Caspase-mediated proteolysis of the polyglutamine disease protein ataxin-3

Caspase-mediated proteolysis of the polyglutamine disease protein

ataxin-3

Sarah J. Shoesmith Berke,*,� Francisca A. Flores Schmied,� Ewout R. Brunt,� Lisa M. Ellerby§and Henry L. Paulson*,�

*Neuroscience Graduate Program and the �Department of Neurology, University of Iowa, Iowa City, Iowa, USA

�Department of Neurology, University Hospital Groningen, Groningen, The Netherlands

§Buck Institute for Research in Aging, Novato, California, USA

Abstract

Spinocerebellar ataxia type-3, also known as Mach-

ado-Joseph Disease, is one of many inherited neurodegen-

erative disorders caused by polyglutamine-encoding CAG

repeat expansions in otherwise unrelated disease genes.

Polyglutamine disorders are characterized by disease protein

misfolding and aggregation; often within the nuclei of affected

neurons. Although the precise mechanism of polyglutamine-

mediated cell death remains elusive, evidence suggests that

proteolysis of polyglutamine disease proteins by caspases

contributes to pathogenesis. Using cellular models we now

show that the endogenous spinocerebellar ataxia type-3 dis-

ease protein, ataxin-3, is proteolyzed in apoptotic paradigms,

resulting in the loss of full-length ataxin-3 and the corres-

ponding appearance of an approximately 28-kDa fragment

containing the glutamine repeat. Broad-spectrum caspase

inhibitors block ataxin-3 proteolysis and studies suggest that

caspase-1 is a primary mediator of cleavage. Site-directed

mutagenesis experiments eliminating three, six or nine

potential caspase cleavage sites in the protein suggest

redundancy in the site(s) at which cleavage can occur, as

previously described for other disease proteins; but also map

a major cleavage event to a cluster of aspartate residues

within the ubiquitin-binding domain of ataxin-3 near the poly-

glutamine tract. Finally, caspase-mediated cleavage of

expanded ataxin-3 resulted in increased ataxin-3 aggregation,

suggesting a potential role for caspase-mediated proteolysis

in spinocerebellar ataxia type-3 pathogenesis.

Keywords: ataxin-3, caspase, proteolysis, Machado-Joseph

disease, polyglutamine disease, spinocerebellar ataxia type-3.

J. Neurochem. (2004) 89, 908–918.

Evidence from many experimental systems links the clea-

vage of polyglutamine (polyQ) disease proteins by cellular

proteases to disease pathogenesis. First, polyQ protein

fragments often aggregate and cause cell death more readily

than their full-length protein counterparts, and are sufficient

to produce disease in various cellular, invertebrate and

vertebrate models (Satyal et al. 2000; Wellington et al. 2000;

Morley et al. 2002; Sanchez et al. 2003). Second, activated

caspases have been detected in numerous models of disease,

and inhibition of caspase activity suppresses disease protein

proteolysis and subsequent pathogenesis in vitro and in vivo

(Ellerby et al. 1999a; Ellerby et al. 1999b; Kim et al. 1999;

Sanchez et al. 1999; Li et al. 2000; Wellington et al. 2000).

Finally, in some polyQ diseases proteolytic fragments have

been observed in susceptible regions of human disease brain

tissue, with the appearance of such fragments preceding the

onset of disease phenotypes (Gafni and Ellerby 2002; Garden

et al. 2002; Wellington et al. 2002).

Caspases are cysteine proteases that cleave proteins at

specific aspartate residues (Earnshaw et al. 1999). Caspase

Received November 25, 2003; revised manuscript received December

18, 2003; accepted December 22, 2003.

Address correspondence and reprint requests to Sarah J Shoesmith

Berke, University of Iowa, Department of Neurology, 240 A EMRB,

Paulson Laboratory, Iowa City, Iowa 52242.

E-mail: [email protected]

Abbreviations used: ALLN, N-acetyl-leucinyl-leucyl-norleucinal; CI,

caspase inhibitor; DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde-

3-phosphate dehydrogenase; HD, Huntington Disease; PARP, poly-

adenosine ribose phosphatase; polyQ, polyglutamine; SCA3/MJD,

Spinocerebellar ataxia type-3/Machado-Joseph Disease; ST, staurospo-

rine.

Journal of Neurochemistry, 2004, 89, 908–918 doi:10.1111/j.1471-4159.2004.02369.x

908 � 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 908–918

involvement in polyQ pathogenesis appears to be complex,

varying in the details among the polyQ diseases. An initiator

role is supported by studies in a mouse model of Huntington

Disease (HD), in which caspase-derived proteolytic frag-

ments were observed prior to behavioral or pathological

signs of disease (Wellington et al. 2002). Continued pro-

duction of toxic polyQ fragments may further propagate

cellular toxicity and aggregation (Ellerby et al. 1999a;

Paulson 2000). Low-grade chronic caspase activation during

disease progression could also have secondary effects on

other cellular targets. Finally, caspases may accelerate

apoptosis leading to the neuronal demise that occurs in this

class of diseases (Ellerby et al. 1999b; Sanchez et al. 1999).

Several polyQ disease proteins are known targets for

caspase cleavage, including huntingtin (caspases -2, -3, -6

and -7), the androgen receptor (caspases -1, -3, -7 and -8)

and atrophin-1 (caspases -1, -3, -7 and -8) (Wellington

et al. 1998; Ellerby et al. 1999a; Ellerby et al. 1999b;

Kim et al. 1999; LaFevre-Bernt and Ellerby 2003).

Caspase involvement varies among the polyQ diseases,

in part because the disease proteins are highly dissimilar,

with the number and position of potential caspase sites

differing for each protein, and in part because different

caspases may be activated in different polyQ diseases.

Moreover, cell-type dependent cleavage by specific casp-

ases or other proteases (Troy and Salvesen 2002) may

explain some of the selective degeneration seen in these

diseases (Zoghbi and Orr 2000), as evidenced by a recent

report in HD showing that caspase-mediated fragments

were found in susceptible human and mouse brain regions

(Wellington et al. 2002).

In this report, we address the possible role of proteolytic

cleavage in spinocerebellar ataxia type-3 (also known as

Machado-Joseph disease or SCA3/MJD). SCA3/MJD is the

most common dominantly inherited ataxia and one of at least

nine neurodegenerative polyQ diseases (Paulson 2000). As in

other polyQ diseases, there is regional selectivity to pathol-

ogy. Although mutant ataxin-3, the SCA3/MJD disease

protein, is expressed widely throughout the brain, only

specific brain regions degenerate, including neuronal popu-

lations within the globus pallidus, midbrain, pons and spinal

cord. While many polyQ proteins are known targets for

caspase-mediated cleavage, characterization of the caspase

cleavage of ataxin-3 has not been thoroughly investigated.

Wellington et al. (Wellington et al. 1998) showed a modest

production of ataxin-3 fragments by caspases in vitro. In

contrast, Evert et al. (1999) reported that mutant ataxin-3 is

not prone to fragmentation in stably transfected cell lines.

Although there have been no descriptions of ataxin-3

fragments in SCA3/MJD human disease tissue, a recently

developed SCA3/MJD mouse model suggests the accumu-

lation of proteolytic fragments (Goti and Colomer 2003). A

possible explanation for these divergent results is that

caspase cleavage depends on protein–protein interactions

(Earnshaw et al. 1999), which may be affected by cellular

environment or by the overexpression of ataxin-3.

Here we show that ataxin-3 is a target for caspase-

mediated cleavage in cell culture. Full-length ataxin-3 is

cleaved during staurosporine (ST)-induced apoptosis in all

cell types tested and leads to the appearance of an ataxin-3

fragment that contains the glutamine repeat in at least one

cell type. In our cellular model, this proteolysis is mediated

predominantly by caspase-1, and site-directed mutagenesis

experiments narrows the major cleavage site to three

aspartate residues clustered in the ubiquitin binding region

of ataxin-3 near the polyQ domain. Interestingly, aggregation

of mutant ataxin-3 was increased in ST treated cells, and

blocked by caspase inhibition, consistent with caspase

involvement in SCA3/MJD pathogenesis.

Experimental procedures

Cell culture and transfection

Methods for cell culture of Cos-7, HeLa and PC6-3 cells have been

described previously (Chai et al. 1999a, b). NIH3T3 cells were

grown in Delbecco’s modified Eagle’s medium (DMEM; Gibco

BRL, Grand Island, NY, USA) supplemented with 10% FBS

(HyClone, Logan, UT, USA) and 100 U/mL/500 lg/mL penicillin/

streptomycin (P/S; Gibco BRL). NT2 cells were grown in Opti-

minimum essential medium (MEM; Gibco BRL) supplemented with

5% FBS and 1% P/S. All cells were maintained at 37�C with 5%

CO2. PC63 cells were differentiated using 100 ng/mL nerve growth

factor (NGF; Promega, Madison, WI, USA) maintained in 4% HS,

2%FBS with 1% P/S. For transient transfections, 50% confluent

dishes were transfected with 1 lg of expression plasmid DNA using

LipofectAMINE PLUSTM Reagent (Invitrogen, Carlsbad, CA,

USA).

Apoptotic paradigms and treatments

Twenty-four hours after plating, apoptosis was induced by incuba-

ting cells with 1 lM ST in serum free media (ST; Sigma; St. Louis,

MO, USA). Control cells were incubated with equivalent volumes

of the vehicle, dimethylsulfoxide (DMSO). To block caspase

activation, Z-VAD-FMK or specific caspase inhibitors to caspases

-1, -2, -3, -5, -6, -8 and -9 were used at 100 lM and 10 lM,respectively (Calbiochem, San Diego, CA, USA). Lactacytsin was

used to inhibit proteasome activity, and N-acetyl-leucinyl-leucyl-

norleucinal (ALLN) to block calpain activity at 10 lM and 100 lM,respectively (Calbiochem). All inhibitors were added at the time of

ST treatment except ALLN, which was added 1 h prior to ST

treatment.

Western blot analysis

Cell lysates were collected 24 h after ST treatment. Both live and

dead cells from the same condition were harvested into Laemmli

buffer (50 mM Tris pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1%

bromophenol blue, 10% glycerol) and then pooled. Lysates were

analyzed by western blot as described previously (Chai et al.

1999b). Ataxin-3 was detected with rabbit polyclonal antibod-

ies, anti-ataxin-3 (1 : 20 000) and 146 pa (1 : 200; gift from

Caspase cleavage of ataxin-3 909

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 908–918

V. Colomer, Johns Hopkins University) mouse monoclonal anti-

bodies, anti-1H9, and anti-2B6 (1 : 2000; gift from Y. Trottier,

Louis Pasteur University), and rabbit polyclonal antibodies anti-HA,

or anti-myc (1 : 1000; Santa Cruz, Santa Cruz, CA, USA). Polya-

denosine ribose phosphatase (PARP) expression was detected using a

rabbit polyclonal antibody, anti-PARP (1 : 500; Santa Cruz), and the

loading control glyceraldehyde-3-phosphate dehydrogenase (GAP-

DH) was assessed with anti-GAPDH mouse monoclonal antibody

(1 : 1000; Research Diagnostics Inc., Flanders, NJ, USA). Secondary

antibodies were peroxidase-conjugated goat anti-rabbit or mouse

antisera (1 : 15 000; Jackson Laboratories, West Grove, PA, USA).

Site-directed mutagenesis

Site-directed mutagenesis was performed using the QuikChange Kit

(Stratagene, La Jolla, CA, USA) to generate the pCMX-HA-ataxin-3

(Q35) D225N, pCMX-HA-ataxin-3(Q35)D225/228 N and pCMX-

HA-ataxin-3(Q35)-6mut constructs (Ellerby et al. 1999a, b; LaFe-

vre-Bernt and Ellerby 2003). The pFLAG-CMV6a-ataxin-3(Q28)-

3mut and pFLAG-CMV6a-ataxin-3(Q35)-9mut constructs were

generated by PCR amplification using pCDNA3-ataxin-3(Q28)

and pCMX-HA-ataxin-3(Q35)-6mut as templates, respectively. The

generated PCR fragments were then subcloned into the pFLAG-

CMV6a vector (Sigma) and sequence verified.

Plasmids

The plasmids, pcDNA3-HA ataxin-3(Q28) and pcDNA3-HA ataxin-

3(Q84) were used to generate doubly tagged ataxin-3 constructs.

Normal and expanded ataxin-3 epitope tagged with myc at the

C-terminus were generated by PCR amplification of an Eco0109

I/Apa I fragment with pcDNA3-HA ataxin-3(Q28) as the template,

then subcloning this fragment into pcDNA3-HA ataxin-3(Q28) and

pcDNA-HA-ataxin-3(Q84). All constructs were sequence verified.

Human disease tissue

We looked for ataxin-3 fragments in two different SCA3/MJD

disease brains. The first brain, designated MJD-LL, was from a

31-year-old female in late stages of SCA3/MJD, described in

(Paulson et al. 1997a). The CAG repeat lengths were approximately

29 and 82 repeats. The second SCA3/MJD brain, S017-01, was

from a 34 years old male who died in advanced-stage disease with

cytosine-adenine-guanine (CAG) repeat lengths of 20 and 75. Tissue

samples were collected at 22 h for MJD-LL and 4 h for S017-01.

Normal disease brain tissue was from a 58 years old female who

died of non-neurologic causes, with a postmortem interval of 16 h.

For western blot analysis, lysates were collected in 50 mM Tris

(pH ¼ 7.5) supplemented with 1% triton-X-100, 1 mM EDTA and

protease inhibitors, pepstatin, leupeptin, and PMSF. The tissue was

then homogenized centrifuged at 75 600 g for 30 min, after which

soluble and pelleted samples were separated for further analysis. For

the soluble protein, the protein concentration was determined using

Protein Assay Reagent (Bio-Rad, Hercules, CA, USA), and 100 lgof protein was lysed in Laemmli buffer. To the pelleted protein, a

similar volume of 50 mM Tris was added, the pellet resuspended,

and half of the sample was boiled at 100�C for 4 min. Both pelleted

samples were then heated at 37�C for 10 min, sonicated, and protein

concentration determined. The desired amount of protein was then

lysed in Laemmli buffer.

Results

Endogenous ataxin-3 is proteolyzed during apoptosis

Caspases cleave many polyQ disease proteins, in some cases

producing cleavage products that may contribute to disease

pathogenesis (Ellerby et al. 1999a; Ellerby et al. 1999b;

Wellington et al. 2002). Evidence for cleavage of ataxin-3

has only been demonstrated in vitro with recombinant

disease protein and purified caspases (Wellington et al.

1998). We analyzed the protein sequence of ataxin-3 and

found nine potential caspase cleavage sites (Fig. 1), seven

amino-terminal to the polyQ domain and two in the

Fig. 1 Potential caspase cleavage site

aspartate residues in ataxin-3 are highly

conserved. Shown are the amino acid

sequences of human, mouse and rat ataxin-

3. The originally published C-terminus of

human ataxin-3 is also shown (Orig C;

Kawaguchi et al. 1994; Schmidt et al.

1998). Red boxes highlight nine potential

caspase cleavage sites. *Highlights aspar-

tates that were mutated to asparagines or

alanine in subsequent experiments. The

blue and green lines mark two identified

ubiquitin interaction motifs in ataxin-3.

910 S. J. Shoesmith Berke et al.


C-terminus of the protein (Kawaguchi et al. 1994), one of

which is only present in an alternatively spliced form of

ataxin-3 (Schmidt et al. 1998). At least eight of these

potential cleavage sites are conserved across mammalian

species, suggesting that caspase-mediated proteolysis of

ataxin-3 may be evolutionarily conserved. Some of these

sites may also be conserved due to their importance in the

ubiquitin binding domain of ataxin-3 (Chai et al. 2003;

Donaldson et al. 2003).

This large number of potential caspase cleavage sites

prompted us to study the proteolysis of endogenous ataxin-3

in cells undergoing apoptotic cell death.We induced apoptosis

in cells with staurosporine (ST), which initiates apoptosis by

inhibiting a broad spectrum of cellular kinases. Since caspase

cleavage is influenced by protein–protein interactions and cell

type (Troy and Salvesen 2002), we examined five different

immortalized cell lines and primary cerebellar neurons for ST-

induced cleavage of endogenous ataxin-3. After 24 h treat-

ment with 1 lM ST, cells were lysed and the lysates analyzed

for ataxin-3 cleavage by western blot analysis. The degree of

ataxin-3 proteolysis varied between cell types (Fig. 2).

NIH3T3 mouse fibroblast and NT2 human neural cells

showed complete proteolysis of full-length ataxin-3 while

Cos-7 primate epithelial cells showed a marked reduction.

HeLa human cervical cells, differentiated PC6-3 neural cells

(a derivative of rat PC12 cells) and primary cerebellar granule

neurons showed a modest reduction of ataxin-3. In Cos-7

cells, loss of full-length ataxin-3 was accompanied by the

appearance of an approximately 28-kDa fragment that often

appeared as a doublet. This fragment was recognized by both

monoclonal (1H9) and polyclonal (anti-ataxin-3) ataxin-3

antibodies (Fig. 4a). Thus, ataxin-3 is cleaved in a cell-type

dependent manner during apoptosis in cell culture.

The appearance of an ataxin-3 fragment in Cos-7 cells

undergoing apoptosis is consistent with the toxic fragment

hypothesis, a model of disease pathogenesis in which the

production of polyQ fragments accelerates toxicity in part by

promoting misfolding or aggregation (Ellerby et al. 1999a;

Paulson 2000). To further investigate ataxin-3 proteolysis

and its potential relationship to disease, we performed

additional studies in Cos-7 cells because this cell type

exhibited both the loss of full-length ataxin-3 and the

consistent appearance of a readily observed fragment. A ST

dose–response curve showed that 1–10 lM ST was effective

at inducing ataxin-3 cleavage and the appearance of this

fragment (Fig. 3a). At much higher concentrations (100 lM),cell death occurred rapidly via necrosis rather than apoptosis

(data not shown), which may explain the relative resistance

of full-length ataxin-3 to cleavage at this concentration

(Fig. 3a). Using an optimal concentration of ST (1 lM), weperformed a time course of ataxin-3 proteolysis, assessing

cleavage and the appearance of the fragment at 0, 2, 6, 12

and 24 h (Fig. 3b). Appearance of the 28 kDa ataxin-3

fragment was optimal at 24 h. Therefore, treatment with

1 lM ST for 24 h was used in all subsequent experiments.

Ataxin-3 proteolysis is caspase-dependent, mediated

predominantly by caspase-1

Caspases are activated during ST treatment (Brophy et al.

2002; Caballero-Benitez and Moran 2003), suggesting that

they might mediate ataxin-3 cleavage. We investigated the

involvement of caspases by treating Cos-7 cells with the

broad-spectrum caspase inhibitor, Z-VAD-FMK. Z-VAD-

FMK (100 lM) inhibited both ataxin-3 cleavage and the

appearance of the 28-kDa fragment (Fig. 4a) suggesting that

one or more caspases were responsible for ST-induced

Fig. 2 Endogenous ataxin-3 is cleaved in various cell types during ST

induced apoptosis. Cells were treated with ST or vehicle control

(DMSO) for 24 h, after which live and dead cells were pooled and

lysed for western blot analysis. Shown here are western blot results

obtained with lysates from various non-neuronal (HeLa, Cos-7,

NIH3T3) and neuronal (hNT2, and differentiated PC6-3 cells) cell lines

and primary rat cerebellar granule neurons. Full-length ataxin-3 is

marked by an arrowhead (c), and the 28 kDa ataxin-3 fragment is

marked by an asterisk (*). The differing apparent molecular weights of

ataxin-3 reflect the divergent polyglutamine tract lengths in various

mammalian species.



ataxin-3 cleavage. To determine which caspase(s) mediated

ataxin-3 cleavage, cells were treated with lower concentra-

tions of relatively specific inhibitors (10 lM) to caspases -1,-2, -3, -5, -6, -8, and -9. The caspase-1 inhibitor was most

effective at inhibiting ataxin-3 proteolysis and fragment

production (Fig. 4b), while inhibitors of caspases -5 and -6

led to mild inhibition, and inhibitors of caspases -2, -3, -8 and

-9 had little or no effect. As a positive control for caspase

activation in Cos-7 cells, we compared cleavage of the

known caspase target, PARP (Caballero-Benitez and Moran

2003) in the same cells (Fig. 4a and b, middle panel). For

these experiments, PARP cleavage was not completely

blocked by the specific or broad-spectrum caspase inhibitors

because of the lower concentrations (10 lM) used, whichwere necessary to reduce cross reactivity between the specific

inhibitors.

These results suggested that ST-induced ataxin-3 proteo-

lysis is mediated by caspases, with caspase-1 being a primary

mediator in Cos-7 cells. However, ST treatment can also

activate other cellular proteases including calpains and the

proteasome, both of which have been implicated in the

proteolysis of other polyQ disease proteins (Kim et al. 2001;

Gafni and Ellerby 2002; Goffredo et al. 2002). To test

whether these other proteases played a role in ataxin-3

cleavage, we treated Cos-7 cells with ST and a specific

proteasome inhibitor (lactacystin) or an inhibitor that blocks

both calpains and the proteasome (ALLN). Neither lactacys-

tin nor ALLN suppressed ataxin-3 cleavage, indicating that

the proteasome complex and calpains do not cleave ataxin-3

in this apoptotic paradigm [Fig. 4c,d; staining with

anti-ataxin-3 showed no change in fragment production

(data not shown)].

Domain mapping of the ataxin-3 fragment

To characterize the ataxin-3 fragment, we performed

antibody profiling with monoclonal and polyclonal atax-

in-3 antibodies (Fig. 5a). The fragment was recognized by

a polyclonal antibody directed against the entire protein,

anti-ataxin-3 (Paulson et al. 1997a), and more weakly by

the monoclonal antibody 1H9 (Trottier et al. 1998). A

polyclonal antibody also recognized the fragment, p146a

(V Colomer, unpublished results) directed against the

originally published C-terminus, but not by monoclonal

antibody 2B6 (data not shown) (Trottier et al. 1998).

These results suggested that a carboxy-terminal portion of

ataxin-3 comprises the fragment. Currently available

antibodies, however, did not allow us to determine

whether either the N- or C-termini of ataxin-3 were

present in the fragment. Moreover, because we are

analyzing endogenous ataxin-3 in Cos-7 cells, which

contains a very short polyQ domain, we could not use

the polyQ-specific antibody 1C2 to determine whether the

fragment contains polyQ.

To further define the cleavage fragment, we transfected

Cos-7 cells with normal (28 glutamines, Q28) or mutant

(Q68 and Q84) ataxin-3 constructs doubly tagged at the

N-terminus with a HA epitope and at the C-terminus

with a myc-epitope (designated HA-ataxin-3(Q28)-myc,

HA-ataxin-3(Q68)-myc, and HA-ataxin-3(Q84)-myc;

Fig. 5(b). As shown in Fig. 5(c), the proteolytic ataxin-3

fragment contained the polyQ region as evidenced both by

an incrementally larger ataxin-3 fragment corresponding to

increasing polyQ lengths, and by its recognition with the

monoclonal antibody 1C2 (Fig. 5c third panel). The

ataxin-3 fragment was also recognized by anti-myc

(Fig. 5c second panel) but not anti-HA antibody (data

not shown), indicating that the fragment contains the

C-terminus of ataxin-3. These results were further con-

firmed in separate experiments probing first with either

1C2 or anti-myc.

Interestingly, in cells overexpressing normal or mutant

ataxin-3, we also noted that significant fragment production

(a)

(b)

Fig. 3 ST induced ataxin-3 cleavage occurs in a dose- and time-

dependent manner. (a) The proteolysis of ataxin-3 observed with

increasing concentrations of ST treatment for 24 h in Cos-7 cells. The

relative resistance of ataxin-3 to cleavage at 100 lM ST is likely due to

the rapid necrotic death that is known to occur at that concentration.

(b) A time course of ataxin-3 cleavage with 1 lM ST treatment. Optimal

concentrations chosen for subsequent experiments were 1 lM ST

treatment for 24 h. As a positive control for caspase activity the blots

were probed with anti-PARP (middle panels), and for loading control

with anti-GAPDH (lower panels).



occurred even without ST treatment. This too could be

blocked with caspase inhibitors (Fig. 5c). While RT-PCR

analysis showed that the originally published ataxin-3 C-

terminus (Kawaguchi et al. 1994) was the only ataxin-3

species expressed in Cos-7 cells, overexpression of ataxin-3

containing the alternative C-terminus (Schmidt et al. 1998)

also produced significant fragment production in the absence

of ST treatment (data not shown). This suggests that

overexpressed ataxin-3 either activates caspases or is more

accessible to cleavage by basal cellular proteases.

Intriguingly, when cells were transfected with expanded

ataxin-3 constructs and subsequently treated with ST, an

increase in ataxin-3 aggregation was observed in the stacking

region of the gel (Fig. 6). This increased aggregation was

suppressed by treatment with Z-VAD-FMK (100 lM), sug-gesting it is a caspase-mediated event. Indeed, treatment with

Z-VAD-FMK reduced ataxin-3 aggregation even in the

absence of the apoptotic stressor ST, suggesting that caspase

cleavage of ataxin-3 contributes to the formation of ataxin-3

aggregates in this cell model (Fig. 6; compare lanes 5 with 8,

9 with 12).

Site-directed mutagenesis of ataxin-3 caspase cleavage

sites

Caspase cleavage sites are four amino acid motifs marked by

an essential aspartate residue, termed the P1 aspartate, at

which cleavage occurs (Earnshaw et al. 1999). Mutation of

the P1 aspartate to an asparagine prevents caspase recogni-

tion and subsequent cleavage (Ellerby et al. 1999a; Ellerby

et al. 1999b; Wellington et al. 2000). The size of the ataxin-3

fragment, together with analysis of likely caspase sites in

ataxin-3, suggested that two predicted cleavage sites corres-

ponding to P1 aspartates at amino acid positions 225 and 228

of ataxin-3 were strong candidate targets (sites LDED and

DEED in Fig. 5a). To test their role in proteolysis, we created

ataxin-3 constructs in which one or both of these aspartates

were mutated to asparagine residues. We transfected Cos-7

cells with wild-type ataxin-3(Q35) or mutated ataxin-3

constructs (ataxin-3(Q35) D225N or D225/228N) and one

day later treated cells with 1 lM ST for 24 h. As shown in

Fig. 7(a), ataxin-3(Q35) and ataxin-3(Q35) D225/228N were

both cleaved during ST-induced apoptosis, suggesting that

the sites at 225 and 228 either do not represent major

cleavage sites or that their elimination promotes cleavage at

nearby secondary sites.

For some polyQ disease proteins, elimination of multiple

P1 aspartate residues is required to block caspase cleavage

(Wellington et al. 1998; 2000). Reasoning that similar caspase

site redundancy may exist in ataxin-3, we created an ataxin-3

construct in which all nine potential amino-terminal aspartate

residues (145, 171, 208, 217, 225, 228, 241, 244 and 248)

were mutated to asparagines or alanine (aspartate 217), termed

(a) (b)

(c) (d)

Fig. 4 Ataxin-3 cleavage is caspase-

dependent and primarily mediated by ca-

spase-1, not the proteasome or calpains.

(a) ST-induced proteolysis of full-length

ataxin-3 (c) and the appearance of the

28 kDa fragment (c) is blocked by treat-

ment with 100 lM Z-VAD-FMK (CI), a broad

spectrum caspase inhibitor. (b) Cells trea-

ted with ST and a panel of inhibitors to

specific caspases (10 lM) or Z-VAD-FMK

(10 lM). In panels c and d, ST-induced

ataxin-3 proteolysis was not blocked by

treatment with the proteasome and calpain

inhibitor, ALLN (100 lM), or the specific

proteasome inhibitor, lactacystin (10 lM).

The fragment is not present in panels c and

d because the 1H9 antibody only weakly

recognizes the fragment. Blots were also

probed with anti-PARP (panels second from

the bottom) and anti-GAPDH (lower pan-

els).



ataxin-3(Q35)-9mut. We transfected ataxin-3(Q28) and atax-

in-3(Q35)-9mut into Cos-7 cells and 24 h later treated with ST.

Ataxin-3(Q35)-9mut was resistant to caspase-mediated clea-

vage during ST treatment (Fig. 7b). To further define which

region of ataxin-3 was primarily involved in cleavage, we

created ataxin-3 constructs with either the first 6 aspartates

(145, 171, 208, 217, 225, 228) or the last 3 aspartates (241,

244, 248) mutated to asparagines or alanine (aspartate 217).

These constructs were expressed in Cos-7 cells, which were

(a)

(b)

(c)

Fig. 5 Ataxin-3 antibody recognition sequences and generated con-

structs. (a) A schematic diagram of ataxin-3 highlighting potential

caspase cleavage sites (A–G) as well as the epitope locations for

ataxin-3 monoclonal antibodies 2B6 and 1H9 and polyclonal antibody

146 pa. The ST-induced ataxin-3 fragment is recognized by 146 pa

and weakly by 1H9, but not by 2B6. (Not drawn to scale; slash marks

indicate that N-terminus is longer than depicted in diagram). (b)

pcDNA-HA-ataxin-3-myc constructs used for the transient transfec-

tions in panel C. Panel C, Cos-7 cells were transfected with the

pcDNA3-HA-ataxin-3-myc constructs and 24 h later treated with ST

(1 lM) and/or Z-VAD-FMK (100 lM). The ataxin-3 fragment (c)

increases in size with corresponding repeat lengths. The fragments

are immunostained by anti-myc, which recognizes the extreme C-

terminus of the proteins (second panel), and 1C2 antibody, which

recognizes expanded polyglutamine tracts (third panel). Anti-PARP

and anti-GAPDH were performed as controls (bottom two panels).

Fig. 6 Aggregation of mutant ataxin-3 is reduced by broad spectrum

caspase inhibitors. Cos-7 cells were transiently transfected with the

normal (Q28) or mutant (Q68 and Q84) ataxin-3 constructs described

in Fig. 5(b). High molecular weight SDS-insoluble ataxin-3 aggregate

running in the stacking gel (b) are increased during 1 lM ST treat-

ment, and suppressed by 100 lM Z-VAD-FMK. The small degree of

mutant ataxin-3 aggregation occurring even in the absence of ST is

also reduced by caspase inhibitor treatment.

(a)

(b)

Fig. 7 Site-directed mutagenesis partially blocked proteolysis of full-

length ataxin-3. Panel A shows results from Cos-7 cells transiently

transfected with either normal ataxin-3(Q35), or ataxin-3 constructs

with D225N or D225/228N. Neither mutation blocked ataxin-3 pro-

teolysis during 1 lM ST treatment. Full-length ataxin-3 (c); ataxin-3

fragment (c). Z-VAD-FMK treatment, 100 lM. In Panel B cells were

transfected with either normal ataxin-3 or ataxin-3 with 3, 6 or 9

aspartate to asparagine or alanine mutations. Ataxin-3(Q28)-3mut and

ataxin-3(Q35)-9mut were relatively resistant to proteolysis, whereas

ataxin-3(Q35)-6mut was readily proteolyzed.



then treated with ST 24 h later. As shown in Fig. 7(b), ataxin-

3(Q28)-3mut was relatively resistant to cleavage, similar to

that of ataxin-3(Q35)-9mut, whereas ataxin-3(Q35)-6mut was

efficiently proteolyzed. These results suggest that a preferred

cleavage site for ST-induced ataxin-3 proteolysis resides

within the cluster of potential caspase sites corresponding to

aspartates 241, 244, and 248.

Proteolytic fragments are not observed in SCA3/MJD

human disease tissue

The above results establish that ataxin-3 is a target for

caspase-mediated cleavage during apoptosis in cultured cells.

To characterize the role of ataxin-3 proteolysis in SCA3/MJD

disease, we looked for evidence of ataxin-3 cleavage in brain

tissue from two SCA3/MJD patients. The first individual

died of end stage SCA3/MJD (Paulson et al. 1997b) while

the second, S01-017, was in advanced-stage SCA3/MJD yet

died of other causes. We were unable to detect ataxin-3

fragments, in either soluble or pelleted fractions, in any of the

brain regions tested. These included the globus pallidus,

midbrain, and pons, three regions known to be susceptible to

degeneration in SCA3/MJD (Fig. 8; data not shown).

Because the observed ataxin-3 fragment in cells contains

the polyQ domain, it is possible that a similar fragment in

human brain would be incorporated into insoluble aggregates

and therefore would not be detectable by western blot

analysis.

Discussion

Here we have presented evidence that the polyQ disease

protein, ataxin-3, is a substrate for caspase-mediated

proteolysis in tissue culture. Using ST to induce apoptosis

in cultured cells, we established that: (1) both endogenous

ataxin-3 and exogenous, overexpressed ataxin-3 are proteo-

lytic targets during apoptosis, resulting in production of a

polyQ-containing fragment of ataxin-3; (2) cleavage is

caspase-mediated, primarily by caspase-1 in Cos-7 cells,

with no evidence for calpain or proteasomal involvement;

(3) apoptotic cleavage of ataxin-3 is abolished by mutating

all nine potential caspase cleavage sites amino terminal to the

polyQ domain, and significantly inhibited by mutating a

cluster of three sites within a ubiquitin interaction motif near

the polyQ domain; (4) caspase-mediated proteolysis of

expanded ataxin-3 leads to increased formation of SDS-

insoluble aggregates; and (5) this aggregation can be

suppressed by treatment with caspase inhibitors. Taken

together, our findings are consistent with a disease model

in which caspase-mediated proteolysis of ataxin-3 contri-

butes to SCA3/MJD pathogenesis.

Importantly, much of our analysis was performed on

endogenous ataxin-3. Thus, our studies establish that, at

normal expression levels in an intact cellular milieu, ataxin-3

is readily proteolyzed by caspases. The only prior evidence

that ataxin-3 is a proteolytic target came from an in vitro

analysis of recombinant proteins, in which several other

polyQ proteins were more robust targets than ataxin-3

(Wellington et al. 1998). We found that ataxin-3 is suscep-

tible to cleavage both in neurons and in various non-neuronal

cell lines. While our results agree with an earlier in vitro

study showing that purified ataxin-3 protein is cleaved by

recombinant caspase-1 (Wellington et al. 1998), we were

unable to detect caspase-3 mediated cleavage in cell culture.

Additionally, our observed fragment in Cos-7 cells was larger

(28 kDa) than the proteolytic fragment noted in vitro

[14 kDa; (Wellington et al. 1998)], which may reflect altered

proteolysis due to the cellular environment. In the cell,

endogenous ataxin-3 presumably engages in a variety of

protein–protein interactions (Wang et al. 2000; Chai et al.

2001; Doss-Pepe et al. 2003) that may modulate its suscep-

tibility to caspases. Indeed, caspase-mediated events are

known to be influenced both by cell type and by protein–

protein interactions (Troy and Salvesen 2002). Further

supporting the importance of cellular context is the fact that

cleavage of ataxin-3 and the appearance of a detectable

cleavage product were not identical across the various cell

lines we tested.

Our studies with ataxin-3 constructs mutated at potential

caspase cleavage sites suggest redundancy in the sites at

which cleavage may occur, as has been described for

huntingtin (Wellington et al. 2000). Of the mutated con-

structs we tested, ataxin-3 mutated at three closely spaced

sites proved to be most resistant to cleavage in Cos-7 cells,

indicating that a favored cleavage event occurs at aspartates

241, 244 and/or 248. This is intriguing because these three

aspartate residues reside within, or next to, a recently

Fig. 8 Proteolytic fragments are not observed in SCA3/MJD human

disease brain tissue. Tissue lysates from different brain regions of a

patient with SCA3/MJD (patient S01-017). Normal (.) and mutant (c)

ataxin-3 protein is present, however, no fragment is observed. Brain

regions are as follows, frontal cortex (FC), caudate (Ca), occipital

cortex (OC), parietal cortex (PC), cingulated gyrus (CG), putamen (P),

substantia nigra (SN), cerebellar cortex (CC), and vermis (V). The

band appearing at approximately 33 kDa is non-specific staining from

the polyclonal ataxin-3 antibody.



discovered ubiquitin interaction motif (Donaldson et al.

2003). Ataxin-3 is now known to be an ubiquitin binding

protein, and likely participates in ubiquitin dependent

pathways (Burnett et al. 2003; Chai et al. 2003; Donaldson

et al. 2003; Doss-Pepe et al. 2003). Cleavage within the

ubiquitin binding domain would eradicate this integral

biochemical property of ataxin-3, thereby likely altering

ubiquitin-dependent events in the cell. During apoptosis,

proteins serving vital cellular functions are often targets for

caspase-mediated cleavage, which in turn accelerates the

process of cellular death. As more is learned about the

cellular functions of ataxin-3 and the precise role played by

ubiquitin binding, the effect of ataxin-3 cleavage on the sick

or dying cell may become clear. Moreover, our observation

that overexpressed ataxin-3 underwent proteolysis even in

the absence of ST treatment suggests ataxin-3 levels may be

closely regulated within the cell. Future studies may define a

relationship between ubiquitin binding of ataxin-3 and its

regulation by proteolysis.

When cells were treated with an apoptotic stressor,

expanded ataxin-3 showed increased aggregation. Import-

antly, in our studies aggregation of expanded ataxin-3 could

be suppressed by treatment with a broad-spectrum caspase

inhibitor, suggesting that caspase-mediated cleavage of

mutant ataxin-3 promotes its aggregation in cells. To

determine whether this proteolysis directly contributes to

SCA3/MJD pathogenesis, further studies will be required in

animal models employing ataxin-3 constructs engineered to

lack the relevant caspase cleavage sites.

Although we were unable to detect ataxin-3 fragments

in disease brain, this does not rule out the involvement of

proteolytic cleavage in SCA3/MJD pathogenesis. As

suggested by the current study and previous reports (Ikeda

et al. 1996; Mangiarini et al. 1996; Paulson et al. 1997b;

Wellington et al. 2000; Kim et al. 2001; Sanchez et al.

2003), polyQ-containing ataxin-3 fragments are prone to

aggregation. In the chronic disease state, such fragments

may be highly insoluble and hence undetectable by

standard SDS-gel immunoblot analysis. In diseased brain,

moreover, soluble ataxin-3 fragments could be degraded

more rapidly and thus be present at low steady state levels.

Our current antibodies and detection methods may not be

sensitive enough to detect low amounts of an ataxin-3

fragment in disease tissue, especially if it is insoluble.

Moreover, in HD, protein extraction utilizing formic acid

was required to detect some polyQ fragments (Lunkes

et al. 2002), suggesting more stringent conditions may be

necessary to detect ataxin-3 fragments in SCA3/MJD

human disease tissue. The presence of ataxin-3 fragments

in an SCA3/MJD mouse model notwithstanding (Goti and

Colomer 2003), it is also possible that ataxin-3 is not

cleaved during SCA3/MJD disease pathogenesis, in which

case no ataxin-3 fragments would be detected in human

disease tissue.

Although a detectable ataxin-3 fragment was not observed

in most cell types, full-length ataxin-3 was readily proteo-

lyzed during apoptosis in all cell types tested. This is

intriguing in light of the fact that ataxin-3 is now known to be

a ubiquitin-binding protein with ubiquitin –protease activity

(Burnett et al. 2003; Chai et al. 2003; Donaldson et al.

2003; Doss-Pepe et al. 2003). Perturbations of the ubiquitin-

proteasome pathway have been shown to accelerate polyQ

disease pathogenesis in cellular and animal models (Berke and

Paulson 2003). Thus, regardless of whether detectable ataxin-

3 fragments are produced, ataxin-3 proteolysis under

conditions of cellular or apoptotic stress may be deleterious

to the cell.

In our cellular model, ataxin-3 cleavage was prevented by

broad-spectrum caspase inhibitors and reduced by specific

caspase inhibitors. Caspase inhibitors have been proposed as

a potential therapy for polyQ diseases and some studies in

mouse models support their potential utility in slowing

disease progression (Ona et al. 1999). However, since

apoptosis is necessary during development and may contrib-

ute to normal brain functioning (Troy and Salvesen 2002),

treatment with caspase inhibitors would need to be tempor-

ally and spatially regulated to be feasible. Our current results

show that site-directed mutagenesis of specific aspartate

residues could only partially block ataxin-3 cleavage. It will

be important to extend this analysis to animal models of

SCA3/MJD to assess whether specific caspase inhibitors can

block ataxin-3 processing and toxicity in disease neurons.

The development of therapeutic strategies for SCA3/MJD

and other polyQ diseases will require further characterization

of the role of caspases in pathogenesis. Caspase activity

profiles should be conducted in SCA3/MJD human disease

tissue and animal models to determine whether caspase

activation plays a primary or secondary role in disease

progression. Although the studies described here in Cos-7

cells suggest that particular attention should be paid to

caspase-1 activity, molecular events in disease neurons may

well differ. Finally, dominant negative caspase constructs,

beginning with caspase-1, could be expressed in various cell

and animal models of SCA3/MJD to determine whether they

can suppress aspects of the disease phenotype, as has been

shown in models of HD (Ona et al. 1999).

Acknowledgements

We thank V. Colomer (Johns Hopkins University) for communica-

ting results prior to publication and for the polyclonal ataxin-3

antibody 146 pa. SJSB is supported by the National Institutes of

Health grant NS043076 and National Institutes of Health/National

Institute on Aging Grant T32 AG 00214, Interdisciplinary Research

Training Program on Aging, University of Iowa. HLP and FFS are

supported by National Institutes of Health grant NS38712. LME is

supported by National Institutes of Health grant NS40251A and the

Muscular Dystrophy Association. We are also grateful to the



Hereditary Disease Foundation and to the Huntington’s Disease

Society of America for their support of these studies. Thanks for

technical support from Jessica Young and Stephanie Propp.

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Caspase-mediated proteolysis of the polyglutamine disease protein ataxin-3

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