SCA17 repeat expansion: Mildly expanded CAG/CAA repeat alleles in neurological disorders and the...

Clinica Chimica Acta xxx (2009) xxx–xxx

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SCA17 repeat expansion: Mildly expanded CAG/CAA repeat alleles in neurologicaldisorders and the functional implications

Chiung-Mei Chen a,1, Li-Ching Lee b,1, Bing-Wen Soong c, Hon-Chung Fung a, Wen-Chuin Hsu a, Pei-Ying Lin b,Hui-Ju Huang b, Fen-Lin Chen b, Cheng-Yueh Lin b, Guey-Jen Lee-Chen b,⁎, Yih-Ru Wu a,⁎a Department of Neurology, Chang Gung Memorial Hospital and Chang-Gung University College of Medicine, Taipei, Taiwanb Department of Life Science, National Taiwan Normal University, Taipei, Taiwanc Department of Neurology, National Yang-Ming University School of Medicine, The Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan

Abbreviations: AD, Alzheimer's disease; PD, Parkinso70 kDa protein 5; HSPA8, heat shock 70 kDa proteinprotein 1; PARK7, Parkinson's disease 7; polyQ, polyglutataxia type 17; TBH, tert-butyl hydroperoxide; TBP, TAT⁎ Corresponding authors. Lee-Chen is to be contacted

National TaiwanNormal University, 88 Ting-Chou Road, STel.: +886 2 29336875; fax: +886 2 29312904. Wu, DeGungMemorial Hospital and Chang-GungUniversity CollNorth Road, Taipei 10507, Taiwan. Tel.: +886 3 328120

E-mail addresses: [email protected] (G.-J. [email protected] (Y.-R. Wu).

1 The first two authors contributed equally to this wo

0009-8981/$ – see front matter © 2009 Elsevier B.V. Aldoi:10.1016/j.cca.2009.12.002

Please cite this article as: Chen C-M, et al, Sthe functional implications, Clin Chim Acta

a b s t r a c t
a r t i c l e i n f o
Article history:
Received 30 May 2009Received in revised form 11 October 2009Accepted 1 December 2009Available online xxxx
Keywords:SCA17TBP expansionClinical studyLymphoblastoid cellsExpression analysis

Background: Spinocerebellar ataxia type 17 (SCA17) involves the expression of a CAG/CAA expansion muta-tion in the gene encoding TATA-box binding protein (TBP), a general transcription initiation factor. The spec-trum of SCA17 clinical presentation is broad.Methods:Wescreened for triplet expansion in the TBP gene in Taiwanese Parkinson's disease (PD), Alzheimer'sdisease (AD) and atypical parkinsonism and investigated the functional implication of expanded alleles usinglymphoblastoid cells as a model.Results: A total of 6 mildly expanded alleles (44–46) were identified in patients group. The frequency of theindividuals carrying expanded alleles in PD (3/602 [0.5%]), AD (2/245 [0.8%]) and atypical parkinsonism (1/44[2.3%]) is not significant as compared to that in the control subjects (0/644 [0.0%]). In lymphoblastoid cells,HSPA5, HSPA8 and HSPB1 expression levels in cells with expanded TBP were significantly lower than that ofthe control cells. Although not significantly, the levels of PARK7 protein isoforms 6.1 and 6.4 are notably
increased in SCA17 lymphoblastoid cells. Treatment of TBH (tert-butyl hydroperoxide) significantly increasescell death in the cells with mildly expanded TBP.Conclusions: Our findings expand the spectrum of SCA17 phenotype andmay contribute to our understandingof the disease.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominantataxia causedby an expandedpolyglutamine (polyQ) in a general tran-scription initiation factor, the TATA-box binding protein (TBP) [1,2].In addition to progressive gait and limb ataxia, the broad phenotypicspectrumof this rare disorder includes seizure, cognitive dysfunctions,psychiatric symptoms, and pyramidal and extrapyramidal featuressuch as spasticity, dystonia, chorea, and parkinsonism (review in [3]).Unrelated expanded alleles found in familial and sporadic ataxia patients

n's disease; HSPA5, heat shock8; HSPB1, heat shock 27 kDaamine; SCA17, spinocerebellarA-box binding protein.at Department of Life Science,ection 4, Taipei 11677, Taiwan.partment of Neurology, Changege ofMedicine, 199 TungHwa0; fax: +886 3 3288849.en),

rk.

l rights reserved.

CA17 repeat expansion: Mil(2009), doi:10.1016/j.cca.20

range from 43–66 repeats [4,5] as opposed to 25–42 repeats in thegeneral population [6]. Reduced penetrance was observed for alleleswith 44–47 repeats [7]. Small TBP expansions accounting for a smallproportion of patients with Parkinson's disease (PD)-like, multiplesystem atrophy, Alzheimer's disease (AD)-like and Huntington'sdisease-like phenotypes have also been shown [8–12].

The transcriptional involvement of TBP is well characterized [13].Nevertheless, how the polyQ domain contributes to the normal func-tion of proteins and the expanded polyQ induces selective neuropa-thology remain unclear. A mechanism for aberrant polyQ gain offunction and increased Cre-dependent transcriptional activity inSCA17 has been suggested [14]. Studies of the impact of polyQexpansion on TBP function further revealed that expanded polyQtracts reduce TBP dimerization and DNA binding, but enhance theinteraction of TBP with TFIIB to down-regulate heat shock 27 kDaprotein 1 (HSPB1) expression [15,16]. Our previous proteomics studyusing a cellularmodel of SCA17 has revealed altered expression of heatshock 70 kDa protein 5 (HSPA5), heat shock 70 kDa protein 8 (HSPA8)and Parkinson's disease 7 (PARK7), suggesting that impaired proteinfolding may contribute the cell dysfunction of SCA17 [17].

Although the pathology of SCA is mainly in cerebellum, most SCAgenes are ubiquitously expressed in central nervous system and

dly expanded CAG/CAA repeat alleles in neurological disorders and09.12.002

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peripheral tissues including peripheral lymphocytes. Lymphoblastoidcells are frequently used as amodel to study SCA [18,19]. Although ourprevious study has shownmildly expanded SCA17 CAG repeats in onePD and two AD patients [10,11], in this study, we examined the lengthof the SCA17 repeats in a larger cohort of ethnic Chinese PD, AD andatypical parkinsonism in Taiwan. Furthermore, we investigated if theHSPA5, HSPA8, HSPB1 and PARK7 expression levels are altered and ifsensitivity to tert-butyl hydroperoxide (TBH), an oxidant, is increasedin lymphoblastoid cells carrying SCA17mildly expanded repeat tracts.

2. Materials and methods

2.1. Subjects

The study group comprised 602 sporadic PD patients (45.6%females, aged 69.4±10.7 years), 245 sporadic AD patients (62.4%females, aged 75.8±8.6 years) and 44 atypical parkinsonism patients(46.7% females, aged 69.9±10.3 years) recruited from the outpatientclinic of Chang Gung Memorial Hospital. All PD patients werediagnosed according to the published criteria [20]. Probable AD wasdiagnosed by consensus based on the criteria for probable AD of theNational Institute of Neurological and Communicative Disorders andStroke and the Alzheimer's Disease and Related Disorders Association(NINCDS–ADRDA) [21]. Subjects with atypical parkinsonism includesubgroups of corticobasal degeneration, progressive supranuclearpalsy, dementiawith Lewy bodies andmultiple system atrophy [22]. Agroup of 644 subjects (47.7% females, aged 59.4±14.6 years) wasenrolled as the normal control group. The age difference wasstatistically significant between cases and controls. All the cases andcontrols in the study were Han Chinese in Taiwan and not related toeach other. Each control was interviewed by one of the neurologists(Y-R Wu and C-M Chen) to rule out the possibility of parkinsonism,dementia, ataxia, and other neurological diseases. Each subject wasinformed of the study goals, and all examinations were performedafter obtaining informed consent.

2.2. Genetic analysis of SCA17 repeats

DNA was extracted from leukocytes, and the SCA17 CAG/CAArepeats were determined by polymerase chain reaction (PCR)amplification [1] and PCR products resolved on a linear polyacryl-amide gel using an automated MegaBACE Analyzer. Allele sizes weredetermined by comparing the migration relative to the standardmolecular weights. DNA sequencing was performed to assess therepeat size and the presence of interruptions. Allele frequencies ateach locus were estimated using the gene count method. A chi-squaregoodness of fit test was used to examine whether the controls used inthis study were in the Hardy–Weinberg equilibrium. For this test, wecombined all the rare genotypes (expected probability, b1%) into 1group so that the expected frequencies of all genotype groups wouldbe N5 [23]; ultimately, there were 13 groups. Fisher's exact test wasused to compare the probability of rare expanded alleles in thegenotypes of the PD, AD and atypical parkinsonism patients with thatof the controls, respectively.

2.3. Lymphoblastoid cell lines and cell viability assay

Lymphoblastoid cell lines from3 SCApatients (P1–P3, 55, 46 and44repeats), one PD patient (P4, 44 repeats), 1 AD patient (P5, 46 repeats)and 1 atypical parkinsonism patient (P6, 46 repeats) as well as age-and gender-matched normal controls (C1–C6, 36 repeats) wereestablished (Food Industry Research and Development Institute,Taiwan) after obtaining informed consent. The 3 SCA patients wererecruited from Taipei Veterans General Hospital. The clinical featuresof the three SCA patients were mainly ataxic gait with or withoutdementia. The rest patients and controls were recruited from Chang

Please cite this article as: Chen C-M, et al, SCA17 repeat expansion: Milthe functional implications, Clin Chim Acta (2009), doi:10.1016/j.cca.20

GungMemorial Hospital. Cellsweremaintained in RPMI 1640medium(Gibco) containing 10% FCS.

To evaluate the cell viability, the cultured cell suspensions (105 in100 µl) in medium with or without 10% FCS were treated with TBH(10–20 µmol/l). After 24 or 48 h, 20 µl 0.4% trypan blue (Gibco) wasmixed with 20 µl cell suspension sampled from each well for 1 h. Cellviability was determined by light microscope. Cells that excludedtrypan blue were considered viable and counted in quadruplicate.

2.4. Western blot analysis

Total protein lysate from lymphoblastoid cells was prepared usinglysis buffer (in PBS) containing 5% glycerol, 0.5% TritonX-100, 1 mmol/l dithiothreitol, and protease inhibitor cocktail (Sigma). Protein lysate(25 µg) was separated on 10% SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and blotted onto nitrocellulose membranes by reverseelectrophoresis. After blocking, the membrane was stained withantibody to TBP (1:3000 dilution, Abcam), HSPA5, HSPA8 (1:500dilution, Santa Cruz Biotechnology, Santa Cruz, CA), HSPB1 (1:500dilution, Santa Cruz Biotech), or C4 (actin) (1:10,000 dilution,Chemicon). The immune complexes were detected using horseradishperoxidase-conjugated donkey anti-goat (Santa Cruz Biotech.) or goatanti-mouse (Jackson ImmunoResearch) IgG antibody (1:10,000dilution) and chemiluminescent substrate.

2.5. Two-dimensional gel electrophoresis and immunoblot analysis

Protein extracts were prepared from lymphoblastoid cells asdescribed [17]. Protein samples (300 µg) were first separated usingImmobiline DryStrip (7 cm, pH 3–10) (GE Healthcare) and furtherseparated by a 12.5% SDS-PAGE. The blottingmembraneswere stainedwith antibody to PARK7 (1:10,000 dilution, Chemicon) and immunecomplexes detected using horseradish peroxidase-conjugated goatanti-rabbit IgG antibody (1:10,000 dilution, Rochland) and chemilu-minescent substrate.

3. Results

3.1. Frequency distributions of SCA17 repeat lengths

PCR amplification of the SCA17 locus yielded a pattern of 2 distinctalleles in 63% of the cases analyzed. Fig. 1 shows the distributions ofthe SCA17 repeat lengths in both alleles in the 602 PD patients, 245 ADpatients, 44 atypical parkinsonism patients, and 644 normal controlsubjects. The controls were found to be in the Hardy–Weinberg equi-librium (χ2=3.27, df=13, P=0.997). At the SCA17 locus, CAG repeatlength varied from 28 to 43 in the control group and from 27 to 46 inthe patient group. In both control and patient groups, the most com-mon length was 36 repeats (52–56%). Mildly expanded CAG repeatswere found in three PD (44, 44 and 46 repeats), two AD (44 and 46repeats) and one atypical parkinsonism (46 repeats) patients. Thefrequency of individuals carrying mildly expanded SCA17 alleles inPD (3/602 [0.5%]; P=0.112), AD (2/245 [0.8%]; P=0.076) or atypicalparkinsonism (1/44 [2.3%]; P=0.064) was not significantly differentfrom that in the control subjects (0/644 [0.0%]).

3.2. Clinical analysis of patients with SCA17 expansions

The clinical features and repeat sequences of one PD (77/F, 46repeats) and two AD (59/F, 45 repeats; 76/F, 46 repeats) patients havebeen described previously [10,11]. Patient H1288 (48/M, PD, case 1)had 44 repeats. The CAG expansion number in patient H1173 (62/F,PD, case 2) was 44. Patient H1387 (76/F, DLBD, case 3) had 46 repeats.The clinical features and the sequences of mildly expanded alleles ofthe newly identified patients with TBP expansions are displayed inTable 1. The relatives of H1288 and H 1387 were not available for the



Fig. 1. Distributions of the SCA17 repeat lengths in controls and patients with PD, AD, or atypical parkinsonism. The percentages of the large alleles (44–46 repeats) (A) or genotypes(B) in each group are shown in parentheses.

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genetic test. The son of H1173 also has the same mildly expandedSCA17 sequence as his mother, but neurological and physical exam-ination revealed no ataxic gait, or any neurological abnormality. Inter-estingly, 99mTc-TRODAT-1 SPECT showed a prominent decrease instriatal DAT binding in two PD patients (H1288 and H1173). In addi-tion to SCA17 expansion, SCA8 CAG expansionwas also seen in patientsH1173 (84 CTA/CTG repeats) and H1387 (97 CTA/CTG repeats) [24].

3.3. Lymphoblastoid cell lines establishment and TBH sensitivity

Lymphoblastoid cells from six patients with TBP expansions,including 3 SCA (P1, 28/F, 36/55 repeats; P2, 78/F, 33/46 repeats; P3,80/M, 37/44 repeats), 1 AD (P4, 76/F, 36/46 repeats), 1 PD (P5, 62/F,36/44 repeats), 1 DLBD (P6, 76/F, 36/46 repeats), and 6 age- and

Table 1Clinical features and repeat sequences in PD (cases 1–2) and atypical parkinsonism (case 3

Subject Sex, onset age, age atexamination (years)

Clinical features at examination

Case 1 (H1288) M, 46, 48 Asymmetric resting tremor,bradykinesia, and rigidity,levodopa responsive

Case 2 (H1173) F, 40, 62 Asymmetric resting tremor,bradykinesia, and rigidity,levodopa responsive;levodopa-induced dyskinesia;dementia at late disease stage

Case 3 (H1387) F, 75, 76 Symmetric resting tremor,bradykinesia, and rigidity;dementia at early diseasestage; visual hallucination

F: female; M: male; TRODAT: 99mTc-TRODAT-1 SPECT; DAT: dopamine transporter.


gender-matched normal controls (C1, 23/F; C2, 70/F; C3, 68/M; C4,70/F; C5, 63/F; C6, 68/F; 36/36 repeats) were established. As shown inFig. 2A, Western blot analysis using TBP antibody detected a singleband (non-expanded repeats) protein in all six control cell lines,whereas a larger TBP protein carrying 44–55 repeats and a small TBPprotein carrying 33–37 repeats were detected in cell lines frompatients with TBP expansions.

The sensitivity of TBP expansions to TBH, a mild oxidative reagent,was examined using lymphoblastoid cell lines from 3 SCA patients(P1–P3) and 3 normal controls (C1–C3). As summarized in Fig. 2B,with 10–20 µmol/l TBH treatment for 0, 24 and 48 h, the percentagesof cells that retained trypan blue were 7.8–9.3% (0 h), 11.6–12.2%(24 h), 12.7–14.8% (48 h) for cells with TBP expansion (P1–P3) and7.9–9.7% (0 h), 10.6–11.7% (24 h), 11.6–12.4% (48 h) for cells without

) patients carrying TBP expansion allele.

Image studies Repeat sequence

Brain MRI: normal,TRODAT: asymmetricreduction of activityof DAT in the bilateralstriatum

(CAG)3(CAA)3(CAG)8CAACAGCAA(CAG)25CAACAG

Brain MRI: normal,TRODAT:asymmetric reductionof activity of DAT in thebilateral striatum


Brain CT: moderatecerebral atrophy




Fig. 2. Lymphoblastoid cell lines and TBH sensitivity assay. (A)Western blot analysis of lymphoblastoid cells from normal controls (C1–C6) and TBP expansion patients (P1–P6) usingTBP antibody. LaneM contains molecular weight markers. (B) Characteristics of cell death induced by TBH treatment. Lymphoblastoid cells from normal controls (C1–C3) and SCA17patients (P1–P3) were treated with 10–20 µM TBH for 0–48 h (open bar, non-treated; gray bar, 10 µM TBH for 24 h; charcoal gray bar, 10 µmol/l TBH for 48 h; striped bar, 20 µmol/l TBH for 24 h; black bar, 20 µmol/l TBH for 48 h). Trypan blue exclusion assay was performed to determine the cell death. The data shown are the mean±SCE of 3 independentexperiments. *Pb0.05, **Pb0.01, ***Pb0.001 compared with non-treated.


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TBP expansion (C1–C3). Although the difference between patient andcontrol groups for the same treatment concentration and time wasnot significant (P=0.109–0.686), the increase of cell death after10 µmol/l TBH treatment for P1 from 7.8% to 9.8% (24 h) and to 10.2%(48 h), P2 from 9.0% to 10.6 % (24 h) and to 11.9% (48 h) and P3 from9.3% to 11.7% (48 h) was significant (P=0.046–0.004) as compared tountreated cells. For control cell lines C1–C3, the increase of cell deathwas not significant (PN0.05) with the same 10 µM TBH treatment.With 20 µM TBH treatment, the increase of cell death for P1 from 7.8%to 11.6% (24 h) and to 12.7% (48 h), P2 from 9.0% to 12.1 % (24 h) andto 14.8% (48 h) and P3 from 9.3% to 12.2% (24 h) and to 13.5% (48 h)was significant (P=0.037–0.000) as compared to untreated cells. Forcontrol cell lines C1–C3, the increase of cell death was only significant(P=0.015–0.004) with 20 µmol/l TBH treatment for 48 h.

3.4. Analysis of HSPA5, HSPA8, HSPB1 and PARK7 expression

As reduced expression of several heat shock proteins was impli-cated in SCA17 pathogenesis [15–17], we examined the expressionlevel of HSPA5, HSPA8 and HSPB1 using Western blotting and appro-priate antibodies (Fig. 3A). When the expressed level in one of thecontrol in each blot (C1 and C4) was set as 100%, HSPA5 protein ex-pression in lymphoblastoid cells expressing TBP-Q44–55 (P1–P6) wassignificantly reduced when compared to the lymphoblastoid cellsexpressing TBP-Q36 (C1–C6) (median: 76% vs. 100%, range: 71–81% vs.90–117%, P=0.001). Similarly, HSPA8 and HSPB1 protein expressionin lymphoblastoid cells expressing expanded TBP were also signifi-cantly reduced when compared to the lymphoblastoid cells expres-sing normal TBP (for HSPA8, median: 85% vs. 100%, range: 80–89% vs.87–115%, P=0.010; for HSPB1, median: 75% vs. 101%, range: 58–89%vs. 87–109%, P=0.001). The relativeHSPA5,HSPA8 andHSPB1proteinlevels in lymphoblastoid cells from controls and patients with TBPexpansions are shown in Fig. 3B.

Previously increased expression of PARK7 was observed in ourSCA17 293 cell model [17]. As different PARK7 isoforms exist [25], weperformed comparative high resolution, two-dimensional gel electro-


phoresis experiments on protein extracts from three SCA17 patientsand age- and gender-matched controls to investigate the expressionand posttranslational modification of PARK7. Immunoblot analysiswith PARK7 antibody revealed the presence of at least four distinctisoforms that have the same apparent molecular mass of 20 kDa butdifferent isoelectric points (5.7, 5.8, 6.1 and 6.4, respectively; Fig. 3C).The predominant form of PARK7 is the 20-kDa/pI 6.4 isoform. Al-though not significant, the increase level of PARK7 pI 6.1 (median:301% vs. 100%, range: 160–387% vs. 89–111%, P=0.104) and pI 6.4(median: 193% vs. 100%, range: 120–246% vs. 62–127%, P=0.116)isoforms in SCA17 lymphoblastoid cells was notable.

4. Discussion

This study examined the length of the SCA17 repeats in Taiwanesepatientswith PD, AD and atypical parkinsonism, and in normal controlsubjects. Mildly expanded SCA17 alleles (44 and 46 repeats) werefound in 6 patients, including 3 PD, 2 AD and 1 atypical parkinsonism(Fig. 1). Recently, Kim and colleagues have also reported 8 PD patientscarrying 43–45 SCA17 repeats, which is in accordance with our find-ing, suggesting that low-range SCA17 expansions tend to have aparkinsonian phenotype [12]. Although the possibility of reducedpenetrance of TBP 44–47 repeats exists [7], and the possibility of co-incidence of the mildly expanded CAG repeats with PD, AD or atypicalparkinsonism cannot be completely excluded, the reported SCA17cases with atypical parkinsonism and PD [12,26] and our resultsstrengthen the previous observation [10,11] that SCA17 CAG repeatexpansion may be a rare cause of PD, AD and atypical parkinsonism.

A role for oxidative damage in polyQ diseases is gathering increas-ing experimental support and oxidative stress may be a predisposingfactor to the late-onset feature of SCA17. Thus we established lym-phoblastoid cell lines and used a mild oxidative reagent TBH [27] andtrypan blue exclusion assay to assess the oxidative vulnerability ofcells carrying expanded TBP. We observed increased susceptibility tooxidative stress that leads to increased cell death in SCA17 lympho-blastoid cells carrying TBP expansion (P1, 55 repeats; P2, 46 repeats;



Fig. 3. Western blot analysis of the lymphoblastoid cells from controls (C1–C6) and patients with SCA17 expansions (P1–P6). (A) Representative images (top panel) and levels ofHSPA5, HSPA8, HSPB1 and PARK7 expression (bottom panel). To normalize, the relative expression level in C1 and C4 is set as 1.0. Data are expressed as the mean±SEM values from4 independent experiments. (B) The relative HSPA5, HSPA8, HSPB1 and PARK7 protein level in lymphoblastoid cells from controls (open diamond) and patients with TBP expansions(black diamond). The * indicates a significant difference between the patient and the control samples (Pb0.05). (C) The level of each PARK7 isoform in SCA17 lymphoblastoid cells(black bar) normalized to the level of the same isoform in control cells (open bar).

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P3, 44 repeats) (Fig. 2). However, the increased vulnerability and celldeath to oxidative stress did not correlate well with the length ofpolyQ, which may be due to the prominent age difference and/orother genetic variations among the three patients.

As a basal transcription factor, TBP is involved in both TATA andTATA-less promoter sites. Transcriptional dysfunction of enhancingTFIIB–TBP interaction to down-regulate the small heat shock proteinHSPB1 (HSP27) expression has been reported [15,16]. Our proteo-mics study of a SCA17 cellular model also demonstrated reducedexpression of heat shock protein HSPA5 and HSPA8 [17]. The HSPB1 isinvolved in stress resistance. In a HD cellular model, HSPB1 was pro-posed to suppress polyQ induced reactive oxygen species that con-tributed to cell death [28]. Defects in the HSPB1 gene are a cause of 2related peripheral neuropathies, Charcot-Marie-Tooth disease type 2and distal hereditary motor neuropathy [29]. The HSPA5 (also knownas 78 kDa glucose-regulated heat shock protein GRP-78) is involved inthe folding and assembly of proteins in the endoplasmic reticulum.Abnormal protein folding due to decreased rate of HSPA5 hydroly-sis and disturbed SIL1-HSPA5 interaction is the cause of Marinesco–Sjogren syndrome that is characterized by ataxia, progressivemyopathy and cataract [30]. The HSPA8 encodes a constitutivelyexpressed 70 kDa heat shock cognate protein (HSC70) binding tonascent polypeptides to facilitate correct folding. Immunohistochem-ical studies on HSPA8 using autopsied brains from patients withmultiple system atrophy suggest that HSPA8 may be associated withthe pathogenesis of multiple system atrophy [31]. The above evidencesuggests that altered expression of HSPB1, HSPA5, and HSPA8 may


contribute to neuronal dysfunction in SCA17. To assess the roles ofthese chaperones in SCA17, we examined the expression levels ofHSPA5, HSPA8 and HSPB1 in lymphoblastoid cells carrying expandedTBP. We have shown that HSPB1, HSPA5 and HSPA8 expression levelswere significantly reduced in transformed lymphoblastoid cells frompatients withmild TBP expansion (Fig. 3B). While direct evidence thatHSPB1, HSPA5 and/or HSPA8 contribute to the pathogenesis of SCA17is not provided in this manuscript, in SCA17 transgenic mice, down-regulated HSPB1 due to decreased general transcription factor TFIIBoccupancy of theHspb1promoter has been shown, and overexpressionof HSPB1 or TFIIB alleviated mutant TBP-induced neuritic defects [15].Furthermore, an expanded polyQ domain that can inhibit the intrinsicassociation of TBP with TATA-box DNA in vitro has been shown toinfluence TBP-dependent gene transcription [16]. Although whetherthe reduced HSPA5 or HSPA8 gene expression is TBP-mediated is notknown, the previous finding together with our results and reports thatthe reduced expressionofHSP27 and/orHSP70 in lymphoblastoid cellsfrom patients with SCA3 and SCA7 [18,19] supports the proposal thatreduction of HSPB1, HSPA5 and/or HSPA8 may contribute to the neu-ronal dysfunction of polyQ-mediated SCA.

In addition to HSPB1, HSPA5 and HSPA8 chaperones, we alsoexamined the up-regulated PARK7, a causative gene of early-onsetPD. PARK7, also known as DJ-1, has a role in the anti-oxidative stressreaction to prevent cell death [32]. Increased expression of PARK7 inHEK-293 cell models expressing expanded TBP has been shown in ourprevious study [17]. However, maybe due to the expression leveldifference (constitutive vs. induced) between the lymphoblastoid and




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HEK-293 cell models and/or the small difference in the polyglutaminelength (44–55 repeats in SCA17 lymphoblastoid cells vs. 61 repeats inHEK-293 cells) compared to the upper limit of normal repeat range,the observed increased expression of PARK7 in SCA17 lymphoblastoidcells with expanded TBP was not significant (Fig. 3C).

Our study however has a couple of limitations. One of them is thesmall number of cell lines used, because both inter and intra-indi-vidual variability could be a confounding variable. Another limitationis that the possibility of co-incidence of the mildly expanded CAGrepeats with PD, AD or atypical parkinsonism cannot be completelyexcluded. However, there are two lines of evidence to support that themildly expanded CAG repeats in PD, AD or atypical parkinsonism arepathogenic. One is that there is no such mildly expanded CAG repeatin 644 controls of our study. Another one comes from that in the studyof Kim et al.: they have shown that CAG expansion in the SCA17 generanged from 43 to 46 in 10 parkinsonian patients, suggesting low-range expansions tend to cause the parkinsonian phenotype [12].Furthermore, they have shown decreased striatal dopamine trans-porter binding in 4 control individuals with 42–44 repeats, suggestingthat an expansion as low as 42 repeats might constitute a suscep-tibility gene for parkinsonism. This finding is also in agreement withthe observation that mildly expanded CAG repeats in both SCA2 andSCA3 genes can cause a pure parkinsonism phenotype in the absenceof prominent ataxia in patients of Asian and African ancestry [33,34].

In summary, our data expand the current phenotype associatedwith SCA17.Our cellmodelsmaybe valuable because they shed insightinto the pathogenesis and provide a basis for developing therapeuticinterventions for this disease.

Acknowledgments

The authors would like to thank the patients and controls forparticipating in this study. This work was supported by grants NSC96-2314-B-182A-099-MY2, NSC97-2311-B-003-008-MY3, and NSC97-2311-B-182A-001-MY3 from the National Science Council, ExecutiveYuan, 96TOP001 fromNational TaiwanNormal University, and CMRGP37056 from Chang Gung Memorial Hospital, Taipei, Taiwan.

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