Neuron, Vol. 36, 1007–1019, December 19, 2002, Copyright 2002 by Cell Press
Parkin Protects against the Toxicity Associated
with Mutant ?-Synuclein: Proteasome Dysfunction
Selectively Affects Catecholaminergic Neurons
C-terminal hydrolase (UCHL1, OMIM 191342) has been
described (Farrer et al., 2000; Leroy et al., 1998), al-
though pathogenicity of this mutation has not been fully
established. However, polymorphic variability in UCHL1
has been associated with altered risk for development
of PD in case-control studies (Maraganore et al., 1999;
Wintermeyer et al., 2000).
Mutations in ?-synuclein have been reported in au-
tosomal dominant pedigrees (OMIM 601508, Kruger et
al., 1998; Polymeropoulos et al., 1997). Several pieces
teasome function may be related. Whether ?-synuclein
turnover is regulated by proteasome function is contro-
versial, with both positive (Bennett et al., 1999; Tofaris
et al., 2001) and negative (Ancolio et al., 2000; Paxinou
et al., 2001) results reported. Forced overexpression of
?-synuclein, especially mutant forms, sensitize PC12
(Stefanis et al., 2001; Tanaka et al., 2001), NT2, and SK-
N-MC (Lee et al., 2001b) neuroblastoma cell lines to
The mechanism by which this occurs is not clear, but
overexpression of mutant ?-synuclein produces an inhi-
bition of proteasome-associated proteolytic activities.
The A30P mutant ?-synuclein inhibits the postacidic
proteasome activity by 25% and the trypsin-like and
chymotrypsin-like activities by slightly smaller amounts,
with wild-type ?-synuclein having a much smaller effect
(Tanaka et al., 2001). The A53T mutant form of ?-synu-
clein also inhibits the chymotrypsin-like activity of the
proteasome (Stefanis et al., 2001). Finally, it has been
in human brain is ubiquitinated by parkin (Shimura et al.,
2001), raising the possibility that loss of parkin function
might result in ?-synuclein accumulation. ?-synuclein-
positive Lewy bodies have been noted in a case of Par-
kin-related PD (Farrer et al., 2001).
Overall, the above studies suggest that proteasome
inhibition might be a common link between the different
genetic triggers of PD. Furthermore, there is evidence
that proteasome function is impaired in sporadic PD
(McNaught and Jenner, 2001). However, the hypothesis
that proteasome dysfunction is an explanation for PD
remains conjecture. For example, as cell loss in PD is
not uniform, any attempt to link proteasome function
to disease should account for selective vulnerability of
specific subgroups of neurons. The selective vulnerabil-
ity of different neuronal types to cell death or formation
of the pathological hallmarks of the disease is complex
(reviewed in Braak and Braak, 2000), but it is clear that
functional loss of dopaminergic neurons in the substan-
tia nigra (SN) pars compacta is important. The move-
ment-related symptoms of PD patients are related to
dopaminergic cell loss, and loss of these cells not only
precedes symptom development, it is also progressive
throughout the course of the disease (Pakkenberg et
between overexpression of ?-synuclein and parkin with
toxicity associated with proteasome inhibition. We have
also used primary cell cultures to distinguish effects on
Leonard Petrucelli,1Casey O’Farrell,1
Paul J. Lockhart,1Melisa Baptista,3
Kathryn Kehoe,1Liselot Vink,1
Peter Choi,2Benjamin Wolozin,2
Matthew Farrer,1John Hardy,3
and Mark R. Cookson3,4
Mayo Clinic Jacksonville
Jacksonville, Florida 32224
2Department of Pharmacology
Loyola University Medical Center
Maywood, Illinois 60153
3Laboratory of Neurogenetics
National Institute on Aging
Bethesda, Maryland 20892
(PD) is that subsets of neurons are vulnerable to a
we show that overexpression of mutant ?-synuclein
increases sensitivity to proteasome inhibitors by de-
kin decreases sensitivity to proteasome inhibitors in
a manner dependent on parkin’s ubiquitin-protein E3
ligase activity, and antisense knockdown of parkin in-
creases sensitivity to proteasome inhibitors. Mutant
?-synuclein also causes selective toxicity to catechol-
aminergic neurons in primary midbrain cultures, an
effect that can be mimicked by the application of pro-
teasome inhibitors. Parkin is capable of rescuing the
tionin these cells.Therefore,parkinand ?-synucleinare
selective cell death in catecholaminergic neurons.
The identification of genes linked to familial forms of
Parkinson’s disease (PD) provides an important tool for
modeling the pathways leading to neurodegeneration
in this disorder. To date, eight linkages have been re-
ported, with three genes identified as causal, or proba-
bly causal, in different families. Two of these encode
proteins whose function is related to ubiquitin-depen-
dent protein degradation through the proteasome (for
review, see Hershko and Ciechanover, 1998). Parkin
(OMIM 600116) is an E2-dependent E3 protein-ubiquitin
ligase (Shimura et al., 2000; Zhang et al., 2000), and
mutations in this gene are generally associated with
recessive early onset parkinsonism (Kitada et al., 1998).
Parkin mutations reported to date appear to be loss-
of-function mutations reducing the ability of parkin to
regulate degradation of substrate removal (Shimura et
al., 2000; Zhang et al., 2000). One mutation in ubiquitin-
Figure 1. Effects of Proteasome Inhibitors on Cell Viability in M17 Cells Overexpressing ?-Synuclein
antibody 42. Untransfected cells (lane 2) or cells transfected with vector alone (lane 3) show moderate expression of ?-synuclein compared
to cells overexpressing wild-type (lane 4), A30P (lane 5), or A53T synuclein (lane 6). Human cerebral cortex extract was used as a positive
control (lane 1). A reprobe of the same blot using ?-actin is shown below to demonstrate similarity of loading across the lanes. Quantitation
of ?-synuclein expression is shown in (B) and is expressed as a ratio of the major synuclein band to ?-actin (n ? 4 serial passages of the
cells, error bars represent the SEM).
(C and D) Overexpression of ?-synuclein produces increased sensitivity to the proteasome inhibitor MG132 (C) or lactacystin (D). Cells were
exposed to either inhibitor for 24 hr, after which cell viability was estimated using the MTT assay (see Experimental Procedures). Cell lines
included untransfected cells (open squares) or cells transfected with empty vector (open circles), WT (open triangles), A30P (closed squares),
or A53T (closed circles) ?-synuclein. Results are expressed as a percentage of untreated cells for each clonal line (n ? 8, each curve is
representative of three or more experiments). Similar increased sensitivity to proteasome inhibition was seen in a second set of clonal lines.
Statistical significance was estimated using two-way ANOVA using cell lines and concentration of inhibitors as independent variables. **p ?
0.001 for differences between cell lines, both inhibitors having a significant effect on viability at p ? 0.001. Representative data from one of
TH-negative neurons. We show that parkin and mutant
associated with impaired proteasome function and that
parkin is capable of reducing toxicity associated with
?-synuclein overexpression. We also show that knock-
down of parkin increases the sensitivity of cells to pro-
tations in parkin would cause cell death by the same
mechanism as gain-of-function ?-synuclein mutations.
Furthermore, the effects of either mutant ?-synuclein or
proteasome inhibition are both selective for TH-positive
Overexpression of ?-Synuclein and Sensitivity
to Proteasome Inhibitors
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about a 5-fold increased level of ?-synuclein compared
with untransfected cells (Figure 1A). Although the ex-
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9-fold higher than untransfected M17 cells and approxi-
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