Loss of leucine-rich repeat kinase 2 causes impairment
of protein degradation pathways, accumulation of
α-synuclein, and apoptotic cell death in aged mice
Youren Tonga, Hiroo Yamaguchia, Emilie Giaimea, Scott Boyleb, Raphael Kopanb, Raymond J. Kelleher IIIc,
and Jie Shena,1
aCenter for Neurologic Diseases, Department of Neurology, Brigham and Women’s Hospital, Program in Neuroscience, Harvard Medical School, Boston, MA
02115;bDepartment of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110; andcCenter for Human Genetic Research,
Department of Neurology, Massachusetts General Hospital, Program in Neuroscience, Harvard Medical School, Boston, MA 02115
Edited* by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved April 19, 2010 (received for review April 7, 2010)
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most
containinga small GTPasedomain anda kinase domain,butits phys-
The dopaminergic system of LRRK2−/−mice appears normal, and
numbers of dopaminergic neurons and levels of striatal dopamine
are unchanged. However, LRRK2−/−kidneys, which suffer the great-
est loss of LRRK compared with other organs, develop striking accu-
mulation and aggregation of α-synuclein and ubiquitinated proteins
at 20 months of age. The autophagy–lysosomal pathway is also im-
paired in the absence of LRRK2, as indicated by accumulation of
more, loss of LRRK2 dramatically increases apoptotic cell death, in-
flammatory responses, and oxidative damage. Collectively, our
findings show that LRRK2 plays an essential and unexpected role in
LRRK2 mutations may cause Parkinson’s disease and cell death via
accumulation and aggregation over time.
degeneration of dopaminergic (DA) neurons and the presence of
intraneuronal cytoplasmic inclusions known as Lewy bodies, of
which α-synuclein is a major constituent (1). Dominantly inherited
mutations in leucine-rich repeat kinase 2 (LRRK2) are the most
common cause of familial PD (2, 3), highlighting the importance
of LRRK2 in PD pathogenesis; however, the normal physiological
role of LRRK2 is unknown. LRRK2 is a large protein containing
a Ras-like small GTPase domain and a MAPKKK-like kinase
domain, and has a functional homolog LRRK1, which shares
similar domain structures (4). Crystal structural and biochemical
studies showed that the GTPase domain forms a dimer; the
pathogenic mutations destabilize the dimer and reduce GTPase
activity (5–7). A recent in vitro study suggested that LRRK2 and
LRRK1 can interact with each other and form a heterodimer (8).
Although no physiological substrate of the LRRK2 kinase activity
has been reported, studies in cultured cells have suggested that
some pathogenic mutations in LRRK2 cause increases in LRRK2
kinase activity (9, 10).
Protein aggregation is thought to play a major role in neuro-
degeneration and PD pathogenesis (11). The strongest evidence
came from studies of α-synuclein. Gene multiplication and mis-
sense mutations in α-synuclein have been identified in early-onset
familial PD with dominant inheritance (12). α-Synuclein is a major
constituent of Lewy bodies (1). Overexpression of either WT or
mutant α-synuclein in transgenic mice causes age-related neuro-
degeneration (13–15). Although patients carrying recessively in-
arkinson’s disease (PD) is the most common movement dis-
order. The neuropathological hallmarks of PD are progressive
herited mutations in Parkin often do not have Lewy bodies (no
bearing mutations in α-synuclein or LRRK2 typically have Lewy
bodies in nigral neurons (16).
To elucidate the normal physiological role of LRRK2 in vivo,
we generated two independent lines of LRRK2germ-line deletion
mice. Although LRRK2−/−mice do not develop apparent neuro-
degeneration and neuropathological changes in the brain, loss of
LRRK2 causes striking age-dependent accumulation and aggre-
gation of α-synuclein (60-fold) and ubiquitinated proteins in the
kidney, in which LRRK2 is normally expressed at high levels (∼6-
foldcompared tothe brain).Theautophagy–lysosomal pathway is
also impaired in the absence of LRRK2, and there are dramatic
increases in apoptotic cell death, inflammatory responses, and
oxidative damage. These results demonstrate an essential cellular
function of LRRK2 during aging in the maintenance of protein
homeostasis and, specifically, α-synuclein through the regulation
of protein degradation pathways.
Generation and Molecular Characterization of LRRK2−/−Mice. We
generated two independent lines of LRRK2 germ-line deletion
mice, targeting the promoter and exon 1 in knockout 1 (KO1)
and exons 29 and 30, which encode the first half of the Ras-like
small GTPase domain, in LRRK2 KO2. The targeting strategies
for the generation of KO1 and KO2 are shown in Fig. 1A and
Fig. S1A, respectively. The linearized targeting vectors were
transfected by electroporation into MKV6.5 ES cells, which were
derived from F1 hybrid of B6 and 129 mice. ES cell clones were
screened by Southern blotting using the 5′ external probe to
identify those carrying the proper homologous recombination
event, which were further confirmed by Southern blotting using
the 3′ external and the neo probes. Southern blotting analysis of
tail genomic DNAs of heterozygous F1 and homozygous F2 (Fig.
1B and Fig. S1B) mice confirmed the germ-line transmission of
the targeted alleles.
We then deleted the floxed PGK-neo cassette by crossing the
homozygous F2 mice with CaMKII-cre transgenic mice, in which
Cre is expressed in male gametes, resulting in excision of the
floxed PGK-neo cassette. Southern blotting confirmed the de-
letion of the floxed PGK-neo selection cassette in homozygous
KO1 (Fig. 1C) and KO2 (Fig. S1C) mice. Northern analyses of
Author contributions: Y.T. and J.S. designed research; Y.T., H.Y., E.G., and S.B. performed
research; Y.T., H.Y., E.G., S.B., R.J.K., and J.S. analyzed data; and Y.T. and J.S. wrote the
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| May 25, 2010
| vol. 107
| no. 21
total RNAs from brains of mice using a cDNA probe specific for
exons 1–5 (KO1) or specific for exons 29–30 (KO2) indicate the
absence of LRRK2 mRNAs (Fig. 1D and Fig. S1D). Western
blotting confirmed the absence of LRRK2 protein in the brain of
KO1 and KO2 mice (Fig. 1E and Fig. S1E). Thus, we conclude
that the KO1 and KO2 alleles are null alleles (−/−). Because
similar results were obtained from both KO1 and KO2 mice, the
data shown below are all from the analysis of KO1 mice.
Absence of DA Neurodegeneration and Neuropathological Changes in
LRRK2−/−Mice. To determine whether the deletion of LRRK2
causes degeneration of dopaminergic neurons, we performed im-
and their projections (Fig. S1G) appeared grossly normal in
LRRK2−/−mice. Quantitative analysis of tyrosine hydroxylase–
immunoreactive neurons in the substantia nigra pars compacta
using stereological methods revealed normal numbers of dopa-
minergic neurons in LRRK2−/−mice at 2 years of age (Fig. S1H).
We also measured the levels of striatal dopamine and its major
metabolites using HPLC. The steady-state striatal dopamine level
was not significantly changed in LRRK2−/−mice at the ages of 3
months, 1 year, and 2 years (Fig. S1I), compared with their WT
littermate controls. There was also no significant alteration in the
striatal levels of major metabolites of dopamine, dihydroxyl-
phenylacetic acid, and homovanillic acid in LRRK2−/−mice at
2 years of age (Fig. S1I), suggesting normal dopamine turnover.
We did not observe abnormal accumulation and aggregation of
α-synuclein(Fig.S2A) and ubiquitin(Fig. S2B) inLRRK2−/−brains
or microgliosis, normally associated with neurodegeneration, in any
brain subregions of LRRK2−/−mice at 2 years of age, as shown by
normal immunoreactivity for glial fibrillary acidic protein (GFAP)
(Fig. S3A) and ionized calcium binding adaptor molecule 1 (Iba1)
(Fig. S3B). Neuronal structures shown by MAP2 and synaptophysin
immunoreactivity appeared normal in LRRK2−/−mice.
Striking Age-Dependent Renal Atrophy in LRRK2−/−
LRRK2−/−mice age, they develop striking age-dependent ab-
normalities in the kidney. At 10 weeks of age, LRRK2−/−kidneys
were normal in morphology and weight, but by 20 months of age,
LRRK2−/−kidneys appeared significantly smaller in size and
much darker in color and weighed 31% less (Fig. 1F), whereas
the body weights of LRRK2−/−mice were not significantly dif-
ferent from those of WT controls. In addition, the surface of the
aged LRRK2−/−kidney appeared rough and granular. These
changes in kidneys were observed in both KO1 and KO2 lines
with 100% penetrance, but not in our LRRK2 R1441C knockin
mice. Despite these dramatic morphological changes in the
kidney, we did not observe any gross changes in other organs of
LRRK2−/−mice up to 2 years of age. Thus, the loss of LRRK2
causes striking age-dependent alteration specifically in the kid-
ney, which may be explained by the fact that LRRK2 is normally
expressed at much higher levels in the kidney (∼6.2-fold) relative
to the brain (Fig. 1G) and other organs (17).
Accumulation and Aggregation of α-Synuclein and Ubiquitinated
Proteins. To uncover the mechanism underlying age-related ab-
normalities developed in older LRRK2−/−mice, we performed
a numberofanalysestolookfor molecular and cellularalterations.
Western blotting showed an ≈60-fold increase in the level of
α-synuclein in Triton X-100–soluble fractions of kidneys from 20-
month-old LRRK2−/−mice (Fig. 2 A and B) , indicating dramatic
accumulation of α-synuclein in the absence of LRRK2, whereas
α-synuclein was normally present at very low levels in the kidney
(∼1/200 compared with the brain). Furthermore, levels of higher-
molecular-weight (HMW) species immunoreactive to α-synuclein–
from these LRRK2−/−kidneys (Fig. 2 A and B), indicating aggre-
gation of α-synuclein in the absence of LRRK2.
Consistent with findings from Western blotting, immunohisto-
chemical analysis showed widely distributed cytosolic α-synuclein–
immunoreactive granular aggregates or inclusions in boxy cells
of renal tubules in the cortical area of LRRK2−/−kidneys at
20 months of age (Fig. 2 C and D), which were not present in
kidneys of WT controls or LRRK2−/−mice at 10 weeks of age.
These data together suggest an age-dependent accumulation
and aggregation of α-synuclein in the absence of LRRK2.
α-Synuclein is phosphorylated at the residue Ser-129 in synu-
cleinopathy lesions, and phosphorylation of α-synuclein at Ser-
129 has been reported to promote aggregation of α-synuclein in
for α-synuclein phosphorylated at Ser-129 also revealed granular
aggregatesorinclusionsinkidneys ofLRRK2−/−miceat 20 months
of age (Fig. 2 C and D) but not in kidneys of WT controls or young
LRRK2−/−mice, indicating increased levels of phospho-Ser-129
α-synuclein, which likely contribute to α-synuclein aggregation.
We further examined whether the ubiquitin–proteasome sys-
tem (UPS)–mediated protein degradation is affected in aged
LRRK2−/−kidneys. Western blotting showed dramatically in-
creased levels of HMW protein species immunoreactive for
ubiquitin-specific antibody in Triton X-100–insoluble fractions of
kidneys from aged LRRK2−/−mice (Fig. 2 A and B), suggesting
accumulation and aggregation of ubiquitinated proteins in the
absence of LRRK2. Consistent with this finding, immunohisto-
egy for generation of LRRK2 KO1 mice. The locations of the 5′ and 3′ external
probes used for Southern blotting are indicated. Restriction site: N, NheI; S,
SphI. (B and C) Southern blotting of tail genomic DNAs using 5′ probe shows
germ-line transmission of targeted allele (B) and KO1 allele (C). Tail genomic
DNAs were digested with NheI. The 11.5-kb band represents the WT allele,
whereas the 5.4-kb and 3.7-kb bands represent the targeted allele and KO1
allele, respectively. (D) Northern blotting of total RNAs from brains of KO1
mice shows absence and reduction of LRRK2 mRNAs in−/−and+/−mice, re-
a probe. 18S rRNAs were used as loading control. (E) Western blotting indi-
cates absence of LRRK2 in the brain of
(F)Compared with WT controls, kidneys (fresh, nonperfused) from 20-month-
old LRRK2−/−mice are significantly smaller and darker and weigh ∼30% less,
and weight to WT controls. n = 8 Kidneys per genotypic group. (G) Quantita-
tive RT-PCR showing relative expression levels of LRRK1 and LRRK2 mRNAs,
after normalized to an internal control TATA-binding protein (TBP) mRNA, in
ns, Not significant. **P < 0.01.
Age-dependent renal atrophy in LRRK2−/−mice. (A) Targeting strat-
−/−mice. Reprobing of the same
| www.pnas.org/cgi/doi/10.1073/pnas.1004676107 Tong et al.
chemical analysis revealed widely distributed cytosolic granules
or inclusions immunoreactive to ubiquitin-specific antibody in
kidneys of aged LRRK2−/−mice (Fig. 2 C and D), which were
barely detectable in WT controls and in kidneys of young
LRRK2−/−mice. These results suggest that UPS-mediated pro-
tein degradation is impaired in the absence of LRRK2.
Impaired Autophagy–Lysosomal Pathway in Aged LRRK2−/−Mice. We
Sudan Black B, which stain macromolecules containing carbohy-
significantly darker pink staining in renal tubules and glomeruli in
the cortex of LRRK2−/−kidneys at 20 months of age (Fig. 3A).
Sudan Black B staining further revealed dark brown granules,
which show bright autofluorescence, suggesting abnormal accu-
mulation of lipofuscin granules in kidneys of aged LRRK2−/−mice
(Fig. 3A). Lipofuscin granules are composed of undigested mate-
rials from lysosomes containing oxidized lipids, carbohydrates and
proteins, and are undegraded aggregates as a result of excessive
oxidation and crosslinking (19). Consistent with this finding, levels
of protein carbonyls, a general marker of oxidative damage, were
also increased in kidneys of 20-month-old LRRK2−/−mice (Fig.
3B). Abnormal accumulation of lipofuscin granules suggests im-
pairment of the autophagy–lysosomal system and has been impli-
cated in neurodegenerative disorders such as PD.
To assess further autophagy function in the absence of LRRK2,
we evaluated markers of autophagosomes, such as microtubule-
associated protein 1 light chain 3 (LC3), a homolog of Atg8, which
is essential for autophagy function in yeast (20, 21). There are two
forms of LC3, I and II. LC3-II is the lipidated form of LC3-I and is
bound to autophagosomal membranes. The amount of LC3-II
correlates with the extent of autophagosome formation, and the
conversion of LC3-I to LC3-II is a reliable indicator of autophagic
activity (22). Western analysis showed that levels of LC3-II are
dramatically decreased in LRRK2−/−kidneys at 20 months of age
(Fig. 3C), indicating impaired autophagosome formation in the
absence of LRRK2. The higher level of LC3-I in LRRK2−/−mice
(Fig. 3C) suggests altered LC3-I to LC3-II conversion, further
supporting reduced autophagic activity in the absence of LRRK2.
proteins in absence of LRRK2. (A) Representative Western blots showing
dramatically increased levels of α-synuclein monomers (soluble α-syn) in
Triton X-100–soluble fractions and HMW α-synuclein species (HMW α-syn)
and ubiquitin-positive proteins (HMW ubi+) in Triton X-100 insoluble frac-
tions in kidneys of 20-month-old LRRK2−/−mice. Ponceau S staining is used as
loading control, as levels of β-actin are altered in LRRK2−/−kidneys. (B)
Quantification of Triton-soluble α-synuclein monomers and insoluble HMW
α-synuclein and ubiquitin-positive proteins (bracketed region) from Western
blots as shown in A. (C) Immunohistochemical analysis shows widely dis-
tributed cytosolic granules or inclusions immunoreactive to α-synuclein-,
phospho–α-synuclein–specific (pS129), or ubiquitin-specific antibody in boxy
cells of renal tubules in kidneys of 20-month-old LRRK2−/−mice. (All scale
bars, 20 μm.) (D) Relative areas of granules immunoreactive to α-synuclein–
specific (α-syn), phospho–α-synuclein–specific (pS129), or ubiquitin (ubi)–
specific antibody as shown in C were estimated using the ImageJ program
(National Institutes of Health). Data in all panels are expressed as mean ±
SEM. *P < 0.05; **P < 0.01; ***P < 0.001.
Accumulation and aggregation of α-synuclein and ubiquitinated
oxidative damage. (A) Periodic Acid-Schiff staining of cross sections of kidneys
shows thicker and darker pink staining in renal tubules and the presence of
widely distributed brown granules in boxy cells of renal tubules of 20-month-
with hematoxylin (blue). Sudan Black B staining reveals, in renal tubules of
LRRK2−/−mice, widely distributed dark brown granules that show bright
autofluorescence (orange for merged fluorescence) in the absence of any pri-
mary and secondary antibodies. (B) Elevated levels of protein carbonyls (Oxy-
mice. Equal loading of total proteins was confirmed by Ponceau S–staining of
the membranes. (C) Western blotting shows dramatically decreased levels of
autophagosome marker LC3-II (some LC3-II signals are detected when more
proteinswere loadedand longer exposure was used) and increased levels ofan
autophagy substrate p62 in Triton X-100–insoluble fractions of kidneys from
LRRK2−/−mice. (D) Immunohistochemical analysis revealed the presence of
granular aggregates or inclusions immunoreactive to p62-specific antibody in
immunoreactive to p62 antibody were estimated using the ImageJ program
(National Institutes of Health). (All scale bars, 20 μm.) Data in all panels are
expressed as mean ± SEM. *P < 0.05; ***P < 0.001.
Impairment of the autophagy–lysosomal pathway and increases in
Tong et al.PNAS
| May 25, 2010
| vol. 107
| no. 21
Inhibition of autophagy by inactivation of Atg7, a protein es-
sential for autophagy function (23), leads to drastically elevated
levels of p62 in Atg7 conditional KO mice (24). We found that
levels of p62 were significantly higher in aged LRRK2−/−mice
(Fig. 3C). Immunohistochemical analysis showed dramatically
increased numbers of p62-immunoreactive granules in the cortex
of kidneys from 20-month-old LRRK2−/−mice (Fig. 3D). In-
terestingly, the strongest p62-immunoreactive granular signals in
kidneys of LRRK2−/−mice appeared to be localized in the
deeper layer of the renal cortex and the boundary region be-
tween the cortex and medulla, where LRRK2 mRNA is most
highly expressed in WT kidneys and LRRK1 mRNAs were al-
most undetectable (25). These results further demonstrate im-
pairment of autophagy function in the absence of LRRK2.
Increased Apoptosis and Inflammation in Aged LRRK2−/−Mice. To
examine the consequence of impaired UPS and autophagy as well
as accumulation and aggregation of α-synuclein, we looked for
possible degeneration in kidneys of LRRK2−/−mice at 20 months
of age, which may explain the 30% weight reduction. Immuno-
histochemical analysis revealed dramatic increases in the number
of apoptotic cells labeled by TUNEL assay and active caspase-3
immunoreactivity in medulla, renal tubules, and glomeruli of
kidneys from 20-month-old LRRK2−/−mice compared with WT
controls (Fig. 4 A and B) . Few cells were immunoreactive for
active caspase-3 in LRRK2−/−mice at 10 weeks of age. Western
blotting showed elevated levels of the active forms of caspase-3
and caspase-6 (Fig. 4C), two effector caspases in apoptosis, pro-
viding further support for increased apoptotic cell death in the
absence of LRRK2.
Because degeneration is often accompanied by inflammatory
responses, we further looked at markers for inflammatory
responses in LRRK2−/−kidneys. Immunohistochemical analysis
revealed striking up-regulation of cathepsin S and complement
C1q (Fig. 4 D and E), widely used markers for inflammatory
responses (26, 27), in kidneys of LRRK2−/−mice at 20 months of
age, whereas levels of these markers in kidneys of LRRK2−/−mice
at 10 weeks of age were normal. Western analysis also confirmed
the increases in the levels of cathepsin S and C1q in kidneys of 20-
month-old LRRK2−/−mice (Fig. 4F). Histological and immuno-
histochemical analyses also showed large clusters of invading B
cells and accumulation of endogenous mouse Ig around these B
cell clusters in kidneys of 20-month-old LRRK2−/−mice.
Mutations in LRRK2 are the most common genetic cause of PD,
but its function in the cell has remained unknown. In the current
study, our genetic analysis revealed an unexpected cellular role
of LRRK2 in protein homeostasis. Specifically, loss of LRRK2
causes dramatic accumulation and aggregation of α-synuclein and
ubiquitinated proteins in an age-dependent manner (Fig. 2) as
well as impairment of the autophagy–lysosomal pathway (Fig. 3).
These age-dependent abnormalities are accompanied by increas-
es in apoptotic cell death, inflammatory responses, and oxidative
damage (Figs. 3 and 4). Interestingly, these cellular changes bear
striking resemblance to processes that are thought to be involved
in PD pathogenesis, such as α-synuclein aggregation (1), apo-
ptosis and autophagic degeneration (28), impairment of the
ubiquitin–proteasome pathway, and accumulation of oxidized
How does loss of LRRK2 lead to these PD-like changes?
Defects in the protein degradation pathways are the likely culprit.
The ubiquitin–proteasome pathway may be affected, as suggested
by dramatic increases in ubiquitinated proteins in the kidney of
by 60-fold accumulation of total α-synuclein and formation of
α-synuclein aggregates in the kidney of aged LRRK2−/−mice (Fig.
2). Furthermore, the autophagy–lysosomal pathway is impaired,
as indicated by reduced levels of LC3-II (Fig. 3), which is con-
impairment of autophagy function is further supported by in-
creased levels of p62 (Fig. 3), consistent with previous findings
showing that suppression of autophagy function by Atg7 in-
activation caused accumulation of p62 (24). Given the fact that
death in various cell types including neurons and T cells (30–32),
impairment of autophagy caused by loss of LRRK2 likely
underlies or contributes to the increased apoptotic cell death in
There is accumulating evidence indicating that there is
a crosstalk between autophagy and the UPS. For example, sup-
pression of autophagy function by Atg5 or Atg7 inactivation
causes accumulation of ubiquitin-positive inclusions and UPS
substrates (24, 30, 31, 33). Deletion of p62, which binds LC3 and
ubiquitin, prevents the formation of ubiquitin-positive protein
aggregates caused by autophagy inactivation (24, 33). Over-
expression of p62 in cultured cells resulted in increased levels of
mutant α-synuclein (A53T) (33). Thus, accumulation and aggre-
gation of α-synuclein in our LRRK2-deficient mice may be a con-
sequence of impaired autophagy and UPS functions. Future
research will be needed to resolve whether LRRK2 controls UPS
function directly or via its regulation of the autophagy pathway.
Despite these striking age-related changes in LRRK2−/−mice
similar phenotypes are missing in the brain (Figs. S1–S3). The
simplest explanation is that LRRK1, a functional homolog of
LRRK2, may compensate functionally for the loss of LRRK2 in
brains of LRRK2−/−mice. LRRK1 mRNAs are broadly expressed
in the developing and adult brain, and the expression level of
LRRK1 in the developing brain is much higher than that of
LRRK2 (25). The specific abnormalities in LRRK2−/−kidneys are
likely due to the much higher level of LRRK2 mRNAs normally
expressed in the kidney (Fig. 1G) compared with other organs (e.
g., ∼6-fold higher than in the brain) (17). There is no compen-
satory up-regulation of LRRK1 in the absence of LRRK2. In-
terestingly, in the deeper cortical region of the kidney, where
LRRK2 mRNAs are most highly expressed and LRRK1 mRNAs
are least abundant (25), we observed the strongest granular ac-
cumulation of p62. These results suggest that the LRRK family
(LRRK1 and LRRK2) plays an essential role in the regulation of
protein homeostasis, and that the LRRK2−/−kidney suffers the
biggest loss of LRRK compared with other organs, and thereby
develops the most striking age-dependent abnormalities as a con-
sequence of impaired protein degradation pathways. Therefore, it
will be necessary to generate LRRK1/LRRK2 double KO to de-
termine whether complete loss of LRRK in neurons, especially
in DA neurons where oxidative stress is elevated, results in age-
related α-synuclein aggregation, autophagy impairment, and neu-
Another important issue that is worth discussing is why loss of
LRRK2 function causes alterations that are strikingly similar to
PD, whereas only dominantly inherited missense mutations have
been found in familial and sporadic PD cases (12). The lack of
deletion and nonsense mutations and the dominant inheritance
argue against a simple loss-of-function pathogenic mechanism,
and suggest that mutant LRRK2 may act in a dominant negative
manner to inhibit function of WT LRRK2. Several lines of re-
cent experimental evidence are in support of this notion. For
example, crystal structural analysis revealed that the ROC (for
Ras of complex proteins) GTPase domain forms a dimer, and
that mutations at the R1441 (R1441C, R1441G, R1441H) and
I1371 (I1371V) residues destabilize the ROC dimer and de-
crease GTPase activity (5), which is thought to up-regulate the
kinase activity (10, 34). Additional biochemical studies provided
further support for LRRK2 functioning as a dimer (6, 7). It was
also reported that tagged LRRK2 and LRRK1 can interact with
| www.pnas.org/cgi/doi/10.1073/pnas.1004676107Tong et al.
each other and form a heterodimer, and that LRRK1 variants
can affect the age of disease onset among LRRK2 mutation
carriers (8). Future studies will be needed to determine whether
all pathogenic missense mutations in LRRK2 affect dimer for-
mation and reduce GTPase activity, which in turn increases its
Consistent with this partial loss-of-function pathogenic mecha-
nism, expression of various mutant forms of LRRK2 in mice, even
at more than 10-fold of overproduction, has so far failed to re-
produce PD-like neuropathological changes, such as cell death
and α-synuclein aggregation (35–38). Interestingly, a recent report
showed that LRRK2 inactivation alleviated rather than exacer-
bated α-synuclein aggregation and neurodegeneration under over-
expression conditions (>16-fold) in α-synuclein transgenic mice
(37). However, it was unclear whether autophagy function is af-
fected by α-synuclein overexpression in these transgenic mice. It
will be interesting to test whether a huge excess of α-synuclein in
the brain of transgenic mice impairs protein degradation path-
ways, and whether autophagy function is the focal point of the
regulation by both α-synuclein overexpression and loss of LRRK2.
In summary, our genetic study demonstrates that LRRK2
controls protein homeostasis in the cell. During the aging pro-
cess, the LRRK2−/−kidney, which suffers the biggest loss of
LRRK, develops impairment of protein degradation pathways,
leading to huge accumulation of α-synuclein and aggregation.
These dramatic molecular and cellular alterations likely underlie
increased cell death and inflammatory responses in these mice.
Thus, inhibition of LRRK2 function may not represent a suitable
therapeutic strategy for the treatment of PD and suppression of
α-synuclein aggregation. Future studies will be needed to de-
termine how LRRK2 regulates the autophagy–lysosomal path-
way and whether its Ras-like GTPase domain is required for this
function. It remains to be tested whether LRRK2 mutations
affect autophagy and UPS functions and whether LRRK2 reg-
ulates the ubiquitin–proteasome pathway directly or indirectly
via autophagy. Crossing LRRK2−/−with α-synuclein−/−mice will
allow us to determine the importance of α-synuclein accumula-
tion and aggregation in the age-dependent cell death and other
degenerative processes caused by loss of LRRK2. Targeting
protein degradation pathways (e.g., enhancing autophagy) may
be a useful venue to pursue for restoring protein homeostasis
and treatment of PD.
Materials and Methods
Generation of LRRK2 KO Mice. To generate two independent lines of LRRK2
KO mice, two targeting vectors were constructed. To generate LRRK2 KO1
mice, a 2.5-kb DNA fragment upstream of the LRRK2 promoter, and a 3.5-kb
DNA fragment encompassing exon 2 as well as part of introns 1 and 2 of
LRRK2, were amplified by PCR using genomic DNA of C57BL/6J (B6) mice in
a BAC clone containing this part of the LRRK2 gene as template. To generate
LRRK2 KO2 mice, a 2.3-kb DNA fragment encompassing exon 28 and a 3.4-kb
DNA fragment encompassing exon 31 were amplified by PCR using tail ge-
nomic DNAs of B6 mice as template. These fragments were confirmed by
DNA sequencing and used as 5′ and 3′ homologous arms in KO1 and KO2
targeting vectors, respectively. Each linearized targeting vector was trans-
fected by electroporation into MKV6.5 ES cells derived from B6/129 F1 mice.
Desired homologous recombination events were confirmed by Southern
blotting with the 5′ and 3′ external probes and the neo probe. ES cells from
two targeted clones for each KO line were injected into B6 blastocysts. The
resulting chimeric mice were mated with B6/129 F1 mice. The homozygous
F2 mice obtained from intercrossing of heterozygous F1 mice carrying the
targeted allele were mated with CaMKII-cre transgenic mice to delete the
floxed PGK-neo selection cassette to obtain heterozygous mice carrying the
KO allele. All animal experimentation followed the protocol approved by
Harvard Center for Animal Resources and Comparative Medicine.
Histological and Immunohistochemical Analysis. Mice were anesthetized by i.p.
injection of sodium pentobarbital 15 min after injection of heparin. Mice
were then transcardially perfused with Ringer’s solution containing 0.25 g/L
heparin and 5 g/L procaine followed by ice-cold 4% paraformaldehyde in
and immunohistochemical analysis shows increased numbers of TUNEL-positive
cells and densely stained active caspase-3–immunoreactive cells, respectively, in
kidneys of 20-month-old LRRK2−/−mice. (B) Quantification of active caspase-3–
(C) Western analysis shows increased levels of active caspase-3 and caspase-6 in
reveals up-regulation of cathepsin S and complement C1q, two commonly used
inflammation markers, in LRRK2−/−mice. (E) Relative areas of granules immuno-
reactiveto cathepsin S-or C1q-specificantibodywereestimated usingthe ImageJ
Increases in apoptotic cell death and inflammatory responses. (A) TUNEL
Tong et al.PNAS
| May 25, 2010
| vol. 107
| no. 21
PBS (pH 7.4). The brain and kidneys were postfixed in 4% paraformalhehyde Download full-text
at 4 °C overnight and then processed for paraffin embedding following
standard procedures. Sections were cut at 16 μm (brain) or 8 μm (kidney). For
histology, sections were stained by Periodic Acid-Schiff stain kit (Dako) or
Sudan Black B. For immunohistochemical analysis, some tissue sections were
subjected to antigen retrieval by microwaving or autoclaving for 10 or 15
min in 10 mM sodium citrate buffer, pH 6.0. Endogenous peroxidase activity
was quenched by incubating in 0.3% H2O2. After blocking, sections were
incubated with primary antibodies overnight at 4 °C, followed by 1-h in-
cubation with biotinylated secondary antibodies and 1-h incubation with
Vectastain Elite ABC reagent, and then developed using chromogenic DAB
substrate (Vector Laboratories). For negative controls, primary antibodies,
alone or together with secondary antibodies, were omitted from the in-
cubation buffer. Stereological DA neuron counting was performed as pre-
viously described (38). Apoptotic cells were detected by a colorimetric TUNEL
staining using an in situ cell death detection kit (Roche) following the
Preparation of Nonionic Detergent-Soluble and Detergent-Insoluble Fractions.
Fresh mouse brains or kidneys were homogenized in ice-cold radioimmu-
noprecipitation assay buffer (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 0.1%
SDS, 1% Triton X-100, 1% sodium deoxycholate, supplemented with pro-
tease inhibitor mixture and phosphatase inhibitor mixtures), followed by
sonication. Homogenates were centrifuged at 14,000 × g for 20 min at 4 °C
to separate supernatants (fractions soluble in 1% Triton X-100). The result-
ing pellets were further lysed with a buffer containing 4% SDS and 20 mM
Hepes, pH 7.5, supplemented with protease inhibitor mixture and phos-
phatase inhibitor mixtures by vortexing and sonication, followed by centri-
fugation at 19,600 × g for 10 min at room temperature to separate the new
supernatants (Triton X-100–insoluble fractions).
Western Blotting Analysis and OxyBlot. Equal amounts of total proteins from
each preparation were loaded and separated in NuPAGE 3–8% Tris-Acetate
gels or 4–12% Bis-Tris gels (Invitrogen) and then transferred to nitrocellulose
membranes. Oxyblots for detecting protein carbonyls were prepared fol-
lowing the manufacturer’s instructions (Chemicon). After blocking and
overnight incubation with primary antibodies, protein bands of interest
were visualized by binding of IRDye-labeled secondary antibodies, and band
intensity was analyzed using Odyssey imaging system (Li-Cor). Antibodies
used are listed in SI Materials and Methods.
StatisticalAnalysis. Statisticalanalysis was performed usingPrism 5 (GraphPad
Software) and Excel (Microsoft). Data are presented as means ± SEM. Sta-
tistical significance was determined based on P values derived from Stu-
dent’s t test or two-way ANOVA.
ACKNOWLEDGMENTS. We thank Huailong Zhao and Lan Wang for technical
assistance. This work was supported by grants from the National Institute of
Neurological Disorders and Stroke and the Michael J. Fox Foundation (to J.S.)
and grants from the National Institute of Diabetes and Digestive and Kidney
Diseases (to R.K. and S.B.).
1. Spillantini MG, et al. (1997) Alpha-synuclein in Lewy bodies. Nature 388:839–840.
2. Paisán-Ruíz C, et al. (2004) Cloning of the gene containing mutations that cause
PARK8-linked Parkinson’s disease. Neuron 44:595–600.
3. Zimprich A, et al. (2004) Mutations in LRRK2 cause autosomal-dominant parkinsonism
with pleomorphic pathology. Neuron 44:601–607.
4. Marín I (2006) The Parkinson disease gene LRRK2: Evolutionary and structural insights.
Mol Biol Evol 23:2423–2433.
5. Deng J, et al. (2008) Structure of the ROC domain from the Parkinson’s disease-
associated leucine-rich repeat kinase 2 reveals a dimeric GTPase. Proc Natl Acad Sci
6. Greggio E, et al. (2008) The Parkinson disease-associated leucine-rich repeat kinase 2
(LRRK2) is a dimer that undergoes intramolecular autophosphorylation. J Biol Chem
7. Sen S, Webber PJ, West AB (2009) Dependence of leucine-rich repeat kinase 2 (LRRK2)
kinase activity on dimerization. J Biol Chem 284:36346–36356.
8. Dachsel JC, et al. (2010) Heterodimerization of Lrrk1-Lrrk2: Implications for LRRK2-
associated Parkinson disease. Mech Ageing Dev 131:210–214.
9. Smith WW, et al. (2006) Kinase activity of mutant LRRK2 mediates neuronal toxicity.
Nat Neurosci 9:1231–1233.
10. West AB, et al. (2005) Parkinson’s disease-associated mutations in leucine-rich repeat
kinase 2 augment kinase activity. Proc Natl Acad Sci USA 102:16842–16847.
11. Rubinsztein DC (2006) The roles of intracellular protein-degradation pathways in
neurodegeneration. Nature 443:780–786.
12. Singleton AB (2005) Altered alpha-synuclein homeostasis causing Parkinson’s disease:
The potential roles of dardarin. Trends Neurosci 28:416–421.
13. Giasson BI, et al. (2002) Neuronal alpha-synucleinopathy with severe movement
disorder in mice expressing A53T human alpha-synuclein. Neuron 34:521–533.
14. Lee MK, et al. (2002) Human alpha-synuclein-harboring familial Parkinson’s disease-
linked Ala-53→Thr mutation causes neurodegenerative disease with alpha-synuclein
aggregation in transgenic mice. Proc Natl Acad Sci USA 99:8968–8973.
15. Chandra S, Gallardo G, Fernández-Chacón R, Schlüter OM, Südhof TC (2005) Alpha-
synuclein cooperates with CSPalpha in preventing neurodegeneration. Cell 123:
16. Hardy J, Lewis P, Revesz T, Lees A, Paisan-Ruiz C (2009) The genetics of Parkinson’s
syndromes: A critical review. Curr Opin Genet Dev 19:254–265.
17. Biskup S, et al. (2007) Dynamic and redundant regulation of LRRK2 and LRRK1
expression. BMC Neurosci, 8:102. Available at: http://www.biomedcentral.com/1471-
18. Fujiwara H, et al. (2002) Alpha-synuclein is phosphorylated in synucleinopathy lesions.
Nat Cell Biol 4:160–164.
19. Keller JN, et al. (2004) Autophagy, proteasomes, lipofuscin, and oxidative stress in the
aging brain. Int J Biochem Cell Biol 36:2376–2391.
20. Kabeya Y, et al. (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in
autophagosome membranes after processing. EMBO J 19:5720–5728.
21. Kirisako T, et al. (1999) Formation process of autophagosome is traced with Apg8/
Aut7p in yeast. J Cell Biol 147:435–446.
22. Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy
research. Cell 140:313–326.
23. Komatsu M, et al. (2005) Impairment of starvation-induced and constitutive
autophagy in Atg7-deficient mice. J Cell Biol 169:425–434.
24. Komatsu M, et al. (2007) Homeostatic levels of p62 control cytoplasmic inclusion body
formation in autophagy-deficient mice. Cell 131:1149–1163.
25. Westerlund M, et al. (2008) Developmental regulation of leucine-rich repeat kinase 1
and 2 expression in the brain and other rodent and human organs: Implications for
Parkinson’s disease. Neuroscience 152:429–436.
26. Beglopoulos V, et al. (2004) Reduced beta-amyloid production and increased
inflammatory responses in presenilin conditional knock-out mice. J Biol Chem 279:
27. Bonifati DM, Kishore U (2007) Role of complement in neurodegeneration and
neuroinflammation. Mol Immunol 44:999–1010.
28. Anglade P, et al. (1997) Apoptosis and autophagy in nigral neurons of patients with
Parkinson’s disease. Histol Histopathol 12:25–31.
29. McNaught KS, Olanow CW, Halliwell B, Isacson O, Jenner P (2001) Failure of the
ubiquitin-proteasome system in Parkinson’s disease. Nat Rev Neurosci 2:589–594.
30. Hara T, et al. (2006) Suppression of basal autophagy in neural cells causes neuro-
degenerative disease in mice. Nature 441:885–889.
31. Komatsu M, et al. (2006) Loss of autophagy in the central nervous system causes
neurodegeneration in mice. Nature 441:880–884.
32. Pua HH, Dzhagalov I, Chuck M, Mizushima N, He YW (2007) A critical role for the
autophagy gene Atg5 in T cell survival and proliferation. J Exp Med 204:25–31.
33. Korolchuk VI, Mansilla A, Menzies FM, Rubinsztein DC (2009) Autophagy inhibition
compromises degradation of ubiquitin-proteasome pathway substrates. Mol Cell 33:
34. West AB, et al. (2007) Parkinson’s disease-associated mutations in LRRK2 link en-
hanced GTP-binding and kinase activities to neuronal toxicity. Hum Mol Genet 16:
35. Li X, et al. (2010) Enhanced striatal dopamine transmission and motor performance
with LRRK2 overexpression in mice is eliminated by familial Parkinson’s disease
mutation G2019S. J Neurosci 30:1788–1797.
36. Li Y, et al. (2009) Mutant LRRK2(R1441G) BAC transgenic mice recapitulate cardinal
features of Parkinson’s disease. Nat Neurosci 12:826–828.
37. Lin X, et al. (2009) Leucine-rich repeat kinase 2 regulates the progression of
neuropathology induced by Parkinson’s-disease-related mutant alpha-synuclein.
38. Tong Y, et al. (2009) R1441C mutation in LRRK2 impairs dopaminergic neurotransmission
in mice. Proc Natl Acad Sci USA 106:14622–14627.
| www.pnas.org/cgi/doi/10.1073/pnas.1004676107Tong et al.