RAB7L1 Interacts with LRRK2
to Modify Intraneuronal Protein Sorting
and Parkinson’s Disease Risk
David A. MacLeod,1,2,4Herve Rhinn,1,2,4Tomoki Kuwahara,1,2,4Ari Zolin,1,2Gilbert Di Paolo,1,2Brian D. McCabe,1,2
Karen S. Marder,1,3Lawrence S. Honig,1,3Lorraine N. Clark,1,2Scott A. Small,1,2and Asa Abeliovich1,2,*
1Department of Neurology and Taub Institute
2Departments of Pathology and Cell Biology
3Gertrude H. Sergievsky Center
Columbia University, Black Building 1208, 650 West 168th Street, New York, NY 10032, USA
4These authors contributed equally to this work
Recent genome-wide association studies have
kinson’s disease (PD) risk. Here we show that the
consequences of variants at 2 such loci, PARK16
and LRRK2, are highly interrelated, both in terms of
their broad impacts on human brain transcriptomes
of unaffected carriers, and in terms of their associa-
tions with PD risk. Deficiency of the PARK16 locus
gene RAB7L1 in primary rodent neurons, or of
a RAB7L1 ortholog in Drosophila dopamine neurons,
recapitulated degeneration observed with expres-
sion of a familial PD mutant form of LRRK2, whereas
RAB7L1 overexpression rescued the LRRK2 mutant
phenotypes. PD-associated defects in RAB7L1 or
LRRK2 led to endolysosomal and Golgi apparatus
sorting defects and deficiency of the VPS35 compo-
nent of the retromer complex. Expression of wild-
type VPS35, but not a familial PD-associated mutant
form, rescued these defects. Taken together, these
studies implicate retromer and lysosomal pathway
alterations in PD risk.
Parkinson’s disease (PD) is a common neurodegenerative
disorder of aging, characterized by slowed movements and
a distinctive tremor at rest (Lang and Lozano, 1998). Defining
pathological features of the disease include neurodegeneration
that is most prominent among midbrain dopamine neurons
(DNs) in the substantia nigra (SN) and Lewy body protein aggre-
gates that are composed in part of alpha-synuclein (a-syn)
protein. As the course of PD is thought to last decades, and as
at the time of autopsy the vast majority of DNs are long lost,
the molecular pursuit of initial etiological events has proven
In rare inherited familial forms of PD, specific causative muta-
tions have been identified, and this has significantly advanced
the field (Abeliovich and Flint Beal, 2006; Hardy et al., 2006).
For instance, autosomal dominantly inherited mutations in
a-syn, including missense mutations and triplication of the
locus, lead to a familial PD variant, implicating a-syn directly
in the disease process. Another familial genetic cause of PD is
the presence of autosomal dominantly inherited mutations in
the leucine-rich repeat kinase-2 (LRRK2) protein, which encodes
a large multidomain protein with GTPase and kinase activities.
Although the precise functions of a-syn and LRRK2 in neurons
remain to be determined, both proteins have been broadly impli-
cated in intraneuronal protein sorting. a-Syn mutations have
been reported to modify synaptic vesicle kinetics (Abeliovich
et al., 2000) as well as trafficking to the Golgi apparatus in a
variety of model systems (Cooper et al., 2006; Thayanidhi
et al., 2010), whereas LRRK2 mutations are implicated in defec-
tive lysosomal protein degradation and macroautophagy, which
is a cellular process that delivers cytosolic proteins and protein
aggregates to the lysosome (Dodson et al., 2012; Heo et al.,
2010; MacLeod et al., 2006), and Golgi apparatus integrity (Stafa
familial PD mutations in VPS35 (Vilarin ˜o-Gu ¨ell et al., 2011; Zim-
prich et al., 2011), which encodes a component of the retromer
complex that guides protein sorting from the endosome-lyso-
some degradation pathway retrogradely to the Golgi apparatus
(Bonifacino and Hurley, 2008; Skinner and Seaman, 2009;
Seaman et al., 1998), suggests that defective protein sorting in
vesicular compartments may play a role in PD.
Several genome-wide association studies (GWAS) have
described common genetic variants (at single nucleotide poly-
morphisms [SNPs]) that modify PD risk in nonfamilial ‘‘sporadic’’
cases (Hamza et al., 2010; Simo ´n-Sa ´nchez et al., 2009; Lill et al.,
2012). Strikingly, a subset of these common variants lie within
genomic loci previously associated with familial disease, such
as a-syn or LRRK2, supporting the notion that common patho-
genic pathways underlie familial and sporadic forms of PD.
However, mechanisms that underlie the impact of nonfamilial
genetic loci on PD risk, or that relate the functions of such loci
to familial PD genes, remain unclear.
Neuron 77, 425–439, February 6, 2013 ª2013 Elsevier Inc. 425
Figure 1. LRRK2 and PARK16 PD Risk-Associated Variants Function in a Common Genetic Pathway
(A) PD risk-associated variants exert functional effects in the CNS of unaffected individuals that is assessed in terms of a global transcriptome impact. Similar to
the one observed in PD-affected brain, it may reflect a predisease prodromal state.
gene (red star in middle panel). This secondarily impacts the brain transcriptome (lower panels), with significant overlap for different PD-risk genotype shows.
(C) Hierarchical clustering dendrogram shows that the gene expression signatures across seven PD-associated variant GPIs (‘‘Risk GPI’’; in unaffected cerebral
cortex Brodmann area 9 [BA9]) are most similar to the signatures seen in PD brain (BA9 or substantia nigra [SN] in red) rather than in other CNS diseases such as
Alzheimer’s disease, Huntington’s disease, bipolar disorder, or schizophrenia. A total of 352 gene transcript expression patterns—corresponding to the inter-
section of the PD risk variants GPIs (Figures S1A–S1C)—were interrogated. Clustering was performed using Pearson’s distance with complete linkage (see
(D) Genetic interaction between PARK16 and LRRK2 alleles revealed by GPI analysis in 185 unaffected brain samples (GEO GSE15222 ‘‘Initial’’) and in an
independentcohortof143unaffectedbrain samples(GEOGSE15745, ‘‘Replication’’),asestablished bytheinteractionfactorbetween pairsofGPIsasindicated,
(legend continued on next page)
RAB7L1/LRRK2/VPS35 Pathway in Parkinson’s Disease
426 Neuron 77, 425–439, February 6, 2013 ª2013 Elsevier Inc.
Here we describe a series of human brain transcriptome,
human genetic, and cell biological studies, that together impli-
cate a PD-associated genetic and cellular pathway. RAB7L1—
one of five genes within the PARK16 nonfamilial PD risk-associ-
PD risk in the human population; this genetic interaction is
apparent even in unaffected individuals who carry both risk
alleles, as quantified in terms of a broad transcriptomic analysis
of brain gene expression. Similarly, these genes together modify
neuronal survival and neurite integrity in model systems. At
a cellular level, defects in this PD-associated RAB7L1-LRRK2
pathway lead to abnormal lysosomal structures and defective
retromer complex function, that normally links the endolysoso-
mal protein degradation system with the Golgi apparatus (Boni-
facino and Hurley, 2008; Skinner and Seaman, 2009; Seaman
et al., 1998). Consistent with a role for such cellular defects in
VPS35, have recently been associated with rare forms of auto-
somal dominantly inherited familial PD (Vilarin ˜o-Gu ¨ell et al.,
2011; Zimprich et al., 2011).
LRRK2 and PARK16 PD Risk Variants Impart a Common
Brain Transcriptome Impact
We sought an unbiased and systematic approach to assess the
phenotypic impacts of commongenetic variantsassociated with
PD risk, particularly in brain tissue from yet unaffected carriers
(Figure 1A), in order to circumvent the limitations of the analysis
of diseased patient autopsy tissue. To this end, we compared
the transcriptome-wide gene expression profiles of brain
tissue samples from cohorts of unaffected individuals who share
either a risk or a protective allele at any given PD risk SNP (Fig-
ure 1B). Such a global phenotypic impact (GPI) quantifies the
effect of disease risk variants onto the transcriptome-wide
gene expression profile in brain. A key aspect of the GPI analysis
herein is that we focus on tissue from unaffected individuals, in
hope of avoiding secondary effects of disease pathology such
as cell loss.
We assessed the transcriptome-wide GPI at 7 PD-associated
loci (SNCA, LRRK2, MAPT, HLA-DRA, PARK16, LAMP3, STK39;
TableS1availableonline)(Simo ´n-Sa ´nchezetal.,2009)inapubli-
cally available gene expression data set from cerebral cortex
autopsy brain tissue of 185 individuals not apparently affected
by a neurodegenerative disease (Gene Expression Omnibus
[GEO] data set GSE15222). The GPIs of the seven loci revealed
a high degree of overlap in terms of the identity of transcripts
altered in expression level and the valence of such alterations:
genes were coordinately altered in their expression by each of
the seven PD-associated loci (over 15-fold greater than ex-
pected by chance; p = 1.5E-5 by resampling statistics; Figures
S1A and S1B; Table S2). This observation of an overlapping
GPIforthesesevenPD-associated lociwas moreoverconfirmed
in an additional independent data set of cerebral frontal cortex
autopsybrain tissueof143individuals (p=1.6E-3byresampling
statistics; derived from GEO data set GSE15745).
Function annotation was performed on the gene expression
changes that underlie the common GPIs among PD risk variants.
Strikingly, among the annotated gene sets most significantly
reduced in expression are ‘‘mitochondria’’ functions (Figures
S1C and S1D), consistent with the well-described association
of defects in mitochondria with PD pathology (Zheng et al.,
2010). Furthermore, the common overlapping transcriptomic
signature of gene expression changes associated with these
scriptome changes observed in the context of PD patient brain
tissue (relative to unaffected brain tissue; Figure S1C), rather
than to other CNS disorders such as Alzheimer’s disease or
schizophrenia (Figure 1C).
LRRK2 and PARK16 Variants Cooperatively Determine
Among the seven analyzed PD risk locus GPIs, those at the
PARK16 and LRRK2 loci were found to be the most similar.
Furthermore, variants at these two loci impacted the transcrip-
tome in a nonadditive manner, signifying a genetic interaction
(as determined by analysis of carriers of both risk variants; Fig-
ure 1D). We thus investigated whether these loci similarly genet-
harboring either a risk (or protective) allele at one of these loci
would modify the association of the second locus with PD risk.
In an initial study on an Ashkenazi Jewish (AJ) population, the
effect of a risk-associated variant at the LRRK2 locus was in
fact highly dependent on the presence of the risk variant at the
PARK16 locus, and vice versa (Figure 1E). Such ‘‘epistasis’’
between the LRRK2 and PARK16 loci regarding PD risk was
replicated by reanalysis of three other independent GWAS,
strongly supporting a common mechanism of action (Figure 1E).
Although to our knowledge, prior studies have not reported
genetic interactions with the common sporadic PD risk-associ-
ated variants at the LRRK2 locus, a GWAS of patients who
harbor rare familial LRRK2 mutations identified a broad 15 Mb
region of Chromosome 1 as harboring a genetic modifier of
age of PD onset (Latourelle et al., 2011). We note that this region
dent sporadic PD GWAS data sets (as above) of the 74 identified
in a linear regression model (see Experimental Procedures). The p value (‘‘p’’) associated with the interaction term as well as its orientation (‘‘Dir.’’) are presented.
Results combined across both cohorts presented (‘‘Combined’’) with the resulting Z-scores and p values for interaction.
genotype in four independent GWAS cohorts: one of Ashkenazi Jews (AJ, n = 417) and three of Caucasians (NGRC, n = 4008; NINDS, n = 537; MAYO, n = 886).
(F) Manhattan plot of the Chr1 region reported as a modifier of age of disease onset in familial PD with LRRK2 mutation (Latourelle et al., 2011). Epistasis was
evaluated for 74 SNPs in four independent sporadic PD GWAS data sets. The x axis represents chromosomal location, y axis represents ?log10 of the combined
p value for epistasis of each SNP with the PD risk SNP rs11176052 at the LRRK2 locus. The PARK16 locus PD-associated SNP rs823114 (arrow) exhibited the
most significant association (p = 4.6 E-6; red line represents the significance threshold after correction for multiple testing).
See also Figure S1 and Tables S1, S2, and S3.
RAB7L1/LRRK2/VPS35 Pathway in Parkinson’s Disease
Neuron 77, 425–439, February 6, 2013 ª2013 Elsevier Inc. 427
complementary to exon1 of RAB7L1) and CTTCAGGGTCAGCTTGCCGTAG
(Rev., complementary to GFP CDS) and Accuprime high-fidelity polymerase
(Invitrogen) following the manufacturer’s instructions with a hybridization at
55?C and an elongation step of 1 min.
Supplemental Information includes six figures, six tables, and Supplemental
Experimental Procedures and can be found with this article online at http://
We thank the New York Brain Bank and Jean Paul Vonsattel for banked tissue
samples. We are grateful to all the contributors who made their data publicly
available at the Gene Expression Omnibus. We thank Oliver Hobert for review-
ing the manuscript. The work was supported by grants from the Michael J. Fox
Foundation (A.A.), the NIH (NS064433 and NS060876 to A.A.; NS060113 to
L.N.C.; NS063660 and UL1 TR000040 to K.S.M.; AG025161 to S.A.S.;
AG08702-21toB.D.M.;and P50AG08702toL.S.H.),the Daiichi-SankyoFoun-
dation and the Japan Society for the Promotion of Science (T.K.), and the Par-
kinson’s Disease Foundation (L. N. C. and K.S.M.). A.A. designed studies and
interpreted data. H.R. designed and performed the human genetics analyses,
and performed the bioinformatics and the splicing experiments. D.A.M. per-
formed the Drosophila and mouse experiments. D.A.M., T. K., and A.Z per-
formed the cell culture experiments. B.D.M. helped designing and interpreting
the Drosophila experiments,L.N.C. the human genetics, G.D.P. and S.A.S. the
retromer and trafficking experiments. A.A. and H.R wrote the manuscript.
Accepted: November 30, 2012
Published: February 6, 2013
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