Neuroscience Letters 410 (2006) 80–84

Digenic parkinsonism: Investigation of the synergistic effects

of PRKN and LRRK2
Justus C. Dächsel a , Ignacio F. Mata a,b , Owen A. Ross a,∗ , Julie P. Taylor a ,
Sarah J. Lincoln a , Kelly M. Hinkle a , Cecilia Huerta b , Renee Ribacoba c ,
Marta Blazquez d , Victoria Alvarez b , Matthew J. Farrer a
a Department of Neuroscience, Mayo Clinic College of Medicine, Jacksonville, Florida, USA
b Laboratorio de Genetica Molecular, Instituto de Investigacion Nefrologica (IRSINFRIAT),
Hospital Universitario Central de Asturias, Oviedo, Spain
c Servicio de Neurologı́a, Hospital Alvarez-Buylla, Mieres, Spain
d Servicio de Neurologı́a, Hospital Universitario Central de Asturias, Oviedo, Spain

Received 5 May 2006; received in revised form 14 June 2006; accepted 30 June 2006

Abstract
The complex genetic etiology of Parkinson’s disease (PD) is indicative of a multifactorial syndrome. A combination of gene–gene and
gene–environment interactions may determine a variable phenotypic outcome. Recently a direct gene/protein interaction between two of the
most common genetic causes of parkinsonism PRKN and LRRK2 has been postulated. We have identified three Spanish patients simultaneously
harboring mutations in PRKN and LRRK2. In comparison to other Spanish patients with a single LRRK2 or PRKN mutation, the three double-
mutation patients reported here do not present with an earlier age-at-onset or a faster progression of disease. Although the clinical findings do
not support a synergistic effect of LRRK2 and PRKN, a potential genetic interplay might be concealed by the modulating effects of other genes.
Nevertheless, this work demonstrates that the presence of mutations in one familial gene should not serve as exclusion criteria in a screen for further
genetic variation. Direct interaction of Lrrk2 and parkin proteins was not observed in co-immunoprecipitation pull down experiments. However,
in vivo studies are required to assess whether there is an indirect link between Lrrk2 and parkin in disease pathogenesis.
© 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Parkin; LRRK2; Gene; Protein interaction

Parkinsonism is a clinicopathological and genetically heteroge-
neous syndrome [17]. This phenomenon is clearly demonstrated
within the leucine-rich repeat kinase 2 (LRRK2) families that
present with autosomal dominant forms of parkinsonism caused
by single point mutations. These kindreds display remarkable
diversity in both clinical and pathological aspects of the dis-
ease [9,20]. This is indicative of a complex neurological disease
with gene–environment and gene–gene interactions. Over the
last decade mutations in six genes have been identified that
cause familial forms of parkinsonism [2]. Genetic variations of
the SNCA, MAPT and LRRK2 genes are reported to cause auto-
somal dominant forms of parkinsonism whereas mutations of
PRKN, DJ-1 and PINK1 genes cause recessive forms.
∗ Corresponding author. Tel.: +1 904 953 7135; fax: +1 904 953 7370.
E-mail address: ross.owen@mayo.edu (O.A. Ross).
While a number of integrated systems have been proposed
to explain the interactions between the protein products of these
loci, little data is available on their joint effects. The ␣-synuclein
and tau proteins synergistically promote and propagate each
other’s polymerization into fibrils in vivo, and tau may seed
the fibrilization of ␣-synuclein [5]. Common variability in the
genes encoding ␣-synuclein and tau, independently and com-
bined, may modulate the risk of developing Parkinson’s disease
(PD) as reported by Mamah et al. [12].
Recently individuals with both LRRK2 and PRKN mutations
have been reported [10,16]. In addition, based on their findings
in a cell culture model, Smith et al. postulated that the Lrrk2 and
parkin proteins interact directly [18]. Herein we investigate the
potential epistatic interaction of these two loci and examine the
evidence for a direct protein–protein interaction.
The study involved a total of 351 patients with idiopathic PD
(mean onset 63.2 ± 14.6 years, 25.9% with a family history of

0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2006.06.068
 J.C. Dächsel et al. / Neuroscience Letters 410 (2006) 80–84 81

Table 1 Co-immunoprecipitation assays were performed to study the

Frequency of LRRK2 or PRKN mutation carriers in the Asturias region of North- potential direct interaction between parkin and Lrrk2. HEK293T
ern Spain
cells were transiently transfected with pcDNA3 vectors encod-
Number of Mean onset S.D. Range Family ing the respective protein using Lipofectamine 2000 as per man-
carriers (years) history (%) ufacturer’s instructions. Two days after transfection cells were
M192V 9 (2.6%) 54 7 44–60 11 harvested and lysed (invitrogen) in 50 mM Tris/HCl, 150 mM
N52/STOP80 4 (1.2%) 50 13 36–60 60 NaCl and 0.1% Triton-X-100. Processed lysates containing
G2019S 9 (2.6%) 61 9 50–77 33 equal amounts of protein were incubated for 4 h at 4 ◦ C with
R1441G 10 (2.8%) 57 13 36–76 70
either V5- or MYC-antibodies preconjugated agarose (SIGMA)
and subsequently washed three times with lysis buffer. The
disease) from the Asturias region of Northern Spain. Patients resulting immunoprecipitate was subjected to SDS-PAGE sep-
were referred by neurologists specializing in movement disor- aration and Western blot analysis using a standard protocol.
ders from three main hospitals (Hospital Universitario Cental de Immunolabeling with the respective primary and secondary anti-
Asturias, Oviedo; Hospital Alvarez-Buylla, Mieresi; and Hospi- bodies was performed.
tal de Cabueñes, Gijón) within the Asturias region. All patients The frequency of Lrrk2 G2019S and R1441G was estimated
fulfilled criteria for a clinical diagnosis of PD with at least two to be ∼2.7% in sporadic PD for both substitutions in this Spanish
of three cardinal signs, tremor, rigidity and bradykinesia, and sample (Table 1) [14,15]. Of 19 LRRK2 carriers, three patients
with a positive response to L-DOPA therapy when administered (15.8% total, 3/19) were found to harbor PRKN point mutations.
[4]. Patients and controls gave their consent to participate in this PRKN dosage has not been examined so this is a minimum fre-
study, which was approved by the Ethical committees of all three quency of double mutation carriers in this sample. Two individ-
hospitals. uals carried the Lrrk2 R1441G substitution, with either parkin
Of the 351 patients, 276 (78.6%) presented with typical late- M192V or N52/STOP80, and one patient carried Lrrk2 G2019S
onset PD (mean onset 69.8 ± 17.6 years, range 51–89). Sixty and parkin M192V. The location and effect on kinase activity
of the late-onset patients (21.7%) reported a family history of of these two Lrrk2 substitutions has previously been described
parkinsonism (defined herein as having one or more affected 1st [6,19].
degree relatives). The remaining 75 patients (21.4%) presented The parkin M192V amino acid substitution is located in the
with early-onset parkinsonism (≤45 years at onset) although so-called “central” or “unique” domain. While mutations in
their symptoms were otherwise consistent with a clinical diagno- this “central” domain are unlikely to affect E3 ligase activity,
sis of PD (mean onset 41.9 ± 14.8 years, range 24–45). A family resulting substitutions such as M192V may potentially inter-
history of parkinsonism was noted in 31 early-onset patients rupt this region’s interaction with p38 [1]. The N52/STOP80
(41.3%). parkin substitution leads to the generation of a premature stop
DNA was extracted from blood using standard protocols and codon at amino acid position 80. No enzymatic activity can be
genotyped for LRRK2 mutations (4321C > G {R1441G} and expected from the resultant truncated protein. A compound het-
6055G > A {G2019S}) using ABI Taqman chemistry (Applied erozygous patient with Lrrk2 R1441G/parkin N52/STOP80 may
Biosystems, Foster City, CA) or restriction enzyme digest as be expected to manifest earlier or more profound symptoms
previously described [14,15]. Samples were screened for the than compound heterozygotes with Lrrk2 R1441G/G2019S and
presence of PRKN point mutations using single-strand confor- parkin M192V amino acid substitutions.
mational polymorphism (SSCP) analysis. Nucleotide changes To look for a possible synergic effect between LRRK2 and
were subsequently confirmed by direct DNA sequencing using PRKN, clinical information was compared between compound
BigDye chemistry on an ABI 3100. Genomic rearrangements of heterozygotes, carriers with a single PRKN or LRRK2 muta-
the PRKN or LRRK2 loci were not examined. tion, and to patients overall (Tables 1 and 2). Interestingly

Table 2
Clinical details of LRRK2 and PRKN carriers
PE040 PE055 PJ089

Gender Male Male Female

Age of onset (years) 57 56 61
Duration (years) 17 12 11
Hoehn-Yahr 2.5 2 2
Months on L-DOPA 168 132 132
Response to L-DOPA ++ ++ +
RT + + −
R + + +
B + + +
Other features Hallucinations − −
Treatment L-DOPA, APM and Ropirinole Pramipexol, propanolol and L-DOPA/Entacapone Sinemet
Genetics Lrrk2 R1441G, parkin M192V Lrrk2 G2019S, parkin M192V Lrrk2 R1441G, parkin N52/STOP80
Family history No No Yes
82 J.C. Dächsel et al. / Neuroscience Letters 410 (2006) 80–84

all three compound heterozygotes presented with an age-at-
onset of approximately 60 years. Only one subject reported
a family history of parkinsonism (PJ089; Lrrk2 R1441G and
parkin N52/STOP80). This patient has a late age-at-onset
(61 years; Table 2) and her daughter, presently in her mid
50s and with only PRKN N52/STOP80, has no symptoms of
disease.
Complementary to our genetic analysis we performed a series
of experiments employing co-immunoprecipitation assays of
Lrrk2 wild-type and mutants. As a positive control, we measured

Fig. 1. Lrrk2 protein does not directly interact with parkin: HEK293T cells were transiently transfected with the vectors indicated followed by immunoprecipitation
with V5 antibodies. (A) Dimerization of Lrrk2: immunolabeling using GFP antibodies (Abcam) shows co-precipitation of Lrrk2-GFP but not GFP alone with
Lrrk2-V5 (invitrogen). (B) Neither wild type Lrrk2 nor Lrrk2-mutants interact with parkin: immunolabeling with parkin antibodies (Cell signaling) fails to detect
MYC-parkin in the V5 precipitate. Lack of interaction between MYC-parkin and lacZ-V5 was used as a negative control. Successful V5 pull down was monitored
by using V5 antibodies (invitrogen).
 J.C. Dächsel et al. / Neuroscience Letters 410 (2006) 80–84
the capacity of Lrrk2 to form dimers or potentially oligomeric
species [6]. While we could confirm Lrrk2 dimerization, a direct
interaction between Lrrk2 and parkin could not be shown. Fur-
thermore, parkin was not detected in the immunoprecipitate of
several Lrrk2-V5 proteins harboring pathogenic amino acid sub-
stitutions. Immunoprecipitation assays were carried out in both
directions using either V5 or MYC (data not shown) antibodies
for the pull down (Fig. 1).
We report clinical and genetic findings in three heterozygous
PRKN and LRRK2 mutation carriers with parkinsonism. Com-
pared to individuals carrying a single LRRK2 or PRKN mutation,
no significant variations regarding the age-at-onset or severity of
disease were observed. A limitation of our study is the number of
LRRK2-PRKN patients identified (n = 3). Three compound het-
erozygous subjects with PRKN-LRRK2 have previously been
reported. Paisan-Ruiz et al. identified a healthy 52-year old car-
rying mutations in PRKN, LRRK2 and GBA genes, while Lesage
et al. identified two patients carrying mutations in both PRKN
and LRRK2 genes [10,16]. One individual harbored a homozy-
gous PRKN exon 2 triplication and the Lrrk2 G2019S mutation;
an additive effect was postulated based on the early age-at-onset
(31 years). However, 31 years as age-at-onset is not uncommon
for carriers of a homozygous PRKN mutation.
The impact of heterozygous PRKN mutations on the devel-
opment of disease remains controversial as the frequency is
relatively high in healthy controls (∼3%) [11]. The frequency of
heterozygous PRKN mutations in this study was 3.8% suggest-
ing these variants may not influence PD susceptibility. However,
studies are now required to assess the affect of each individual
PRKN variant in the heterozygous state on disease onset and
phenotypic presentation. Interestingly, homozygous and com-
pound heterozygous PRKN mutations are the major cause of
early-onset parkinsonism (onset <45 years) but also have been
found in late-onset PD [3,13]. Similarly, while LRRK2 muta-
tions are typically associated with late onset PD they have also
been noted in early-onset parkinsonism [7]. Of note, homozy-
gous LRRK2 carriers do not have an earlier onset or more severe
progression than heterozygous carriers [8].
Co-immunoprecipitation experiments did not demonstrate a
direct interaction between Lrrk2 and parkin proteins. We did not
confirm the results of Smith et al. despite using the same cell
line [18], however, transgene expression level and the respective
cellular response to exogenous protein are unpredictable. Tran-
sient expression of exogenous proteins in cultured cells does
not always accurately reflect a relevant, physiological interac-
tion with positive findings requiring in vivo confirmation. It may
be a scenario whereby modifying genes/proteins may conceal a
interaction even if their interplay is not direct, both are clearly
involved in dopaminergic dysfunction.
The clinical heterogeneity of parkinsonism is probably deter-
mined by the joint effect of genetic background, gene–gene and
gene–environment interactions. Additional pathogenic, suscep-
tibility, and modifier disease genes remain to be identified. It is
clear from this study that the presence of a mutation in one gene
should not be an exclusion criteria for further genetic screening.
To fully resolve the importance of gene–gene interactions in
disease a comprehensive screening of all known parkinsonism-
83
related, loci is now required to identify those individuals who
harbor compound mutations in different genes.
Acknowledgements
We are grateful to the following organizations for their sup-
port of this work: Mayo Clinic Jacksonville a M.K. Udall
Parkinson’s Disease Research Centre of Excellence (NINDS
P50 NS40256), NIA (P01 AG17216) Spanish Fondo de Investi-
gaciones Sanitarias grant (PI020022) and Asociacion Parkinson
Asturias. We thank Minnie Schreiber for technical assistance.
We would also like to thank the donors and their families with-
out whom this work would not have been possible.
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