Dopaminergic Neuronal Loss, Reduced Neurite
Complexity and Autophagic Abnormalities in Transgenic
Mice Expressing G2019S Mutant LRRK2
David Ramonet1., Joa ˜o Paulo L. Daher2,3,4., Brian M. Lin2,3, Klodjan Stafa1, Jaekwang Kim5,6, Rebecca
Banerjee7, Marie Westerlund8, Olga Pletnikova5, Liliane Glauser1, Lichuan Yang7, Ying Liu5, Deborah A.
Swing10, M. Flint Beal7, Juan C. Troncoso5, J. Michael McCaffery9, Nancy A. Jenkins10¤, Neal G.
Copeland10¤, Dagmar Galter8, Bobby Thomas7, Michael K. Lee5,6, Ted M. Dawson2,3,11, Valina L.
Dawson2,3,11,12*, Darren J. Moore1*
1Brain Mind Institute, School of Life Sciences, Ecole Polytechnique Fe ´de ´rale de Lausanne (EPFL), Lausanne, Switzerland, 2NeuroRegeneration and Stem Cell Programs,
Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 3Department of Neurology, Johns Hopkins
University School of Medicine, Baltimore, Maryland, United States of America, 4Department of Pathology, School of Medicine, Fluminense Federal University, Nitero ´i,
Brazil, 5Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 6Institute for Translational Neuroscience,
Department of Neuroscience, University of Minnesota, Minneapolis, Minnesota, United States of America, 7Department of Neurology and Neuroscience, Weill Medical
College of Cornell University, New York, New York, United States of America, 8Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden, 9Integrated
Imaging Center and Department of Biology, Johns Hopkins University, Baltimore, Maryland, United States of America, 10Mouse Cancer Genetics Program, NCI-Frederick
Cancer Research and Development Center, Frederick, Maryland, United States of America, 11Department of Neuroscience, Johns Hopkins University School of Medicine,
Baltimore, Maryland, United States of America, 12Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson’s disease
(PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and
neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing
wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in
nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and
characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S.
Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway
dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities
in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons.
These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial
mutations precipitate neuronal degeneration and PD.
Citation: Ramonet D, Daher JPL, 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(4): e18568. doi:10.1371/journal.pone.0018568
Editor: Huaibin Cai, National Institute of Health, United States of America
Received November 24, 2010; Accepted March 4, 2011; Published April 6, 2011
Copyright: ? 2011 Ramonet 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: This work was supported by the National Parkinson Foundation (D.J.M., V.L.D.); Swiss National Science Foundation (grant no. 310030_127478 to D.J.M.);
Parkinson Schweiz (D.J.M.); American Parkinson Disease Association (D.J.M.); Ecole Polytechnique Fe ´de ´rale de Lausanne (D.J.M.); Swedish Research Council (D.G.);
and the National Institutes of Health (grant no. NS060885 and NS062165 to B.T., and NS03877 to T.M.D. and V.L.D.). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (DJM); email@example.com (VLD)
. These authors contributed equally to this work.
¤ Current address: Institute of Molecular and Cell Biology, Singapore, Singapore
Mutations in the LRRK2 gene (PARK8, OMIM 609007) cause
late-onset, autosomal dominant familial Parkinson’s disease (PD)
with a clinical and neurochemical phenotype that is largely
indistinguishable from sporadic PD [1–3]. At least six disease-
segregating mutations have been identified in LRRK2-linked
families, including the R1441C/G/H, Y1699C, G2019S and
I2020T variants [4–5]. Of these, G2019S is the most common
variant that uniquely contributes to both familial and sporadic PD
[6–9]. LRRK2-linked PD is characterized by the degeneration of
substantia nigra dopaminergic neurons and gliosis together with
heterogeneous protein inclusion pathology [3,10]. How mutations
in LRRK2 precipitate neuronal degeneration and pathology in PD
is not known.
LRRK2 encodes a multi-domain protein belonging to the
ROCO family characterized by a Ras of Complex (ROC) GTPase
domain and a C-terminal of ROC (COR) domain in conjunction
with a kinase domain with similarity to RIP kinases [11–12].
LRRK2 contains both GTPase and kinase activities and certain
familial mutations can modify one or other of these enzymatic
activities [5,11,13–19]. Familial mutations consistently enhance
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LRRK2-induced neuronal toxicity in vitro in a GTP-binding- and
kinase-dependent manner [13,19-22], suggesting a gain-of-func-
tion mechanism for familial mutations. Whether LRRK2
mutations can also induce neuronal toxicity in vivo has not been
demonstrated. LRRK2 expression has been shown to regulate
neuronal morphology in vitro where familial LRRK2 mutants
induce a reduction of neurite length and branching, and LRRK2
deficiency produces opposing effects . Autophagy may
mediate neurite shortening induced by G2019S LRRK2 expres-
sion since inhibition of autophagy reverses, and activation
potentiates, the effects of G2019S LRRK2 on neurites .
These observations suggest a potential role for autophagy in
mediating the pathogenic actions of LRRK2 mutations.
A number of models have been developed to probe the normal
function of LRRK2 in vivo, and to dissect the pathogenic actions of
familial mutations. Genetic disruption of LRRK2 or its paralogs in
Caenorhabditis elegans [24–25], Drosophila melanogaster  and mice
[27–28] suggest that LRRK2 is not essential for the survival of
dopaminergic neurons. However, transgenic expression of human
LRRK2 bearing the G2019S mutation in Drosophila causes adult-
onset, selective degeneration of dopaminergic neurons, L-DOPA-
responsive locomotor impairment and early mortality [29–30].
LRRK2 transgenic mice have been developed recently to model
LRRK2-linked PD [31–35]. BAC transgenic mice expressing
R1441G mutant LRRK2 exhibit reduced striatal dopamine
release, L-DOPA-sensitive motor deficits, dopaminergic neuritic
atrophy/dystrophy and increased tau phosphorylation .
Additionally, BAC mice expressing G2019S mutant LRRK2 or
R1441C knock-in mice display impairments of nigrostriatal
dopaminergic neurotransmission and tau processing [31,34–35].
These mouse models have provided important insight into the
pathogenic effects of familial LRRK2 mutations in vivo and further
support a gain-of-function mechanism for these mutations.
However, the current mouse models do not exhibit overt neuronal
loss and have failed to recapitulate the progressive degeneration of
nigrostriatal dopaminergic neurons; the hallmark pathology
underlying the clinical motor symptoms of PD.
To model the effects of familial mutations in vivo, we have developed
LRRK2 transgenic mice bearing the PD-associated R1441C and
G2019S mutations or wild-type LRRK2. Here, we demonstrate that
the expression of G2019S LRRK2 induces the progressive degener-
ation of nigrostriatal dopaminergic neurons in mice. G2019S LRRK2
expression also produces autophagic and mitochondrial abnormalities
in the mouse brain, and reduces dopaminergic neurite complexity in
primary cultures. Our study provides new insight into the pathogenic
actions of familial LRRK2 mutations in vivo related to the pathogenesis
induced by the expression of G2019S mutant LRRK2.
Generation of Transgenic Mice Expressing Mutant
The expression of full-length human LRRK2 variants was
placed under the control of a CMV-enhanced human platelet-
(Figure 1A). This hybrid promoter drives long-term neuronal-
specific transgene expression in the rat brain including substantia
nigra dopaminergic neurons [36–38]. Transgenic mice were
generated expressing human LRRK2 harboring the familial PD
mutations, R1441C and G2019S, in addition to WT LRRK2. We
identified 73 founder mice by genomic PCR with 59 and 39 primer
pairs (Figure 1A). Quantitative PCR using genomic DNA revealed
the relative transgene copy number between founder mice (data
not shown). Of the initial founders, 24 lines with medium-high
transgene copy number transmitted the transgene to F1 progeny
following breeding to C57BL/6J mice. Semi-quantitative RT-
PCR revealed the expression levels of human LRRK2 mRNA in
hemi-brains of F1 mice (Figure 1B). We selected 4 lines for each
LRRK2 variant with the highest transgene expression and
determined human LRRK2 protein levels in hemi-brain extracts
by Western blotting with pan- or human-specific LRRK2
antibodies. LRRK2 transgenic mice express human LRRK2 at
3-5-fold the level of endogenous LRRK2 (Figure 1C and S1).
Figure 1. Generation of LRRK2 transgenic mice. A, Schematic showing the CMVE-PDGFb-LRRK2 transgene and the positions of familial PD
mutations. PCR primers for 59 (P1/P2) and 39 (P3/P4) genotyping are indicated. B, Semi-quantitative RT-PCR analysis of human LRRK2 mRNA
expression in 2–3 month LRRK2 transgenic lines. Mouse b-actin mRNA is used as a loading control. The absence (2) or presence (+) of RT enzyme in
the reaction is indicated. C, Western blot analysis of soluble extracts from hemi-brains of 2–3 month LRRK2 transgenic mice (TG), non-transgenic mice
(NTG) or LRRK2 knockout (KO) mice using LRRK2-specific antibodies, JH5514 (human/mouse) or human-specific NB300-267. b-tubulin is used a
control for protein loading. Bar chart showing densitometric quantitation of total LRRK2 levels (JH5514 antibody) in each transgenic line. LRRK2 levels
are normalized to b-tubulin levels and expressed as a percent of NTG mice.
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We selected the highest expressing LRRK2 transgenic lines with
similar protein levels for the R1441C (line 574) and G2019S (line
340) variants for further detailed analysis. WT-LRRK2 transgenic
mice (line 249) express human LRRK2 mRNA and protein at
lower levels than mutant LRRK2 lines and as such were only
examined in some experiments (Figure 2A–B). The pattern of
human LRRK2 mRNA expression was determined in the brains
of transgenic mice by in situ hybridization with oligonucleotide
probes (Figure 2A–B and S2). G2019S-LRRK2 mRNA is
expressed throughout the mouse brain with highest expression in
the olfactory bulb, cerebral cortex, hippocampus, striatum and
cerebellum (Figure 2A) and clear expression in neurons of the
substantia nigra pars compacta (Figure 2B and S2). We could
further confirm the overexpression of G2019S LRRK2 protein
(,2.7-fold over endogenous LRRK2 levels) specifically within
tyrosine hydroxylase (TH)-positive dopaminergic neurons of the
substantia nigra pars compacta from transgenic mice by confocal
fluorescence microscopy using a pan-LRRK2 antibody that
detects both mouse and human LRRK2 (Figure 2C). The
expression pattern of human G2019S-LRRK2 mRNA is broadly
similar to endogenous LRRK2 mRNA in the mouse brain
(Figure 2A–B and S3A). In general, human LRRK2 expression
does not influence the expression level or pattern of endogenous
LRRK2 mRNA in the brain, spleen or kidney of transgenic mice
(Figure S3). Unexpectedly, R1441C-LRRK2 mRNA expression is
detected at highest levels in the cerebral cortex and cerebellum but
is not appreciably expressed in the striatum, hippocampus or
ventral midbrain of transgenic mice (Figure 2A–B). Similarly, WT
LRRK2 mRNA is widely expressed in the brains of transgenic
mice but at markedly lower levels than G2019S LRRK2 mRNA
and not appreciably within the ventral midbrain (Figure 2A–B).
Therefore, only G2019S LRRK2 transgenic mice appear to be
Figure 2. Localization of human LRRK2 in the brain of transgenic mice. A, In situ hybridization with33P-labeled antisense oligonucleotide
probes specific to human LRRK2 mRNA. Autoradiographs of human LRRK2 in WT (line 249), G2019S (line 340) and R1441C (line 574) transgenic mice
at 2–3 months, at the level of the olfactory bulb (Olf), striatum/cortex (Str/Ctx), hippocampus/cortex (Hip/Ctx) and cerebellum (Cb). B, Localization of
LRRK2 (mouse or human), and endogenous TH or a-synuclein mRNAs for comparison in adjacent midbrain sections of 2–3 month-old WT, G2019S
and R1441C LRRK2 transgenic mice. C, Confocal microscopic images of LRRK2 (mouse + human; MJFF2/c41-2 antibody) and tyrosine hydroxylase (TH)
immunofluorescence in the substantia nigra of 4–5 month G2019S LRRK2 transgenic (TG) mice and their non-transgenic (NTG) littermates. Bar
chart showing LRRK2+ fluorescence intensity localized within nigral TH+ dopaminergic neurons of TG and NTG mice. Bars present the mean 6 SEM
(n=3 mice/genotype). *P,0.001 comparing TG and NTG as indicated. Scale bar: 25 mm (C).
Dopaminergic Neuronal Loss in G2019S LRRK2 Mice
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useful for assessing the impact of human LRRK2 expression on
the nigrostriatal dopaminergic pathway. In general, LRRK2
transgenic mice are viable, fertile and produce normal numbers of
progeny. LRRK2 mice are generally unremarkable with no
obvious behavioral abnormalities, and no differences in body
weight or survival compared to their non-transgenic littermates up
to 24 months of age (data not shown).
Progressive Dopaminergic Neuronal Loss in G2019S
LRRK2 Transgenic Mice
To determine whether the expression of G2019S-LRRK2 in
mice induces the degeneration of nigrostriatal dopaminergic
neurons with age, cohorts of LRRK2 transgenic mice were aged
to 19–21 months. The numbers of TH+ and Nissl+ neurons in the
substantia nigra pars compacta were counted using unbiased
stereological methods (Figure 3). Remarkably, G2019S-LRRK2
mice exhibit a significant ,18% loss of TH+ dopaminergic
neurons and a corresponding ,17% loss of Nissl+ nigral neurons
compared to their non-transgenic littermates (Figure 3C), indicat-
ing dopaminergic neuronal degeneration rather than a loss of
dopaminergic phenotype. At 1-2 months, G2019S-LRRK2 mice
display normal numbers of TH+ and Nissl+ nigral neurons
suggesting that neuronal loss occurs in a progressive manner
(Figure 3B). We also observe a corresponding significant ,14%
reduction of TH+ dopaminergic neuritic density in the adjacent
substantia nigra pars reticulata of 19–20 month-old G2019S-
LRRK2 mice compared to their non-transgenic littermates
(Figure 3D). The loss of dopaminergic neuritic density could
result directly from the loss of dopaminergic neurons and/or a
reduction in neuritic complexity. As expected, R1441C-LRRK2
Figure 3. Progressive loss of substantia nigra dopaminergic neurons in G2019S LRRK2 transgenic mice. A, Example of TH and Nissl
staining in the substantia nigra of NTG and G2019S LRRK2 TG mice (line 340) at 19–20 months. B and C, Stereological counts for TH+ and Nissl+
neurons in the pars compacta region of NTG and G2019S LRRK2 TG mice at (B) 1–2 months (n=7–8 mice/genotype) and (C ) 19–20 months (n=5
mice/genotype). D, Stereological measurement of TH+ dopaminergic neuritic density in the pars reticulata of NTG or TG G2019S mice at 19–20
months, expressed as average length of TH+ fibers (mm) per mm3section area (n=5–6 mice/genotype). E, Stereological counts of TH+/Nissl+ neurons
in the pars compacta of 20–21 month NTG or R1441C LRRK2 TG mice (line 574, n=6–8 mice/genotype). F, Stereological counts of TH+ neurons in the
VTA region of 19–21 month R1441C or G2019S TG mice and their NTG littermates (n=5 mice/genotype for G2019S or n=7–8 for R1441C). Bars
present the mean 6 SEM. *P,0.02 and **P,0.005 comparing TG with NTG as indicated.
Dopaminergic Neuronal Loss in G2019S LRRK2 Mice
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mice display a normal number of nigral TH+ and Nissl+ neurons
at 20–21 months (Figure 3E) consistent with the observed lack of
transgene expression in the substantia nigra in this mouse line
(Figure 2B). Furthermore, dopaminergic neuronal loss is not
observed in a second lower-expressing G2019S-LRRK2 line (line
1128) at a similar advanced age implying that neurodegeneration
in the 340 mouse line is most likely due to higher transgene
expression (Figure S4). Two WT-LRRK2 transgenic mouse lines
(lines 249 and 27) and a second R1441C-LRRK2 line (line 546)
also fail to reveal dopaminergic neuronal loss (Figure S4) consistent
with lower expression levels and/or restricted expression patterns
of human LRRK2 in these mouse lines (Figure 1C and 2A–B).
These observations would suggest that dopaminergic neurodegen-
eration in aged G2019S-LRRK2 mice (line 340) results from
higher levels of expression of human LRRK2 directly in nigral
dopaminergic neurons that may reach a critical threshold
necessary for degeneration. To address the selective vulnerability
of nigral dopaminergic neuronal loss in G2019S-LRRK2 mice,
TH+ neurons were also counted in the adjacent ventral
tegemental area (VTA) which contains a similar population of
dopaminergic neurons projecting to the nucleus accumbens
(mesolimbic pathway) and the frontal cortex (mesocortical
pathway). G2019S- and R1441C-LRRK2 transgenic mice reveal
normal numbers of TH+ dopaminergic VTA neurons at 19–21
months despite detectable expression of G2019S-LRRK2 mRNA
in VTA neurons (Figure 3F and S2). Unexpectedly, the density of
TH+ dopaminergic nerve terminals in the striatum of G2019S-
LRRK2 mice at 19–20 months is normal compared to their non-
transgenic littermates, which suggests a compensatory re-sprouting
of the remaining dopaminergic neuronal processes (Figure S5).
Collectively, our data demonstrates that G2019S-LRRK2 trans-
genic mice exhibit a progressive and relatively selective degener-
ation of nigrostriatal dopaminergic neurons.
Dopamine Levels in G2019S LRRK2 Transgenic Mice
To further examine the impact of G2019S mutant LRRK2
expression on the nigrostriatal dopaminergic pathway, the levels of
striatal dopamine and its metabolites, 3,4-dihydroxyphenylacetic
acid (DOPAC) and homovanillic acid (HVA), were measured by
HPLC analysis (Figure 4). At 14–15 months of age, G2019S-
LRRK2 mice reveal normal levels of dopamine, DOPAC and
HVA and normal dopamine turnover in the striatum and cerebral
cortex (Figure 4A–D). This finding is largely consistent with the
normal density of striatal dopaminergic nerve terminals at 19–20
months of age (Figure S5). Sufficient cohorts of G2019S-LRRK2
mice were not available to assess striatal dopamine content at later
ages that parallel nigral dopaminergic cell loss. In the olfactory
bulb, the levels of DOPAC and HVA are significantly reduced
resulting in a modest enhancement of dopamine turnover
compared to non-transgenic littermates (Figure 4A–D). Notably,
G2019S-LRRK2 mRNA is expressed at highest levels in the
olfactory bulb relative to the striatum or cerebral cortex
(Figure 2A). G2019S-LRRK2 mice also exhibit a small yet
significant increase in the levels of serotonin (5-HT) and its
metabolite, 5-HIAA, in the prefrontal cortex (Figure S6A). As
expected, the levels of striatal dopamine, DOPAC and HVA are
Figure 4. HPLC analysis of dopamine and its metabolites in G2019S LRRK2 transgenic mice. A–D, Levels of (A) dopamine (DA) and its
metabolites, (B) DOPAC and (C ) HVA, and (D) dopamine turnover ([DOPAC+HVA]/DA) in the striatum, olfactory bulb and cerebral cortex of 14–15
month-old G2019S LRRK2 TG mice (line 340) and their NTG littermates by HPLC analysis (n=8/genotype). Bars present the mean 6 SEM. *P,0.05 or
**P,0.005 comparing TG with NTG as indicated.
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normal in 19–20 month-old R1441C-LRRK2 mice (Figure S6B)
consistent with the absence of detectable transgene expression in
the nigrostriatal pathway (Figure 2B). However, aged R1441C-
LRRK2 mice exhibit a significant reduction of cortical dopamine,
DOPAC, HVA and norepinephrine (NE) levels compared to their
non-transgenic littermates (Figure S6B) consistent with most
prominent expression of R1441C-LRRK2 mRNA in the cerebral
cortex (Figure 2A). The reduction of cortical catecholamine levels
induced by R1441C-LRRK2 expression may indicate abnormal-
ities in mesocortical dopaminergic neurotransmission. WT-
LRRK2 mice (line 249) exhibit normal levels of dopamine,
DOPAC and HVA in the striatum, cerebral cortex and olfactory
bulb at 16–17 months of age (Figure S6C). Taken together,
G2019S-LRRK2 mice reveal modestly enhanced dopamine
turnover in the olfactory bulb but normal levels of striatal
dopamine and its metabolites, whereas R1441C-LRRK2 mice
reveal reduced levels of cortical catecholamines.
Normal Locomotor Activity and Prepulse Inhibition in
G2019S LRRK2 Transgenic Mice
To investigate whether G2019S mutant LRRK2 expression
influences motor performance, we measured locomotor activity in
the open field quadrant (Figure 5). G2019S-LRRK2 mice display
normal locomotor activity in the open field at 6 and 15 months of
age (Figure 5A–B). In contrast, R1441C-LRRK2 mice exhibit a
significant reduction in horizontal and vertical locomotor activity
at 15 months compared to their non-transgenic littermate mice
that is not evident at 6 months (Fig. 5C–D). We also assessed
prepulse inhibition of the acoustic startle reflex, a measure of
sensorimotor gating that can be modulated in part by dopami-
nergic neurotransmission . However, G2019S- and R1441C-
LRRK2 transgenic mice do not perform differently from their
non-transgenic littermates when tested at 6 months (data not
shown) and 15 months of age (Figure 5E–F). Our data
demonstrate that G2019S-LRRK2 expression does not influence
locomotor activity or prepulse inhibition of the acoustic startle
reflex in aged mice. Furthermore, our data suggests that R1441C-
LRRK2 mice exhibit a progressive impairment of locomotor
Reduced Complexity of Dopaminergic Neurites in
Primary Midbrain Cultures from G2019S LRRK2
LRRK2 has been shown to regulate the morphology of
neuronal processes in cultured primary cortical neurons [20,40].
To further explore this phenotype in our LRRK2 transgenic mice,
primary midbrain cultures were prepared from the ventral
mesencephalon of G2019S-LRRK2 transgenic mice (line 340)
and their non-transgenic littermates. These cultures typically
contain 5–10% of TH+ dopaminergic neurons (unpublished
observation). Sholl analysis was conducted to provide a measure
of neuritic complexity (Figure 6 and Table 1). At days-in-vitro (DIV)
3, developing dopaminergic neurons from G2019S-LRRK2 mice
exhibit significantly reduced overall neurite complexity manifest-
ing as shorter neurites but with modestly increased neurite
branching compared to non-transgenic neurons (Figure 6B and
Table 1). However, at DIV 7 when dopaminergic neurite
outgrowth is fully established, G2019S-LRRK2 dopaminergic
neurons reveal a dramatic reduction of neurite length and
branching with an overall reduction in neurite complexity
(Figure 6A and 6C, Table 1). We did not perform similar
measurements on cultured dopaminergic neurons derived from
R1441C-LRRK2 mice (line 574) since these transgenic mice do
not exhibit transgene expression within the nigrostriatal dopami-
nergic pathway (Figure 2B). Collectively, our data demonstrate
that G2019S-LRRK2 expression dramatically reduces the neuritic
complexity of cultured dopaminergic neurons.
Autophagic Abnormalities in G2019S LRRK2 Transgenic
LRRK2-linked PD is associated with heterogeneous neuropathol-
ogy, including Lewy bodies, neurofibrillary tau pathology, ubiqui-
tin-positive inclusions or in some cases the absence of inclusions
[3,10,41]. To investigate pathology in G2019S-LRRK2 mice (line
340), immunohistochemistrywith a numberofpathological markers
wasconductedinthe ventralmidbrain,striatumand cerebral cortex
at 23–24 months of age. G2019S-LRRK2 mice do not reveal
abnormalities in the distribution of a-synuclein, ubiquitin, tau and
do not exhibit abnormal staining for phospho-a-synuclein (pS129) or
phospho-tau (pS396/pS404) (Figure S7).
Transmission electron microscopy (TEM) was performed on the
cerebral cortex and striatum from G2019S-LRRK2 mice (line
340) at 17–18 months to investigate more subtle pathological
abnormalities. Cytopathological abnormalities are observed in
G2019S-LRRK2 mice including enlarged vacuolar structures with
multiple membranes resembling autophagic vacuoles including
early and late autophagosomes present in neuronal soma and
regions enriched for axons and synapses (Figure 7A–D and S8).
Autophagic vacuoles are frequently observed within neuronal
soma and axonal processes (Figure S9). We also observe
condensed aggregated mitochondria in neuronal soma consistent
with increased mitochondrial autophagy, in addition to damaged
mitochondria (Figure 7E–G and S10). Similar yet less pronounced
cytopathology is observed in the cortex of R1441C-LRRK2 mice
(line 574) at 20–23 months (data not shown). Quantitation of TEM
cytopathology within the cortex reveals that G2019S LRRK2
transgenic mice exhibit a significant increase in the density of
autophagic vacuoles and the proportion of abnormal condensed
mitochondria compared to their non-transgenic littermates
(Figure 7G). R1441C LRRK2 mice also display a smaller yet
significant increase in the density of autophagic vacuoles
(Figure 7G). A significant accumulation of autophagic vacuoles is
also evident in the striatum of G2019S LRRK2 mice (Figure S8).
Our data demonstrate that G2019S- and R1441C-LRRK2
transgenic mice exhibit autophagic abnormalities in the brain
with advanced age.
The major finding of this study is the observation that the
expression of human G2019S LRRK2 induces the progressive
degeneration of nigrostriatal pathway dopaminergic neurons in vivo
in transgenic mice. Accompanying the loss of dopaminergic
neurons are autophagic and mitochondrial abnormalities through-
out the mouse brain, as revealed by electron microscopy, and
reduced neurite complexity of cultured midbrain dopaminergic
neurons. G2019S LRRK2 expression in aged mice, however, fails
to influence the levels of striatal dopamine and its metabolites,
affect locomotor activity or produce abnormal protein inclusion
pathology. In contrast, aged R1441C LRRK2 transgenic mice
with a restricted pattern of transgene expression display reduced
levels of cortical catecholamines, a progressive impairment of
locomotor activity and the accumulation of autophagic vacuoles in
the cerebral cortex. Collectively, our study reveals a number of
intriguing phenotypes caused by the expression of LRRK2
harboring the PD-associated G2019S and R1441C mutations in
Dopaminergic Neuronal Loss in G2019S LRRK2 Mice
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vivo. Importantly, similar to LRRK2-linked PD, expression of
G2019S mutant LRRK2 is sufficient to precipitate modest nigral
dopaminergic neuronal loss with advanced age.
It is unlikely that a ,20% loss of dopaminergic neurons would
be sufficient to produce motor impairment or striatal dopamine
deficits in aged G2019S LRRK2 mice especially since the density
of dopaminergic striatal nerve terminals appears normal at this age
possibly due to the compensatory re-sprouting of existing nerve
terminals. We do not observe dopaminergic neuronal loss in five
additional LRRK2 transgenic lines (WT, R1441C or G2019S) at
Figure 5. Behavioral analysis of LRRK2 transgenic mice. A–D, In the open field G2019S LRRK2 TG mice (line 340) exhibit normal horizontal
(A) and vertical (B) locomotor activity compared to NTG littermates at 6 and 15 months (n=7–9 mice/genotype). R1441C LRRK2 TG mice (line 574)
reveal normal locomotor activity at 6 months but deficits at 15 months (C and D) compared to NTG mice (n=6-9 mice/genotype). Data represent the
number of beam breaks during the first 15 min period. E–F, Normal pre-pulse inhibition (PPI) of the acoustic startle response in LRRK2 transgenic
mice. E and F, G2019S and R1441C LRRK2 TG mice display normal PPI of the acoustic startle reflex at 15 months compared to their NTG littermates,
with increasing pre-pulse tones of 74–90 dB (n=6–8 mice/genotype). Data are expressed as % PPI relative to no pre-pulse tone. Bars present the
mean 6 SEM. *P,0.05 comparing TG with NTG as indicated.
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Figure 6. Reduced neuritic complexity of G2019S LRRK2 dopaminergic neurons in vitro. A, Example of neuritic morphology of
immunoreactive TH+ and MAP+ dopaminergic neurons in primary midbrain cultures derived from G2019S LRRK2 TG mice (line 340) and their NTG
littermates at DIV 7. TH+ neuronal soma (arrows) and neurites (arrowheads) are indicated. B–C, Sholl analysis of TH+ dopaminergic neurites plotting
the mean number of dendritic intersections with circles of increasing radii at DIV 3 (B) and DIV 7 (C ). Data represents the mean number of dend-
ritic intersections within each circular interval (mm) from independent cultures derived from 4 mice per genotype. Bars present the mean 6 SEM
(DIV 3: NTG, n=94 and TG, n=150; DIV 7: NTG, n=54 and TG, n=71 neurons). *P,0.05 comparing TG with NTG as indicated.
Dopaminergic Neuronal Loss in G2019S LRRK2 Mice
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similar advanced ages. The neuronal loss in G2019S LRRK2 mice
(line 340) most likely reflects higher levels of transgene expression
and detectable expression in dopaminergic neurons of the
substantia nigra in this mouse line. The loss of nigral dopaminergic
neurons is also relatively selective as dopaminergic neurons of the
adjacent VTA (A10 nucleus) are spared despite expression of
G2019S LRRK2 in this population. As our LRRK2 transgenic
lines do not share matched transgene expression within the
substantia nigra, it is difficult to determine whether the equivalent
expression of WT or R1441C mutant LRRK2 would also be
sufficient to precipitate neuronal loss similar to G2019S LRRK2.
While we could clearly demonstrate the expression of G2019S
LRRK2 in nigral dopaminergic neurons, this was not possible for
WT or R1441C LRRK2 mice. A further caveat is that WT and
R1441C LRRK2 are generally expressed at lower levels than
G2019S LRRK2 in the brains of transgenic mice. At this juncture
it is unclear whether nigral dopaminergic degeneration in G2019S
LRRK2 transgenic mice is a result of the pathogenic actions of the
G2019S mutation or is due to the overexpression of human
LRRK2. That five additional LRRK2 transgenic lines with
varying transgene expression patterns and levels do not display
nigral neuronal loss perhaps suggests that human LRRK2
overexpression per se is not sufficient to precipitate neuronal
degeneration. BAC and inducible transgenic mouse models
overexpressing human LRRK2 variants also do not display nigral
neuronal loss although it is unclear to what degree transgenes in
these mice are expressed in substantia nigra dopaminergic neurons
[32–34]. Further supporting the pathogenicity of the G2019S
mutation in our transgenic mice are the recent observations that
viral-mediated expression of human G2019S LRRK2 in the
substantia nigra dopaminergic neurons of rodents causes the
progressive degeneration of these neurons whereas the equivalent
expression of WT LRRK2 or GFP failed to induce neuronal
degeneration [42,43]. These observations support a specific
pathogenic effect of G2019S LRRK2 on nigral dopaminergic
neurons in our LRRK2 transgenic mice.
While certain mutant LRRK2 BAC transgenic models exhibit
subtle tau pathology or processing abnormalities in the absence of
frank neuronal loss [31–32,34], our G2019S LRRK2 mice lack
obvious protein inclusion pathology that is observed in PD brains
bearing LRRK2 mutations, including a-synuclein, tau or ubiquitin
inclusions . This finding suggests that dopaminergic neuronal
degeneration in our G2019S LRRK2 model may occur
independently from abnormal inclusion pathology or protein
aggregation. We cannot exclude the possibility that submicro-
scopic protein aggregates contribute to neuronal loss or that
inclusions are rapidly removed following neuronal death. In a rat
LRRK2 adenoviral model, we recently found that tau hyperpho-
sphorylation in nigral dopaminergic neurites is induced equally by
the expression of WT and G2019S human LRRK2 in a transient
manner, whereas only G2019S LRRK2 expression induced
dopaminergic neuronal loss. Thus, the induction of tau pathology
and neuronal loss in dopaminergic neurons by human LRRK2
expression can be dissociated at least in this rat viral model .
Although LRRK2 mutations are predominantly associated with
Lewy body pathology in PD brains [44–45], some LRRK2
mutations produce nigral degeneration without additional pathol-
ogy  suggesting that protein aggregation may not be an
absolute requirement for mutant LRRK2-induced neurodegener-
ation. The data obtained in our G2019S LRRK2 mice supports
Coincident with neuronal loss in G2019S LRRK2 mice, we
observe the accumulation of autophagic vacuoles and evidence of
increased mitochondrial autophagy and damage in the brains of
transgenic mice by electron microscopy. Of particular interest is
that both R1441C and G2019S LRRK2 transgenic mice
consistently display autophagic abnormalities in the cerebral
cortex; a region where each transgene is prominently expressed
in these models. At least in vitro, alterations in autophagy have been
reported to modulate G2019S LRRK2-induced neurite shortening
and the expression of R1441G LRRK2 leads to the accumulation
of autophagic vacuoles and multivesicular bodies [23,46]. In a
yeast model of LRRK2-induced cytotoxicity there is an accumu-
lation of autophagic vacuoles together with associated defects in
endocytic vesicular trafficking . Overexpression of G2019S
LRRK2 in cultured neurons induces the accumulation of swollen
lysosomes, multivesicular bodies, distended vacuolated mitochon-
dria and phosphorylated tau-positive spheroid axonal inclusions
. Taken together, our data demonstrate for the first time that
mutant LRRK2 expression in vivo can cause abnormalities in the
autophagy pathway. It is not clear at present whether the
abnormal accumulation of autophagic vacuoles induced by
G2019S and R1441C LRRK2 expression in vivo reflects the
impairment or activation of the autophagy pathway, or instead
Table 1. Sholl analysis of TH+ dopaminergic neurons from G2019S LRRK2 mice (line 340).
Type of measurementDefinitionDIV NTG G2019S
(-log slope of regression fit)
Overall measure of
Av. density of branching
per primary dendrite
Rmax (mm) Av. radius where neuritic
intersections are maximal
7 482.03647.61 176.05627.40**
Av. radius where longest
neurite extends to
7 1326.72664.53 606.10655.23**
**P,0.001 versus NTG. Abbreviations: DIV, days in vitro; NTG, non-transgenic; Av., average.
Dopaminergic Neuronal Loss in G2019S LRRK2 Mice
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Figure 7. Transmission electron microscopic (TEM) analysis of LRRK2 transgenic mice. TEM analysis of cerebral cortex tissue from 17–18
month G2019S LRRK2 transgenic mice (line 340). A–D, Vacuoles with multiple membranes resembling autophagosomes or autophagic vacuoles
(indicated by *) are observed within regions enriched in axons and/or synapses (A–C ) or within neuronal soma (D). E and F, Clusters of condensed
mitochondria within the neuronal soma (indicated by arrows) reminiscent of mitochondria that are undergoing autophagocytosis. Synapses
(arrowheads), axons (ax) and normal mitochondria (m) are indicated. G, Quantitation of the density of autophagic vacuoles and the proportion of
normal, damaged or condensed/aggregated mitochondria in equivalent regions of cingulate cortex from 17–26 month G2019S and R1441C LRRK2
transgenic (TG) mice relative to their non-transgenic (NTG) littermates. Bars represent the mean 6 SEM (n=3 mice/genotype). *P,0.05 comparing TG
and NTG mice as indicated. Scale bars: 2 mm (A, E, F) or 1 mm (B–D).
Dopaminergic Neuronal Loss in G2019S LRRK2 Mice
PLoS ONE | www.plosone.org10 April 2011 | Volume 6 | Issue 4 | e18568
manifests through impaired autophagic flux due to the down-
stream inhibition of autolysosome formation and/or lysosomal
function. Whether or not such autophagic alterations underlie
dopaminergic neuronal death in G2019S LRRK2 transgenic mice
is unclear but certainly warrants further attention. At least in
cultured neuronal cells, G2019S LRRK2-induced neurite short-
ening is mediated, in part, by the autophagy pathway and can be
exacerbated by activation of autophagy through treatment with
rapamycin . It will be important to determine whether similar
vesicular abnormalities are also observed in PD brains with LRRK2
G2019S LRRK2 expression reduces the neuritic complexity of
cultured dopaminergic neurons derived from G2019S LRRK2
transgenic mice. A similar reduction in neurite length and
branching of cortical neurons has also been demonstrated in vitro
following expression of mutant LRRK2 [20,40,48]. In cultured
neurons, the reduced neuritic complexity results from the reduced
outgrowth of neurites rather than from neurite retraction since the
neuritic phenotype is already apparent in developing neurons
before neurites have fully extended . However, the reduced
complexity of dopaminergic neurites would appear to be a
phenotype specific to cultured neurons since we find no evidence
for abnormal dopaminergic neurons in the brains of young
G2019S LRRK2 mice. Alterations in neuritic complexity may
become apparent due to the rapid neurite outgrowth experienced
by neurons under culture conditions, a situation which is not likely
mimicked in vivo. It is not yet clear whether reduced neuritic
complexity is a precursor to neuronal death following LRRK2
overexpression or whether this neuritic phenotype reflects an
independent physiological function of LRRK2 in regulating
Together, our data provide evidence that expression of human
LRRK2 harboring the G2019S mutation in vivo is sufficient to
recreate the slowly progressive degeneration of dopaminergic
neurons that forms the hallmark pathology of familial and sporadic
PD. LRRK2 transgenic mice fail, however, to reveal additional
key phenotypes related to LRRK2-linked or sporadic PD
supporting previous observations that mouse models based upon
monogenic causes of familial PD are not sufficient alone to
recapitulate the full spectrum of disease . Our study reveals a
potential role for alterations in autophagy and neuritic morphol-
ogy in mediating the pathogenic effects of LRRK2 mutations in vivo.
The mouse models presented here together with similar recent
studies [31–35] reveal a role for familial LRRK2 mutations in
mediating the dysfunction of the nigrostriatal dopaminergic
pathway. The current LRRK2 mouse models will provide
important tools for understanding the mechanism(s) through
which familial mutations precipitate neuronal degeneration and
Materials and Methods
Mice were housed and treated in strict accordance with the
NIH Guide for the Care and Use of Laboratory Animals. All animal
procedures were approved by the Institutional Animal Care and
Use Committees of the Johns Hopkins Medical Institutions
(Animal Welfare Assurance No. A3272-01), or were conducted
in accordance with the Swiss legislation (Canton de Vaud, Animal
Authorization No. 2293) and the European Community Council
directive (86/609/EEC) for the care and use of laboratory
animals. Mice were maintained in a pathogen-free facility and
exposed to a 12 h light/dark cycle with food and water provided
Generation of LRRK2 transgenic mice
To generate LRRK2 transgenic mice, we used a pGL3-Basic
vector (Promega, Madison, WI, USA) containing the ,1.5 kb
human PDGFb promoter preceded by a ,400 bp CMV immediate-
early enhancer inserted between 59 KpnI and 39 HindIII sites. The
pGL3-CMVE-PDGFb construct was kindly provided by Dr. S.
Wang (National University of Singapore, Singapore) . The
HindIII site was converted to a NheI site by digestion, Klenow fill-in
and blunt-end ligation. A ,200 bp chimeric intron from vector pCI
(Promega) was inserted into the NheI site. A luciferase cDNA was
removed by digestion with NheI and XbaI and replaced by a NheI-
flanked humanLRRK2 cDNA.The ,7.6 kb LRRK2cDNAfrom a
pcDNA3.1-LRRK2-Myc-His vector  was previously modified to
introduce a 59 Kozak sequence and 39 stop codon (TAA) between
flanking NheI sites. A ,10.5 kb transgene cassette was excised by
digestion with KpnI and DrdI and purified by GELase extraction
(Epicentre Biotechnologies, Madison, WI, USA). Transgene DNA
was microinjected into the pronucleus of single-cell embryos
(C57BL/6J x C3H/HeJ F1 hybrids) and implanted into pseudo-
pregnant female mice. Founder mice were identified by genomic
PCR with transgene-specific primers flanking the CMVE-PDGFb
region (P1, 59-ATTACCATGGTTCGAGGTGA-39 and P2, 59-
CAAGTGTCTGCAGGAAGGTT-39) and the LRRK2-SV40 re-
gion (P3, 59-TGGTTTGTCCAAACTCATCA-39 and P4, 59-CG-
TTTGGGACATCAATCTTC-39) producing a ,170 bp product
together with mouse GAPDH primers (F1, 59-TGTTTGTG-
ATGGGTGTGAAC-39 and R1, 59-TACTTGGCAGGTTTCTC-
CAG-39) producing a ,380 bp internal control. Founder mice were
bred with C57BL/6J mice to produce F1 hemizygous mice for
expression analysis. In general, LRRK2 transgenic mice were
maintained as hemizygotes by backcrossing to the C57BL/6J strain
for 3-4 generations.
For RT-PCR, total RNA was purified from mouse hemi-brains
using an RNeasy kit (Qiagen, Valencia, CA, USA) and digested
with DNase I (Qiagen). RNA was reverse transcribed using a
SuperScript III First-Strand Synthesis system (Invitrogen, Carlsbad,
CA,USA)witholigo(dT)20. cDNAderived from 50 ngoftotalRNA
was amplified by PCR with primers specific to human LRRK2
(TGLRK3-F, GAAGATTGATGTCCCAAACG and TGLRK3-
R, GAACTGGAGGAGGCCATATT) or b-actin (sense, 59-GC-
TCGTCGTCGACAACGGCTC-39, and antisense, 59-CAAA-
CATGATCTGGGTCATCTTCTC-39) producing products of
220 bp and 353 bp, respectively. For semi-quantitative analysis,
increasing PCR cycles and densitometry were used to construct
PCR amplification curves to identify the exponential phase. For
each PCR, 30 cycles for LRRK2 and 25 cycles for b-actin were
compared for semi-quantitative analysis.
In situ hybridization
In situ hybridization was performed as previously described [50,51]
on fresh-frozen brain sections from 2-3 month-old mice using species-
specific33P-labeled oligonucleotide probes complementary to human
LRRK2 (nt 4436-4483; NM_198578.3), mouse LRRK2 (nt 6036-
6085; NM_025730.2), mouse TH (nt 197-248; NM_009377.1) or
mouse a-synuclein (nt 562-611; NM_00104245.1). mRNA signals
were revealed by exposure of slides to autoradiographic film or by
development in photo-emulsion for microscopic inspection.
Western blot analysis
Protein extracts were prepared from hemi-brains of 2-4 month-
old mice as previously described  by homogenization in TNE
buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA,
0.5% NP-40, 1 X Complete protease inhibitor cocktail [Roche], 1
X phosphatase inhibitor cocktail I and II [Sigma-Aldrich, St.
Dopaminergic Neuronal Loss in G2019S LRRK2 Mice
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Louis, MO, USA]). Protein concentration was determined by
BCA method (Pierce Biotechnology, Rockford, IL, USA) and
75 mg of protein was resolved by SDS-PAGE, transferred to
nitrocellulose and probed with rabbit anti-LRRK2 antibodies
recognizing human/mouse (JH5514  or monoclonal c81-8,
Epitomics, Inc, Burlingame, CA) or human-specific (NB300-267,
Novus Biologicals, Littleton, CO, USA ) epitopes, or mouse
b-tubulin (TUB 2.1, Sigma-Aldrich) antibody. Refer to www.
pdonlineresearch.org for detailed characterization of rabbit
monoclonal LRRK2 antibody (clone c81-8, Epitomics, Inc).
Quantitation of total LRRK2 and b-tubulin protein levels by
densitometry was conducted using LabImage 1D software
(Kapelan Bio-Imaging Solutions, Leipzig, Germany) on Western
blot images captured using a FujiFilm LAS-4000 Luminescent
Image Analysis system.
Measurement of biogenic amines by HPLC
HPLC with electrochemical detection was employed to measure
the concentration of the biogenic amines, dopamine (DA), 3,4-
dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA),
5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-
HIAA) and norepinephrine (NE) as previously described .
Samples were prepared from dissected brain regions and injected
onto a C-18 8064.6 mm column (ESA Inc., Chelmsford, MA,
USA) with detection using a 2-channel Coulochem II electro-
chemical detector (ES Inc.). Data were processed on an
EZChrome Elite Client Workstation (ESA Inc.) with biogenic
amine concentration expressed as ng per mg protein.
TH immunohistochemistry and stereological assessments
Mice were anesthetized with sodium
pentobarbital (150 mg/kg) before intracardial perfusion with cold
PBS and 4% paraformaldehyde (PFA). Brains were removed, post-
fixed with PFA overnight and cryopreserved in 30% sucrose in
PBS at 4uC for 48 h. Coronal midbrain sections (40-mm) were
prepared and processed for immunohistochemistry with rabbit
anti-TH antibody (Novus Biologicals) and Nissl counterstain as
previously described .
Stereological assessment of TH+ +/Nissl+ + neurons.
iased stereological methodology was employed as described
previously [55,56] to count TH+ and Nissl+ neurons in the left
and right pars compacta region of every fourth section throughout
the ventral midbrain. Stereological counts were obtained using a
computer-assisted image analysis system consisting of an Axiophot
2 photomicroscope (Carl Zeiss Inc., Thornwood, NY, USA)
equipped with a computer-controlled motorized stage (Ludl
Electronics, Hawthorne, NY, USA), a Hitachi HV C20 video
camera, and interfaced with a StereoInvestigator system (MBF
Bioscience, Williston, VT, USA) with optical fractionator probe.
TH+ neurons in the VTA were counted by stereology in a similar
manner. In general, we used a 40640 mm counting frame, a 1 mm
guard, 1006100 mm sampling grid, and a dissector height of
8 mm. Investigators were blinded to the genotype of each mouse.
Stereological assessment of TH+ + fibers.
length/density of TH+ fibers in the substantia nigra pars
reticulata, we performed stereological length estimation with
spherical probes on every fourth midbrain section, as previously
described . Briefly, virtual spherical probes were placed within
a 40-mm thick section and the intersection of TH+ fibers with the
sphere were counted. The lengths were measured at 50 random
locations throughout the reference space. At each focal plane,
concentric circles of progressively increasing and decreasing
diameters were superimposed, and the intersections with the
TH+ fibers and circles were counted (Q). To minimize surface
To determine the
artifacts, a guard volume of 1 mm was used. This method allows
the determination of the total length density (LV) and the total
length (L). To reduce the effects of variations in the area selection,
LVwas used for comparison between groups.
Coronal sections (30-mm) from cortex, striatum and midbrain
were prepared and processed for immunohistochemistry for
various pathological markers as previously described [53,54].
Briefly, sections were quenched of endogenous peroxidase activity,
permeabilized, and incubated with primary antibodies for a-
synuclein (Syn-1; BD Biosciences, San Jose, CA, USA), phospho-
Ser129-a-synuclein (Wako Chemicals, Richmond, VA, USA),
ubiquitin (DAKO, Carpinteria, CA, USA), tau (TAU-5; BD
Biosciences), phospho-Ser396/404-tau (PHF1; kindly provided by
Prof. Peter Davies, Albert Einstein College of Medicine, New
York) and glial fibrillary acidic protein (GFAP; Sigma-Aldrich).
Sections were processed with biotinylated anti-rabbit IgG or anti-
mouse IgG antibodies and avidin-biotin-complex coupled to horse
radish peroxidase (Vectastain ABC; Vector Laboratories, Burlin-
game, CA, USA), and visualized with 3,39-Diaminobenzidine
(DAB) reagent (Sigma-Aldrich).
Immunofluorescence and confocal microscopy
Coronal midbrain sections (30 mm) were prepared from 4-5
month G2019S LRRK2 transgenic mice (TG; line 340) and non-
transgenic littermate mice (NTG). Sections were processed for
immunohistochemistry with rabbit monoclonal anti-LRRK2 anti-
body (clone c41-2, Epitomics; refer to www.pdonlineresearch.org
for detailed characterization of this antibody) recognizing human
and mouse epitopes, and mouse monoclonal anti-TH antibody
(clone TH-2, Sigma-Aldrich), and anti-rabbit IgG-AlexaFluor-488
and anti-mouse IgG-AlexaFluor-633 antibodies (Invitrogen). Fluo-
rescent images were captured using a Leica SP2 inverted confocal
microscope in x, y and z planes. Relative LRRK2+ fluorescence
intensity localized within substantia nigra TH+ neurons was
determined using NIH ImageJ software by analysis of fluorescence
signal in every fourth section throughout the entire midbrain region
of each mouse.
Transmission electron microscopy (TEM)
For TEM analysis, mice were perfused with fixative (3% PFA,
1.5% glutaraldehyde, 100 mM cacodylate, 2.5% sucrose, pH 7.4)
and post-fixed for 1 h. Cortex and striatal sections were processed
as described . Sections were post-fixed in Palade’s OsO4, en
bloc stained in Kellenberger’s uranyl acetate, dehydrated, and flat-
embedded in epon. 80 nm en face sections were prepared on a
Leica UCT ultramicrotome, collected onto 400 mesh high
transmission nickel grids, and post-stained with lead and uranyl
acetate. Images were collected on a Philips EM 410 TEM
equipped with a Soft Imaging System Megaview III digital
For quantitation of cytopathology, images were collected on a
Philips FEI CM10 TEM equipped with a 11-megapixel Morada
soft imaging system camera (Olympus) at the Interdisciplinary
Centre for Electron Microscopy (CIME) at EPFL. TEM images
were sampled at random over equivalent areas of 8006800 mm
from representative regions of cingulate cortex. We analyzed
between 50-100 images for each animal representing an average
total area of 23486323 mm2per animal. The total number of
autophagic vacuoles and mitochondria were measured within each
image. The mean number of autophagic vacuoles per mm2area
was determined for LRRK2 transgenic (TG) and non-transgenic
(NTG) mice. Mitochondria were subclassified as either morpho-
Dopaminergic Neuronal Loss in G2019S LRRK2 Mice
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logically normal (class I), damaged (class II) or abnormally
condensed/aggregated based upon previously established criteria
( and refer to Figure S10). Class I and II mitochondria were
distinguished based on the appearance of the electron transparent
cristae with class II representing damaged pre-apoptotic mito-
chondria, whereas condensed mitochondria were identified as
mitochondrial aggregates with condensed cristae. An average of
8906200 mitochondria were counted per animal. Mitochondrial
subclasses were expressed as a percent of the total number of
mitochondria for each animal.
Primary midbrain cultures and analysis of neuritic
Whole brains were dissected from P0 mice and the ventral
mesencephalic region containing the substantia nigra (A9) and
VTA (A10) was stereoscopically isolated and dissociated in media
containing papain (20 U/ml). The cells were grown on coverslips
pre-coated with mouse laminin (33 mg/ml; Invitrogen) and poly-
D-lysine (20 ng/ml; Becton Dickinson) in media consisting of
Neurobasal (Invitrogen), B27 supplement (2% w/v), L-glutamine
(500 mM) and penicillin/streptomycin (100 U/ml). At DIV 3, the
cells were treated with b-D-arabinofuranoside (10 mM) to inhibit
glial cell division.
Cultures were fixed with 4% PFA and processed for
immunocytochemistry with rabbit anti-TH (Novus Biologicals)
and mouse anti-MAP2 (Sigma-Aldrich) antibodies and anti-rabbit
IgG-AlexaFluor-546 and anti-mouse IgG-AlexaFluor-488 anti-
bodies (Invitrogen). Fluorescent images were captured using a
Leica DMI 4000 inverted fluorescence microscope (Leica) at 10x
magnification. Sholl analysis was performed on TH+/MAP+
neurons using NIH ImageJ software with a Sholl analysis plug-in
(v1.0) developed by the laboratory of Anirvan Ghosh (http://
www.biology.ucsd.edu/labs/ghosh/software/index.html)  to
quantify neuritic complexity. Briefly, we constructed continuous
concentric circles centered upon the neuronal soma to measure the
number of intersections of dendrites with circles of increasing radii.
Sholl analysis was performed using the semi-log method and
measurements are shown in Table 1.
Open field test.
open field quadrant was assessed over a 60 min period using four
activity chambers with 16616 infrared beams (San Diego
Instruments, San Diego, CA, USA) . Horizontal and vertical
activities were automatically recorded. Activity recorded during
the first 15 min period was used for analysis.
Acoustic startle response.
(San Diego Instruments) were used for measuring startle reactivity
and plasticity as previously described . Briefly, the experi-
mental session consisted of a 5 min acclimatization period to a 70-
dB background noise (continuous throughout the session), followed
by a habituation session with the presentation of 10640-ms 120-
dB white noise stimuli at a 20-s inter-stimulus interval. Mice were
left for 5 min in the chamber without acoustic stimuli and then
subjected to the pre-pulse inhibition (PPI) session. For each PPI
session, mice were exposed to various trials: 1) pulse-alone trial
(120-dB, 100-ms broadband burst), 2) no stimuli trial, and 3) five
prepulse-pulse combinations consisting of a 20-ms broadband
burst used as a pre-pulse and presented 80-ms before the pulse
(120 dB) using one of the five pre-pulse intensities (74, 78, 82, 86
and 90 dB). Each session consisted of six presentations of each
type of trial presented in a pseudorandom order. PPI was
calculated as % PPI: 1006 (mean startle amplitude on pulse
alone trials – mean startle amplitude on prepulse-pulse trials/
Novelty-induced locomotor activity in the
Two identical startle chambers
mean startle amplitude on pulse alone trials) for each animal and
data were plotted at mean % PPI for each prepulse type.
Data were analyzed by two-tailed, unpaired Student’s t-test by
comparison of non-transgenic and transgenic mice for each
condition or data interval. P,0.05 was considered significant.
transgenic mice. Western blot analysis of soluble brain extracts
(75 mg protein) derived from hemi-brains of 3-4 month G2019S
(line 340) and R1441C (line 574) LRRK2 transgenic mice (TG)
and their non-transgenic littermates (NTG). Blots were probed
with a pan-LRRK2 antibody (clone c81-8/MJFF4) recognizing
mouse and human LRRK2, or with b-tubulin as a protein loading
control. Densitometric analysis was conducted to quantify the fold
overexpression of human LRRK2 relative to endogenous mouse
LRRK2. Total LRRK2 levels were normalized to b-tubulin levels
and expressed as a percent of the corresponding NTG control.
Bars represent the mean from n = 2 mice per genotype.
Quantitation of LRRK2 protein levels in LRRK2
ventral midbrain of G2019S LRRK2 transgenic mice. In situ
hybridization of human or mouse LRRK2 and endogenous a-
synuclein mRNAs with species-specific33P-labeled oligonucleotide
probes in the substantia nigra pars compacta (SNpc, A9), hippo-
campus and ventral tegmental area (VTA, A10) of 2-3 month
G2019S LRRK2 transgenic mice (line 340). mRNA signals were
revealed by development of sections in photo-emulsion and
counter-staining with Cresyl violet. Notice that the mRNA signal
for human LRRK2 is greater than that of endogenous LRRK2 in
the SNpc, and that human LRRK2 is detected in VTA neurons
whereas mouse LRRK2 is not. Scale bars: 10 mm (SNpc), 50 mm
(hippocampus), 20 mm (VTA). DG, dentate gyrus.
Cellular localization of human LRRK2 mRNA in the
in situ hybridization with species-specific
oligonucleotide probes. A, Localization of mouse LRRK2 mRNA
throughout the brains of 2-3 month WT (line 249), R1441C (line
574) and G2019S (line 340) LRRK2 transgenic mice. B, Expression
pattern of mouse or human LRRK2 mRNA in the spleen and
kidney of WT, R1441C and G2019S LRRK2 transgenic mice.
Noticetheendogenous expression ofLRRK2throughout the spleen
and kidney. Olf, olfactory bulb; Str, striatum; Ctx, cerebral cortex;
Hip, hippocampus; Cb, cerebellum.
Expression analysis of LRRK2 transgenic mice by
substantia nigra pars compacta of aged LRRK2 transgenic mice.
Unbiased stereological analysis of TH+ and Nissl+ neurons in the
pars compacta fails to reveal dopaminergic neuronal loss in 15-21
month WT (lines 249 and 27), R1441C (line 546) and G2019S
(line 1128) LRRK2 transgenic mice (TG) compared to their age-
matched non-transgenic littermates (NTG). Bars represent the
mean 6 SEM. Numbers of mice used per genotype: lines 249 (n =
5), 27 (n = 3-4), 546 (n = 4-6) and 1128 (n = 6). There are no
statistically significant differences between TG and NTG groups.
Stereological analysis of dopaminergic neurons in the
LRRK2 transgenic mice. TH+ immunoreactivity in the striatum
of 19-20 month G2019S LRRK2 mice (TG, line 340) compared to
Striatal dopaminergic nerve terminals in G2019S
Dopaminergic Neuronal Loss in G2019S LRRK2 Mice
PLoS ONE | www.plosone.org13 April 2011 | Volume 6 | Issue 4 | e18568
their non-transgenic littermate mice (NTG). The optical density of
TH+ immunoreactivity was quantified by densitometric analysis of
every fourth section throughout the left and right striatum for each
mouse using NIH ImageJ software. Bars represent the mean 6
SEM (n = 5-6 mice/genotype).
transgenic mice. A, Biogenic amine levels in the prefrontal cortex
of 14-15 month G2019S LRRK2 mice (line 340) compared to their
NTG littermates by HPLC (n = 8 mice/genotype). B, Biogenic
amine levels in the striatum and cerebral cortex of 19-20 month
R1441C LRRK2 mice (line 574) compared to their NTG
littermates by HPLC (n = 7-11 mice/genotype). C, Levels of
dopamine and its metabolites, DOPAC and HVA, in the striatum,
olfactory bulb and cerebral cortex of 16-17 month WT LRRK2
mice (line 249) compared to their non-transgenic (NTG) littermates
by HPLC (n = 6=7 mice/genotype). Bars represent the mean 6
SEM. *P,0.05 or **P,0.01 comparing TG with NTG for each
biogenic amine as indicated.
HPLC analysis of biogenic amines in LRRK2
LRRK2 transgenic mice. Sections containing the substantia nigra
and cerebral cortex from 23-24 month-old G2019S LRRK2
transgenic (TG, line 340) or non-transgenic (NTG) mice were
stained by immunohistochemistry with antibodies for (A) mouse a-
synuclein, (B) phospho-a-synuclein (pSer129), (C) mouse ubiquitin,
(D) mouse tau, (E) phospho-tau (PHF-1; pSer396/Ser404) and (F)
GFAP, and by histological staining with hematoxylin (G). There
are no distinguishable differences between NTG and TG mice.
Scale bar: 100 mm.
Lack of PD-related neuropathology in aged G2019S
G2019S LRRK2 transgenic mice. Transmission electron micro-
scopic analysis of striatal tissue from 17-18 month-old G2019S
LRRK2 transgenic mice (line 340) revealing the accumulation of
autophagic vacuoles (indicated by *) within (A-B) neuronal soma
and (C-D) axonal-rich regions. Nuclei (nuc), axons (ax) and normal
mitochondria (m) are indicated. (E) Quantitation of the density of
autophagic vacuoles in equivalent regions of striatum from 17-18
month-old G2019S LRRK2 transgenic (TG) mice relative to their
non-transgenic (NTG) littermates. Bars represent the mean 6 SEM
(n = 3 mice/genotype). *P,0.01 comparing TG and NTG mice.
Scale bars: 200 nm (A-D).
and axonal processes in G2019S LRRK2 transgenic mice.
Transmission electron microscopic images of cerebral cortex tissue
from 17-26 month-old G2019S LRRK2 transgenic mice (line 340)
highlighting the accumulation of autophagic vacuoles (indicated
by *) within (A-B) neuronal soma and (C-D) axonal processes.
Nuclei (nuc), axons (ax) and normal mitochondria (m) are indicated.
Scale bars: 1 mm (A-D).
Autophagic vacuoles accumulate in neuronal soma
transgenic mice. Transmission electron microscopic images
showing representative examples of a morphologically normal
mitochondrion (class I), a damaged mitochondrion (class II), or
abnormal condensed mitochondrial aggregates in the cerebral
cortex of 17-26 month G2019S LRRK2 transgenic mice (line
340). Scale bar: 250 nm.
Mitochondrial abnormalities in G2019S LRRK2
The authors are grateful to Dustin Dikeman and Michael Johnson for
valuable technical assistance, and Dr. Mikhail V. Pletnikov for advice on
behavioral analysis (all Johns Hopkins). We thank Dr. Graham Knott and
members of the EPFL Bio-EM facility for assistance with electron
microscopy. TMD is the Leonard and Madlyn Abramson Professor in
Neurodegenerative Diseases at Johns Hopkins University.
Conceived and designed the experiments: DR JPLD TMD VLD DJM.
Performed the experiments: DR JPLD BML KS JK RB MW OP LG LY
YL DAS DG DJM. Analyzed the data: DR JPLD OP MFB JCT JMM DG
BT MKL TMD VLD DJM. Contributed reagents/materials/analysis
tools: JPLD NAJ NGC TMD VLD DJM. Wrote the paper: TMD VLD
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