Journal of Parkinson’s Disease 7 (2017) 219–233
DOI 10.3233/JPD-161020
IOS Press
219
Review
VPS35, the Retromer Complex
and Parkinson’s Disease
Erin T. Williams
a,b
, Xi Chen
a
and Darren J. Moore
a,∗
a
Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, MI, USA
b
Van Andel Institute Graduate School, Van Andel Research Institute, Grand Rapids, MI, USA
Accepted 13 January 2017
Abstract. Mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene encoding a core component of the retromer
complex, have recently emerged as a new cause of late-onset, autosomal dominant familial Parkinson’s disease (PD). A single
missense mutation, AspD620Asn (D620N), has so far been unambiguously identified to cause PD in multiple individuals and
families worldwide. The exact molecular mechanism(s) by which VPS35 mutations induce progressive neurodegeneration
in PD are not yet known. Understanding these mechanisms, as well as the perturbed cellular pathways downstream of
mutant VPS35, is important for the development of appropriate therapeutic strategies. In this review, we focus on the
current knowledge surrounding VPS35 and its role in PD. We provide a critical discussion of the emerging data regarding
the mechanisms underlying mutant VPS35-mediated neurodegeneration gleaned from genetic cell and animal models and
highlight recent advances that may provide insight into the interplay between VPS35 and several other PD-linked gene
products (i.e. ␣-synuclein, LRRK2 and parkin) in PD. Present data support a role for perturbed VPS35 and retromer function
in the pathogenesis of PD.
Keywords: VPS35, retromer, Parkinson’s disease (PD), endosomal sorting, mitochondria, autophagy, lysosome, ␣-synuclein,
LRRK2, parkin
INTRODUCTION
Parkinson’s disease (PD), a common progressive
neurodegenerative movement disorder, belongs to the
family of synucleinopathies that are characterized
by the accumulation of aggregated ␣-synuclein pro-
tein. PD affects approximately 1.8% of individuals
over the age of 65 years, increasing to ∼5% over 85
years [1, 2]. PD is predominantly an idiopathic dis-
ease with the largest risk factor simply being age,
however, up to 10% of cases occur in a familial
manner with both autosomal dominant and recessive
transmission [3]. PD is clinically characterized by
the development of the cardinal motor symptoms,
∗
Correspondence to: Darren J. Moore, Ph.D., Center for Neu-
rodegenerative Science, Van Andel Research Institute, 333
Bostwick Ave NE, Grand Rapids, MI 49503, USA. Tel.: +1 616
234 5346; E-mail: Darren.Moore@vai.org.
bradykinesia, resting tremor, rigidity and postural
instability, owing to the relatively selective degener-
ation of nigrostriatal pathway dopaminergic neurons
[1, 2]. Neuropathologically, PD is typically character-
ized by dopaminergic neuronal loss in the substantia
nigra pars compacta (resulting in reduced dopamine
levels in the caudate-putamen) accompanied by reac-
tive gliosis, and by the presence of intracytoplasmic,
eosinophilic inclusions termed Lewy bodies in sur-
viving brainstem neurons, a major component of
which is fibrillar forms of ␣-synuclein [1, 2].
The etiology of PD remains obscure. In the past
two decades, our understanding of the mechanisms
underlying the pathogenesis of PD has undergone a
remarkable transformation, mainly due to the iden-
tification of distinct genetic loci at which mutations
are linked to disease [3]. Familial PD is caused by
mutations that are inherited in an autosomal dominant
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220 E.T. Williams et al. / VPS35, the Retromer Complex and Parkinson’s Disease
(SNCA [4], LRRK2 [5, 6], VPS35 [7, 8], RAB39B
[9], TMEM230 [10]) or autosomal recessive (Parkin
[11], PINK1 [12], DJ-1 [13], ATP13A2 [14], PLA2G6
[15], FBX07 [16], DNAJC6 [17], SYNJ1 [18, 19])
manner. In addition to being implicated in the devel-
opment of inherited PD, common genetic variation
at the SNCA and LRRK2 loci confer risk for devel-
oping idiopathic PD [3, 20]. Furthermore, mutations
in the lysosomal gene product, glucocerebrosidase
(GBA), represent a major risk factor for PD [3].
These gene products and their disease-associated
mutations have been shown to regulate several cel-
lular pathways, including mitochondrial turnover,
synaptic vesicle exocytosis/endocytosis, endosomal
sorting, autophagy and lysosomal function [21–23].
Accordingly, a common theme has emerged that
involves perturbations in organelle/vesicle traffick-
ing, recycling and turnover that may be central to the
pathophysiology of PD.
IDENTIFICATION OF VPS35 MUTATIONS
IN PD
In 2008, Wider and colleagues initially reported
a Swiss family with late-onset, autosomal domi-
nant PD with a mean age of disease onset of 51
years. The clinical phenotype was slowly progres-
sive, tremor-predominant and levodopa-responsive
parkinsonism [24]. In 2011, Vilari
˜
no-G
¨
uell and
coworkers applied next-generation sequencing tech-
nology to this Swiss PD family. Utilizing exome
sequencing an aspartic acid to asparagine mutation
at residue 620 (D620N) (p.Asp620Asn, c.1858G>A)
was identified in the vacuolar protein sorting 35
ortholog (VPS35, PARK17, OMIM 614203) gene in
all six affected family members who were avail-
able for genetic testing [7]. The D620N mutation
was also identified in PD families from the United
States, Tunisia and Israel (Yemenite Jews) and in one
idiopathic PD subject of Yemenite Jewish origin [7].
An independent study identified three Austrian fam-
ilies harboring the D620N mutation in VPS35 that
presented with levodopa-responsive PD occasionally
accompanied by action tremor [8].
Following the initial reports of VPS35 mutations,
other groups have been able to identify the D620N
mutation in a number of individuals and families with
PD worldwide (Table 1) [25–31]. At this time, only
the VPS35 D620N mutation has been confirmed as
pathogenic. The VPS35 D620N mutation has been
identified predominantly in families of Caucasian
descent with autosomal dominant PD. In contrast,
VPS35 mutations are rare in Asian populations with
the exception of Japanese populations [32]. The fre-
quency of the VPS35 D620N mutation in patients
with familial PD is estimated to be 0.1 to 1% [25].
Analyses of the entire VPS35 gene sequence has
also revealed a proline to serine substitution at amino
acid 316 (P316S) in two affected siblings with
PD from a US family (Table 1) [7]. However, the
pathogenicity of the P316S variant is uncertain since
it was also found in an unaffected sibling in the same
Table 1
Summary of the distribution and frequency of VPS35 variants linked to Parkinson’s disease
Mutation Region where mutation was present Frequency in PD cohorts Found in controls? References
D620N Switzerland 24/14126 No [7, 8, 26–28, 86]
Austria
United States
Tunisia
Yemenite Jews
United Kingdom
France
Japan
Germany
Others
P316S United States 2/106 Yes (1/3309) [7]
R524W Austria 1/860 No [8]
L774M Austria 8/9730 Yes (3/7527) [8, 27]
Germany
R32S Spain 1/134 Unknown [31]
I560T Belgium 1/592 No [29]
H599R Belgium 1/592 No [29]
M607V Belgium 1/592 No [29]
G51S Korea 5/9495 Yes (2/6513) [27, 30]
Norway
Others
E.T. Williams et al. / VPS35, the Retromer Complex and Parkinson’s Disease 221
family. Additional human genetic and functional
studies are therefore warranted to establish whether
the P316S variant represents a pathogenic mutation,
a risk variant or a rare benign polymorphism. Sev-
eral additional rare variants have also been identified
(i.e. R32S, R524W, I560T, H599R and M607V) in
individual PD subjects however their pathogenic-
ity remains inconclusive (Table 1) [25]. Therefore,
the D620N mutation represents the only confirmed
pathogenic VPS35 variant identified to date.
The clinical symptoms and neuroimaging (i.e. DAT
SPECT or Fluorodopa PET) of VPS35-linked PD
subjects suggests a classical disease spectrum similar
to idiopathic PD [7, 8, 24, 25]. PD subjects harbor-
ing VPS35 mutations present clinically with at least 3
of 4 cardinal motor symptoms. Subjects occasionally
exhibit action tremor and mild cognitive impairment.
All of the reported subjects have responded to lev-
odopa therapy [25, 33]. Given that VPS35-linked PD
is clinically and neurochemically indistinguishable
from idiopathic PD [3, 33], it would be important
to evaluate the neuropathology of PD subjects with
VPS35 mutations to confirm whether nigral neurode-
generation and Lewy body pathology similarly form
part of the disease spectrum. The neuropathological
features of VPS35-linked PD are not yet known since
only a single D620N mutation carrier with PD has
been assessed at autopsy [24]. The incomplete neu-
ropathological examination of only parts of the cortex
and basal ganglia (excluding the brainstem) in this
subject did not reveal any signs of Lewy body dis-
ease or ␣-synuclein immunoreactivity in these areas
[24]. Therefore, it remains to be determined whether
VPS35 mutations lead to PD with classical brainstem
Lewy body pathology.
VPS35 AND THE RETROMER
The VPS35 protein functions as a core subunit
of a heteropentameric complex referred to as the
retromer (Fig. 1) [34]. Originally identified in yeast,
the retromer is a protein complex that associates
with the endosome to facilitate both endosome-to-
Golgi complex and endosome-to-plasma membrane
transport and recycling of transmembrane protein
cargo [35–37]. In the seminal studies that identi-
fied and characterized the retromer, sorting of the
VPS10 receptor between the endosome and trans-
Golgi network (TGN) by the retromer was shown to
be important for the delivery of acid hydrolases to the
endosome for their eventual delivery to the lysosome
[36, 37]. The retromer is conserved from yeast to
mammals in addition to being involved in discrete
cellular pathways that may play a role in disease [34].
The retromer is commonly divided into two sub-
complexes: a cargo-selective complex trimer (CSC)
and a sorting nexin (SNX) dimer (Fig. 1). The
CSC is composed of VPS26, VPS29 and VPS35
and is responsible for binding to and sorting pro-
tein cargo [34, 38]. Of the CSC proteins, VPS35
is the largest, being composed of 796 amino acids.
Structural studies of VPS35 reveal a highly flexible
protein that forms an ␣-solenoid fold that extends
throughout the entire length of the protein. The ␣-
solenoid fold is important in the binding of VPS29,
whose structure includes a metallophosphoesterase
fold that binds to the C-terminal end of VPS35
[39]. The N-terminal portion of VPS35 is responsi-
ble for binding to VPS26 through a PRLYL motif
[40]. The CSC associates with a SNX dimer that
canonically consists of SNX1 or SNX2 and SNX5
or SNX6 in mammalian cells (SNX5 and SNX17
in yeast). These SNX proteins are members of the
SNX-BAR family which consist of both a Bin-
Amphiphysin-Rvs (BAR) and phox homology (PX)
domain that aid in retromer association with the endo-
somal membrane [34, 39]. Specifically, the BAR
domain aids in sensing membrane curvature and pos-
sibly in membrane remodeling [35]. The PX domain
binds to phosphatidylinositol 3-phosphate (PI3P) on
the membrane. PI3P production occurs through the
activity of the class III phosphoinositide 3-kinase
(PI3K) VPS34, which is regulated by Beclin1 (or
VPS30 in yeast) and RAB5 [41, 42]. Once acti-
vated, VPS34 phosphorylates phosphatidylinositol
resulting in the production of PI3P in the endoso-
mal membrane, which can recruit downstream targets
including RAB7A and SNX proteins [41]. Struc-
turally, the CSC cannot sustain a sufficiently strong
interaction with the SNX dimer to establish associ-
ation with the endosome, so RAB7A serves as an
additional anchor for retromer association with the
endosomal membrane (Fig. 1) [34].
In addition to its major association with a SNX
dimer and RAB7A, a small proportion of the CSC
can interact with other proteins that demonstrate
the importance of the retromer in discrete endoso-
mal sorting pathways. For example, the retromer can
associate with the Wiskott-Aldrich syndrome and
SCAR homolog (WASH) complex, which is com-
posed of WASH1, strumpellin, FAM21, CCDC53
and KIAA1033/SWIP, and adaptor proteins such
as SNX27, to facilitate both the endosome-to-TGN
222 E.T. Williams et al. / VPS35, the Retromer Complex and Parkinson’s Disease
Fig. 1. Retromer-regulated retrograde transport of endosomal-associated protein cargo. The retromer, along with retromer-associated proteins
that assist in membrane binding (Snx3 and Rab7a), is responsible for the retrograde transport of several cargo proteins from the endosomal
network to either the trans-Golgi network (TGN) or the plasma membrane. VPS35, along with VPS26 and VPS29, sit at the endosomal
membrane and recognize cargo (transmembrane proteins) to be sorted. Two canonical cargo proteins that the retromer is responsible for
transporting are the mannose-6-phosphate receptor (CI-MPR) and the 2-adrenergic receptor (2-AR). These two examples demonstrate
the two major routes of transport that are facilitated by the retromer [34]. CI-MPR is responsible for delivering acid hydrolases (cathepsin D,
for example) to the endosome for eventual delivery to the lysosome. While other mechanisms are responsible for delivering CI-MPR with
its ligand to the endosome, the retromer facilitates the retrieval of CI-MPR to the TGN to bind more ligand, which will eventually make its
way to the endosome once again [85]. On the other hand, 2-AR is recycled from the endosome to the plasma membrane where it will stay
until activated. Although the retromer is not responsible for the initial endocytosis of 2-AR at the plasma membrane, its role in recycling
the receptor back to the plasma membrane prevents it from lysosomal degradation [48].
and endosome-to-plasma membrane transport of
cargo [43–45]. The WASH complex functions to
facilitate the formation of actin patches on the
endosomal membrane that generate distinct domains
to which cargo are partitioned for transport to
the TGN or plasma membrane. The WASH com-
plex associates with the retromer via an interaction
between the unstructured tail of FAM21 and the C-
terminus of VPS35, and this interaction mediates the
endosome-to-TGN retrieval of select cargo such as
the cation-independent mannose 6-phosphate recep-
tor (CI-MPR) [44]. Alternatively, the WASH complex
can associate with the retromer and SNX27 to facil-
itate the endosome-to-plasma membrane retrieval of
specific cargo including the 2-adrenergic recep-
tor (2AR), the glucose transporter 1 (GLUT1) and
several metal ion transporters [35, 45]. All of the
interactions with the retromer reported thus far are
dependent on the association of the SNX-BAR dimer
with the CSC, however, SNX3 which lacks a BAR
domain, can bind to the CSC and facilitate endosome-
to-TGN retrieval primarily of the Wntless protein
independent of the SNX-dimer [46, 47]. Therefore,
the interactions with and within the retromer are mul-
tiple and diverse with different subcomplexes most
likely serving to mediate the retrieval of specific cargo
to distinct vesicular compartments.
Retromer function is clearly important for the
transport and recycling of numerous transmem-
brane cargo from the endosome to the TGN or
plasma membrane in a diverse number of cells
and tissues, however, the distinct role of the
retromer in cells of the central nervous system
(CNS) is incompletely understood. Choy et al.
[48] identified the retromer as an important mech-
anism for membrane protein transport to the
dendritic processes of neurons. Choy and colleagues
demonstrated that the retromer was responsible
E.T. Williams et al. / VPS35, the Retromer Complex and Parkinson’s Disease 223
for the distribution of membrane proteins to
the post-synaptic compartment (␣-amino-3-hydroxy-
5-methyl-4-isoxazolepropionic acid [AMPA] and
N-methyl-D-aspartate [NMDA] receptors) and to
extra-synaptic locations (2AR) along the dendrite
[48]. Whether the dendritic sorting of receptors is
altered in neurodegenerative disease, specifically in
PD, remains unclear. However, over the last few
years, several studies have suggested a specific role
for the retromer in PD.
FUNCTIONAL INSIGHT INTO
PD-ASSOCIATED VPS35 MUTATIONS
Evidence for VPS35-induced pathogenicity
While the identification of disease-associated mis-
sense mutations in VPS35 suggests dysfunction of the
retromer complex as a contributor to PD, our under-
standing of the molecular and cellular mechanisms
of VPS35-dependent neurodegeneration is still rather
limited. The absence of truncation, rearrangement or
deletion mutations in the VPS35 gene and the domi-
nant inheritance pattern of mutations in PD, suggest
that heterozygous VPS35 mutations could potentially
act via either i) a toxic gain-of-function mechanism,
ii) a dominant-negative mechanism (with a partial or
full loss-of-function effect) or iii) via a haploinsuffi-
cient mechanism (creating a partial loss-of-function).
Distinguishing between these possibilities for the
pathogenic effects of disease-causing mutations is
rarely simple and is often rather complicated espe-
cially when more than one mechanism may be
involved. Experimental studies using cell-based and
in vivo models (i.e. mice, rats, worms and flies;
Table 2) of mutant VPS35-induced neuronal damage
have so far provided important clues to the poten-
tial function(s) of VPS35, the cellular pathways that
are potentially perturbed in PD, and have identified
functional interactions with known PD-linked gene
products.
VPS35 and the retromer are essential for nor-
mal cellular function and viability, however, several
studies have demonstrated that both silencing and
overexpression of VPS35 is detrimental to cellu-
lar health. For example, Drosophila overexpressing
both human ␣-synuclein and RNAi directed against
VPS35 demonstrated that depletion of VPS35
resulted in defective ␣-synuclein lysosomal degra-
dation, mainly due to the impaired recycling of
the retromer cargo CI-MPR and the reduced deliv-
ery of its ligand cathepsin D to lysosomes [49].
This sorting defect resulted in the enhanced accu-
mulation of insoluble ␣-synuclein and exacerbated
␣-synuclein-induced locomotor deficits and com-
pound eye disorganization in flies [49]. These data
suggest that reduced retromer levels may disrupt the
normal lysosomal degradation of ␣-synuclein leading
to its inappropriate accumulation. Such a mecha-
nism is of particular interest to PD as it may offer
one explanation for the accumulation of aggregated
␣-synuclein in Lewy bodies. Similarly, the endoso-
mal sorting of CI-MPR was reported to be disrupted
by the D620N VPS35 mutation in mammalian cells
[50–52], although alterations in CI-MPR trafficking
have not been consistently reported by all studies
including in cultured primary neurons [53–55]. The
impact of the D620N VPS35 mutation on the sort-
ing of other retromer cargo has not been extensively
studied, with reports suggesting no alterations in the
sorting of sortilin-1 and SorLA in primary corti-
cal neurons or patient-derived fibroblasts [53, 54].
In mouse cortical neurons and human iPSC-derived
dopaminergic neurons, D620N VPS35 has been
reported to alter the localization of the AMPA gluta-
mate receptor, GluR1, to dendritic spines suggesting
a role for the retromer in post-synaptic receptor traf-
ficking [53]. Consistent among most studies is the
observation that the D620N mutation does not alter
the subcellular localization of VPS35, the assembly
of the CSC or the stability of retromer components in
different cellular models [51, 53–55]. Therefore, the
D620N mutation is unlikely to influence all retromer
functions but rather may have discrete effects involv-
ing select cargo and/or subcellular compartments.
Whether or not the altered sorting of CI-MPR, GluR1
or other retromer cargo by D620N VPS35 is required
or relevant for neurodegeneration remains to be for-
mally established.
To understand the mechanism through which PD-
associated mutations in VPS35 contribute to disease
pathogenesis, one study demonstrated that the viral-
mediated overexpression of human wild-type (WT)
or D620N VPS35 in primary cortical neurons equiva-
lently led to neuronal cell death and impaired neurite
outgrowth [54]. WT and D620N VPS35 overexpres-
sion also markedly increased neuronal vulnerability
to PD-relevant cellular stressors, including the mito-
chondrial Complex-I inhibitors MPP
+
and rotenone
[54]. Therefore, both WT and D620N VPS35 expres-
sion induce equivalent levels of neuronal damage, and
are therefore similarly functional in this model, sup-
porting either a gain-of-function mechanism for the
D620N mutation or potentially a dominant-negative