UCH-L1 aggresome formation in response to proteasome impairment indicates a role in inclusion formation in Parkinson's disease

UCH-L1 aggresome formation in response to proteasome

impairment indicates a role in inclusion formation in Parkinson’s

disease

Helen C. Ardley, Gina B. Scott, Stephen A. Rose, Nancy G. S. Tan and Philip A. Robinson

Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James’s University Hospital, Leeds, UK

Abstract

Aggresomes are associated with many neurodegenerative

disorders, including Parkinson’s disease, and polyglutamine

disorders such as Huntington’s disease. These inclusions

commonly contain ubiquitylated proteins. The stage at which

these proteins are ubiquitylated remains unclear. A malfunc-

tion of the ubiquitin/proteasome system (UPS) may be asso-

ciated with their formation. Conversely, it may reflect an

unsuccessful attempt by the cell to remove them. Previously,

we demonstrated that overexpression of Parkin, a ubiquitin-

protein ligase associated with autosomal recessive juvenile

Parkinsonism, generates aggresome-like inclusions in UPS

compromised cells. Mutations in the de-ubiquitylating enzyme,

UCH-L1, cause a rare form of Parkinsonism. We now dem-

onstrate that overexpression of UCH-L1 also forms ribbon-like

aggresomes in response to proteasomal inhibition. Disease-

associated mutations, which affect enzymatic activities,

significantly increased the number of inclusions. UCH-L1

aggresomes co-localized with ubiquitylated proteins, HSP70,

c-tubulin and, to a lesser extent, the 20S proteasome and the

chaperone BiP. Similar to Parkin inclusions, we found UCH-L1

aggresomes to be surrounded by a tubulin rather than a

vimentin cage-like structure. Furthermore, UCH-L1 aggre-

gates with Parkin and a-synuclein in some, but not all inclu-

sions, suggesting the heterogeneous nature of these inclusion

bodies. This study provides additional evidence that aggre-

gation-prone proteins are likely to recruit UPS components in

an attempt to clear proteins from failing proteasomes. Fur-

thermore, UCH-L1 accumulation is likely to play a pathological

role in inclusion formation in Parkinson’s disease.

Keywords: de-ubiquitylating enzyme, inclusion bodies, neu-

rodegeneration, Parkinson’s disease, ubiquitin.

J. Neurochem. (2004) 90, 379–391.

Neurodegenerative disorders such as Parkinson’s (PD),

Alzheimer’s (AD) and Huntington’s (HD) diseases are

characterized by the selective and symmetric loss of neurones

in specific regions of the brain. This loss affects motor,

sensory or cognitive systems, causing progressively increas-

ing disability leading to the death of the patient. These

disorders are pathologically characterized by neuronal pro-

teinaceous inclusions of insoluble, unfolded, ubiquitylated

polypeptides that fail to be degraded by the 26S proteasome

(Lowe et al. 1988; Lang and Lozano 1998; Kaytor and

Warren 1999; Sherman and Goldberg 2001). Their apparent

stability may, in part, be due to the decreased levels of 26S

proteasomal activity, which is associated with increasing age

(Goto et al. 2001; McNaught et al. 2002a).

The molecular changes underlying the initiation and

progression of sporadic idiopathic disease are poorly under-

stood. However, genetic studies have provided strong

evidence that implicate abnormal processing of a variety of

cellular proteins via the ubiquitin/proteasome system (UPS)

in these disorders, particularly in the development of PD.

Indeed, of the familial forms of PD, a-synuclein, Parkin,UCH-L1 and DJ-1 are associated with the UPS (Wilkinson

et al. 1989; Shimura et al. 2000, 2001; Choi et al. 2001;

Miller et al. 2003; Tofaris et al. 2003). Parkin and UCH-L1

are components of the system, a-synuclein and DJ-1 are

targets.

Received December 16, 2003; revised manuscript received March 1,

2004; accepted March 1, 2004.

Address correspondence and reprint requests to Dr Helen Ardley,

Molecular Medicine Unit, Clinical Sciences Building, St James’s Uni-

versity Hospital, Leeds LS9 7TF, UK. E-mail: [email protected]

Abbreviations used: AD, Alzheimer’s disease; DUB, deubiquitylating

enzyme; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating

enzyme; E3, ubiquitin-protein ligase; HA, influenza haemagglutinin;

HD, Huntington’s disease; PD, Parkinson’s disease; UPS, ubiquitin/

proteasome system.

Journal of Neurochemistry, 2004, 90, 379–391 doi:10.1111/j.1471-4159.2004.02485.x

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 90, 379–391 379

The UPS is the major route of intracellular protein

degradation. Ubiquitin ‘tagging’ of a target protein is an

energy-dependent, four-step pathway that operates in all

eukaryotic cell types (for reviews see Pickart 2001; Weissman

2001; Doherty et al. 2002). Ubiquitin activated by a ubiquitin-

activating enzyme (E1) is transferred via a ubiquitin-conju-

gating enzyme (E2) and then, with the aid of a ubiquitin-

protein ligase (E3), to an e-amino group of a lysine residue in atarget protein. The C-terminally-conjugated ubiquitin can then

itself serve as a ubiquitylation substrate such that repeated

ubiquitylation leads to polyubiquitin chain formation. Poly-

ubiquitylated conjugates may be degraded by the large,

multicomponent, multicatalytic 26S proteasome into small

peptides. These products can be processed further for antigen

presentation or hydrolysed by other cellular peptidases

(Serwold et al. 2002). The functional role for the large

numbers of de-ubiquitylating enzymes (DUBs) in the regula-

tion of this process is not yet clear. They may provide a proof-

reading mechanism by limiting the length of the ubiquitin

chain to less than four ubiquitin molecules, thus preventing

selection for proteasomal degradation. They may also hinder

protein aggregation that could occur if the polyubiquitin

chains became too long. A number of DUBs, including UCH-

L1 (ubiquitin C-terminal hydrolase-L1, also known as

PGP9.5), synUSP (USP31), ataxin-3 and Drosophila fats

facets, are abundant in brain tissue and are known to be

essential for normal neuronal function (Wilkinson et al. 1989;

Huang et al. 1995; Burnett et al. 2003; Tian et al. 2003).

UCH-L1 is linked with inclusions associated with several

neurodegenerative disorders including PD, AD and the

Rosenthal fibres associated with cerebellar astrocytomas

(Lowe et al. 1990). It is a small but abundant neuronal DUB

that accounts for 1–2% of soluble neuronal cell protein. It is

also highly abundant in testis (Wilkinson et al. 1989; Chung

and Baek 1999) and is up-regulated in cancers (Hibi et al.

1999; Yamazaki et al. 2002; Takase et al. 2003). It releases

ubiquitin monomers from small ubiquitylated peptides

(Wilkinson et al. 1989). Indeed, UCH-L1 can associate

with, and stabilize, ubiquitin in neuronal cells (Osaka et al.

2003). Surprisingly, UCH-L1 can also act as an E3 when it

dimerizes. One of its substrates in vitro is a-synuclein.However, this latter activity remains to be confirmed in vivo

(Liu et al. 2002).

Disease-causing mutations in UCH-L1 appear to be very

rare (Maraganore et al. 1999; Zhang et al. 2000b). The I93M

mutation was identified as the cause of autosomal dominant

PD in a German kindred (Leroy et al. 1998). I93M has a

severely diminished hydrolase activity and lower E3 activity

compared with wild-type UCH-L1. By contrast, an S18Y

polymorphism of UCH-L1 has been identified that may be

associated with a decreased susceptibility to sporadic idio-

pathic PD in a dose-dependent manner (Maraganore et al.

1999; Zhang et al. 2000a; Elbaz et al. 2003). Additionally,

S18Y may have modest protective effects in HD patients

(Naze et al. 2002). This variant form is characterized by a

greater hydrolase but lower E3 activity than wild-type UCH-

L1. The effects of the I93M mutation and S18Y polymorph-

ism on incidence of PD can therefore be partly explained by

changing function (Leroy et al. 1998; Liu et al. 2002;

Nishikawa et al. 2003).

UCH-L1 is found in the Lewy body protein aggregates

associated with PD (Lowe et al. 1990). Lewy bodies amass a

range of normal and abnormal proteins, many of which are

ubiquitylated (Pollen et al. 1993). Although a-synuclein is

the main constituent of these inclusions (Spillantini et al.

1997), multiple UPS components are also detected (Lowe

et al. 1990; Ii et al. 1997; Shimura et al. 1999; Schlossma-

cher et al. 2002). Lewy bodies represent a specialized

‘aggresome-related’ inclusion specific to dopaminergic

neurones of the substantia nigra (McNaught et al. 2002a,

2002b). Aggresome formation is proposed to occur when the

capacity of the proteasome is exceeded by substrate expres-

sion (Johnston et al. 1998; Garcia-Mata et al. 1999; Waelter

et al. 2001). Indeed, overexpression of neurodegenerative

disease-associated proteins such as Parkin, a-synuclein or

Huntingtin can cause aggresome formation in proteasome-

impaired cells (Rideout et al. 2001; Waelter et al. 2001; Junn

et al. 2002; Lee et al. 2002; Ardley et al. 2003). These in

vitro systems are proving to be valuable aids for analysing

the potential of novel therapeutics. For example, compounds

with properties that may prevent the formation of patholo-

gical proteinaceous aggregates associated with HD have been

identified (Apostol et al. 2003).

UCH-L1 is present in Lewy bodies in sporadic disease

(Lowe et al. 1990). Unfortunately, it has not yet been

established whether they are present in the remaining

dopaminergic neurones of patients with UCH-L1-associated

PD (Dekker et al. 2003; McNaught and Olanow 2003). Since

other PD-associated proteins such as Parkin and a-synucleinare prone to aggregation either in their mutant forms (Ardley

et al. 2003; Cookson et al. 2003) or when proteasomal

activity is impaired (Rideout et al. 2001; Junn et al. 2002;

Lee et al. 2002; Ardley et al. 2003), we propose to test the

hypothesis that disease-associated forms of UCH-L1 would

also form aggresomes. Here, we demonstrate that overex-

pression of UCH-L1 does produce ubiquitylated inclusions

in cultured cells. Moreover, proteasome inhibition is not

required for the PD-associated I93M mutant to produce

inclusions. This study provides further evidence that ineffi-

ciency of the UPS due to mutation and/or malfunction has a

major pathological role in Lewy body formation in PD.

Materials and methods

Generation of plasmid constructs

A pcDNA3-HA expression vector was generated by ligation of an

oligonucleotide coding for the influenza haemagglutinin (HA)

380 H. C. Ardley et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 90, 379–391

epitope (YPYDVPDYA) into pcDNA3 (Invitrogen, Paisley, UK).

UCH-L1 coding sequence was amplified by PCR from brain cDNA

using Pfu Turbo DNA polymerase (Stratagene, Amsterdam, The

Netherlands), purified by agarose gel electrophoresis and ligated

into Bam HI and Xho I digested pcDNA3-HA or Green Fluorescent

Protein (GFP) expression vector pJMA2eGFP, (Askham et al. 2000)

to generate UCHL1-HA and UCHL1-GFP, respectively. The

construction of FLAG-Parkin has been described previously (Ardley

et al. 2003).

Mutant UCH-L1 constructs were generated using the Quik-

Change site-directed mutagenesis kit (Stratagene) according to the

manufacturer’s protocol.

Antibodies

Mouse monoclonal antibodies to b-actin (AC-15), c-tubulin (GTU-88) and vimentin (clone V9) were obtained from Sigma (Poole,

Dorset, UK). Mouse monoclonals to BiP and HSP70, ubiquitylated

proteins (clone FK2), HA (262K) and a-synuclein were obtained

from BD Biosciences (Cowley, Oxford, UK), Affiniti Research

Products (Exeter, UK), Cell Signalling Technologies (Beverly, MA,

USA) and Zymed (South San Francisco, CA, USA), respectively.

Rabbit polyclonal antibody to ubiquitin was obtained from Dako

(Glostrup, Denmark). Rabbit polyclonal antibodies to 20S protea-

some and UCH-L1 were purchased from Affiniti Research Products.

Rat anti-a-tubulin was obtained from Serotec (Kidlington, Oxford,

UK). Affinity purified rabbit anti-Parkin peptide antibodies have

been described previously (Ardley et al. 2003).

Cell culture and transfection

COS-7 cells were grown in Dulbecco’s minimal essential medium

(DMEM) with Glutamax (Invitrogen). SH-SY5Y neuroblastoma

cells were maintained in a 1 : 1 mixture of minimal essential medium

and Ham’s F12 medium. All cells were grown at 37�C in 5% CO2

and supplemented with 10% (v/v) fetal calf serum, 100 U/mL

penicillin and 100 lg/mL streptomycin. Transfections were per-

formed, using LIPOFECTAMINE 2000 (Invitrogen) according to the

manufacturer’s protocol, in medium appropriate for each particular

cell type but without antibiotics or serum, with the exception of

COS-7 cells which were supplemented with 2% (v/v) fetal calf serum.

Stress-inducing reagents were added to cell cultures 28 h post-

transfection for 16 h unless stated otherwise. Such reagents included

5 lM MG132 (Calbiochem, Nottingham, UK), 10 lM lactacystin

(Affinti Research Products), 1 lM calpeptin (Calbiochem), 10 lg/mLtunicamycin, 10 mg/mL brefeldin A or 10 lg/mL nocodazole

(all Sigma). Control experiments using the carrier solvents dimeth-

ylsulfoxide (DMSO) or ethanol were performed where appropriate.

Immunofluorescence and microscopy

Cellswere seeded onto sterile glass coverslips in a 6-well culture plate.

Following attachment, cells were transfected as described above with

1.0 lg of the appropriate construct(s). Cells were fixed with methanolat ) 20�C for 5 min. Immunostaining of cells was performed as

previously described (Ardley et al. 2001, 2003) using appropriate

dilutions of antibody solutions as follows: mouse monoclonals to

c-tubulin (GTU-88) 1 : 1000; vimentin (clone V9), 1 : 80; BiP

1 : 200; HSP70 1 : 400; ubiquitylated proteins (clone FK2), 1 : 50;

HA (262K), 1 : 200; and a-synuclein, 1 : 100; rabbit polyclonals to

ubiquitin, 1 : 50; 20S proteasome, 1 : 100; Parkin, 1 : 50; and rat

anti-a-tubulin, 1 : 500. Images were obtained using a Leica TCS SP

(Wetzlar, Germany) confocal imaging system or a Zeiss Axiovert

(Welwyn Garden City, UK) microscope and camera with Hamamatsu

Orca 2 (Hamamatsu City, Japan) ER imaging software.

Analysis of inclusion body formation

A Zeiss inverted microscope with 40· objective lens was used to

observe inclusion body formation. The number of transfected cells

with or without inclusion bodies was counted in three randomly

chosen microscope fields in different areas of the slide. A minimum

of 300 transfected cells were analysed for each sample. Experiments

were repeated at least three times, and counts were made in a

blinded manner. One-way ANOVA (Bonferroni) statistical analysis

was performed using SSPS 11.0 for Windows (Chicago, IL, USA).

Preparation of cell lysates and immunoprecipitation

Transfections for solubility and immunoprecipitation analysis were

performed in 75 cm2 flasks, using 3.0 lg of each DNA construct

and 25.0 lL LIPOFECTAMINE 2000, essentially as previously

described (Ardley et al. 2001, 2003). COS-7 cells were either mock

transfected or transfected with pcDNA3-HA, or UCHL1-HA wild-

type or mutant construct. At 28 h post-transfection, cells were

treated with drugs as outlined above where appropriate. At 44 h

post-transfection, cells were harvested for immunoprecipitation and/

or western blot analysis as previously described (Ardley et al. 2001,

2003). For ubiquitin western blots, proteins were transferred to a

nitrocellulose membrane which was then autoclaved (121�C for

20 min at 10 3421 Pa (103.421 kilopascals) prior to incubation with

anti-ubiquitin antibodies. Immunoblots were probed using appro-

priate dilutions of antibody solutions as follows: mouse monoclo-

nals to HA (262K), 1 : 1000 and b-actin, 1 : 3000; rabbit

polyclonals to ubiquitin, 1 : 1000 and UCH-L1, 1 : 3000.

Results

Assessment of the effects of cellular stress on the

subcellular localization of endogenous and overexpressed

UCH-L1 in neuronal and non-neuronal cell lines

UCH-L1 is a highly abundant protein that is located in Lewy

bodies of PD patients (Wilkinson et al. 1989; Lowe et al.

1990). Furthermore, overexpression of other PD-associated

proteins, such as Parkin and a-synuclein, have been found tolead to aggregation in cultured cells subjected to proteasomal

and mitochondrial inhibitors (Rideout et al. 2001; Junn et al.

2002; Lee et al. 2002; Ardley et al. 2003). We hypothesized

that any alteration in UCH-L1 protein levels, brought about

by excess protein or mutation, may lead to inclusion

formation inside cells. We therefore investigated the effects

of different cellular stresses on the subcellular localization of

UCH-L1 in both neuronal and non-neuronal cell lines

overexpressing the protein.

COS-7 or SH-SY5Y cells, transfected with C-terminal

HA-tagged (Fig. 1a left-hand panel) or GFP-tagged (Fig. 1a,

middle panels) UCH-L1, produced identical patterns of

expression. Although staining was essentially cytoplasmic,

UCH-L1 aggresome formation 381


Fig. 1 Effects of cellular stress on UCH-L1 localization in mammalian

cells. (a) UCHL1-HA or UCHL1-GFP were transfected into COS-7 or

SH-SY5Y cells where indicated. At 44 h post-transfection, cells were

washed and fixed with methanol at ) 20�C for 5 min prior to immuno-

staining with antibodies that bind to the appropriate tag (green) [either

monoclonal HA (left-hand column) or rabbit anti-GFP (middle columns)

or antibodies to endogenous UCH-L1 (right-hand column)], and count-

erstained with DAPI to visualize nuclei (red or blue). (b) COS-7 cells

were transfected with UCHL1-HA. At 28 h post-transfection, cells were

incubated for 16 h in the presence of either: left-hand panel: 5 lM

MG132, 10 lM lactacystin (Lact), 10 lg/mL tunicamycin (Tuni), 10 mg/

mL brefeldin A (Bref A) or carrier [DMSO or ethanol (EtOH)]; right-hand

panel: 10 lM lactacystin, 1 lM calpeptin (Calpep), lactacystin and cal-

peptin or carrier (DMSO). The presence of UCH-L1 was then assessed

by immunofluorescence using mouse monoclonal anti-HA antibodies.

Bars represent the percentage of cells containing inclusion bodies. A

minimum of 300 transfected cells were analysed for each sample. Data

shown represent the results of three independent sets of experiments.

Error bars indicate the standard error from the mean of these experi-

ments. Asterisk(s) indicate a significant difference between the per-

centage of untreated cells versus the percentage of treated cells.

*p < 0.001. (c) COS-7 or SH-SY5Y (as indicated) cells were transfected

with UCH-L1 tagged proteins as indicated or stained for endogenous

UCH-L1 (COS-7 cells, right-hand panel) and incubated in the presence

of either 5 lM MG132 or 10 lM lactacystin for 16 h. Cells were stained as

described in (a). Arrows in (c) indicate the position of inclusions. Scale

bars 20 lm. (d) COS-7 cells were either transfected with UCHL1-HA or

mock transfected as indicated and treated as described in (b), left-hand

panel. Protein lysates were prepared in immunoprecipitation buffer

(Ardley et al. 2001) and 10 lg of each total cell lysate were separated by

SDS-PAGE. Western blotting analysis was performed with either mouse

anti-HA or rabbit polyclonal anti-UCH-L1 (upper panels) antibodies, or

anti-b-Actin (lower panels) antibodies as indicated. The positions of

individual molecular mass markers are indicated.



high levels of expression were often observed at the nuclear

periphery. In addition, a small percentage of cells displayed

additional nuclear or endoplasmic reticulum (ER)-like stain-

ing patterns. Similar staining patterns were obtained when

staining for endogenous UCH-L1 in COS-7 cells (Fig. 1a,

right-hand panel), indicating that the observed staining

patterns were not a function of the attached tag.

We next subjected COS-7 and SH-SY5Y cells to a variety

of stress-inducing agents in order to establish whether these

compounds would affect the subcellular distribution of UCH-

L1. At 28 h after transfection with UCHL1-HA, cells were

treated with MG132 (a reversible proteasome inhibitor),

lactacystin (an irreversible proteasome inhibitor), tunicamy-

cin (N-glycosylation inhibitor, which induces the unfolded

protein response), brefeldin A (an inhibitor of movement

from the ER to Golgi apparatus) or carrier (DMSO or

ethanol). Similar to previous studies (reviewed by Lee and

Goldberg 1998), none of the treatments described appeared

to significantly affect cell viability in the time frame of our

experiments as visualized by the number of adherent cells

and the presence of intact nuclei. Furthermore, normal UCH-

L1 expression patterns were observed after exposure to

tunicamycin or brefeldin A. By contrast, UCH-L1 expression

patterns were significantly altered after inhibition of protea-

somal activity with either MG132 or lactacystin treatment. At

least 67% of MG132-treated and 28% of lactacystin-treated

cells contained inclusions (Fig. 1b, left-hand graph). These

inclusions were observed in COS-7 and SH-SY5Y cells with

both HA- and GFP-tagged UCH-L1 constructs (Fig. 1c).

Although inclusions were observed in UCHL1-GFP trans-

fected cells fixed without antibody staining, structures were

less distinct (data not shown). Mock or vector alone

transfected cells did not produce a significant number of

inclusions (data not shown). MG132 treatment caused

redistribution of endogenous UCH-L1, mainly to the

nucleus. However, protein aggregates or inclusions were

not observed in these cells (Fig. 1c, right-hand panel). To

confirm that the elevated number of inclusions observed in

MG132-treated cells compared with lactacystin-treated cells

was not due to inhibition of other proteases such as calpains,

we performed additional experiments using the calpain

inhibitor, calpeptin, in the presence or absence of lactacystin

(Fig. 1b, right-hand panel). Calpeptin treatment did not

produce inclusions within cells. Furthermore, addition of

calpeptin and lactacystin to cells did not increase the number

of inclusions observed (22% in lactacystin-treated cells

compared with 21% in cells treated with both inhibitors). As

the synthetic peptide inhibitor, MG132, and the natural

inhibitor, lactacystin, but not the calpain inhibitor, calpeptin,

caused UCH-L1 inclusion formation, the data suggest that

these protein aggregates resulted from an impairment of

proteasome function, and that the number of inclusions in

MG132 cells is unlikely to be a consequence of additional

protease inhibition.

We did not observe any alteration in the levels of either

UCHL1-HA protein or endogenous UCH-L1 after exposure

to any of the drugs tested (Fig. 1d, and data not shown).

The majority of MG132-induced UCH-L1 inclusions were

found in the cytoplasm (Fig. 2a, left-hand panel). However, a

small number of cells (< 10%) displayed either nuclear

(Fig. 2a, middle panel), or cytoplasmic and nuclear (Fig. 2a,

right-hand panel), staining patterns. Detailed analysis of

these inclusions revealed a ‘honeycomb’ or ‘chain-like’

structure (Fig. 2b).

The solubility of UCH-L1-HA inclusions in COS-7 cells

was also assessed (Fig. 2c). Prior to proteasome inhibition,

UCH-L1 was detected exclusively in the soluble fraction of

cell lysates (Fig. 2c). However, a substantial fraction of

UCH-L1 was redistributed to the insoluble fraction after the

inhibition of proteasomal activity (Fig. 2c). High molecular

weight species, characteristic of the presence of polyubiquit-

ylated products, were not observed in the insoluble fraction

(Fig. 2c).

The familial Parkinson’s disease-causing I93M mutation

produces inclusions without inhibition of proteasomal

activity

We next analysed whether UCH-L1 point mutations I93M

and S18Y caused changes to its cellular distribution. I93M is

proposed to be responsible for disease in a German PD

family (Leroy et al. 1998), whereas the S18Y polymorphism

may be protective against PD (Maraganore et al. 1999;

Zhang et al. 2000a; Elbaz et al. 2003).

In the absence of MG132, overexpression of the UCH-L1

I93M mutant produced significantly more inclusions than

either wild-type or the S18Y polymorphism (50% compared

with 7% and 11%, respectively, p < 0.001) (Fig. 3a and

Table 1). In the presence of MG132, both I93M and S18Y

gave higher levels of inclusions compared with those cells

overexpressing native UCH-L1. Indeed, inclusions were

observed in 99% of cells transfected with the I93M mutant.

The overwhelming majority of inclusions produced in cells

transfected with either I93M or S18Y were observed in the

cytoplasm (Table 1). Nuclear inclusions were not observed

with the S18Y polymorphism. No significant difference in

cell viability was observed with the different constructs.

Both S18Y and I93M UCH-L1 mutant proteins were

detected exclusively in the soluble fraction of cell lysates

(Fig. 3b). By contrast, both S18Y and I93M UCH-L1 mutant

proteins were detected in the insoluble fraction when

proteasomal activity was inhibited. These results mirror those

observed with wild-type UCH-L1. There was no obvious sign

of high molecular weight products either in the presence or

absence of proteasome inhibitor (data not shown).

As the S18Y polymorphism is thought to be protective, we

also investigated whether co-transfection of S18Y with the

I93M mutant would reduce the number of inclusions formed

in the co-transfected cells compared with I93M alone



(Fig. 3c). The double transfection appeared to reduce

significantly the number of inclusions observed (p < 0.01),

although the numbers were not reduced compared with those

of S18Y alone.

Wild-type and mutant forms of UCH-L1 are

immunoprecipitated with polyubiquitylated

species when proteasomal activity is inhibited

We investigated whether insoluble UCH-L1 aggregates were

ubiquitylated as it had previously been noted that many

aggregation-prone proteins are ubiquitylated when protea-

somal activity is inhibited. HA-tagged, normal and mutant

UCH-L1 constructs were immunoprecipitated using HA

antibodies from cell lysates prepared from transfected cells

(Fig. 3d). High molecular weight ubiquitylated species were

observed after immunoprecipitation of all three HA-tagged

UCH-L1 constructs. By contrast, they were not observed

either in the absence of MG132, or in mock or vector alone

transfected cells (Fig. 3d, top panel left-hand side). Further-

more, monoubiquitylated UCH-L1 was not detected. The

presence of multiple ubiquitin species in total cell lysates

indicated that lack of monoubiquitylated species was not due

to a lack of ubiquitin availability in these samples (Fig. 3d,

top panel right-hand side).

Stripping and re-probing of the blot with antibodies to

UCH-L1 (Fig. 3d middle panels) or HA (Fig. 3c lower

panels) confirmed the specificity of the immunoprecipitation.

Elevated levels of the UCH-L1 constructs in the immuno-

precipitated samples after incubation with MG132 were

observed. This increase was not apparent in non-immuno-

precipitated lysates.

Co-localization of UPS and chaperone system

components to UCH-L1 inclusions

Many neurological-associated inclusions are ubiquitin-posit-

ive and stain for other components of the UPS and chaperone

system (Lowe et al. 1988; Pollen et al. 1993; Saudou et al.

1998; Cummings et al. 1999). Therefore, we analysed the

expression patterns of UCH-L1 and other components of the

UPS or chaperone system in cells overexpressing UCH-L1 in

which proteasomal activity was inhibited (Fig. 4). Prior to

proteasome inhibition, all UPS and chaperone components

localized to regions as previously described (Bush et al.

1997; Waelter et al. 2001; Ardley et al. 2003; Muqit et al.

2004). UCH-L1 co-localized with ubiquitin throughout the

inclusion (Fig. 4a). Indeed, consistent with our immunopre-

cipitation data, co-localization of UCH-L1 with an antibody

that recognizes only ubiquitin conjugates (Fujimuro et al.

1994) indicates that the inclusions comprise ubiquitylated

proteins rather than free ubiquitin moieties (Fig. 4b). A

predominant nuclear staining pattern was observed with anti-

20S proteasomal antibodies when proteasomal activity was

inhibited. Only weak 20S staining was localized within the

UCH-L1 inclusions (Fig. 4c).

Fig. 2 Characteristics of UCH-L1 inclusions. COS-7 cells were

transfected with UCHL1-HA. At 28 h post-transfection, cells were

incubated in the presence (a-c) or absence (c) of 5 lM MG132 for

16 h. (a) and (b) Cells were fixed and stained as described in Fig. 1.

(a) UCH-L1-induced inclusions appear to localize to the cytoplasm

(left-hand panel), nuclei (middle panel) or both (right-hand panel).

Arrows indicate large cytoplasmic inclusions; arrowheads, nuclear

inclusions. (b) Magnification of the aggregated UCH-L1 indicates a

‘honeycomb’-like structure. Boxed area in left-hand panel is magni-

fied ·3 in right-hand panel. Scale bars 20 lm. (c) Inhibition of pro-

teasomal activity leads to the formation of insoluble UCH-L1

aggregates. Protein lysates were prepared as described in Fig. 1. A

10 lg aliquot of each soluble (S) and insoluble (I) fraction was sep-

arated by SDS-PAGE and analysed by western blotting with either

mouse anti-HA (upper panel), or anti-b-Actin (lower panel), antibodies.

The positions of individual molecular mass markers are indicated.



Proteasome inhibition can cause an increase in the level of

chaperones including BiP and HSP70 (Kuznetsov et al. 1996;

Bush et al. 1997; Ardley et al. 2003). Moreover, BiP and

HSP70 have been observed in PD-related inclusions (Junn

et al. 2002; Muqit et al. 2004; Tanaka et al. 2004). Therefore,

we investigated whether BiP or HSP70 were also found in

UCH-L1 inclusions. BiP co-localized with UCH-L1 within

inclusions but in discrete foci (Fig. 4d, arrowheads indicate

regions of co-localization). HSP70 co-localized with UCH-L1

within inclusions, although HSP70 was also observed within

the nucleus of the cells (Fig. 4e). HSP70 containing inclu-

sions were also observed in untransfected cells after MG132

treatment (Fig. 4e, cell indicated by an asterisk).

Parkin and a-synuclein co-localization in UCH-L1

inclusions

Both a-synuclein and Parkin are found in Lewy bodies in

idiopathic PD. Therefore, we investigated whether these

proteins co-localized within UCH-L1 inclusions. A signifi-

cant proportion of endogenous a-synuclein was found to co-localize with inclusions in cells overexpressing UCH-L1

(Fig. 5a).

Previously, we were unable to detect endogenous Parkin

in COS-7 cells (Ardley et al. 2003). Therefore, overexpres-

sion of UCHL1-HA and FLAG-Parkin was performed to

analyse any regions of co-localization within inclusions

(Fig. 5b). Most cells expressing both proteins generated

inclusions that contained both UCH-L1 and Parkin

co-localizing in the aggregate [Fig. 5b(i)]. However, a small

proportion of these cells contained inclusions where each

protein localized to different areas. For example, in

Fig. 5b(ii), UCH-L1 staining was found within the central

core region of the inclusion with partial Parkin

co-localization. By contrast, the majority of Parkin staining

was found at the periphery of the inclusion. Of interest, we

also noted the presence of a few cells that contained

aggregates of one protein only, despite the overexpression

of both. For example, in Fig. 5b(iii), Parkin expression was

observed throughout the cytoplasm despite the presence of

UCH-L1 inclusions.

Aggresome characteristics of UCH-L1 inclusions

We wished to establish whether UCH-L1 aggregates had

aggresome-like characteristics similar to those observed

previously with Parkin (Fig. 5b and Ardley et al. 2003).

First, we investigated whether UCH-L1 inclusions displayed

the microtubule-dependent properties characteristic of classi-

cal aggresomes (Fig. 6a) (Johnston et al. 1998; Garcia-Mata

et al. 1999). COS-7 cells overexpressing UCHL1-GFP were

treated with MG132, the microtubule disruption reagent,

nocodazole, or both, prior to fixation and immunostaining.

Co-staining with a-tubulin antibodies was performed in orderto confirm disruption of microtubules. Prior to drug treatment,

UCH-L1 displayed a mostly cytoplasmic localization. It did

not co-localize with a-tubulin. By contrast, in the presence ofMG132, bundling of a-tubulin around the aggregate was

observed in the majority of cells containing UCH-L1

(a)

(c)

(b)

(d)

Fig. 3 Effect of Parkinson’s disease-associated UCH-L1 mutations on

inclusion formation. COS-7 cells were transfected with UCHL1-HA

(WT) or HA tagged mutant protein (S18Y, I93M). At 28 h post-trans-

fection, cells were incubated in the presence or absence of 5 lM

MG132 for 16 h as indicated. (a) I93M overexpression causes inclu-

sion formation in the presence or absence of MG132. The expression

of UCH-L1 was analysed by immunofluorescence using anti-HA anti-

bodies. Cells were scored for the presence of inclusion bodies. Light

grey bars indicate untreated cells; dark grey bars indicate MG132-

treated cells. Error bars indicate the standard error from the mean.

Asterisk(s) indicate a significant difference between the number of

inclusions observed with each construct compared with wild-type

UCHL1-HA in the presence or absence of MG132: *p < 0.05;

**p < 0.001. (b) UCH-L1 and its mutants form insoluble inclusions only

in the presence of proteasome inhibitors. Cell lysates were prepared

and analysed as described for Fig. 2. Blots were probed with mouse

anti-HA (upper panel) and anti-b-Actin (lower panel) antibodies.

(c) Overexpression of S18Y with I93M may protect against inclusion

formation. The number of cells containing inclusions was scored as

described in (a) in cells expressing either S18Y, I93M or both (S18Y/

I93M). Error bars indicate the standard error from the mean. Aster-

isk(s) indicate a significant difference between the number of inclu-

sions observed with each construct compared with I93M in the

absence of MG132: *p < 0.01; **p < 0.001. (d) UCH-L1 and its

mutants associate with polyubiquitylated species in proteasomal

inhibited cells. Cell lysates were immunoprecipitated using anti-HA

antibodies and size fractionated by SDS PAGE. A 20 lL aliquot of

each total cell lysate was also analysed in parallel (no IP). The

membrane was probed with anti-ubiquitin antibodies (a-Ub). The

success of the immunoprecipitation was ascertained by western

blotting with anti-HA or anti-UCH-L1 antibodies (lower panels).

Endogenous UCH-L1 expression was observed in the total cell lysates

from the mock and vector alone samples. The positions of individual

molecular mass markers are indicated.



inclusions (Fig. 6a, arrowheads). In nocodazole-treated cells,

UCH-L1 inclusions were not detected in the majority of cells.

Conversely, UCH-L1 inclusion bodies were observed in cells

overexpressing UCH-L1 after the addition of both MG132

and nocodazole (Fig. 6a).

Aggresome formation routinely causes disruption of

c-tubulin within cells (Johnston et al. 1998; Garcia-Mata

et al. 1999). UCH-L1 transfected cells were cultured in the

presence of MG132 and stained with c-tubulin antibodies inorder to assess the effect of UCH-L1 aggregates on c-tubulindistribution. UCH-L1 co-localized with c-tubulin at the

centrosome in some cells, but not others [Fig. 6b, compare

the lower cell where the yellow staining indicates that

UCH-L1 co-localizes with c-tubulin to the centrosome

(arrowheads) with the upper cell where UCH-L1 is found

around the centrosome as indicated by red c-tubulin stainingand green UCH-L1 staining].

Aggresomes (Johnston et al. 1998; Garcia-Mata et al.

1999) and aggresome-like inclusions (Meriin et al. 2001;

Lee et al. 2002) are often surrounded by a vimentin cage-like

structure. Co-immunostaining of UCH-L1 transfected cells

with anti-GFP and anti-vimentin antibodies (Fig. 6c)

revealed disruption of vimentin filament staining, but little

co-localization of UCH-L1 and vimentin.

Discussion

The purpose of this study was twofold. We wished first, to

address the question of whether mutants of UCH-L1 are

prone to aggregate into inclusions and second, to identify the

impairment or activation of which cellular function(s) may be

responsible for UCH-L1 inclusion formation. Such informa-

tion may help to explain its presence in Lewy bodies in

idiopathic and hereditary forms of PD.

Inhibition of ER to Golgi transport, or induction of the

unfolded protein response, did not result in UCH-L1

inclusion formation. By contrast, inclusions were formed

in cells overexpressing UCH-L1 when the proteasomal

activity was inhibited. These inclusions were predominantly

cytoplasmic, ubiquitylated and insoluble structures with a

chain-like appearance. MG132 produced far more inclusions

than treatment of cells with lactacystin. However, the

number of inclusions in lactacystin-treated cells did not

increase to levels observed with MG132 when together with

the calpain inhibitor, calpeptin. These data suggested that

the elevated number of inclusions with MG132 is unlikely

to be due to inhibition of other proteases such as calpain.

This is perhaps not surprising given that inhibition of such

protease activities requires at least a 10-fold higher

concentration of MG132 than for the proteasome (Tsubuki

et al. 1996). MG132 is the most potent inhibitor of the

proteasome (Bush et al. 1997; Kisselev and Goldberg

2001). This is likely to explain the apparent discrepancies

in the potency of MG132 and lactacystin to generate

inclusions (67% of cells contained inclusions compared

with 28%). MG132, unlike lactacystin, is able to enter cells

rapidly and it does not require transition to an active form.

These properties allow MG132 to be used at lower

concentrations and its effects are seen more rapidly than

in lactacystin-treated cells.

Unlike our UCH-L1 overexpressing cells, endogenous

UCH-L1 positive inclusions were not observed in COS-7

cells when proteasomal activity was inhibited. This may

indicate differences in response in different cell types, or it

may indicate that prolonged proteasomal impairment, as

observed in aged organisms, is required for the endogenous

protein to aggregate.

We have previously observed that different Parkin muta-

tions, which are known to disrupt its ubiquitin-protein ligase

activity, demonstrate different aggregation potentials (Ardley

et al. 2003). Furthermore, Parkin mutants often cause rapid

inclusion formation in the absence of proteasome inhibitors

Table 1 Analysis of wild-type and mutant UCH-L1 inclusion formation in the presence or absence of proteasomal inhibitors. COS-7 cells trans-

fected with HA tagged wild-type (UCH-L1) or UCH-L1 mutants (S18Y; I93M) were incubated in the presence or absence of MG132 for 16 h at 28 h

post-transfection. The percentage of cells containing inclusions was assessed by immunofluoresence using anti-HA antibodies as described for

Fig. 1. Experiments were repeated at least three times and a minimum of 300 transfected cells were analysed for each sample. ± SE ¼ ± standard

error of the mean; ‘cytoplasmic’, ‘nuclear’ and ‘both’ refer to the localization of the observed inclusions

Construct

Presence

of MG132

% of cells.

Containing inclusions

(Mean ± SE) Cytoplasmic

Both cytoplasmic

and nuclear Nuclear

UCH-L1 – 7 ± 1.0 7 ± 1.0 0 0

UCH-L1 + 70 ± 3.2 64 ± 2.9 4.5 ± 0.3 1.5 ± 1.2

S18Y – 11 ± 1.6 11 ± 1.6 0 0

S18Y + 80 ± 2.7 74 ± 3.4 6.0 ± 0.9 0

I93M – 50 ± 2.6 50 ± 2.6 0 0

I93M + 99 ± 0.5 91 ± 1.2 7.5 ± 1.9 1.5 ± 0.6



(Ardley et al. 2003; Cookson et al. 2003). We have now

demonstrated that different UCH-L1 mutants behave simi-

larly. The I93M mutant has an inherent ability to form

inclusions that was not observed with either the wild-type or

the S18Y isoform. However, inhibition of proteasomal

activity then resulted in a dramatic increase in the number

of cells containing inclusions with both wild-type and mutant

isoforms. Indeed, inclusions were present in virtually all

I93M-expressing cells. Interestingly, the protective S18Y

polymorphism appears to be capable of reducing inclusion

formation in cells co-transfected with the PD mutant I93M,

suggesting that S18Y may rescue I93M cells from inclusion

formation. Although as both proteins were HA-tagged we

cannot rule out the possibility that the S18Y construct was

preferentially expressed in the co-transfected cells, western

blotting data do not support this proposal. These findings are

particularly relevant to PD onset as proteasomal activity in

neuronal cells decreases with age (Goto et al. 2001) and is

notably lower in PD patients (McNaught and Jenner 2001;

McNaught et al. 2003).

(a)

(b)

(c)

(d)

(e)

Fig. 4 Co-localization of UCH-L1 with

components of the UPS and chaperone

systems. COS-7 cells were transfected with

the UCHL1-HA (a and c) or UCHL1-GFP

(b, d and e) expression construct and cul-

tured in the presence of MG132 for 16 h.

Cells were probed with the following: (a)

anti-HA and anti-ubiquitin (Ub) antibodies;

(b) anti-GFP and anti-ubiquitylated proteins

(PolyUb) antibodies; (c) anti-HA and anti-

20S antibodies; (d) anti-GFP and anti-BiP

antibodies; (e) anti-GFP and anti-HSP70

antibodies. Staining as follows: left-hand

panel: HA or GFP (green) to identify

UCH-L1; middle panel: ubiquitin, polyubi-

quitylated proteins, 20S, BiP or HSP70

(red); right-hand panel represents the

overlay of each set with the addition of

DAPI staining (blue) to identify the nucleus.

Regions of co-localization within cells stain

yellow. Arrows indicate the positions of

inclusions. Arrowheads in (d) indicate

co-localization within parts of the inclusion.

The asterisk indicates an untransfected cell.

Scale bars 20 lm.



(a)

(b)

Fig. 5 Co-localization of UCH-L1 with Parkinson’s disease-associated

proteins. (a) COS-7 cells were transfected with UCHL1-GFP and

cultured in the presence of MG132 for 16 h. Cells were fixed and

stained with anti-GFP (green) and monoclonal anti-a-synuclein (red).

(b) COS-7 cells were transfected with UCHL1-HA and FLAG-Parkin

and cultured in the presence of MG132 for 16 h. Cells were fixed and

stained with anti-HA (red) to identify UCH-L1 and anti-Parkin (green)

to identify FLAG-Parkin. UCH-L1 and Parkin co-localize throughout

the inclusion in some cells (i), partially in some cells (ii), or not at all (iii)

in cells expressing both proteins. The right-hand panel in (a), and all

panels in (b), represents the overlay of each set with the addition of

DAPI staining (blue) to identify the nucleus. Regions of co-localization

within cells stain yellow. Arrows indicate co-localization within inclu-

sions; arrowhead indicates presence of an UCH-L1 inclusion in cell

which is also overexpressing Parkin. Scale bars 20 lm.

(a)

(b)

(c)

Fig. 6 UCH-L1 inclusions exhibit properties of ‘ribbon-like’ aggre-

somes. COS-7 cells were transfected with UCHL1-GFP. (a) UCH-L1

aggregates are stable in the presence of nocodazole, but cause

bundling of a-tubulin. Cells were cultured in the presence of MG132,

nocodazole or both for 16 h as indicated. Cells were fixed and stained

with anti-GFP (green), anti-a-tubulin (red) and DAPI (blue). Bundling of

a-tubulin is observed in MG132 cells as indicated by arrowheads. (b)

Large UCH-L1 inclusions co-localize with c-tubulin at the centrosome.

Cells were cultured in the presence of MG132 for 16 h. Cells were

fixed and stained with anti-GFP (green), anti-c-tubulin (red) and DAPI

(blue). Arrowheads indicate the position of the centrosome in each cell.

(c) UCH-L1 inclusions are not surrounded by a vimentin cage. Cells

were cultured in the presence of MG132 for 16 h. Cells were fixed and

stained with anti-GFP (green), anti-vimentin (red) and DAPI (blue). In

(a)-(c) arrows indicate the position of inclusions. Scale bars 20 lm.



Interestingly, the I93M mutant form of UCH-L1 only

produced insoluble protein when proteasomal activity was

inhibited, i.e. when it is ubiquitylated, but cannot be degraded

by the proteasome. Under these conditions, western blot

analysis of UCH-L1 immunoprecipitated from transfected cell

lysates revealed multiple high molecular weight ubiquitylated

species. Their presence apparently correlated with the number

of inclusions that we observed by immunofluorescence

(compare Figs 3a and 3b). These high molecular weight

species may represent endogenous ubiquitylated UCH-L1

targets, or they may indicate that UCH-L1 itself is a UPS

target. Indeed, polyubiquitylation of the cytokine-inducible

DUB, DUB-1 A, has recently been demonstrated (Baek et al.

2004). Conversely, ubiquitylated species may represent an

artificial state brought about by overexpression of UCH-L1

but which may be recapitulated in neurodegenerative disor-

ders when the proteasomal activity is compromised.

Although the vast majority of inclusions were cytoplasmic,

a small number of cells contained either nuclear inclusions, or

both cytoplasmic and nuclear inclusions, with wild-type

UCH-L1 or the I93M mutant. By contrast, the S18Y isoform

produced no nuclear inclusions. Whether this has some

correlation with the proposed protective function of the S18Y

mutant is not known. However, it may be particularly relevant

to HD where the majority of inclusions are found in the

nucleus (Davies et al. 1997; DiFiglia et al. 1997).

Recently, Osaka et al. (2003) reported that UCH-L1 binds

to, and stabilizes, monoubiquitylated products in neurones.

However, we did not detect any monoubiquitylated species

in our immunoprecipitates or elevated levels of ubiquitin in

the cell lysates. Differences in blotting techniques, transfec-

tion efficiencies or cell type may account for these apparent

discrepancies between the two studies.

The PD-associated proteins Parkin and a-synuclein are

components of Lewy bodies (Spillantini et al. 1997; Shimura

et al. 1999). We demonstrated that both proteins co-localize

with UCH-L1 in some, but not all, inclusions. This is

particularly interesting given that a-synuclein can be targetedto the 26S proteasome by both Parkin and UCH-L1 (Choi

et al. 2001; Shimura et al. 2001; Liu et al. 2002), and

suggests that the protein composition of individual Lewy

bodies is likely to be different. Ubiquitylated proteins and

chaperone proteins were also found to co-localize with UCH-

L1 inclusions. In addition, a weak association with 20S

proteasomal subunits was observed. Our data suggest that

UCH-L1 inclusions may represent aggresomes. This type of

proteinaceous aggregate forms in response to overloading of

the proteasome (Johnston et al. 1998). However, UCH-L1

inclusions were not disrupted by nocodazole and caused

bundling of a-tubulin to form a ‘tubulin-cage’ similar to

findings with Parkin (Ardley et al. 2003). Furthermore,

inclusions co-localized with c-tubulin in many cells and

caused disruption of the vimentin network. Collectively,

these results indicate that UCH-L1 inclusion bodies may not

be classical aggresomes, but resemble the ‘ribbon-like’

aggresomes recently described by Garcia-Mata et al. (2002).

Taken together with previous work on Parkin and other

PD-related inclusions (Rideout et al. 2001; Junn et al. 2002;

Lee et al. 2002; Ardley et al. 2003), this study further

indicates the importance of recruitment of UPS and a

chaperone system in aggresome formation. The presence of

different UPS and chaperone components in specific inclu-

sion bodies strongly suggests that the recruitment of these

proteins to the aggregates is a late event in their formation,

and reflects the cell’s attempt to remove excess proteins.

In summary, our data indicate that UCH-L1 is prone to

aggregate especially when proteasomal activity is reduced,

findings similar to those observed with other PD-associated

proteins such as Parkin and a-synuclein (Rideout et al. 2001;Junn et al. 2002; Lee et al. 2002; Ardley et al. 2003;

Cookson et al. 2003; Muqit et al. 2004).

It is currently unclear whether PD patients with the I93M

mutation or the S18Y genotype display Lewy bodies

(McNaught and Olanow 2003; Dekker et al. 2003). In the

current study, overexpression of the PD-associated I93M

UCH-L1 variant, which has diminished DUB activity, caused

inclusion formation when proteasomal activity was at normal

levels. Moreover, co-expression with S18Y could partially

rescue this phenotype. These observations suggest that

inclusions may be expected in the German kindred charac-

terized by the I93M mutation but perhaps not in those with

the S18Y polymorphism unless proteasomal activity is

impaired.

This proposal is in agreement with the protective effect of

S18Y against PD and HD observed in many population

studies (Maraganore et al. 1999; Zhang et al. 2000a; Elbaz

et al. 2002; Naze et al. 2002). It will now be interesting to

establish the UCH-L1 genotype/phenotype relationship of

these inclusions and compare them with those found in other

neurodegenerative disorders where UCH-L1 may be protect-

ive and, in particular, inclusions associated with HD.

Interestingly, the recently described DUB, synUSP, local-

izes to the post-synaptic density and post-synaptic lipid rafts

of neuronal cells (Tian et al. 2003). Moreover, synUSP maps

to chromosome 1p36.2 within the region of two identified

PD loci, PARK6 (Valente et al. 2001) and PARK9 (Hamp-

shire et al. 2001). Given the cellular localization of synUSP,

and the UCH-L1 association with PD, synUSP may represent

a strong candidate responsible for disease in PARK6 and/or

PARK9 families. Furthermore, it will be extremely interest-

ing to determine whether synUSP is present in Lewy bodies

and whether it is also prone to aggresome formation.

Acknowledgements

HCA is supported by a Research into Ageing, Fellowship Award.

Work in the authors’ laboratory is also supported by The Wellcome

Trust, The Royal Society and Yorkshire Cancer Research.



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UCH-L1 aggresome formation in response to proteasome impairment indicates a role in inclusion formation in Parkinson's disease

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UCH-L1 aggresome formation in response to proteasome impairment indicates a role in inclusion formation in Parkinson's disease