Herzig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: All authors are employees of Novartis Pharma AG who funded this study. There are no patents, products in development or marketed
products to declare. This does not alter the authors’ adherence to all PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.
* E-mail: email@example.com
Parkinson’s disease (PD) is a common neurodegenerative
movement disorder with clinical features including bradykinesia,
rigidity and resting tremor. PD histopathological hallmarks are the
loss of dopaminergic neurons in the substantia nigra and Lewy
pathology. The latter is characterized by fibrillar a-synuclein (aSN)
aggregates that are microscopically visible and referred to as Lewy
bodies (LB) and Lewy neurites. This a-synuclein proteinopathy is
often widespread and affects not only dopaminergic neurons in the
substantia nigra but also neurons in other brainstem nuclei, the
cortex, the spinal cord and the gastrointestinal nervous system
The first mutation causing PD was discovered in the aSN-
encoding gene (SNCA) . Since then, many more PD loci
including leucine-rich repeat kinase-2 (LRRK2) have been
discovered through linkage analysis or genome-wide association
studies (GWAS) [5,6,7,8,9,10,11,12,13]. Furthermore, polymor-
phic variants of genes including SNCA, LRRK2 and microtubule-
associated protein Tau (MAPT) have emerged as susceptibility
factors associated with an increased risk to develop PD
LRRK2 mutations cause late-onset autosomal dominant PD
that is clinically indistinguishable from idiopathic PD. They
account for approx. 4–5% of familiar and 1–2% of sporadic PD
[5,6,7,8,9,10,19,20]. In addition, LRRK2 has been implicated as a
susceptibility factor in other diseases like Crohn’s disease
[21,22,23], cancer [24,25] and leprosy  which could suggest
unrecognized links between these disease pathophysiologies .
The most prominent PD-associated mutation G2019S was shown
to result in increased kinase activity [28,29,30] and induce
neuronal toxicity [31,32,33]. Such findings support the hypothesis
that enhanced LRRK2 kinase function might suffice to evoke
neuropathophysiological changes. They also raised hope that
LRRK2 kinase inhibitors might be capable of halting disease
progression in LRRK2(G2019S) and perhaps other LRRK2
mutation carriers. Although the enhanced kinase function of the
LRRK2(G2019S) mutant is the prime suspect mechanism for
carriers with this mutation to develop PD, the discovery by us and
others of LRRK2-dependent phenotypes in kidney suggest that
also steady-state abundance of the LRRK2 protein might play a
determining role [30,34].
In patients with LRRK2 mutations and clinically manifest PD,
the associated neuropathology is heterogeneous ranging from LB
pathology with a variable burden of Tau neurofibrillary tangles
(NFTs) to Tau-only pathology and no inclusions [10,35,36,37,38].
R1441C carriers seem to lack LB pathology and an initial report
described pathological variability even within the same family
. In contrast, most autopsies of LRRK2(G2019S) mutation
carriers with PD show LB pathology (e.g. 19/22, ; 3/3, )
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although the brain regions displaying Lewy pathology are variable.
For example, extensive cortical involvement was reported in 7/19
cases whereas 12 out of the same 19 cases had extensive brainstem
pathology ( and discussion therein). So genetic factors other
than LRRK2 itself and environmental risk factors might
exacerbate PD related changes in LRRK2(G2019S) patients and
determine also the extent of cortical involvement.
One critical question concerning the molecular pathogenesis in
LRRK2(G2019S) PD patients is whether the SNCA gene and aSN
protein levels have a contributory role. It is known, for example,
that expansion of SNCA Rep1, an upstream polymorphic
microsatellite of the SNCA gene, is associated with elevated risk
for sporadic PD [41,42,43]. Also, Rep1 regulates SNCA expres-
sion by enhancing its transcription in the nervous system [44,45].
So far, human genetic data have not been disclosed to suggest
synergistic effects in PD pathogenesis of the LRRK2(G2019S)
allele and aSN levels as dictated by SNCA gene polymorphisms. In
mice, two groups have reported co- and over-expression of
LRRK2 and aSN [46,47]. Lin et al.  showed that under
control of a strongly forebrain-selective CaMKII-tTA promoter,
over-expression of a tetO-LRRK2(WT) or tetO-LRRK2(G2019S)
transgene failed to cause neurodegeneration. However, both
LRRK2 variants accelerated the progression of a tetO-aSN
transgene-mediated neuronal loss and a-synucleinopathy in
CaMKII-tTA/tetO-LRRK2/tetO-aSN transgenic mice and ex-
acerbated the accompanying astrocytosis and microgliosis. The
most prominent effects were reported in striatum but cortex was
also affected. These findings contrast with recent results reported
by Daher et al. . Co-expression of aSN(A53T) and
LRRK2(G2019S) under control of a hindbrain selective prion
promoter had minimal impact on the lethal neurodegenerative
phenotype and predominantly hindbrain-selective a-synuclein-
related pathology that developed in the aSN(A53T) mice. The
latter study therefore failed to provide support for a pathophys-
iological interaction of LRRK2 and aSN in mouse hindbrain
neurons. Here, we experimentally approached this question
differently by generating double transgenic mice co-expressing
under the control of mouse Thy1-regulatory sequences high levels
of aSN and LRRK2 in a large population of both forebrain and
Results and Discussion
To analyze whether increased levels of LRRK2 compromise
neuronal integrity in vivo and trigger endogenous aSN-gene-driven
or exacerbate transgene-driven a-synucleinopathy, we generated
(hLRRK2(WT)) or thePD-associated
(hLRRK2(G2019S)). Both LRRK2 cDNAs were expressed under
the control of Thy1 regulatory sequences which direct widespread
expression in neurons in cortex, brainstem and spinal cord
(Figure 1A and [48,49,50]). Out of five hLRRK2(WT) lines, four
lines showed either no or variable transgene expression. One line
selected for further studies showed strong and stable LRRK2 over-
expression (Figure S1 and data not shown). From seven
hLRRK2(G2019S) founders, we obtained two lines with high
and stable LRRK2 expression (Figure 1C,S1 and data not
shown). One of the latter lines and the one hLRRK2(WT) line
were used for further studies.
In situ hybridizations using an anti-sense riboprobe correspond-
ing to nucleotides 3537–4024 of the human LRRK2 transcript
(Figure 1B,S2) and Western blot analysis (Figure 1C,S1)
confirmed high and widespread expression of the LRRK2
transgene inallbrain areas,
except forthe cerebellum
(Figure 1B,1C,S1,S2). Protein and transgene expression levels
of the hLRRK2(WT) and hLRRK2(G2019S) transgenes were
very similar in cortex, hippocampus and brainstem (Figure
S1,S2) but slightly lower for the former in spinal cord (Figure S1)
and striatum (Figure S1,S2).
No obvious brain pathology developed in either line up to the
age of 19 months (oldest age analyzed; data not shown). Others
have reported similar negative findings in aCamKII-LRRK2
transgenics  whereas high LRRK2 levels targeted to the
dopaminergic neurons seem to affect their integrity and viability
[33,51,52]. We could not assess this in our lines because like most
Thy1 based lines, also our lines lack expression in substantia nigra
dopaminergic neurons. An attempt to generate lines expressing
LRRK2 under control of the tyrosine hydroxylase promoter failed
(data not shown).
When we assessed behavioral performance of 3–4 months old
mice on the rotarod, we surprisingly found that motor skill
learning, expressed as the latency to fall, was significantly better in
male hLRRK2(G2019S) mice as compared to their male wildtype
littermates (Figure 2A). Similar but statistically insignificant
trends of LRRK2-transgene dependent improved rotarod perfor-
(Figure 2A) and male as well as female hLRRK2(WT) mice
(data not shown). By the age of 10 months, the beneficial rotarod
effects of high LRRK2 levels had waned (data not shown).
Motility, measured as distance travelled in a homecage-like
environment, was also enhanced in the first 30 min in 7 month-
old but not in aged mice (Figure 2B). Although suggestive of
some beneficial role of high LRRK2 levels on motor performance,
it is not possible to correlate the transient behavioral performance
changes with LRRK2 transgene expression in any particular brain
area. Simply, because many areas relevant to motor behavior
(Figure 1B,1C,S1,S2). Others have reported either changes
[47,53,54] or no changes [51,55] in motor behaviors in mice over-
expressing LRRK2(G2019S). Investigators used both different
LRRK2 variants as well as transgenes with different expression
profiles in heterogeneous mouse genetic backgrounds. Therefore,
we believe it is not possible to draw firm conclusions unlike when
comparing mouse lines made via a LRRK2 gene-specific knock-in
mutagenesis approach as we demonstrated recently . Further-
more, no significant changes were detected in other motor
behavior-relevant tests including cocaine-induced hyperlocomo-
tion (data not shown), behavior in the open field (Figure S3C),
homecage running wheel performance (Figure S3F) or move-
ment measured using an actimeter device (data not shown).
Likewise, no changes were observed in anxiety-relevant tests in the
open field, the dark/light box and the elevated plus-maze (Figure
S3D,S3E and data not shown) or hippocampus-dependent spatial
reference learning in the Morris watermaze (Figure S3A,S3B).
Taken together, it seems clear that high levels of G2019S or
wildtype LRRK2 protein are well tolerated in many forebrain,
hindbrain and brainstem neurons in vivo with little bearing on
neuronal network functions required to perform a variety of
behavioral tasks. At first, this seems quite surprising. LRRK2 has
been implicated in key neuronal processes such as synaptic vesicle
trafficking, exo- and endocytosis [54,56,57], the shaping and
branching of neurites [51,58,59,60,61,62,63], autophagy/lyso-
somes [30,51,58,61] and neurogenesis . The underlying
molecular mechanisms remain to be understood but the diversity
of functions suggests that LRRK2 is either involved in multiple
independent signaling pathways or part of a central signaling
complex with multiple in- and outputs. The difficulties thus far to
discover clear LRRK2-dependent brain phenotypes in mice seems
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to suggest that LRRK2 roles are subject to compensation and
success in unmasking these may dependent on choosing the right
Next, we analyzed whether increased levels of LRRK2
compromise neuronal integrity in vivo by triggering neuropatho-
physiological changes via endogenous aSN or Tau. Brains of aged
hLRRK2(G2019S) mice (15 months) showed no differences in the
levels of aSN, P-S129-aSN, Tau and P202-Tau as compared to
wildtype littermate brain (Figure 2C). Note, P-S129-aSN levels in
wildtype mouse brain are very low and variable and this pattern
did not change in LRRK2 over-expressing mice. The levels of Tau
and in particular P202-Tau are also variable from animal to
animal. Overall, P202-Tau levels seemed slightly higher in
hLRRK2(G2019S) mouse brains but robust increases as described
by others [33,54,61,63] were not observed and the effects we
observed remained statistically insignificant. In summary, in our
hands, excessive levels of wildtype or mutant LRRK2 failed to
induce histopathological hallmarks of a-synucleinopathy and
tauopathy in Thy1-transgene targeted mouse neurons. Altogether,
these findings suggest that high LRRK2 levels do not compromise
endogenous aSN and Tau homeostasis. This contrasts with
findings reported by others who documented alterations in aSN
[33,52,54,55,61,63,64,65,66]. Perhaps cellular context is a key
determining factor in this process. Findings in postmortem brains
of LRRK2 mutation carriers with PD show occasionally tauopathy
and much more frequently a-synucleinopathy although more
precise estimates of their prevalence still await larger number of
cases to be investigated [10,35,36,37,38,39,40,67,68,69]. Whether
these proteinopathies occur mainly in neuronal subtypes which
orthologues in the mouse are not targeted by Thy1 transgenes
remains unresolved but seems rather unlikely. In agreement with
results reported by Daher et al.  and Lin et al. , also our
results fail to provide clear evidence that links LRRK2 protein
abundance to alterations in endogenous aSN or Tau homeostasis.
It can be argued that the failure to detect such links might be
due to the fact that normal endogenous aSN and Tau levels are
insufficient to detect LRRK2-mediated effects on these proteins.
Therefore, we tested whether high LRRK2 levels can exacer-
bate transgene-driven a-synucleinopathy. To this end, double-
transgenic mice were generated that co-express, in a large
number ofneurons hLRRK2(G2019S)
Figure 1. hLRRK2(G2019S) transgene mRNA and protein expression in the mouse brain. (A) Schematic representation of wildtype and
G2019S-mutant human LRRK2-encoding cDNA inserted into the murine Thy1 expression cassette (mThy1). (B) Transgene hLRRK2(G2019S) mRNA
expression pattern comparing transgenic and Ntg mouse brain regions and visualized using a DIG-labeled cDNA probe. (C) Immunoblots showing
expression of endogenous and transgene LRRK2 protein in different brain regions of Ntg and TG [hLRRK2(G2019S)] mice. Note, LRRK2 is indicated by
arrowheads and dependent on the brain region, different unspecific cross-reacting proteins are detected as well. LRRK2 knock-out (KO) cortex is
included as a negative control. Ntg: non-transgenic wildtype littermate control.
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Figure 2. Motor assessment and aSN/Tau protein characterization in hLRRK2(G2019S) mice. (A) Motor skill learning of 4-month-old male
and 6-month-old female hLRRK2(G2019S) and Ntg controls in the 3-step accelerated rotarod task over four consecutive days. The number of mice per
genotype is indicated. Three batches of animals were included in this graph (single transgenic and Ntg animals from experiments shown in Figure 3B
as well as a separate batch). p-values were determined by repeated measure ANOVA (group effect males: F(1,119)=9.42, p=0.003, group effect
females: F(1,52)=3.74, p=0.059). (B) Novelty-induced horizontal locomotor activity of 7.3- and 28.2-month-old hLRRK2(G2019S) and Ntg mice. Bar
graphs show the sum of the distance travelled from 5–30 min and from 35–60 min. The number of mice per genotype is indicated. p-values were
determined either by repeated measure ANOVA (group effect males 7.3 M: F(1,16)=4.044, p=0.061; group effect males 28.2 M: F(1,16)=0.093,
p=0.764) or by two-tailed, unequal variances Student’s t-test. (C) Western blotting of forebrain homogenates from 15-month-old hLRRK2(G2019S)
(TG) and Ntg male mice. Lower panel: Shown are levels of mouse a-synuclein (aSN) and phospho-a-synuclein Ser129 (paSN) as well as mouse
microtubule-associated protein Tau and phospho-Tau Ser202/Thr205 (pTau). b-actin (bAc) was used as loading control and for normalization. Upper
panel shows the results of the immunoblot quantifications. Circles represent individual mice, the means (% normalized to Ntg) are indicated as
horizontal bars. p-values were determined by two-tailed, unequal variances Student’s t-test. Ntg: non-transgenic wildtype littermate control.
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together with high levels of the familial PD-causing aSN(A53T)
mutant also under the control of the exact same Thy-1
transgene used to express the LRRK2 variants. These lines
(Figure 3A). Immunofluorescence studies revealed extensive
co-localization within many neurons in different brain regions of
transgene-derived LRRK2 and aSN (Figure S4). As reported
earlier, haSN(A53T) mice developed motor coordination deficits
(Figure 3B and ) earlier than maSN(WT) mice (Figure 3B
and ). Co-expression of LRRK2 had no influence on the
declining motor skill learning phenotype of male and female
haSN(A53T) mice whereas in maSN(WT)/hLRRK2(G2019S)
double transgenics, it had a small but significant benefit on
(Figure 3B). A similar sex-dependant benefit of high LRRK2
levels was notedinsingle
(Figure 2A). Therefore, LRRK2-mediated effects in the double
transgenic background are likely not due to a reversal of aSN-
transgene-induced motor deficits precluding us from pursuing
similar assessments in maSN(WT)/hLRRK2(WT) double trans-
Around 6 months of age, haSN(A53T) and maSN(WT) mice
began to show severe motor deficits, hypokinesia and weight loss.
At this stage, animals were sacrificed in accordance with local
regulations. The age-of-onset of this late-stage phenotype varied
from animal to animal between 6 and 11 months with no gender
effect (Figure 3C). High LRRK2 levels did not modify the
progression and features of the late-stage phenotype with one
exception: a delay in onset was observed in female haSN(A53T)/
hLRRK2(G2019S) double transgenics as compared with haS-
N(A53T) single transgenic female littermates (Figure 3C). No
significant difference in life expectancy was observed in their male
counterparts and in both sexes of maSN(WT)/hLRRK2(G2019S)
and haSN(A53T)/hLRRK2(WT) mice (Figure 3C). These results
are consistent with those of Daher et al. who also reported no
effect on life expectancy in their double transgenic mice . The
in our hands observed female sex-restricted effect specific to the
haSN(A53T)/hLRRK2(G2019S) double transgenic line are most
likely caused by a integration-site effect although other possibilities
can not be ruled out.
End-stage haSN(A53T) mice, as described earlier , and
double transgenic haSN(A53T)/hLRRK2(G2019S) mice both
showed a massive and widespread microgliosis (IBA1-positive
microglia) in hindbrain structures (Figure 4A–D) as compared
to control non-transgenic littermates and single transgenic
hLRRK2(G2019S) mice (Figure 4F–I). Quantitative analysis
(Figure 4E) which is in accordance with the results on gliosis
in hindbrain structures published by Daher et al.  but
contrasts with the exacerbated forebrain (mainly striatum) gliosis
observed in double versus single transgenics reported by Lin et
al. . If anything, a non-significant trend (p=0.179) towards
reduced microgliosis was observed in double as compared to
single transgenics. We had a similar finding in maSN(WT) and
maSN(WT)/hLRRK2(G2019S) mice (Figure S5). End-stage
astrogliosis as described earlier [48,49] was also similar in
maSN(WT) and haSN(A53T) mice with and without co-
expressing of LRRK2 (Figure S6 and data not shown). It
seems likely, that the discrepancy between the accelerated
LRRK2-dependent aSN-pathology and gliosis mainly in the
striatum as reported by Lin et al.  contrary to its lack in
other forebrain, hindbrain and brainstem regions as reported
here by us and others  might have its cause in the much
higher levels of CaMKII promoter-driven transgene expression
Lin et al. achieved in the striatum .
Biochemical analysis showed that end-stage haSN(A53T) but
not their wildtype non-transgenic littermates had accumulated
significant levels of S129-phosphorylated aSN oligomers and
aSN truncated species in both the soluble and the insoluble
fraction of spinal cord extracts (Figure 5; please note, non-
transgenic wildtype controls (Ntg) and KO lanes were taken
from a blot shown also in Figure S7). The forebrain was
largely devoid of aSN oligomers (Figure 5 and ). We
further confirmed these findings by also using novel time-
resolved Foerster-resonance energy transfer (TR-FRET) based
assays that detect either total aSN or specifically aSN oligomers
(Figure 6 and Bidinosti et al., submitted). In the spinal cord,
aSN oligomers appear in an age-dependent fashion and become
prominent only close to end-stage disease when the mice also
display fulminate neuronal aSN and ubiquitin histopathology
[48,50]. Most importantly, none of these aSN disease-related
protein patterns on gel or in situ changed as a result of LRRK2
co-expression. Again, these results largely agree with the
findings in hindbrain regions reported by Daher et al. 
but they differ from the LRRK2-promoting effects on aggrega-
tion and somatic accumulation in mainly the striatum reported
by Lin et al. . It is however important to note that our
Thy-1-based transgenes are poorly expressed in the striatum
and the prion-based transgenes used by Daher et al.  seem
more hindbrain-selective. Therefore, altogether these findings
tempt us to speculate that LRRK2-mediated exacerbation of
aSN neuropathology might be highly cell-type and brain-region
dependent. Our data provide support for no such direct
interplay in many forebrain and brainstem neurons that express
Analysis on denaturing gels showed that P-129S-aSN levels in
the soluble fraction are much lower in spinal cord as compared
to forebrain (Figure 5). The opposite holds for P-129S-aSN
levels in the insoluble fraction (Figure 5) which is not surprising
and in line with our previously published immunohistological
analysis showing abundant localization of P-S129-aSN in
microscopically visible aggregates inside affected spinal and
brainstem neurons . Resolving the same soluble protein
fractions on native gels showed that P-S129-aSN was present in
forebrain but not in brainstem (Figure S8). All these analysis
basically indicate that P-S129-aSN in the histopathologically
affected brain areas is present mainly in insoluble aggregates
which is in line with our earlier reported immunohistochemical,
electron microscopy (EM) and immuno-EM analysis . High
LRRK2 levels in the double transgenics did not significantly
change the levels and protein patterns of monomeric and P-
S129- aSN, neither in the soluble nor in the insoluble fractions
(Figure 5). End-stage haSN(A53T)/hLRRK2(G2019S) mice
with ages ranging from 6–12 months also revealed no qualitative
and quantitative differences in aSN protein patterns (Figure S9).
Neither did TR-FRET analysis in endstage haSN(A53T)/
hLRRK2(G2019S) andmaSN(WT)/hLRRK2(G2019S) mice
(Figure 6,S10). Also immunohistological analysis failed to reveal
obvious differences in aSN staining patterns when comparing
haSN(A53T) single and haSN(A53T)/hLRRK2(G2019S) double
transgenic mice (Figure S11). Therefore, regardless of the
variable age at which mice reached end-stage disease, a-
synucleinopathy features in individual animals were essentially
strikingly similar and indistinguishable between single and double
transgenics. Of course, we may have missed effects that might
have occurred in a specific timeframe in younger animals that
predominantly contain monomeric aSN [48,50]. A detailed
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analysis of time-dependent micro- and astrogliosis might reveal
further insight but will be technically very challenging because of
the variable age at end-stage disease. It is also difficult to exclude
that the aSN transgenic models used override and therefore mask
LRRK2 modifying effects on aSN. Lastly, we should not exclude
that reduced rather than increased LRRK2 abundance can
Figure 3. High LRRK2 transgene levels do not exacerbate a-synuclein-driven phenotypes. (A) Schematic representation of the four
different transgenic lines used to generate double transgenics. (B) 3-Step accelerated rotarod performance of females and males comparing single
and double transgenics. The different genotypes and the number of mice per genotype are indicated. p-values were determined by repeated
measures ANOVA (group effects for the respective panels: 1: F(1,22)=0.483, p=0.494; 2: F(1,26)=0.000, p=0.983; 3: F(1,11)=0.738, p=0.409; 4:
F(1,22)=2.048, p=0.166; 5: F(1,16)=1.255, p=0.279; 6: F(1,27)=5.171, p=0.031). (C) Kaplan-Meier curves showing the time-of-sacrifice when mice
had to be killed because of too severe motor deficits (1=100% and 0=0% of mice alive). The different genotypes, gender, number of mice per
genotype and the p-values (nonparametric Kaplan-Meier) are indicated.
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modify a-synucleinopathy. Recent findings in LRRK2 knockout,
G2019S knock-in and kinase-dead knock-in mice suggest that
LRRK2 steady-state levels can change in LRRK2-variant, tissue
and brain-region dependent fashions . Not reported before,
we have observed a consistent reduction in LRRK2 protein
levels in the striatum but not e.g. the cerebellum of LRRK2
G2019S knock-in mice (Figure S12). In organs like the kidney,
we did see a change in the abundance of the kinase-dead
LRRK2 variant but not for the G2019S variant . Thus, cell-
type specific differences in the regulation of LRRK2 variants and
their steady-state levels might eventually turn out to be a
determining factor in PD pathogenesis.
Figure 4. Microgliosis in end-stage haSN(A53T) transgenic mouse brain is unaltered by high LRRK2 levels. DAB-immunohistochemistry
for Iba1 shows activated microglia on a representative sagittal brain section of a haSN(A53T) mouse (A and 206higher magnification from brainstem
in B) and a haSN(A53T)/hLRRK2(G2019S) double transgenic mouse (C and 206higher magnification from brainstem in D). (E) Quantification of the
brainstem results. Values represent % of the area in the brainstem that is covered by Iba1-positive microglia. p-value (p=0.179) was determined by
two-tailed, unequal variances Student’s t-test. Dots represent quantifications of single individuals. Control images obtained from a separate
experiment but from littermate hLRRK2(G2019S) single transgenic (F and 206higher magnification from brainstem in G) and from non-transgenic
wildtype littermate control (Ntg) (H and 206higher magnification from brainstem in I) mouse.
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Figure 5. aSN and phospho-S129-aSN protein levels in spinal cord and forebrain of end-stage disease single and double transgenic
mice. Tris-soluble and -insoluble fractions of spinal cord and forebrain lysates were immunoblotted and stained with antibodies detecting total a-
synuclein (aSN) or specifically phosphorylated S129-aSN (paSN). b-actin (bAc) levels were measured as loading control and for normalization. For
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Materials and Methods
All experiments were carried out in accordance with the
authorization guidelines of the Swiss federal and cantonal
veterinary offices for the care and use of laboratory animals.
Studies described in this report were approved by the Swiss
cantonal veterinary office and performed according to Novartis
animal license number 2063.
Human LRRK2 wildtype or G2019S mutated cDNA (7665 bp)
was cloned into the Thy1 cassette  and transgenic Thy1-
hLRRK2 mice were generated by pronuclear injection (C57Bl/6
mouse eggs) of linearized (NotI) and purified minigene. Transgenic
mice were selected by PCR analysis of tail DNA with primers
Thy1for2 (59-GGG CTG ACC TGG ACA TTA GG-39) and
PARK8rev2 (59-GGC GAA TTC TGC AGA TAT CC-39), which
amplified with a standard PCR protocol an 870 bp DNA
Thy1-haSN(A53T)  and Thy1-maSN  were genotyped
by using primers HP45 (59-ACA CCC CTA AAG CAT ACA
GTC AGA CC-39) and HP42 (59-TGG GCA CAT TGG AAC
TGA GCA CTT-39), amplified DNA fragment: 1200 bp. Double
transgenic mice Thy1-hLRRK2/Thy1-aSN were identified by
PCR analysis of tail DNA using two independent rounds of PCR,
one for Thy1-hLRRK2 and Thy1-aSN, respectively. Transgenic
mice were kept in C57BL/6 background. For analyses males and
females were used.
Alternatively, single and double transgenic mice were selected
by standard Taqman PCR analysis of tail or ear DNA with
primers and fluorescently labeled probes: Thy1for2 (59-GGG
CTG ACC TGG ACA TTA GG-39), Thy1rev2 (59-GGT CTG
ACT GTA TGC TTT AGG G-39) and Thy1-probe (59-FAM-
CCA GAG ACT GGC TAC ACA GCG ATA TGA C-TAMRA-
39). As endogenous control PCR: a-synuclein (aSN-59 (59-GCT
GGA AAG ACA AAA GAG GG-39), aSN-39 (59-ATT CTC TCA
CCT CCA CAC AG-39), aSN-probe (59-FAM or YYE-TGG
CTG GTG TGT GGT GTC TGA TT-TAMRA-39)) or LRRK2
(LRRK2.for (59-TGT ATC CCA ATG CTG CCA TC-39),
LRRK2.rev (59-CTA TAT CTC CTA GAC CCA CAC-39),
LRRK2.Ex41.probe (59-YYE-TGG GAA TAA AGA CAT CAG
AGG GCA C-TAMRA-39)). Rotorgene 3000 was used for PCR
reaction (95uC 10 min, 40 cycles of 95uC 15 sec and 60uC 1 min).
All work was carried out at 4uC. Mouse brain was added to 5 ml
homogenization buffer (20 mM Tris-HCl, pH 7.4, 0.25 M
sucrose, 1 mM EDTA, 1 mM EGTA, 0.5 mM PMSF, 5 mg/ml
pepstatin A and leupeptin, okadaic acid 0.01 mM, calyculin
0.1 mg/ml, 16phosphatase inhibitor cocktail (PIERCE)) without
any detergent, then homogenized (Precellys24, Bertin Technolo-
gies), incubated on ice for 30 min and then centrifuged at
13000 rpm for 20 minutes at 4uC. The protein concentration of
the supernatant was determined using a BCA protein assay kit
(Bio-Rad Laboratories). For immunoblot analysis, 10 mg/slot of
supernatant (treated with NuPAGE LDS sample buffer (46) and
NuPAGE sample reducing agent (106) at 95uC for 5 min) was
analyzed by PAGE (NuPAGE Novex Bis-Tris gels (4–12%), Tris-
Acetate (3–8%), Invitrogen). The pellet fraction was dissolved in
reference, LRRK2 levels detected via immunoblot are shown comparing single and double-transgenics. Different a-synuclein protein species/forms
are marked as follows: mo, monomer; ol, oligomer; tr, truncated. For reference, in the upper panels the performance and specificity of the antibodies
are illustrated in the two right lanes comparing WT and KO (aSN knock-out) brain samples and were added to indicate unspecific cross-reactive
proteins (taken from Figure S7). Graphs represent quantifications of monomeric aSN and paSN/aSN, all normalized to bAc. Circles represent individual
mice, the means are indicated as horizontal bars and % are normalized to the levels in haSN(A53T) single transgenics. p-values were determined by
two-tailed, unequal variances Student’s t-test. Genotypes: aSN=haSN(A53T), aSN/LRRK2=haSN(A53T)/hLRRK2(G2019S), Ntg=non-transgenic
wildtype littermate control and KO=aSN knock-out mice.
Figure 6. aSN oligomers, detected by TR-FRET, are dramatically
increased in the spinal cord of mice with end-stage disease,
relative to forebrain, independently of LRRK2(G2019S) expres-
sion. TR-FRET analysis for total or oligomeric a-synuclein (aSN) was
performed in 384-well microtiter plates on homogenates of the
indicated brain regions. Antibody combinations used in TR-FRET were
NovSyn2-Tb/NovSyn3-d2 for total aSN, and NovSyn3-Tb/NovSyn3-d2
for oligomeric aSN. dF is the percent increase of the aSN signal above
buffer background. Individual samples were measured in duplicate and
the average used to calculate the plotted group means. p-values
(asterisk indicate p,0.05) were determined by 1way ANOVA (Kruskal-
LRRK2 and Alpha-Synuclein
PLoS ONE | www.plosone.org9 May 2012 | Volume 7 | Issue 5 | e36581
the same homogenization buffer as above without detergent
(106volume of the tissue weight) and then treated like and used
according to supernatant samples. For blotting XCell II blot
module (Invitrogen) was used. Detection was performed using the
following antibodies: Mouse monoclonal antibodies a-synuclein
(1:5000, BD Biosciences), phosphorylated a-synuclein (1:5000,
WAKO) ); rabbit anti-LRRK2 home-made (1:500); mouse anti-
Tau (1:2000, TAU-5, Biosource); mouse anti-P202-Tau (1:500,
AT8, MN1020, Pierce); rabbit anti-TH (1:5000, Chemicon
AB152); rabbit anti-DARPP32 (1:5000, Chemicon AB1656);
rabbit anti-GAD (1:5000, Chemicon AB1511). Membranes were
washed 4 times for 5 min at room temperature in PBS containing
0.05% or 0.1% Tween20 and then incubated for 45 min (light
protected) with secondary antibodies (Alexa Fluor 680, F(ab’)2
fragment of goat anti-mouse (Invitrogen); IRDye 800 CW anti-
rabbit IgG (Li-cor); both 1:5000) in Odyssey Blocking Buffer
(diluted 1:1 in PBS, containing 0.1% or 0.05% Tween20).
Membranes were again washed 46for 5 min at room temperature
in PBS containing 0.05% or 0.1% Tween20, then washed 26for
5 min at room temperature in PBS only and finally scanned on the
Odyssey Li-cor System. Fluorescence intensities quantification was
performed by standardization to b-actin levels using a mouse
monoclonal anti b-actin clone AC-15 antibody (1:50000, Sigma
In situ Hybridisation
20 mm sagittal sections were prepared from fresh frozen mouse
brains, fixed for 1 h in PBS buffered 4% paraformaldehyde and
subjected to an automated in situ hybridisation procedure using
the Ventana/Roche DiscoveryXT technology. Biefly, slides were
postfixed for 4 minutes with VENTANA RiboPrebTMsolution
and conditioned by heat pre-treatment (12 min at 98uC in
RiboCC) followed by mild proteolysis (4 min at 37uC with
VENTANA protease 3). Sections were then hybridized for 6 h at
65uC with 0.1 mg/ml digoxigenin-labelled anti-sense riboprobe
(corresponding to nucleotides 3537 to 4024 of the human LRRK2
gene sequence) diluted in a hybridisation solution containing one
part VENTANA RiboHybTMand two parts 26SSC, followed by
high stringency washes with 26SSC at 75uC for 368 min, and
post-fixation for 8 min in VENTANA RiboFixTM. To visualize
hybridisation signals, sections were incubated for 28 min with
alkaline phosphatase labelled sheep anti-digoxygenin Fab frag-
ments (Roche Diagnostics) diluted 1:500 in VENTANA Discovery
antibody diluent, and subjected to an alkaline phosphatase-
catalyzed colour reaction with NBT/BCIP (VENTANA Blue-
MapTMkit) for 9 h.
Antibodies used for immunohistochemistry and immunofluo-
rescence staining: Primary antibodies: Rabbit anti-alpha synu-
clein (Chemicon AB5038; 1:1000), Mouse anti-alpha synuclein
(abcam 4B12; 1:100), monoclonal mouse anti-alpha synuclein
monoclonal rabbit anti-LRRK2 (Epitomics c41-2 #3514-1);
ubiquitin (DAKO Z0458; 1:200), Rabbit anti-Iba1 (WAKO
Chemicals #019-19741; 1:000); Secondary antibodies: Bioti-
nylated goat anti-mouse (Jackson ImmunoResearch; 1:1000),
Biotinylated goat anti-rabbit (Jackson ImmunoResearch; 1:1000);
Alexa488-labeled anti-rabbit IgG (Invitrogen); Alexa594-labeled
anti-mouse IgG (Invitrogen).
Manual immunohistochemistry: Paraffin sections (4 mm thick)
of immersion fixed mouse brains were dewaxed in xylene,
rehydrated in decreasing ethanol solutions, rinsed in double-
onbehalf of Novartis);
distilled water, rinsed in PBS (Phosphate-buffered saline, pH 7.4),
subjected to antigen unmasking (only for GFAP and ubiquitin) and
incubated for 1 h in blocking solution (from PerkinElmer Kit
NEL-741B). Sections were then incubated with primary antibody
(diluted in blocking solution) over night at 4uC. Slides were rinsed
463 min in PBS, incubated for 60 min at room temperature with
biotinylated secondary antibody diluted in blocking solution,
rinsed 463 min in PBS, incubated for 30 minutes ABC reagent
(PerkinElmer TSA plus Kit) and rinsed 265 min in PBS. Staining
was performed with AEC kit (Zymed ZUC054) according to the
instructions of the supplier. Sections were counterstained for 2 min
with Mayer’s Haematoxylin, immersed in ammonia water for
10 sec, rinsed with running tap water for 5 min, dehydrated in
increasing ethanol solutions and xylene and mounted with Eukitt.
Manual immunofluorescence double staining to investigate co-
localisation of LRRK2 and a-synuclein: 4 mm sagittal paraffin
sections were de-waxed, rinsed 36for 5 min in PBS, subjected to
antigen unmasking by microwaving, incubated for 1 h with PBS
containing 2% goat serum, then over night at 4uC with a mixture
of mouse anti-a-synuclein (Novartis; 1:10000) and rabbit anti-
LRRK2 (MJFF2 c41, Epitomics; 1:200) diluted in PBS/2% goat
serum, washed 36 in PBS, stained with a mixture of Alexa488-
labeled goat anti-rabbit and Alexa594-labeled goat anti-mouse
each diluted 1:500 in PBS, and mounted with with Prolong Gold
containing DAPI nucleic acid counterstain (Invitrogen).
Antigen unmasking procedures to enhance staining of
LRRK2 and GFAP.
Following de-paraffinization and rehydra-
tion, slides were microwaved for 10 min at 90uC (T/T MEGA
Milestone) in 0.1 M sodium citrate buffer pH 5.8, rinsed in PBS
and transferred into blocking solution. The following steps of the
immunostaining procedure were performed as described above.
Following deparaffinization and rehydra-
tion slides were incubated for 15 min at 37uC in 0.05% pronase in
0.5 M Tis/HCl pH 7.6. Slides were then rinsed in PBS and
transferred into blocking solution. The following steps of the
immunostaining procedure were performed as described above.
Automated immunostaining of Iba1.
paraffin sections of immersion fixed mouse brains were mounted
on SuperFrost+ slides and subjected to an automated immuno-
staining procedure using the Discovery XT technology (Ventana/
Roche diagnostics). Briefly, sections were de-paraffinized, rehy-
drated, subjected to antigen retrieval by heating with CC1 cell
conditioning buffer (Ventana/Roche Diagnostics), incubated for
60 min at room temperature with primary antibody diluted in
antibody diluent (Ventana/Roche Diagnostics), incubated with
biotinylated goat anti-rabbit secondary antibody diluted in
Ventana antibody dilution, reacted with DABMab kit (Ventana/
Roche Diagnistics) and counterstained with blueing reagent
Microglia and astroglia were quantified by using systematic-
random series of brain sections at three different anatomical planes
per animal which were analyzed by a MCID image analyzer
(Imaging Research, Brock University, Ontario, Canada, Program
Version M7 elite). The microscopic image was digitized by use of a
Roper black and white CCD TV camera and stored with
112461124 pixel resolution at 256 gray levels. The pixel size was
calibrated using an object micrometer at 56magnification (Leica
Neoplan Objective). Using a motor driven microscope stage for
exact positioning of adjacent object fields the entire brainstem of
each section was analyzed. For each object field the anatomical
area was defined by manual outline. For each individual section
the sample area was defined by manual threshold setting (grey
level). Isolated tissue artifacts were excluded by manual outline.
4 mm para-sagittal
LRRK2 and Alpha-Synuclein
PLoS ONE | www.plosone.org10May 2012 | Volume 7 | Issue 5 | e36581
Data were analyzed as individual counts (microglia or astroglia) to
the area in %.
TR-FRET Immunoassay Analysis
Antibodies recognizing C-terminal a-synuclein epitopes were
raised in-house and are denoted as NovSyn2 and NovSyn3.
Antibodies were chemically coupled with either Lumi4H-Tb
(Terbium cryptate) donor fluorophore or a second generation d2
acceptor fluorophore (CisBio Bioassays). The development and
characterization total or oligomeric a-synuclein TR-FRET assays
is described elsewhere (Bidinosti et al., unpublished) and relies on
the FRET-dependent signal generated upon coincident binding of
two fluorophore-conjugated antibodies to a single a-synculein
molecule or oligomer. The antibody combination of NovSyn2-
Tb/NovSyn3-d2 binds two unique epitopes potentially accessible
on all a-synuclein species. NovSyn3-Tb/NovSyn3-d2 however is
specific for oligomeric a-synuclein via binding to multiple copies of
a single epitope which are uniquely present in oligomeric species.
For conducting the assay, brain regions were homogenized in 10
volumes (w/v) of lysis buffer (PBS containing 1% Triton X-100
and Complete Protease Inhibitor cocktail (Roche)). Total protein
content was determined by BCA assay (Pierce) and 5 ml/well of
total protein-normalized homogenates were loaded onto 384-well
low-volume polystyrene microtiter plates in technical duplicates.
Detection antibodies were diluted in analysis buffer (50 mM
NaHPO4, 400 mM NaF, 0.1% BSA, and 0.05% Tween-20) and
1 ml of this solution was added such that each well contained
0.3 ng of donor fluorophore-conjugated antibody and 3 ng of
acceptor fluorophore-conjugated antibody. The plates were then
incubated overnight at 4uC. Sample fluorescence was measured
with an EnVision Multilabel Reader (PerkinElmer) and FRET-
dependent acceptor fluorescence (665 nm) was normalized to
FRET-independent donor fluorescence (620 nm) for each sample.
The relative total or oligomeric a-synculein content is reported as
the percent increase of each sample 665 nm/620 nm ratio over
that of buffer alone (dF).
on a computerized treadmill (TSE rotarod system). The 3-step
rotarod program consists of a modified rotarod program of three
different running speeds (12 rpm, 24 rpm and 36 rpm) each for
30 sec with intervals of acceleration lasting for 10 sec. Starting
speed is 4 rpm. Rotarod performance was assessed by evaluating
the two best trials out of three performed in one day.
To measure forelimb grip strength, mice are
allowed to grasp a handle connected to a force-measuring device
(San Diego Instruments) and then pulled back with their tails until
they release the handle. The best out of four consecutive trials is
To measure exploratory behavior (pattern and
activity), mice were placed in an open field box (70 cm670 cm,
height of walls: 30 cm) subdivided into nine quadrants with one
middle quandrant. The horizontal distance travelled during 5 min
was recorded by an EthoVision 3.0 system (Noldus).
The dark/light box consists of a dark and a
bright compartment. Mice were placed in the bright compartment
and given the opportunity to move to the dark box for 5 min.
Parameters measured by EthoVision 3.0 (Noldus) were the time
spent in the bright compartment and the latency of first entry to
the dark compartment. The number of transitions and the latency
of first exit back to the bright compartment were measured
To measure motor coordination mice were placed
the floor) consists of four arms (length: 27 cm) arranged in right
angles to each other. Two opposite arms have walls (height:
15 cm) and the two others are open. Mice are placed in the middle
and are allowed to move freely for 5 min. The time spent in the
open arms is recorded by EthoVision 3.0 (Noldus) and the number
of entries to open arms by visual inspection.
Locomotor activity was measured with the
TSE system (process control type 302013-CD, software: Motili-
ta ¨tsmesssystem 4.2) in motility cages (Makrolon Typ III with a lid
and without embedding). Cages were changed after each animal.
Running wheels (med Associates) were
placed to the homecage (Makrolon Typ III, 22637615 cm, with
Filtertop) and the mice were allowed to freely access the wheels for
24 hfor10days(averageddataover24 h,software:WheelAnalysis
SOF-861, environmental sensor included, med Associates).
Morris water maze.
Mice were placed in a pool (white
plastic, Ø 149 cm) filled with 24uC warm water (clouded with 2 l
of skimmed milk 40 min before the start of the experiment) and
was allowed for 2 min to find the escape platform (Ø 8 cm) that
was localized 16 cm to the pools9 edge and 22 cm to pools bottom,
5 mm below waterline always at at the NO quadrant. Cues were
available at the walls. A session consisted of 4 trials (each max
2 min, 15 min between trials) from positions N, S, E, W, each day
the start position was shifted. The room lights were switched off
and indirect light was turned on 30 min before the start of the
trials. For the probetrial the platform was removed and the mice
were placed SW into the pool (one trial only) for 2 min. For
recording and analysis Noldus EthoVision 3.0 was used.
The elevated plus-maze (80 cm from
The animals were housed in a temperature-controlled room
that was maintained on a 12 h light/dark cycle. Food and water
were available ad libitum.
Data are expressed as average 6 SEM. Rotarod and horizontal
locomoter activity curves were analyzed by repeated measure
ANOVA. The survival curves were analyzed by nonparametric
Kaplan-Meier test. TR-FRET was analyzed by 1way ANOVA
(Kruskal-Wallis test). Statistical analyses of the other graphs were
performed using two-tailed, unequal variances Students t-test.
and hLRRK2(WT) mice. Immunoblots of protein extracts from
different brain regions of non-transgenic wildtype littermate
control (Ntg), hLRRK2(G2019S) and hLRRK2(WT) mice (5–7
months old) detecting LRRK2. b-actin served as loading control.
Ntg and LRRK2 knock-out (KO) served as controls to indicate
LRRK2 antibody specificity for the cerebellum immunoblot
(experiment performed separately). Note that the abundance of
some unspecific but LRRK2 antibody cross-reacting proteins
seems slightly increased. We don’t know the reason for this and the
identity of these proteins remains unknown. Nonetheless, the
Western analysis of cerebellar extracts comparing LRRK2 KO
versus non-transgenic tissue clearly shows that these proteins are
unrelated to LRRK2.
LRRK2 protein levels in hLRRK2(G2019S)
gene mRNA expression in the mouse brain. Transgene
hLRRK2(G2019S) and hLRRK2(WT) mRNA expression pattern
comparing transgenic and non-transgenic wildtype littermate
hLRRK2(G2019S) and hLRRK2(WT) trans-
LRRK2 and Alpha-Synuclein
PLoS ONE | www.plosone.org11 May 2012 | Volume 7 | Issue 5 | e36581
control (Ntg) mouse brain regions visualized using a DIG-labeled
cDNA probe. Note the weak expression of hLRRK2(WT)
transgene in striatum that was confirmed in immunoblot analysis.
hLRRK2(G2019S) mouse line. Performance is shown of
non-transgenic wildtype littermate control (Ntg) and transgenic
hLRRK2(G2019S) male mice in (A) the water maze learning task
(one session per day consisted of four trials) at 3.5 months of age;
(B) Probetrial of the water maze. Platform is located in the north-
east (NE) quadrant (NW: north-west; SE: south-east; SW: south-
west); (C) open field behavior expressed as total distance moved
(age of the animals: 2 months); (D) dark/light box behavior
expressed as the latency before entering the dark compartment
(age of the animals: 2 months); (E) the elevated plus-maze task with
results expressed as time spent in the open arms (age of the
animals: 2 months) and (F) on the running wheel shown as activity
over time during 24 hrs in the homecage (age of the animals: 23
months). Genotypes and n values are indicated.
haSN(WT)/hLRRK2(G2019S) mouse brain. Immunofluo-
rescence for aSN (red) and LRRK2 (green) of sagittal brain section
of Ntg and haSN(A53T)/hLRRK2(G2019S) mice for (A) cortex
(206magnification) and (B) brainstem (406magnification). Co-
localization of aSN and LRRK2 (overlay of red and green) is
depicted by white arrows; gray arrows point to aSN-positive,
Co-localizationofLRRK2 andaSN in
hLRRK2(G2019S) end-stage mouse brain. DAB-immuno-
histochemistry for Iba1 shows activated microglia on a represen-
tative sagittal brain section of a maSN(WT) single (A and
206magnification of brainstem in B) and a haSN(A53T)/
hLRRK2(G2019S) double transgenic mouse (C and 206magni-
fication of brainstem in D). (E) Quantification of the area in the
brainstem that is covered by Iba1-positive microglia plotted as %
of total area. Dots represent quantifications of individual mice.
Control images are shown in Figure 4.
Microgliosis in maSN(WT) and maSN(WT)/
astrocytosis in haSN(A53T) end-stage mouse brain. DAB-
immunohistochemistry for GFAP shows astrocytosis on a repre-
setative sagittal brain section of (A) maSN(WT), (B) haSN(A53T),
brainstem is shown in (D)), (E) maSN(WT)/hLRRK2(G2019S),
(F) hLRRK2(G2019S) single transgenic and (G) non-transgenic
wildtype littermate control (Ntg) mice. Please note, experiments for
(E–G) were performed separately.
High neuronal levels of LRRK2 do not worsen
blots of spinal cord extracts from non-transgenic wildtype
littermate control (Ntg), haSN(A53T) and aSN knock-out (KO)
mice detecting total a-synuclein (aSN), phosphorylated S129-aSN
(P-S129-aSN), and unspecific protein species cross-reacting with
each antibody (aSN KO lanes). Parts of these results are shown
also in Figure 5 for illustration purposes.
Specificity of aSN antibodies used. Immuno-
Immunoblotting results are shown using antibodies detecting total
a-synuclein (aSN) and phosphorylated S129-aSN (P-S129-aSN)
aSN protein species resolved on native gels.
(arrows) in soluble protein extracts of forebrain and brainstem
comparing non-transgenic wildtype littermate control (Ntg) and
haSN(A53T) mice. unsp.: refers to non-aSN proteins cross-
reacting with antibody.
and Tris-insoluble (pellet) fractions of spinal cord lysates. Blots were
indicates the time when each mouse had reached the stage of illness
that required us to killthe animal.
aSN and phospho-S129-aSN levels in spinal
different agesof end-stage athaSN(A53T)/
increased in the spinal cord of end-stage maSN(WT)
mice, independently of hLRRK2(G2019S) co-expression.
TR-FRET analysis was performed as in Figure 6 on homogenates
of the indicated brain regions. Each sample was measured in
duplicates. p-values (asterisks indicate p,0.01) were determined
by 1way ANOVA (Kruskal-Wallis test).
aSN oligomers, detected by TR-FRET, are
haSN(A53T) and haSN(A53T)/hLRRK2(G2019S) mice.
For each genotype, a representative section (106magnification)
stained against aSN is shown of the brainstem (reticular
formation), midbrain (deep mesencephalic nucleus), hippocampus
and cortex (primary motor cortex) of a haSN(A53T) and a
aSN brain histopathology in end-stage
LRRK2(G2019S) knock-in mouse striatum. (A) Immuno-
blotting results of striatal lysates of Ntg and KI (LRRK2(G2019S)
knock-in) female mice are shown (age: 7.5 months). Protein levels
determined included LRRK2, DARPP-32 and TH. b-actin (bAc)
was used as loading control and for normalization. Circles
represent individual mice; the means are indicated as horizontal
bars and % are normalized to the protein levels in Ntg. (B)
Immunoblotting results for LRRK2 and GAD65/67 of cerebellar
lysates from Ntg and KI (LRRK2(G2019S) knock-in) female mice
(age: 7.5 months) and quantification of the results. b-actin (bAc)
was used as loading control and for normalization. Circles
represent individual mice, the means are indicated as horizontal
bars and % are normalized to levels in Ntg mice. (C)
Quantification of LRRK2 immunoblot results comparing levels
in Ntg and KI (LRRK2(G2019S) knock-in) lysates of dorsal,
ventral (males, 6.5 months old) and total (dorsal + ventral; females,
5.5 months old) striatum. Values are expressed as % of level in
Ntg. Bars indicates SEM. LRRK2 levels were normalized to b-
actin and DARPP-32 (which is specifically expressed in the
LRRK2-expressing GABAergic projections neurons of the stria-
tum). The number of mice per group is indicated. p-values were
determined by two-tailed, unequal variances Student’s t-test. Ntg:
non-transgenic wildtype littermate control.
ReducedLRRK2 protein levels in
Conceived and designed the experiments: MCH MB AW PS GR PHvdP
DRS. Performed the experiments: MCH MB TS TH CS SD NV DS CB
FM DRS. Analyzed the data: MCH MB TS TH CS SD CB PS GR DRS.
Wrote the paper: MCH MB PS GR PHvdP DRS.
LRRK2 and Alpha-Synuclein
PLoS ONE | www.plosone.org12 May 2012 | Volume 7 | Issue 5 | e36581
1. Esposito E, Di Matteo V, Di Giovanni G (2007) Death in the substantia nigra: a
motor tragedy. Expert Rev Neurother 7: 677–697.
Jellinger KA (2009) Formation and development of Lewy pathology: a critical
update. J Neurol 256 Suppl 3: 270–279.
Braak H, Rub U, Gai WP, Del Tredici K (2003) Idiopathic Parkinson’s disease:
possible routes by which vulnerable neuronal types may be subject to
neuroinvasion by an unknown pathogen. J Neural Transm 110: 517–536.
Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, et al. (1997)
Mutation in the alpha-synuclein gene identified in families with Parkinson’s
disease. Science 276: 2045–2047.
Paisan-Ruiz C, Jain S, Evans EW, Gilks WP, Simon J, et al. (2004) Cloning of
the gene containing mutations that cause PARK8-linked Parkinson’s disease.
Neuron 44: 595–600.
Nichols WC, Pankratz N, Hernandez D, Paisan-Ruiz C, Jain S, et al. (2005)
Genetic screening for a single common LRRK2 mutation in familial Parkinson’s
disease. Lancet 365: 410–412.
Nichols WC, Elsaesser VE, Pankratz N, Pauciulo MW, Marek DK, et al. (2007)
LRRK2 mutation analysis in Parkinson disease families with evidence of linkage
to PARK8. Neurology 69: 1737–1744.
Satake W, Nakabayashi Y, Mizuta I, Hirota Y, Ito C, et al. (2009) Genome-wide
association study identifies common variants at four loci as genetic risk factors
for Parkinson’s disease. Nat Genet 41: 1303–1307.
Simon-Sanchez J, Schulte C, Bras JM, Sharma M, Gibbs JR, et al. (2009)
Genome-wide association study reveals genetic risk underlying Parkinson’s
disease. Nat Genet 41: 1308–1312.
10. Zimprich A, Biskup S, Leitner P, Lichtner P, Farrer M, et al. (2004) Mutations in
LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology.
Neuron 44: 601–607.
11. International Parkinson’s Disease Genomics Consortium, Wellcome Trust Case
Control Consortium 2 (2011) A two-stage meta-analysis identifies several new
loci for Parkinson’s disease. PLoS Genet 7: e1002142.
12. Do CB, Tung JY, Dorfman E, Kiefer AK, Drabant EM, et al. Web-based
genome-wide association study identifies two novel loci and a substantial genetic
component for Parkinson’s disease. PLoS Genet 7: e1002141.
13. Hamza TH, Zabetian CP, Tenesa A, Laederach A, Montimurro J, et al.
Common genetic variation in the HLA region is associated with late-onset
sporadic Parkinson’s disease. Nat Genet 42: 781–785.
14. Mata IF, Yearout D, Alvarez V, Coto E, de Mena L, et al. (2011) Replication of
MAPT and SNCA, but not PARK16–18, as susceptibility genes for Parkinson’s
disease. Mov Disord 26: 819–823.
15. Rhodes SL, Sinsheimer JS, Bordelon Y, Bronstein JM, Ritz B (2011) Replication
of GWAS associations for GAK and MAPT in Parkinson’s disease. Ann Hum
Genet 75: 195–200.
16. Edwards TL, Scott WK, Almonte C, Burt A, Powell EH, et al. (2010) Genome-
wide association study confirms SNPs in SNCA and the MAPT region as
common risk factors for Parkinson disease. Ann Hum Genet 74: 97–109.
17. An XK, Peng R, Li T, Burgunder JM, Wu Y, et al. (2008) LRRK2 Gly2385Arg
variant is a risk factor of Parkinson’s disease among Han-Chinese from mainland
China. Eur J Neurol 15: 301–305.
18. Farrer MJ, Stone JT, Lin CH, Dachsel JC, Hulihan MM, et al. (2007) Lrrk2
G2385R is an ancestral risk factor for Parkinson’s disease in Asia. Parkinsonism
Relat Disord 13: 89–92.
19. Healy DG, Falchi M, O’Sullivan SS, Bonifati V, Durr A, et al. (2008)
Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated
Parkinson’s disease: a case-control study. Lancet Neurol 7: 583–590.
20. Gilks WP, Abou-Sleiman PM, Gandhi S, Jain S, Singleton A, et al. (2005) A
common LRRK2 mutation in idiopathic Parkinson’s disease. Lancet 365:
21. Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, et al. (2008) Genome-
wide association defines more than 30 distinct susceptibility loci for Crohn’s
disease. Nat Genet 40: 955–962.
22. Torkvist L, Halfvarson J, Ong RT, Lordal M, Sjoqvist U, et al. (2010) Analysis
of 39 Crohn’s disease risk loci in Swedish inflammatory bowel disease patients.
Inflamm Bowel Dis 16: 907–909.
23. Liu Z, Lee J, Krummey S, Lu W, Cai H, et al. (2011) The kinase LRRK2 is a
regulator of the transcription factor NFAT that modulates the severity of
inflammatory bowel disease. Nat Immunol 12: 1063–1070.
24. Looyenga BD, Furge KA, Dykema KJ, Koeman J, Swiatek PJ, et al. (2011)
Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required
for oncogenic MET signaling in papillary renal and thyroid carcinomas. Proc
Natl Acad Sci U S A 108: 1439–1444.
25. Saunders-Pullman R, Barrett MJ, Stanley KM, Luciano MS, Shanker V, et al.
(2010) LRRK2 G2019S mutations are associated with an increased cancer risk
in Parkinson disease. Mov Disord 25: 2536–2541.
26. Zhang FR, Huang W, Chen SM, Sun LD, Liu H, et al. (2009) Genomewide
association study of leprosy. N Engl J Med 361: 2609–2618.
27. Lewis PA, Manzoni C (2012) LRRK2 and Human Disease: A Complicated
Question or a Question of Complexes? Sci Signal 5: pe2.
28. West AB, Moore DJ, Biskup S, Bugayenko A, Smith WW, et al. (2005)
Parkinson’s disease-associated mutations in leucine-rich repeat kinase 2 augment
kinase activity. Proc Natl Acad Sci U S A 102: 16842–16847.
29. Kumar A, Greggio E, Beilina A, Kaganovich A, Chan D, et al. (2010) The
Parkinson’s disease associated LRRK2 exhibits weaker in vitro phosphorylation
of 4E-BP compared to autophosphorylation. PLoS One 5: e8730.
30. Herzig MC, Kolly C, Persohn E, Theil D, Schweizer T, et al. (2011) LRRK2
protein levels are determined by kinase function and are crucial for kidney and
lung homeostasis in mice. Hum Mol Genet 20: 4209–4223.
31. Iaccarino C, Crosio C, Vitale C, Sanna G, Carri MT, et al. (2007) Apoptotic
mechanisms in mutant LRRK2-mediated cell death. Hum Mol Genet 16:
32. Smith WW, Pei Z, Jiang H, Dawson VL, Dawson TM, et al. (2006) Kinase
activity of mutant LRRK2 mediates neuronal toxicity. Nat Neurosci 9:
33. Dusonchet J, Kochubey O, Stafa K, Young SM Jr., Zufferey R, et al. (2011) A
rat model of progressive nigral neurodegeneration induced by the Parkinson’s
disease-associated G2019S mutation in LRRK2. J Neurosci 31: 907–912.
34. Tong Y, Yamaguchi H, Giaime E, Boyle S, Kopan R, et al. (2010) Loss of
leucine-rich repeat kinase 2 causes impairment of protein degradation pathways,
accumulation of alpha-synuclein, and apoptotic cell death in aged mice. Proc
Natl Acad Sci U S A 107: 9879–9884.
35. Gaig C, Marti MJ, Ezquerra M, Rey MJ, Cardozo A, et al. (2007) G2019S
LRRK2 mutation causing Parkinson’s disease without Lewy bodies. J Neurol
Neurosurg Psychiatry 78: 626–628.
36. Rajput A, Dickson DW, Robinson CA, Ross OA, Dachsel JC, et al. (2006)
Parkinsonism, Lrrk2 G2019S, and tau neuropathology. Neurology 67:
37. Marti-Masso JF, Ruiz-Martinez J, Bolano MJ, Ruiz I, Gorostidi A, et al. (2009)
Neuropathology of Parkinson’s disease with the R1441G mutation in LRRK2.
Mov Disord 24: 1998–2001.
38. Giordana MT, D’Agostino C, Albani G, Mauro A, Di Fonzo A, et al. (2007)
Neuropathology of Parkinson’s disease associated with the LRRK2 Ile1371Val
mutation. Mov Disord 22: 275–278.
39. Wider C, Dickson DW, Wszolek ZK (2010) Leucine-rich repeat kinase 2 gene-
associated disease: redefining genotype-phenotype correlation. Neurodegener
Dis 7: 175–179.
40. Poulopoulos M, Cortes E, Vonsattel JP, Fahn S, Waters C, et al. (2011) Clinical
and Pathological Characteristics of LRRK2 G2019S Patients with PD. J Mol
41. Tan EK (2007) The role of common genetic risk variants in Parkinson disease.
Clin Genet 72: 387–393.
42. Maraganore DM, de Andrade M, Elbaz A, Farrer MJ, Ioannidis JP, et al. (2006)
Collaborative analysis of alpha-synuclein gene promoter variability and
Parkinson disease. Jama 296: 661–670.
43. Farrer M, Maraganore DM, Lockhart P, Singleton A, Lesnick TG, et al. (2001)
alpha-Synuclein gene haplotypes are associated with Parkinson’s disease. Hum
Mol Genet 10: 1847–1851.
44. Chiba-Falek O, Nussbaum RL (2001) Effect of allelic variation at the NACP-
Rep1 repeat upstream of the alpha-synuclein gene (SNCA) on transcription in a
cell culture luciferase reporter system. Hum Mol Genet 10: 3101–3109.
45. Cronin KD, Ge D, Manninger P, Linnertz C, Rossoshek A, et al. (2009)
Expansion of the Parkinson disease-associated SNCA-Rep1 allele upregulates
human alpha-synuclein in transgenic mouse brain. Hum Mol Genet 18:
46. Daher JP, Pletnikova O, Biskup S, Musso A, Gellhaar S, et al. (2012)
Neurodegenerative phenotypes in an A53T alpha-synuclein transgenic mouse
model are independent of LRRK2. Hum Mol Genet.
47. Lin X, Parisiadou L, Gu XL, Wang L, Shim H, et al. (2009) Leucine-rich repeat
kinase 2 regulates the progression of neuropathology induced by Parkinson’s-
disease-related mutant alpha-synuclein. Neuron 64: 807–827.
48. Rieker C, Dev KK, Lehnhoff K, Barbieri S, Ksiazek I, et al. (2011)
Neuropathology in mice expressing mouse alpha-synuclein. PLoS One 6:
49. van der Putten H, Wiederhold KH, Probst A, Barbieri S, Mistl C, et al. (2000)
Neuropathology in mice expressing human alpha-synuclein. J Neurosci 20:
50. Shimshek DR, Mueller M, Wiessner C, Schweizer T, van der Putten PH (2010)
The HSP70 molecular chaperone is not beneficial in a mouse model of alpha-
synucleinopathy. PLoS One 5: e10014.
51. Ramonet D, Daher JP, Lin BM, Stafa K, Kim J, et al. (2011) Dopaminergic
neuronal loss, reduced neurite complexity and autophagic abnormalities in
transgenic mice expressing G2019S mutant LRRK2. PLoS One 6: e18568.
52. Li Y, Liu W, Oo TF, Wang L, Tang Y, et al. (2009) Mutant LRRK2(R1441G)
BAC transgenic mice recapitulate cardinal features of Parkinson’s disease. Nat
Neurosci 12: 826–828.
53. Zhou H, Huang C, Tong J, Hong WC, Liu YJ, et al. (2011) Temporal
expression of mutant LRRK2 in adult rats impairs dopamine reuptake. Int J Biol
Sci 7: 753–761.
54. Melrose HL, Dachsel JC, Behrouz B, Lincoln SJ, Yue M, et al. (2010) Impaired
dopaminergic neurotransmission and microtubule-associated protein tau
alterations in human LRRK2 transgenic mice. Neurobiol Dis 40: 503–517.
55. Li X, Patel JC, Wang J, Avshalumov MV, Nicholson C, et al. (2010) Enhanced
striatal dopamine transmission and motor performance with LRRK2 overex-
LRRK2 and Alpha-Synuclein
PLoS ONE | www.plosone.org 13May 2012 | Volume 7 | Issue 5 | e36581
pression in mice is eliminated by familial Parkinson’s disease mutation G2019S.
J Neurosci 30: 1788–1797.
56. Piccoli G, Condliffe SB, Bauer M, Giesert F, Boldt K, et al. (2011) LRRK2
controls synaptic vesicle storage and mobilization within the recycling pool.
J Neurosci 31: 2225–2237.
57. Shin N, Jeong H, Kwon J, Heo HY, Kwon JJ, et al. (2008) LRRK2 regulates
synaptic vesicle endocytosis. Exp Cell Res 314: 2055–2065.
58. Plowey ED, Cherra SJ 3rd, Liu YJ, Chu CT (2008) Role of autophagy in
G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells.
J Neurochem 105: 1048–1056.
59. Winner B, Melrose HL, Zhao C, Hinkle KM, Yue M, et al. (2011) Adult
neurogenesis and neurite outgrowth are impaired in LRRK2 G2019S mice.
Neurobiol Dis 41: 706–716.
60. Lee S, Liu HP, Lin WY, Guo H, Lu B (2010) LRRK2 kinase regulates synaptic
morphology through distinct substrates at the presynaptic and postsynaptic
compartments of the Drosophila neuromuscular junction. J Neurosci 30:
61. MacLeod D, Dowman J, Hammond R, Leete T, Inoue K, et al. (2006) The
familial Parkinsonism gene LRRK2 regulates neurite process morphology.
Neuron 52: 587–593.
62. Parisiadou L, Xie C, Cho HJ, Lin X, Gu XL, et al. (2009) Phosphorylation of
ezrin/radixin/moesin proteins by LRRK2 promotes the rearrangement of actin
cytoskeleton in neuronal morphogenesis. J Neurosci 29: 13971–13980.
63. Lin CH, Tsai PI, Wu RM, Chien CT (2010) LRRK2 G2019S mutation induces
dendrite degeneration through mislocalization and phosphorylation of tau by
recruiting autoactivated GSK3ss. J Neurosci 30: 13138–13149.
64. Carballo-Carbajal I, Weber-Endress S, Rovelli G, Chan D, Wolozin B, et al.
(2010) Leucine-rich repeat kinase 2 induces alpha-synuclein expression via the
extracellular signal-regulated kinase pathway. Cell Signal 22: 821–827.
65. Kondo K, Obitsu S, Teshima R (2011) alpha-Synuclein aggregation and
transmission are enhanced by leucine-rich repeat kinase 2 in human
neuroblastoma SH-SY5Y cells. Biol Pharm Bull 34: 1078–1083.
66. Qing H, Wong W, McGeer EG, McGeer PL (2009) Lrrk2 phosphorylates alpha
synuclein at serine 129: Parkinson disease implications. Biochem Biophys Res
Commun 387: 149–152.
67. Alegre-Abarrategui J, Ansorge O, Esiri M, Wade-Martins R (2008) LRRK2 is a
component of granular alpha-synuclein pathology in the brainstem of
Parkinson’s disease. Neuropathol Appl Neurobiol 34: 272–283.
68. Giasson BI, Covy JP, Bonini NM, Hurtig HI, Farrer MJ, et al. (2006)
Biochemical and pathological characterization of Lrrk2. Ann Neurol 59:
69. Vitte J, Traver S, Maues De Paula A, Lesage S, Rovelli G, et al. (2010) Leucine-
rich repeat kinase 2 is associated with the endoplasmic reticulum in
dopaminergic neurons and accumulates in the core of Lewy bodies in Parkinson
disease. J Neuropathol Exp Neurol 69: 959–972.
LRRK2 and Alpha-Synuclein
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