Leucine-rich repeat kinase 2 (LRRK2) interacts
with parkin, and mutant LRRK2 induces
Wanli W. Smith*†, Zhong Pei*†, Haibing Jiang*, Darren J. Moore‡§, Yideng Liang*, Andrew B. West‡§,
Valina L. Dawson‡§¶, Ted M. Dawson‡§, and Christopher A. Ross*‡?
Department of *Psychiatry, Division of Neurobiology, Departments of‡Neurology and Neuroscience, and¶Physiology, and§Institute for Cell Engineering,
The Johns Hopkins University School of Medicine, Baltimore, MD 21287
Edited by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 28, 2005 (received for review
September 14, 2005)
Parkinson’s disease (PD) is a disorder of movement, cognition, and
emotion, and it is characterized pathologically by neuronal degen-
eration with Lewy bodies, which are cytoplasmic inclusion bodies
containing deposits of aggregated proteins. Most PD cases appear
to be sporadic, but genetic forms of the disease, caused by
mutations in ?-synuclein, parkin, and other genes, have helped
elucidate pathogenesis. Mutations in leucine-rich repeat kinase 2
(LRRK2) cause autosomal-dominant Parkinsonism with clinical fea-
tures of PD and with pleomorphic pathology including deposits of
aggregated protein. To study expression and interactions of
LRRK2, we synthesized cDNAs and generated expression con-
structs coding for human WT and mutant LRRK2 proteins. Expres-
is predominately cytoplasmic, as is endogenous protein by subcel-
lular fractionation. Using coimmunoprecipitation, we find that
LRRK2, expressed in cells in culture, interacts with parkin but not
with ?-synuclein, DJ-1, or tau. A small proportion of the cells
overexpressing LRRK2 contain protein aggregates, and this pro-
portion is greatly increased by coexpression of parkin. In addition,
parkin increases ubiquitination of aggregated protein. Also, mu-
tant LRRK2 causes neuronal degeneration in both SH-SY5Y cells
and primary neurons. This cell model may be useful for studies of
PD cellular pathogenesis and therapeutics. These findings suggest
a gain-of-function mechanism in the pathogenesis of LRRK2-linked
PD and suggest that LRRK2 may be involved in a pathogenic
pathway with other PD-related proteins such as parkin, which may
help illuminate both familial and sporadic PD.
cell death ? Parkinson’s disease ? protein aggregation ? protein
interaction ? ubiquitin
kinesia and pathologically by loss of dopamine neurons in substan-
tia nigra and other brain regions (1–4). There are characteristic
ubiquitinated inclusions (proteinaceous aggregates), termed Lewy
bodies. In PD and related disorders, aggregates are located in the
substantia nigra and other brain regions (1, 4, 5). The pathogenesis
of PD remains incompletely understood, but it appears to involve
mutations have been shown to cause Parkinsonism with features
comparable with PD include ?-synuclein, parkin, DJ-1, and PINK1
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene can
cause autosomal-dominant PD (14–16). The gene product has also
been termed ‘‘Dardarin.’’ Several different point mutations segre-
gate with the PD phenotype in families, including the original
cases (23). Patients with LRRK2 mutations have typical relatively
late onset Parkinsonism with features comparable with idiopathic
PD, with asymmetric rest tremor, bradykinesia, rigidity, and a good
response to 3,4-dihyroxy-L-phenylalanine (L-DOPA) (15, 24). The
arkinson’s disease (PD) is a progressive neurodegenerative
disorder characterized clinically by tremor, rigidity, and brady-
pathology of cases with LRRK2 mutations is pleomorphic (15, 17).
There are typical features of neuronal loss and gliosis in the
substantia nigra. In some cases, there are Lewy bodies and Lewy
neurites, sometimes located diffusely in the brain. In other cases,
there are protein deposits, mostly in the cytoplasm, with some
heterogeneity both within and between families.
domain (C-terminal of Roc), a leucine-rich repeat consisting of 12
[mitogen-activated protein kinase kinase kinase (MAPKKK), de-
scribed as a tyrosine kinase], a WD40 domain, and an ankryin
domain (14, 15, 25). Point mutations have been found in almost all
of the identified domains. The distribution of mutations in several
different domains, as well as the lack of deletions or truncations,
along with dominant inheritance, are consistent with a gain-of-
In this study, we investigated the cellular localization of LRRK2
and its possible interactions with other PD-related gene products,
and we sought to determine whether LRRK2 may be involved in
cell toxicity. We found that LRRK2 had a predominantly cytoplas-
mic localization. LRRK2 interacted with parkin but not
?-synuclein, DJ-1, or tau. Mutant LRRK2 dramatically decreased
cell viability in culture with either SH-SY5Y cells or primary
neurons. These findings contribute to the understanding of
LRRK2-linked PD pathogenesis.
Materials and Methods
Materials. Media, N2 and N27 supplements for cell culture, Lipo-
fectamine Plus reagent, and anti-hemagglutinin (HA) polyclonal
Ab were obtained from Invitrogen. Hoechst 33342 was obtained
from Molecular Probes. Anti-ubiquitin polyclonal Ab was obtained
from DAKO. Anti-myc and anti-HA mAbs were obtained from
Santa Cruz Biotechnology and Roche Molecular Biochemicals.
CyTM3-conjugated goat anti-mouse IgG and FITC-conjugated
goat anti-rabbit IgG were obtained from Jackson ImmunoRe-
search. Anti-FLAG Ab was obtained from Sigma. Anti-histone 2B
and anti-tubulin Abs were obtained from Upstate Biotechnology
(Lake Placid, NY).
LRRK2 cDNA Synthesis, Plasmids, and Transfection. WT and mutant
LRRK2 cDNAs were synthesized by DNA 2.0, Inc. (Menlo Park,
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: PD, Parkinson’s disease; LRRK2, leucine-rich repeat kinase 2; IP, immuno-
precipitation; Htt, huntingtin; HA, hemagglutinin; Q, polyglutamine.
†W.W.S. and Z.P. contributed equally to this work.
8-121, 600 North Wolfe Street, Baltimore, MD 21287. E-mail: firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
December 20, 2005 ?
vol. 102 ?
CA) according to the published sequence (GenBank accession no.
AY792511; ref. 15). We added three FLAG tags to the N terminus
of LRRK2 cDNA and inserted the construct into the pcDNA3.1
vector. We also generated the version of C-terminal myc-tagged
WT LRRK2 construct for coimmunoprecipitation experiments.
We constructed a series of FLAG-tagged truncated LRRK2 plas-
mids using WT LRRK2 as a template by PCR. All resulting
constructs were confirmed by sequencing. Plasmids expressing
HA-?-synuclein, HA-parkin, myc-parkin, FLAG-parkin, and full-
length huntingtin (htt) with 23 polyglutamine (Q) have been
described (26–28). Transient transfections were performed with
Lipofectamine Plus according to the manufacturer’s protocol.
Cell Cultures, Immunoprecipitation (IP), and Western Blot Analysis.
Human HEK 293T and SH-SY5Y cells were grown in the media as
described (29, 30). We synthesized two peptides to generate Abs
against two distinct regions of human LRRK2 (amino acids 1245–
1259 and 2396–2408). Cells were harvested in the lysis buffer as
protein assay to ensure equal protein loading. IP experiments from
transfected cell lysates were performed with anti-FLAG, or an-
ti-HA Ab and protein G Plus?protein A–agarose (Amersham
Pharmacia Biotech). The resulting immunoprecipitates and cell
lysates were resolved on 4–12% NuPAGE Bis-Tris gels and trans-
membranes were blocked in TBST (10 mM Tris?HCl, pH 7.4?150
mM NaCl?0.1% Tween 20) containing 5% nonfat milk and then
probed with different Abs. Proteins were detected by using en-
hanced chemiluminescence reagents (NEN).
Subcellular Fractionation of Endogenous LRRK2 in SH-SY5Y Cells.
SH-SY5Y cells were harvested in lysis buffer containing 0.25 M
sucrose, 25 mM KCl, 5 mM MgCl2, 50 mM triethanolamine, 1 mM
PMSF, 1 mM DTT, and complete protease inhibitors. Cells were
incubated on ice for 30 min, and complete lysis of the plasma
membrane was visualized by using 2? trypan blue solution con-
taining Hoechst 33342. Lysates were centrifuged at 800 ? g for 15
min to generate supernatant (cytosol) and crude pellet (containing
nuclear) fractions. The pellet was purified further by the sucrose
cushion buffer (lysis buffer with 1.8 M sucrose) and centrifugation
at 30,000 ? g for 1 h. Protein content was determined by using the
BCA (bicinchoninic acid) protein assay kit (Pierce).
Mouse Primary Cortical Neuronal Cultures and Electroporation Trans-
fection. Mouse primary cortical neuronal cultures were derived
from CD-1 outbred mice (The Jackson Laboratory) at embryonic
day 15 or 16. Cortices were dissociated as described in ref. 29. After
dissociation, neurons were plated on laminin- and poly-D-lysine-
coated plates (BD Biosource, San Diego) and cultured in neuro-
penicillin?streptomycin. Under these culture conditions, 95% of
cells were neurons. Transfection of LRRK2 constructs into mouse
primary cortical neurons was carried out by using Nucleofector
(Amaxa Biosystem, Cologne, Germany), which is an electropora-
tion-based method, as described in ref. 30.
Measurement of Cell Viability and Cell Death. SH-SY5Ycellviability
assays were conducted as described in ref. 29. Cells were cotrans-
fected with pcDNA3.1-GFP, along with vector pcDNA3.1, full-
length htt with 23Q, and pcDNA3.1-FLAG-LRRK2 constructs (at
1:15) for 24 h in 10% FBS OPTI-I media and then changed to
DMEM with N2 supplement for 24 h. Primary neurons were
cotransfected with LRRK2 constructs or full-length htt with 23Q,
cells (neurons) were counted from 40 randomly selected fields by
an investigator, who was kept unaware of the experimental condi-
tion, using fluorescence microscopy. Viable cells (neurons) were
the length of the cell body. The percentage of GFP-positive viable
cells (neurons) in each experimental group relative to those of cells
(neurons) transfected with vector and GFP was calculated.
Hoechst 33342?propidium iodide labeling of cells to detect
29. TUNEL staining was performed as described (30) by using the
Texas red in situ cell-death-detection kit (Roche Molecular Bio-
chemicals). SH-SY5Y cells were cotransfected with various con-
N2 supplement for 24 h, and then subjected anti-FLAG immuno-
chemical staining. TUNEL staining was then performed according
from 20 randomly selected fields from each experimental group by
using conventional fluorescence microscopy by an investigator who
was kept unaware of the experimental condition.
Immunocytochemistry. Cells were fixed with 4% paraformaldehyde,
permeabilized with 0.2% Triton X-100, and processed as described
(31). Cell preparations were incubated with primary Abs: anti-
FLAG, anti-myc, or anti-ubiquitin, then with secondary Abs:
Cy3-conjugated anti-mouse or FITC-conjugated anti-rabbit; nuclei
were stained with Hoechst 33342, and all signals were analyzed by
LRRK2 expression construct in pcDNA3.1 vector. The WT construct was de-
signed with or without a FLAG tag. Unique restriction sites were engineered
transiently expressing various LRRK2 constructs or empty vector were ana-
lyzed by Western blotting using anti-FLAG and anti-LRRK2-(1245–1259) Abs.
LRRK2 proteins migrated at ?280 kDa. Similar results were obtained by using
anti-LRRK2-(2396–2408) Ab (data not shown). (C) Subcellular fractionations
derived from SH-SY5Y cells were analyzed by Western blot analysis using
anti-LRRK2-(1245–1259) Abs to detect the endogenous LRRK2. (D) Represen-
tative merged confocal images of transiently transfected HEK 293T cells.
Nuclear Hoechst 33342 staining is indicated blue, and green indicates FLAG-
FITC Ab staining for LRRK2.
Cytoplasmic expression of LRRK2. (A) Schematic representation of
Smith et al. PNAS ?
December 20, 2005 ?
vol. 102 ?
no. 51 ?
Zeiss). The number of cells with cytoplasmic aggregates were
counted in 20 randomly selected fields with ?1,000 cells in each
experimental condition. Counts were done by an investigator who
was kept unaware of the experimental condition.
Data Analysis. Quantitative data are expressed as arithmetic
means ? SE based on at least three separate experiments per-
formed in duplicate. The difference between two groups was
statistically analyzed by Student’s t test or one-way ANOVA.
Significance was defined at P ? 0.05.
Cytoplasmic Expression of LRRK2. To study the expression of
LRRK2, we generated a cDNA expression vector containing the
full-length LRRK2 ORF with codons optimized for mammalian
were generated, including full-length WT LRRK2 with no tag and
constructs with N-terminal FLAG tag or C-terminal myc tag. Also,
the following three of the reported mutants were generated:
R1441C in the ROC domain, Y1699C in the COR domain, and
G2019S (the most common one) in the mitogen-activated protein
We synthesized two peptides to generate Abs against two distinct
Expression of LRRK2 in HEK 293T cells yielded a protein of
?280 kDa detected with either Abs to the FLAG tag or to LRRK2
(Fig. 1B). The WT LRRK2 protein without FLAG-tag ran at a
similar position (Fig. 1B). Cellular expression of WT or mutant
LRRK2 appeared predominately diffuse cytoplasmic (Fig. 1D),
although a minority of cells had a punctate label suggestive of
aggregates (see below). There was no apparent change in the
cytoplasmic distribution for any of the mutants in HEK 293T cells.
Subcellular fractionations derived from SH-SY5Y cells confirmed
that LRRK2 was excluded from the nucleus and present in cytosol
Interaction of LRRK2 with Parkin. Because LRRK2 showed predom-
inately cytoplasmic expression, we investigated whether it inter-
acted with other cytoplasmic PD-related gene products. By coim-
munoprecipitation assays, LRRK2 showed a specific interaction
with parkin but not with ?-synuclein (Fig. 2), DJ-1, or tau (data not
shown). Fig. 2A shows IP with FLAG-LRRK2 and detection of
either HA-?-synuclein or HA-parkin. Parkin coimmunoprecipi-
tated with LRRK2, but ?-synuclein did not. Conversely, either
?-synuclein or parkin was immunoprecipitated by using HA, fol-
lowed by anti-FLAG LRRK2 immunoblotting (Fig. 2B). LRRK2
was detected only with IP of HA-parkin. Even when the blot was
considerably overexposed, there was no detection of LRRK2 using
not alter the interaction between LRRK2 and parkin (data not
To determine which regions of parkin and LRRK2 were respon-
sible for the interaction, a series of constructs containing different
domains of parkin tagged with myc were cotransfected with full-
length LRRK2. As shown in Fig. 3, only parkin constructs con-
taining the RING2 domain showed an interaction with LRRK2.
Conversely, when different domains of FLAG-LRRK2 were co-
transfected with full-length HA-parkin, there was a strong inter-
action between the COR domain of LRRK2 and parkin (data not
prepared from cells transfected with various constructs as indicated were sub-
immunoblotting. The experiment was repeated three times with similar results.
LRRK2 interaction with parkin in HEK 293T cells. (A and B) Lysates
domain of parkin. Lysates prepared from HEK 293T cells cotransfected with
FLAG-LRRK2 and various myc-tagged parkin domain constructs were sub-
jected to IP with anti-FLAG, followed by anti-myc immunoblotting. Putative
functional domains of parkin used in the mapping experiments are shown.
Ub-H, Ubiquitin homology domain. Cell lysates were subject to Bradford
protein assay to ensure equal protein loading. Parkin fragments had variable
levels, even when we loaded the same amount of total protein, presumably
because these nonphysiological forms are not stable. On a qualitative basis,
the RING2 domain of parkin interacts most strongly with LRRK2
LRRK2 interacts preferentially with the C-terminal R2 ring-finger
www.pnas.org?cgi?doi?10.1073?pnas.0508052102Smith et al.
Increase of Cytoplasmic Aggregates Containing LRRK2 with Coexpres-
sion of Parkin. AlthoughthepredominantlocalizationofLRRK2in
transfected cells is diffusely cytoplasmic, a minority of cells show
apparent aggregates (Fig. 4A). There was no consistent change in
the percentage of cells with aggregates using the mutants (data not
shown). However, when LRRK2 was cotransfected with parkin,
there was a substantial increase in the percentage of cells with
LRRK2 was transfected with ?-synuclein or full-length htt with
Because parkin is an E3 ubiquitin ligase (27, 32–34), we sought
to determine whether the aggregates in these cells contain ubiqui-
tinated proteins. As shown in Fig. 5 B and C, there was a consid-
erable increase in the percentage of cells containing ubiquitinated
aggregates when LRRK2 was cotransfected with parkin. We also
LRRK2 labeling (Fig. 5A). We did not find evidence that LRRK2
might be directly ubiquitinated by parkin (data not shown). In
cotransfection experiments, LRRK2 caused a 25-fold increase in
the autoubiquitination activity of parkin in the context of a 3-fold
increase in parkin protein level (Fig. 5D).
Neuronal Degeneration Caused by Expression of Mutant LRRK2. To
determine whether mutant LRRK2 could alter cell viability, SH-
SY5Y cells were cotransfected with GFP and various LRRK2
constructs by Lipofectamine. Viable cells were defined as having at
least one smooth extension with twice the length of the cell body,
and they were counted by an investigator who was kept unaware of
the experimental condition. Transfection efficiencies were similar
in all groups. There was mild, nonsignificant, decreased viability
cell toxicity compared with WT LRRK2, control protein (full-
length htt with 23Q), and vector (Fig. 6 A and B). We confirmed
these results using mouse primary cortical neurons and electropo-
primary cultures compared with WT LRRK2, control protein
(full-length htt with 23Q), and vector (Fig. 6 C and D). Expression
of mutant LRRK2 in SH-SY5Y cells caused highly condensed and
fragmented nuclei as measured by Hoechst 33342?propidium io-
dide staining and significantly increased the TUNEL-positive cells
(Fig. 6 E and F), indicating that mutant LRRK2 induced primarily
apoptotic cell death. Coexpression of parkin did not significantly
protect against mutant LRRK2-induced neuronal degeneration
(data not shown).
LRRK2. (A and B) Cells were transfected with WT FLAG-LRRK2 with or without
myc-parkin for 72 h and subjected to immunocytochemical assay with anti-myc
and anti-FLAG Abs. (A) Representative images of each experimental group. (B)
Cells with cytoplasmic aggregates containing FLAG-LRRK2 labeling were
counted. Data are shown as means ? SE for three separate experiments per-
formed in duplicate.*, P ? 0.05 vs. cells transfected with FLAG-LRRK2 alone.
Expression of parkin increases cytoplasmic aggregates containing
or without myc-parkin for 72 h and subjected to immunocytochemical assay
with anti-FLAG and anti-myc (A) or anti-FLAG and anti-ubiquitin (B) Abs. (C)
Cells with cytoplasmic aggregates containing both ubiquitin and FLAG label-
ing were counted. Data are shown as means ? SE for three separate experi-
ments performed in duplicate.*, P ? 0.05 vs. cells transfected with FLAG-
LRRK2 alone. (D) LRRK2 enhances parkin autoubiquitination. Cells
a control for 48 h and then were subjected to IP with anti-FLAG Ab. Immu-
noprecipitates were analyzed by Western blotting with anti-HA Ab, and
inputs were probed with anti-FLAG or anti-actin Abs. Note that LRRK2 en-
hances the formation of high-molecular-weight parkin–ubiquitin protein
conjugates corresponding to polyubiquitinated forms of parkin. These exper-
iments were replicated three times with similar results.
Expression of parkin increases ubiquitinated cytoplasmic aggregates
Smith et al. PNAS ?
December 20, 2005 ?
vol. 102 ?
no. 51 ?
In this study, we found that LRRK2 protein was predominately
cytoplasmic. Using coimmunoprecipitation, we found that
LRRK2 interacted with parkin, but not with ?-synuclein, DJ-1,
or tau. LRRK2 interacted preferentially with the C-terminal R2
RING-finger domain of parkin, and parkin interacted with the
COR domain of LRRK2. Coexpression of LRRK2 and parkin
increased cytoplasmic protein aggregates that contain LRRK2
and enhanced the ubiquitination of these aggregates. Last,
expression of mutant LRRK2 caused neuronal degeneration in
both SH-SY5Y cells and mouse primary neurons. These findings
suggest a gain-of-function mechanism in the pathogenesis of
The cytoplasmic localization of LRRK2 protein is similar to that
of several other PD-related gene products, including ?-synuclein
although at low levels (14). The cytoplasmic localization is consis-
tent with the LRRK2 amino acid sequence, which does not contain
any predicted hydrophobic membrane spanning domains or tar-
geting sequences for other cellular organelles (15). However, we
of mitochondrial, endoplasmic reticulum, or other membranes.
?-synuclein, DJ-1, or tau protein. Parkin is an E3 ubiquitin ligase,
PD pathogenesis (11, 27, 37–39). Parkin associates with and ubiq-
uitinates synphilin-1 (27), a protein that interacts with ?-synuclein
(28) and is highly enriched in Lewy bodies (40). Also, parkin can
interact with other proteins, such as p38?JTv-1 (38).
in the RING2 domain of parkin. This site is also the site of parkin
interaction with synphilin-1 (27). When expressed in cells, LRRK2
had a tendency to aggregate, and aggregates were strikingly in-
creased when LRRK2 was coexpressed with parkin but not other
proteins. We also found that cytoplasmic aggregates were ubiqui-
tinated in a parkin-dependent fashion. Previously, we found that
parkin promotes the formation of ubiquitinated aggregates with
cotransfected ?-synuclein and synphilin-1 (31). Other studies also
found that parkin can accumulate in aggresomes under conditions
of proteasome impairment (41, 42). These observations are con-
sistent with the idea of a role for the ubiquitin proteasome pathway
in PD (43, 44). In this study, the effect of parkin on the ubiquiti-
for 24 h. GFP-positive cells with 2-fold continuous extensions were counted by using fluorescence microscopy. Shown are representative photomicrographs for each
that of cells cotransfected with empty vector and GFP. The percentage of GFP-positive viable cells in each experimental group relative to those of cells cotransfected
with vector and GFP was calculated.*, P ? 0.05 vs. cells cotransfected with WT LRRK2 and GFP. (C) Mouse primary cortical neurons were cotransfected with
pcDNA3.1-GFP along with various constructs as in A at 1:15 for 48 h by electroporation. GFP-positive viable neurons with 2-fold continuous neurites were counted by
of each experimental group normalized to that of cells cotransfected with empty vector and GFP as in B.*, P ? 0.05 vs. cells cotransfected with WT LRRK2 and GFP.
(E and F) SH-SY5Y cells were cotransfected with various constructs for 24 h in 10% FBS OPTI-MEM I media and then incubated with DMEM containing N2 supplement
of TUNEL-positive cells in total LRRK2 transfected cells.*, P ? 0.05 vs. cells transfected with WT LRRK2.
Mutant LRRK2 causes neuronal degeneration. (A) SH-SY5Y cells were cotransfected with pcDNA3.1-GFP along with either vector pcDNA3.1, full-length htt
www.pnas.org?cgi?doi?10.1073?pnas.0508052102Smith et al.
nation of aggregates may involve, in part, stimulation of the
ubiquitin ligase activity of parkin by LRRK2. Preliminary data
(data not shown) suggest LRRK2 can be ubiquitinated but not by
parkin. Thus, parkin may ubiquitinate other proteins within the
aggregates. Patients with LRRK2 mutations have pleomorphic
brain pathology, including several different kinds of protein aggre-
gates, suggesting that our findings in cell culture may be relevant to
LRRK2-linked pathogenesis. It is not yet known whether LRRK2
protein is contained in these aggregates in PD patients. A caveat to
this study is that most of the data were obtained by using overex-
pression of exogenous proteins and would need to be extended to
endogenous proteins when reagents are available. LRRK2 appears
to be expressed at relatively low levels in brain, and these studies
specific to detect such low levels of protein.
viability in both SH-SY5Y cells and primary neurons and caused
apoptotic cell death in SH-SY5Y cells. In previous experiments
using comparable systems, we have shown highly specific cell
kind of transient transfection cell model can give specific disease-
related results (26, 30). WT LRRK2 also caused some mild
in toxicity under some circumstances, particularly when combined
with exogenous cellular stressors, raising the possibility that these
effects could be relevant for sporadic PD as well. We did not find
any strong evidence for parkin to protect against LRRK2 toxicity,
consistent with recent studies suggesting that the role of parkin in
the formation of Lewy bodies and cellular protection may be
independent (36, 45, 46).
PD is similar to other neurodegenerative diseases with both
protein aggregation and neuronal cell death, although the relation
study of ?-synuclein has provided insight into both genetic and
potential to identify the contribution of LRRK2 to neuronal cell
death and aggregate formation, and to the molecular pathogenesis
of both familial and sporadic PD, which may suggest approaches to
therapeutic development. For PD, like Alzheimer’s disease, the
identification of rare genetic forms has led to the identification of
critical gene products that are likely to be involved in the patho-
genesis of sporadic disease. Protein interactions are believed to be
central to Alzheimer’s disease pathogenesis. For example, the
presenilin gene product has a key role in the proteolysis of amyloid
precursor protein (APP), yielding the toxic amyloid-? peptide
Our results indicate that LRRK2 is a cytoplasmic protein and
that mutant LRRK2 can be directly toxic to cells, although these
observations would need to be extended to endogenous protein in
useful for studies of PD pathogenesis and, possibly, therapeutics.
LRRK2 interacted with parkin, and parkin can potentiate the
formation of aggregates containing LRRK2 and ubiquitin. These
findings may be relevant to the formation of Lewy bodies or other
protein aggregates in PD and related disorders. Identification of
into the molecular pathway of PD pathogenesis and the targets for
We thank Dr. Akira Sawa for helpful discussions and Brad Harris for
technical help. This work was supported by National Institute of Neu-
rological Disorders and Stroke Grants NS38377, NS054817, and 16375;
the Udall PD Research Center; the National Institutes of Health; the
National Parkinson’s Foundation; and the American Parkinson’s Dis-
ease Association. T.M.D. is the Leonard and Madlyn Abramson Pro-
fessor in Neurodegenerative Diseases.
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