Genetic and Dlolecular analysis of the Spinocerebellar. ataxia type 7 (SCA7) disease gene. Jenni Jonasson

Genetic and Dlolecular analysis of the Spinocerebellar ataxia type 7 (SCA7) disease gene Jenni Jonasson Departments of Clinical Genetics/Cell and Mo...
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Genetic and Dlolecular analysis of the Spinocerebellar ataxia type 7 (SCA7) disease gene

Jenni Jonasson

Departments of Clinical Genetics/Cell and Molecular Biology, and Microbiology Umeå University Umeå 2000

Cover:

Heterotopic Purkinje cells in the cerebellum of a SCA7 patient, showing nuclear expression of ataxin-7.

Printed in Sweden by Solfjädern Offset AB Umeå 2000 ISBN: 91-7191-770-5

TABLE OF CONTENTS Page ABBREVIATIONS

5

ABSTRACT

7

ORIGINAL PUBLICATIONS

9

AlMS OF THIS THESIS

10

INTRODUCTION

11

Spinocerebellar ataxia type 7 (SCA7): manifestation and classification of the disease

11

Symptoms and characteristics of SCA7

11

Classification SCA7 and related spinocerebellar ataxias

14

Relative frequencies ofADCA subtypes

17

Genetics of SCA7

18

Genetic characteristics of SCA7

18

Anticipation and CAG repeat expansion

19

The SCA7 gene

21

Mechanisms of polyglutamine disease

23

Findings supporting a gain-of-function mechanism

24

Nuclear localisation and protein aggregation

27

Altered gene expression

29

Evidencefor misfolding and processing ofpolyglutamine disease proteins: The toxic fragment theory

31

Cell-type specific neurodegeneration

33

3

RESULTS AND DISCUSSION

36

Genetic analysis of the SCA7locus

36

Chromosomallocalisation of the SCA7 gene

36

Isolation of SCA7 candidate genes

37

Molecular analysis of the SCA7 gene

39

Expanded CAG repeats in Swedish SCA7 patients

39

Effect of CAG repeat size on the SCA7 phenotype

40

Maternal versus paternal transmission: effect of the CAG repeat expansion

42

DNA-based diagnosis of SCA7

43

A founder effect for SCA7 in Scandinavia

44

Haplotype analysis infour Swedish SCA7 families

44

Afounder mutationfor SCA7 in Scandinavia

46

Expression of ataxin-7 in normal and pathological tissue

48

Specificity ofSCA7pepl-15

49

Expression of ataxin-7 in cerebellum, olivary nuclei and retina

49

Ataxin-7 expression in cerebral cortex and hippocampus

51

Expression of ataxin-7 in peripheral tissue

52

CONCLUSIONS

54

ACKNOWLEDGEMENTS

55

REFERENCES

57

PAPERS I-IV

4

ABBREVIATIONS

alACT

al-antichymotrypsin

aa

amino acid

AD

Alzheimer'"s disease

ADCA

autosomal dominant cerebellar ataxia

AR

androgen receptor

BCL

bipolar celllayer

CACNL1A4 the alA voltage-dependent calcium channel subunit gene cDNA

complementary deoxyribonucleic acid

cM

centi MorganIs

CNLI

ceroid lipofuchsinosis, neuronall

CNS

central nervous system

DNA

deoxyribonucleic acid

DRPLA

dentatorubral pallidoluysian atrophy

EAAT4

exitatory amino acid transporter type 4

ERO

electroretinogram

EST

expressed sequence tag

FTD-3

frontotemporal dementia, located on chromosome 3

HAP-I

hutingtin-associated protein l

HD

Huntington"'s disease

HDJIHSDJ

human DNAJ chaperone homologue

HIP-I

huntingtin-interacting protein l

HIP-2

huntingtin-interacting protein-2

HPRT

hypoxantine phosphoribosyltransferase

INL

inner nuclear layer

INPP5A

type l inositol polyphosphatase 5-phosphate

5

IP3RI

inositol triphosphate receptor type l

kb

kilo base pairls

kD

kilo Dalton/s

LANP

leucin-rich acidic nuclear protein

MJD

Machado-Joseph disease

mRNA

messenger ribonucleic acid

mTRP3

mouse transient receptor potential type 3

NES

nuclear export signal

NII

neuronal intranuclear inclusions

NLS

nuclear localisation signal

ONL

outer nuclear layer

OPCA

olivoponto cerebellar atrophy

ORF

open reading frame

P35

abaculovirai anti-apoptotic gene

PAC

PI artificial chromosome

PCCMT

prenylcysteine carboxymethyltransferase

PDA

patent ductus arteriosus

PML

promyolytic leukemia

PR

photoreceptorls

SERCA

sarcoplasmic endoplasmic reticulum ATPase type 2

SBMA

spinal and bulbar muscular atrophy

SCA

spinocerebellar ataxia

UTR

untranslated region

6

ABSTRACT

Spinocerebellar ataxia type 7 (SCA7) is a hereditary neurodegenerative disorder affecting the cerebellum, pons and retina. SCA7 patients present with gait ataxia and visual impairment as the main symptoms. Anticipation, commonly observed in SCA7 families, is a phenomenon where an earlier age at onset and a more severe progression of disease is seen in successive generations. In order to identify the gene responsible for SCA7, we performed linkage analysis on a Swedish SCA7 kindred. Evidence for linkage of the SCA7 disease locus to a 32 cM region on chromosome 3p12-21.1, between markers D3S1547 and D3S1274, was established. A number of neurodegenerative disorders associated with anticipation are caused by expanded (CAG)n repeats in their respective disease genes. In order to isolate the SCA7 disease gene we, therefore, screened a human infant brain stem cDNA library for CAG repeat containing clones, mapping to chromosome 3. Four candidate clones were isolated and analysed, but could all be excluded as the SCA7 disease gene. In 1997, the SCA7 disease gene was identified and, as expected, shown to harbour a CAG repeat, expanded in SCA7 patients. Analysis of the SCA7 CAG repeat region in Swedish SCA7 patients demonstrated that CAG repeat size was negatively correlated to age at onset of disease. Furthermore, patients with larger repeats presented with visual impairment, whereas patients with smaller repeats presented with ataxia as the initial symptom. SCA7 is the most common autosomal dominant cerebellar ataxia in Sweden and Finland, but rare in other populations. In order to investigate if the relatively high frequency of SCA7 in these countries is the result of a founder effect in the region, a haplotype analysis was performed on all SCA7 families available. All

7

families shared a common haplotype of at least 1.9 cM surrounding the SCA7 locus. In addition, strong linkage disequilibrium was demonstrated for marker D3S1287 closely linked to the SCA7 gene, suggesting a founder effect for the SCA7 mutation in Sweden and Finland. The function of the SCA7 protein, ataxin-7, is not known and it does not show significant homologies to any previously known proteins. In order to gain insight into the function of ataxin-7 we analysed the expression of ataxin-7 in brain and peripheral tissue from SCA7 patients and controis. In brain, expression was found to be mainly neuronal with a nuclear subcellular localisation. Ataxin-7 expression was found throughout the CNS, not restricted to sites of pathology. We also confirmed previously reported findings of neuronal intranuclear inclusions (NIls) in the brains of SCA7 patients. Based on our findings, we conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression pattern in affected and non-affected tissue or the distribution pattern of aggregated protein.

8

ORIGINAL PUBLICATIONS

This thesis is based on the following original publications, which will be referred to by their roman numerais: I.

Monica Holmberg, Jenni Johansson, Lars Forsgren, Jan Heijbel, Ola Sandgren and Gösta Holmgren. (1995) Localization of autosomaldominant cerebellar ataxia associated with retinal degeneration and anticipation to chromosome 3p12-21.1. Human Molecular Genetics, vol 4, no 8, 1441-1445

II.

Jenni Johansson, Lars Forsgren, Ola Sandgren, Alexis Brice, Gösta Holmgren and Monica Holmberg. (1998) Expanded CAG repeats in Swedish Spinocerebellar ataxia type 7 (SCA7) patients: effect of repeat length on the 171-176. clinical manifestation. Human Molecular Genetics, vol 7, no 2,

III.

Jenni Jonasson, Vesa Juvonen, Pertti Sistonen, Jaakko Ignatius, Daniel Johansson, Erik J. Björck, Jan Wahlström, Atle Melberg, Gösta Holmgren, Lars Forsgren and Monica Holmberg. (2000) Evidence for a common Spinocerebellar ataxia type 7 (SCA7) founder mutation in Scandinavia. (Submitted)

IV. Jenni Jonasson*, Patricia Hart*, Thomas Brännström, Anna-Lena Ström,

Lars Forsgren and Monica Holmberg. (2000) Expression of ataxin-7 in CNS and peripheral tissue of normal and SCA7 individuals. (Manuscript) *These authors contributed equally to this work

9

AlMS OF THIS THESIS

The aims of this thesis were to identify and characterise the gene causing Spinocerebellar ataxia type 7, and to gain understanding of the genetic and pathological events underlying this disease. Specific goals were to:

• Determine the chromosomallocalisation of the SCA7 disease gene

• Isolate candidate genes for SCA7

• Examine the SCA7 mutation in relation to disease phenotype

• Evaluate the possibility of a founder effect for SCA7 in Scandinavia

• Analyse the expression pattern of the SCA7 protein, ataxin-7, in relation to pathology

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INTRODUCTION Spinocerebellar ataxia type 7 (SCA7): manifestation and classification of the disease Symptoms and characteristics of SCA7 Spinocerebellar ataxia type 7 (SCA7) is a severe hereditary neurodegenerative disease characterised by progressive ataxia and decreased visual acuity due to atrophy of the cerebellum and retina respectively. SCA7 is a relatively rare condition, with areported prevalenee of less than 1/100,000 (Oouw et al., 1998). In SCA7, the ataxia manifests as a disturbed co-ordination of movements, resulting in an unsteady, wide-based gait (gait ataxia), speech problems (dysarthria) and difficulties in swallowing (dysphagia). Patients generally show involvement of the upper motor neurons, manifesting as hyperreflexia, extensor plantar reflexes (Babinski"'s sign) and/or spasticity. Oculomotor symptoms are also present, including paralysis of the eye muscles (ophthalmoplegia) and occasionally, involuntary, jerky eye movements (nystagmus). Ahallmark among the many symptoms present in SCA7 patients is progressive decreased visual acuity, leading to blindness. At early stages of disease, patients display a defective blue/yellow colour discrimination, and electroretinogram (ERO) examination usually reveals impairment of photoreceptor (cone and rod) function. Patients further present with pigmentation of the maeula, a structure in the retina of importance in controlling acuity of central vision, which progresses to degeneration of the macula and severe atrophy of the retina. Figure 1 (this thesis) shows fundi photographs of two SCA7 patients at different stages of disease, demonstrating the severe progressive retinal degeneration of this disorder. The severity and combination of the symptoms described above varies

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Figure 1. Fundus photographs of (a) a non-affected individual, age 76 years, (b) a SCA? patient, age 51 years~ disease duration 1O years, and (e) a SCA7 patient age 50 years, disease duration 31 years. Photographs, courtesy of Dr. Ola Sandgren, Department of Ophthalmology, University Hospital of Umeå, Umeå, Sweden.

between SCA7 patients, even within the same family (Harding, 1982; Martin et al., 1994). The symptoms that occur most frequently, in 75 to 100% of the SCA7 patients, include gait ataxia, visual impairment, dysarthria, hyperreflexia and slow eye movements (table 1, this thesis). Decreased vibration sense, dysphagia, ophthalmoplegia,

Babinski~s

sign, sphincter disturbances, spasticity

and muscle atrophy are symptoms that occur at a more moderate frequency (25 to 75%). Finally, symptoms present in less than 25% of the patients involve decreased hearing, postural tremor, Parkinsonian symptoms, facial myokymia, mental deterioration, nystagmus and scoliosis (table 1, this thesis). It has been

12

demonstrated that the occurrence of specific symptoms such as decreased visual acuity, ophthalmoplegia, Babinski"s sign and scoliosis, are significantly more frequent in patients with early age at onset, and that disease duration is significantly longer in patients with dysphagia and sphincter disturbances (David et al., 1998). Age at onset of SCA7 symptoms ranges from early childhood to the fifth decade, in rare cases even later (papers II and III). In cases of childhood onset the disease symptoms and progression of disease are particularly aggressive. Infantile cases of SCA7 show neurological signs similar to adult onset patients, but also exhibit non-neurological symptoms, including muscle hypotonia and patent ductus arteriosus (PDA, an opening between the left lung arteryand the aorta that usually closes at birth). Furthermore, these children fail to thrive, and the rapid disease progression lead to death within a few months (Benton et al., 1998; Enevoldson et al., 1994 and paper II). Neuropathological findings in SCA7 patients varies between affected individuals, but commonly include atrophy of the cerebellum, pyramidal pathways, brainstem, and spinal cord, as weIl as severe degeneration of the retina (Gouw et al., 1994; Martin et al., 1994). The retinal degeneration seen in SCA7 is a feature that distinguishes this disorder the other autosomal dominant spinocerebellar ataxias known to date. Neuropathological examination of the eye reveals atrophy of the choroid and degeneration of pigment epithelial cells, bipolar cells, ganglion cells and both types of photoreceptor cells of the retina (Gouw et al., 1994; Martin et al., 1994).

13

Table 1. Symptoms of SCA7 patients Paper I (n=ll)

David et al. (1998) (n=71)

Frequently occurring symptoms cerebellar gait ataxia 100% dysarthria 100% decreased visual acuity 80% slow eye movements hyperreflexia 60%

100% 98% 83% 88% 78%

Occurring at moderate frequency decreased vibration sense 40% dysphagia 18% ophthalmoplegia 30% extensor plantar reflexes 30% sphincter disturbances 60% spasticity 30% muscle atrophy

62% 58% 53% 52% 50% 41% 25%

Less frequently occurring decreased hearing postural tremor Parkinsonian symptoms facial myokymia mental deterioration 9% scoliosis nystagmus 9%

24% 19% 18% 16% 11% 11%

Classification SCA7 and related spinocerebellar ataxias SCA7 belongs to a genetically heterogeneous group of hereditary neurodegenerative disorders, all characterised by late onset cerebellar ataxia. These disorders have been denoted spinocerebellar ataxias (SCAs), and to date, as manyas eleven different gene loci have been identified, including SCAl-8 and SCA10-12 (figure 2, this thesis),

14

SCA? ....

1

2

3

7

8

9

4

10

5

6

11

12

I~CA4 ~.:.i. I· ;:0"/........

• .

·:~.SCA8

13

1~.

SCA11

SCA3I --MJD

14

15

=.

";:

.":

i

-

.-'.

.•.··•

16

~))+~CA6

.

: :.

,

.:". :.....•.•:•.......:.. ::.:... •......

••..

19

.:

.

:

.......

20

y

Figure 2. Chromosomallocation of Spinocerebellar ataxias (SCAs) 1-8 and 1012. Original photograph, courtesy of Dr !rina Golovleva, Department of Clinical Genetics, University Hospital of Umeå, Umeå, Sweden.

15

(Benomar et al., 1995; Gardner, 1994; Gispert et al., 1993; Gouw et al., 1995; Holmberg et al., 1995; Holmes et al., 1999; Jodice et al., 1997; Koob et al., 1999; Ranum et al., 1991; Ranum et al., 1994; Stevanin et al., 1993; Takiyama et al., 1993; Worth et al., 1999; Zhuchenko et al., 1997; Zu et al., 1999). Earlier classifications of late onset cerebellar ataxias, based on clinical and pathological signs, are found in the literature. In 1970, Konigsmark and Weiner described families with Olivopontocerebellar atrophy (OPCA) and suggested a classification dividing them into five subtypes, OPCA I-V (Konigsmark and Weiner, 1970). According to this classification, SCA7 would fall into the OPCA III subtype, where olivopontocerebellar atrophy is associated with an "atypical retinitis pigmentosa" (Konigsmark and Weiner, 1970). In 1982, neurologist Anita Harding classified the autosomal dominant late onset cerebellar ataxias based on their inheritance pattern and clinical manifestation (Harding, 1982). The disorders were denoted autosomal dominant cerebellar ataxias (ADCA), and four main subtypes were defined. ADCA I, including genetically identified SCA1, 2, 4, and SCA3IMJD, was reported to associate progressive cerebellar ataxia with different combinations of ophthalmoplegia, optic atrophy, dementia, extrapyramidal signs and amyotrophy. In ADCA II, which includes SCA7, cerebellar ataxia associated with pigmentary macular degeneration is the main features. ADCA III is referred to as a "pure" cerebellar syndrome, not involving any ocular or extrapyramidal features, or dementia. Of the genetic subtypes identified so far, SCA5, 6, 8, 10 and 11, fall into the group of ADCA III. It should, however, be noted that Stevanin et al. chose to regard SCA10 as a subtype separate from ADCA III, as two out of ten patients reported also displayed seizures (Stevanin et al., 2000; Zu et al., 1999). Likewise, families with SCA6 are occasionally placed in the ADCA I subgroup due to presence of symptoms that are not of cerebellar origin (Dichgans et al., 1999;

16

Matsuyama et al., 1997). ADCA IV associates ataxia with deafness, myoclonus and peripheral neuropathy (Harding, 1982). This rare condition has only been described in one family with three affected members (May and White, 1968) and in a pair of identical twins (Chayasirisobhon and Walters, 1984), therefore no chromosomallocation has to date been identified for ADCA IV.

Relative frequencies ofADCA subtypes

Autosomal dominant cerebellar ataxias (ADCAs) are estimated to have a prevalence less than 10/100,000, although precise numbers are not available (Stevanin et al., 2000). Although the different genetic subtypes of ADCA generallyare found in populations world wide, their relative frequencies differ depending on the population studied. Generally, SCA7 is considered to be a rare form of ADCA. In studies of ADCA families of various ethnical origin, SCA7 has been reported to constitute only 4.5 to 11.6% of the ADCAs diagnosed (Benton et al., 1998; Moseley et al., 1998), whereas several studies of defined populations have failed to identify patients with SCA7 (Lopes-Cendes et al., 1997; Matsumura et al., 1996; Schols et al., 1997; Silviera et al., 1998). In contrast, SCA7 is the major form of ADCA diagnosed in Sweden and Finland (paper III). Similar discrepancies have been reported for other forms of ADCA. In a comparative study of 202 Japanese and 177 Caucasian families with ADCA, Takano et al. found SCA3IMJD to be the most common form in both populations, representing 43% and 30% respectively (Takano et al., 1998). SCA1 and SCA2 were however found to be more frequent in Caucasians (15% and 14% respectively) than in the Japanese (3% and 5% respectively). SCA6, on the other hand, was more frequent in the Caucasian population, representing 11 % of the ADCAs, compared to 5% in Japanese population (Takano et al., 1998). SCA3IMJD has been reported as the most frequent ADCA subtype in a

17

number of other populations, including families from Portugal, Japan, Brazil and Germany, where it represented 30-74% of all ADCA patients examined. It is likely that these differences can be accounted for by regional founder effects, as indicated by the high prevalence of SCA2 in Cuba (4/10,000) and of SCA3IMJD in the Azores in Portugal (1/4,000), where this disease was first

described ( as referenced in Stevanin et al., 2000). Indeed, linkage disequilibrium, indicative of a regional, single or major, founder mutation, has been demonstrated for several forms of SCA, including SCA7 (Stevanin et al., 1999 and paper III).

Genetics of SCA7

Genetic characteristics of SCA7 SCA7 is an autosomal dominant disease belonging to the ADCA II subtype, according to the classification described above (Harding, 1982). ADCA II was initially believed to be genetically homogenous since it was mapped to the same chromosomal region, chromosome 3p12-21.1, in three independent reports including families from Sweden (paper I), Belgium, Morocco and France (Benomar et al., 1995) as weIl as in Caucasian and Afro-American families from the USA (Gouw et al., 1995). However, a recent publication by Giunti et al. described a family with three affected members which exhibited clinical symptoms suggestive of ADCA II (Giunti et al., 1999). This family was not linked to the SCA7 locus on chromosome 3p, indicating that additionalloci for ADCA II exists. Families with SCA7 generally show genetic anticipation, presenting as an earlier age at onset and a more severe progression of the disease in successive generations (Benomar et al., 1995; Gouw et al., 1994; Gouw et al., 1995; Harding, 1982; Holmberg et al., 1995; Martin et al., 1994). In SCA7, age at

18

onset of symptoms ranges from three months to 75 years (papers I, II and III) and disease duration, from disease onset to death, is significantly shorter in young onset cases compared to late onset cases (David et al., 1998). In infantile cases of SCA7, patients can show severe symptoms as earlyas 3 months with a rapid progression of disease leading to death at age 6-7 months. The reported cases of extreme, infantile SCA7 are all the results of paternal transmissions of the disease gene (paper II and Benton et al., 1998; Enevoldson et al., 1994). Although the ratio of females:males affected with SCA7 is 1: 1, a majority of the patients inherit the disease from their mother. In a study of 81 parent to child transmissions, the resulting female:male ratio was as high as 4: 1 (Gouw et al., 1998).

Anticipation and CAG repeat expansion The genetic anticipation observed in SCA7 is a phenomenon that has been reported in a number of hereditary neurodegenerative disorders, including Huntington"'s disease (HD), spinal and bulbar muscular atrophy (SBMAlKennedy'"s disease) and dentatorubral pallidoluysian atrophy (DRPLA) as weIl as additional forms of spinocerebellar ataxia. Genetic anticipation has also been described in several non-neurological conditions, and over the years it has been debated whether anticipation reflects a phenomenon of true biological importance, or merely constitutes the result of ascertainment biases (Penrose, 1948, as reviewed in McInnis, 1996). Recent data have however provided a molecular explanation for the occurrence of genetic anticipation in the hereditary neurodegenerative disorders mentioned above. In 1991, the gene causing spinobulbar muscular atrophy (SBMAlKennedy,s disease) was isolated and shown to contain a new type of dynamic mutation. The gene, encoding the androgen receptor (AR), contained a trinucleotide repeat with the sequence

19

CAG that was shown to be expanded in patients with SBMA. The normal AR allele displayed 17 to 26 CAG repeats, whereas SBMA patients carried between 40 and 52 CAG repeats on their pathological allele (La Spada et al., 1991). Since then, expanded CAG repeats have been demonstrated to be involved in a number of neurodegenerative disorders, including SCA7, SCAl-3/MJD, SCA6, HD and DRPLA (David et al., 1997; Del-Favero et al., 1998; Group, 1993; Imbert et al., 1996; Jodice et al., 1997; Kawaguchi et al., 1994; Koide et al., 1994; Koob et al., 1998; La Spada et al., 1991; Orr et al., 1993; Pulst et al., 1996; Sanpei et al., 1996; Zhuchenko et al., 1997). The size range of the CAG repeats differ between the disease genes, both for the normal and expanded alleles (table 2, this thesis). In SCA2 normal alleles range between 13 and 33 CAG repeats, while the disease-causing alleles have more than 32 CAG. In SCA3/MJD gene on the other hand, unaffected individuals can have between 12 and 41 CAG repeats whereas patients all have an allele with 40 repeats or more. Therefore, SCA3 alleles with 40 CAG repeats can be found in neurologically healthy individuals, whereas SCA2 alleles with 40 CAG repeats are weIl within the disease-causing range for this disease (table 2, this thesis). Neurologically unaffected individuals with relatively small expanded CAG repeats, so called intermediate alleles (lA), have been observed for all the CAG repeat disorders except SCA6, SBMA and DRPLA. Intermediate alleles are however rare, and have been reported to result in reduced penetrance. Generally lA are found in elderly relatives of sporadic (or familial) de novo cases of disease (Giunti et al., 1999; McNeil et al., 1997; Myers et al., 1993; Rubinsztein et al., 1996; Stevanin et al., 1998 and paper III). For all these disorders, except SCA6, there is a strong negative correlation between the size of the CAG repeat and the age at onset of disease. This observation, in combination with the reported tendency for the CAG repeats to

20

expand upon germline transmission, provides an explanation for the molecular basis underlying genetic anticipation in these disorders.

The SCA7 gene Even before the disease gene was eloned, several findings suggested that SCA7 could be caused by expanded CAG repeats. First, SCA7 families showed pronounced anticipation and clinical similarities to other neurodegenerative CAG repeat disorders (paper I). Second, the presenee of expanded CAG repeats in SCA7 patients was detected by the repeat expansion detection (RED) method (Lindblad et al., 1996). In addition, Trottier et al. detected a 130 kD protein in ADCA II patients using an antibody (lC2) specific to polyglutamine domains of pathological size (Trottier et al., 1995). The assumption of expanded CAG repeats as the disease causing mutation was applied by three independent groups in the cloning of the SCA7 gene (David et al., 1997; Del-Favero et al., 1998; Koob et al., 1998). In 1997 the SCA7 disease gene was successfully isolated and shown, as expected, to contain a CAG repeat. Analysis of the SCA7 CAG repeat in patients and controls showed that healthy controls displayed a CAG repeat size in normal chromosomes ranging from 7 to 17 repeat units, while all affected individuals carried more than 38 repeats (David et al., 1997; Del-Favero et al., 1998; Koob et al., 1998 and paper II). The SCA7 cDNA sequence contained a 2727 bp open reading frame (ORF) (David et al., 1997; Del-Favero et al., 1998), encoding a 892 amino acid (aa) polypeptide with an expected molecular weight of 95 kD (David et al., 1997). The CAG repeat is situated in the first translated exon of the SCA7 gene and a putative nuclear localisation signal (NLS) is located at aa 378-394. Apart from

21

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Table 2. Summary of diseases caused by expanded CAG repeats

Disease

Chromosomal

Normal

Disease-

localisation

alleles

causing

Protein

Subeellular localisation of normal protein

alleles SCA1

6p23

6-44

39-83

ataxin-1

predominantly nuclear

SCA2

12q24.1

13-33

32->200

ataxin-2

predominantly cytoplasmic

SCA3IMJD

14q32.1

12-41

40-89

ataxin-3

predominantly cytoplasmic

SCA6

19p13

7-18

21-27

the alA voltage depen-

membrane

dent calcium channel SCA7

3p12-13

4-40

37-306

ataxin-7

nuclear

HD

4p16.3

6-39

36-121

huntingtin

nuclear and cytoplasmic

SBMA

Xq13-21

11-33

38-66

the androgen receptor

nuclear and cytoplasmic

DRPLA

12p13.31

6-35

51-88

atrophin-1

predominantly cytoplasmic

the CAG repeat region, no significant homologies to other polyglutamine disease genes or other known genes have been reported (David et al., 1997). Some minor inconsistencies can been noted between the two SCA7 sequences reported. David et al. published a 729 bp long whereas Del-Favero et al. reported a 137 bp

3~-UTR

3~-UTR

without a polyA tail,

containing a polyA tail. In

addition, the last 15 bp in the cDNA differed between the two publications, resulting in a substitution of the last five amino acids PKARP (David et al., 1997) with the sequence VGNGL (Del-Favero et al., 1998). However, the subsequent cloning of the SCA7 genomic sequence confirmed that the 3 ~ sequence reported by David et al. was correct, and that the 137 bp sequence found by Del-Favero et al. was an artefact in cDNA synthesis, due to the presence of a (A)n sequence in intron 12 (Michalik et al., 1999). The SCA7 gene covers 140 kb of genomic sequence and consists of 13 exons, two of which reside in the 5 ~ -UTR. The exons range in size from 69 to 979 bp, and the introns vary from 233 bp to about 40 kb (Michalik et al., 1999). Michalik et al. also identified a PAC clone containing 18 kb of sequence upstream of the SCA7 exonllikely to contain regulatory sequences, but the SCA7 promoter has yet to be defined (Michalik et al., 1999) .

Mechanisms of polyglutamine disease The polyglutamine disorders identified so far, including the spinocerebellar ataxias (SCAI-3/MJD, SCA6-7), DRPLA, HD and SBMA, share a number of features (i-iv): (i) All, except for the X-linked recessive SBMA, are autosomal dominantly inherited. (ii) The CAG repeat is situated in the coding region of the respective disease genes and is translated into a polyglutamine stretch in the respective proteins. The term polyglutamine disorders is therefore commonly used for this group of neurodegenerative disorders (Servadio et al., 1995;

23

Trottier et al., 1995). (iii) In all polyglutamine disorders (except SCA6) there appears to be a size threshold of around 35-40 repeat units that the CAG repeat must exceed in order to cause disease. (iv) The non-mutated allele is usually polymorphic and below the pathological threshold in size, but for many of these disorders a small size overlap between pathologic and non-pathologic alleles exists. In SCA6, disease symptoms appear in patients with relatively small repeats (21-27 U) in the implicated disease gene, CACNL1A4, encoding the a1A-voltage-dependent calcium channel. In addition, six different mutations in the CACNL1A4 gene, all disrupting the calcium channel function, have been shown to be the cause of two allelic neurological conditions, familial hemiplegic migraine and episodic ataxia type-2 (Ophoff et al., 1996). It is therefore speculated that the mechanisms underlying SCA6 are different from the other polyglutamine disorders (Zhuchenko et al., 1997).

Findings supporting a gain-of-function mechanism Despite the clinically and genetically shared features, the different genes causing polyglutamine disease do not show any significant sequence homologies, apart from the CAG repeat region. Moreover, the subcellular distributions of the respective proteins are different (table 2, this thesis). The normal atrophin-1, ataxin-2 and ataxin-3 proteins have been reported to have a predominantly cytoplasmic localisation (Koyano et al., 1999; Paulson et al., 1997; Yazawa et al., 1995). Huntingtin, the androgen receptor protein and ataxin-l, on the other hand, are found both in the nucleus and the cytoplasm, although ataxin-1 is predominantly nuclear (De Rooij et al., 1996; Merry and Fischbeck, 1998; Servadio et al., 1995). In contrast, normal ataxin-7 has so far only been detected in the nucleus (Mauger et al., 1999 and paper IV). Little is known about the normal function of the different proteins causing

24

polyglutamine disease, apart from the androgen receptor which is implicated in SBMA pathology. The androgen receptor is a ligand-activated transcription factor belonging to the steroid/thyroid hormone receptor family. The polyglutamine domain is located in the N-terminal part of the protein, where the transactivation function has been shown to reside. However, the effect of CAG expansion on transactivation capacity is unclear (Merry and Fischbeck, 1998). The lack of homology between the polyglutamine proteins, as weIl as the differences in subcellular localisation suggests that the wild-type proteins are not related in function. It has therefore been proposed that the expanded polyglutamine domain itselfplays a direct role in pathology, and cause disease by a gain-of-function mechanism common to the different disorders. Several findings support this hypothesis. First, patients with deletions in the Huntington disease gene and the androgen receptor have been described that do not exhibit a HD or SBMA phenotype respectively (Gusella et al., 1985; Quigley et al., 1992). Instead, patients with Wolf-Hirschhom syndrome, resulting from a partial loss of chromosome 4p including the HD gene, demonstrate severe growth retardation, microcephaly, and developmental closure defects (Gusella et al., 1985). Patients with loss of androgen receptor function suffer from testicular feminization as a result of androgen insensitivity, and do not display a neurological phenotype (Quigley et al., 1992). However, SBMA patients often do showapartial insensitivity to androgens, indicating that a loss-of-function is also a part of the mechanism in this disease. Secondly, HD null mice show an embryonic lethal phenotype (Duyao et al., 1995; Nasir et al., 1995; Zeitlin et al., 1995). In two out of the three HD knock-out models described, heterozygots displayed no phenotype, arguing against a haploinsufficiency or dominant-negative mechanism (Duyao et al., 1995; Zeitlin et al., 1995). In contrast, Nasir et al. reported their heterozygous mice to have an

25

increased motor activity and neurodegeneration of the subthalamic nucleus (Nasir et al., 1995). This was, however, suggested to be due to the expression of erroneous transcript of the targeted allele, speculated to exert a dominant negative effect (Nasir et al., 1995). Thirdly, in contrast to the HD nuIl mice, children homozygous for the HD mutation (Le. carrying the HD linked allele from both of their affected parents) are live bom. In addition, homozygotes do not exhibit a more severe phenotype than heterozygotes (Myers et al., 1989; Wexler et al., 1987). Transgenic mouse models of SCA1, SCA3IMJD and HD, mimicking the respective human conditions, also support the hypothesis of a gain-of-function mechanism underlying polyglutamine disease. Transgenic overexpression of mutant (82 glutamines) fulllength ataxin-1 in the cerebellar Purkinje cells of mice resulted in progressive ataxia, whereas mice carrying a normal sized repeat (30 glutamines) remained phenotypically unaffected (Burright et al., 1995). Pathological changes including heterotopic Purkinje cells, severe loss of Purkinje cells and gliosis of the molecular layer, similar to that seen in SCA1 patients, was observed in heterozygous mice overexpressing mutant ataxin-1 (Burright et al., 1995; Clark et al., 1997). In 1996, Ikeda et al. described transgenic mice expressing normal (26 or 35) or expanded (79) glutamines in fulllength and truncated forms of ataxin-3. Interestingly, ataxia was present only in mice carrying the truncated protein with 79 glutamines (Ikeda et al., 1996). That same year, Mangiarini et al. reported on transgenic mice expressing truncated huntingtin with 115-150 glutamines from the human HD promoter (Mangiarini et al., 1996). These mice showed a severe progressive neurological phenotype similar to human Huntington~s

disease, including chorea-like movements, stereotypic involuntary

movements, loss of body weight and seizures. Expression level of the mutant

26

transcript in these mice was only 75% of endogenous huntingtin, in contrast to the overexpressing SCA1 mice. Neuropathological examination in the HD mutant mice revealed no apparent areas of malformation or degeneration in the CNS, although the brains of mutant mice were consistently smaller (Mangiarini et al., 1996). These two reports of truncated protein resulting in neurological disease both suggested that the polyglutamine domain is directly involved in the pathological process. It can be speculated that the polyglutamine domain is more exposed in a truncated protein, and therefore more toxic than in the context of a full-Iength protein. Perhaps the most striking evidence for a gain-of-function mechanism of expanded CAG repeats, came from the study of transgenie mice expressing 146 CAG repeats introduced into the hypoxantine phosphoribosyltransferase (HPRT) gene, a gene normally not carrying this motif (Ordway et al., 1997). This ectopic expression of a polyglutamine tract produced mice with a neurological phenotype, including handling-induce seizures, clasping of hind legs when held by the tail, tremor, mild ataxia and a reduced life span (Ordway et al., 1997). The proposed gain-of-function mechanism also seem to be conserved in invertebrates, as severe neurodegeneration has been reported in Drosophila, when expressing a truncated ataxin-3 with 78 glutamine residues (Warrick et al., 1998).

Nuclear localisation and protein aggregation It is today generally accepted that polyglutamine disorders are caused by a common gain-of-function mechanism, and a number of different events have been suggested to underlie the neuronal dysfunction and neurodegeneration. Some, including nuclear localisation of the mutant protein have been proven

27

crucial, whereas others, including protein aggregation and proteolytic cleavage, have been topics of intense debate. In 1997, it was shown that brain from HD transgenic mice contained intracellular aggregates of mutant huntingtin. The aggregates were found in the nucleus of neurons in those areas of the brain that are affected in HD patients and stained positive for huntingtin as weIl as ubiquitin (Davies et al., 1997). The same type of aggregates, denoted neuronal nuclear inclusions (NIls), have since been found in brains of patients of all polyglutamine disorders, including HD SCAl-3IMJD, SCA7, DRPLA, and SBMA. In all cases a fraction of the NIls also stained positive for ubiquitin (Becher and Ross, 1998; Davies et al., 1997; Di Figlia et al., 1997; Holmberg et al., 1998; Koyano et al., 1999; Li et al., 1998; Paulson et al., 1997; Skinner et al., 1997). For HD and SCA3IMJD, the inclusions were reported to be most frequent in those regions that constitute the primary sites of pathology in the respective disorders, and was thus proposed to be the cause of neuronal dysfunction (Di Figlia et al., 1997; Paulson et al., 1997). NIls were also reported to be more frequent in juvenile onset than in adult onset cases (Di Figlia et al., 1997). Although initial studies all implied that NIls were an important component in the pathology of polyglutamine disorders, these suggestions have been challenged by more recent studies. Numerous reports demonstrate that NIls might actually constitute a paraliei process that is not related to the central mechanism leading to neuronal dysfunction. For SCA7 and DRPLA is has been shown that NIls are present throughout the CNS and not restricted to areas of pathology (Hayashi et al., 1998; Holmberg et al., 1998 and paper IV). The presence of nuclear inclusions have also been described in non-neuronal cells of SCA7 and SBMA patients (Li et al., 1998 and paper IV) and in a mouse model of HD (Sathasivam et al., 1999). In addition, mutant huntingtin was shown to

28

induce neurodegeneration in cultured striatal neurons, but huntingtin-induced cell death could not be correlated to the presence of NIls. The authors showed that blocking the aggregation of huntingtin using a dominant-negative mutant of the ubiquitin-conjugating enzyme, resulted in an increased cell death of transfected neurons (Saudou et al., 1998). Likewise, in an animal model of SCA1, transgenic mice with a deletion in the self-associating region of ataxin-1 failed to form NIls, but still demonstrated an ataxic phenotype (Klement et al., 1998). In addition to challenging the theory of protein aggregation as mediator of polyglutamine toxicity, the studies by Saudou et al. and Klement et al. defined a crucial step in the disease process. Saudou et al. showed that by adding a nuclear export signal (NES) to the mutant peptide, they were able not only to retain the mutant protein in the cytoplasm, but also to completely block huntingtin-induced cell death (Saudou et al., 1998). Klement et al. presented SCA1 transgenic mice expressing ataxin-1 with 82 glutamines and a mutated NLS from the Purkinje cell specific promoter Pcp-2. Ataxin-1-NLS mutant mice displayed no phenotype, in contrast to mice expressing the same mutation from this promoter with an intact NLS, despite comparable levels of transgene expression (Burright et al., 1995; Klement et al., 1998). These two reports clearly demonstrated the importance of nuclear localisation of the mutant protein, regardless of protein aggregation, in the pathological process.

Altered gene expression

It has been speculated that the neuronal dysfunction in polyglutamine diseases may be the result of altered interactions with nuclear proteins, leading to altered gene expression. Ataxin-l, ataxin-3 and ataxin-7 have all been shown to associate with the nuclear matrix (Kaytor et al., 1999; Skinner et al., 1997; Tait

29

et al., 1998). In addition, ataxin-1 and ataxin-7 have both been demonstrated to colocalize with the promyolytic leukaemia (PML) protein, a transcriptional coactivatior associated with the nuclear matrix (Kaytor et al., 1999; Skinner et al., 1997). Recently, Lin et al., provided evidence that altered gene expression is an early step in pathogenesis of SCA1. In the cerebellum of SCA1 transgenic mice carrying 82 glutamines, the authors identified six neuronal genes that were downregulated and one that was upregulated (Lin et al., 2000). The six genes that were downregulated were identified as prenylcysteine carboxymethyltransferase (PCCMT), inositol triphosphate receptor type 1 (IP3R1), sarcoplasmic endoplasmic reticulum ATPase type 2 (SERCA2), type 1 inositol polyphosphate 5-phosphate (INPP5A), mouse transient receptor potential type 3 (mTRP3) and exitatory amino acid transporter type 4 (EAAT4). The gene found to be upregulated in SCA1 mice was identified as the mouse homologue of human a1-antichymotrypsin (a1ACT). PCCMT, IP3R1, SERCA2, INPP5A, mTRP3 and EAAT4 were all downregulated before onset of morphological changes or neurological symptoms, thus demonstrating the earliest change involved in SCA1 pathology detected so far. Three of the genes, PCCMT, IP3R1 and SERCA2, were also shown to be downregulated in Purkinje cells of human SCA1 patients. Four out of the six genes identified, IP3R1, SERCA2, INPP5A and mTRP3, are all involved in regulation of intracellular calcium leveis, and the authors suggested the possibility that the constant presence of mutant ataxin-1 might exert a stress signal eventually leading to disrupted calcium homeostasis (Lin et al., 2000). PCCMT is a protein involved in posttranslational modification of a number of important proteins, such as the Ras-like GTPases and nuclear lamins. Downregulation of EAAT4, a Purkinje cell-specific glutamate transporter residing on dendritic spines, was speculated to promote exitotoxic cell death by

30

interfering with its normal function in restricting glutamate spillover to neighbouring synapses (Lin et al., 2000). Unlike the six genes downregulated, increased expression of a1ACT was detected regardless of subcellular localisation of mutant ataxin-l. Increased levels of a1ACT were also found in the pons of a SCA1 patient, and has previously been demonstrated in patients with Huntington"'s disease, Alzheimer disease (AD) as weIl as in normal ageing (Lin et al., 2000).

Evidencefor misfolding and processing ofpolyglutamine disease proteins: The toxic fragment theory

The presence of misfolded proteins in the cell exerts a stress signal, resulting in expression of molecular chaperones and in proteolytic processing of the misfolded proteins. The general intracellular mechanism by which the cell regulates and degrades short-lived or misfolded proteins is via the ubiquitinproteasome pathway. This process involves the conjugation of multiple molecules of ubiquitin to the protein in question and the subsequent hydrolysis of the protein by the proteasome. The involvement of misfolded mutant protein in polyglutamine disorders is supported by the findings of ubiquitin, proteasome components and molecular chaperones in the NII in brains of patients polyglutamine disease, as weIl as in various transfection studies showing aggregation of polyglutamine containing proteins (Becher and Ross, 1998; Chai et al., 1999; Cummings et al., 1998; Davies et al., 1997; Di Figlia et al., 1997; Holmberg et al., 1998; Koyano et al., 1999; Li et al., 1998; Paulson et al., 1997; Skinner et al., 1997; Stenoien et al., 1999). It has been speculated that the degradation resistance of the NIls could result in sequestering of proteasome components and/or prevent the recycling of

31

ubiquitin, thereby inducing a stress response in the cell, resulting in the expression ofmolecular chaperones (Cummings et al., 1998). Cummings et. al demonstrated that overexpression of the chaperone HDJ2IHSDJ in HeLa cells cotransfected with ataxin-1 carrying 92 glutamine residues resulted in a decreased number of NIls formed (Cummings et al., 1998). The involvement of the proteasome in NII formation was shown by Chai et al., by expressing a truncated form of ataxin-3 carrying 49 glutamines in HeLa cells. This relatively small mutation resulted in a low frequency of protein aggregation «5%). However, treatment of the cells with the specific proteasome inhibitor lactacystin dramatically increased NII formation (Chai et al., 1999). Already in 1996, Ikeda et al. suggested the possibility that polyglutaminecontaining proteins are subjected to post-translational processing via proteolytic cleavage (Ikeda et al., 1996). It has since been demonstrated by Western blot analysis, that HD and SCA2 brain extracts contain proteolytically processed fragments of huntingtin and ataxin-2 respectively, not present in controls (Di Figlia et al., 1997; Koyano et al., 1999). In addition, NIls in HD and SBMA brains have been speculated to contain processed protein, as NIls stain positive using amino-terminal, but not carboxy-terminal, antibodies (DiFiglia et al., 1995; Li et al., 1998). It has further been speculated that processing of polyglutamine containing proteins by caspases could produce a toxic fragment, which accumulates in the cell causing a stress response. This stress response would then lead to activation of more caspases, production of further toxic fragments, and so on, in a feedback loop that eventually leads to cell death (Wellington et al., 1998). This hypothesis is theory supported by the finding that crossing of HD mutant mice to mice with a dominant-negative caspase-1 mutation, results in a delayed onset

32

and extended survival in double-mutant offspring (Ona et al., 1999). Additional studies have indicated the involvement of activated caspases in polyglutamine disease. Warrick et al. showed that expression of mutant fragments of ataxin-3 with 78 glutamines targeted to the eye in Drosophila resulted in a progressive degenerative phenotype with severe effect on eye morphology. The phenotype was partially rescued by crossing the mutants to flies expressing P35, a baculoviral antiapoptotic gene reported to inhibit all caspases to date known in Drosophila (Warrick et al., 1998). In contrast to this, Jackson et al. were not able to rescue the photoreceptor degeneration seen in Drosophila that expressed truncated huntingtin with 120 glutamines when crossing transgenic flies with Drosophila expressing 1-3 copies of P35 (Jackson et al., 1998). It is not clear whether these discrepancies in cell death inhibition are due to the differences in protein context, the different expression levels of P35 or the possibility that P35 might be able to rescue developmentally induced neuronal death, but not cell death occurring in adult tissue (Jackson et al., 1998).

Cell-type specific neurodegeneration The polyglutamine disorders show overlapping but specific patterns of neurodegeneration, still the respective disease genes seem to be widely expressed in both eNS and peripheral tissues (Jou and Myers, 1995; Li et al., 1993; Nagafuchi et al., 1994; Servadio et al., 1995; Sharp et al., 1995; Strong et al., 1993; Trottier et al., 1995). The basis ofthese differences in cell specific neurodegeneration between the disorders has been a topic of intense debate. Even though there is no obvious difference in protein expression levels between brain regions severely affected and regions showing less degeneration in a particular disease, the levels of HD and DRPLA mRNA are higher in brain than in other tissues, and also higher in neurons than in non-neuronal cells in brain

33

(Li et al., 1993; Nagafuchi et al., 1994; Sharp et al., 1995; Strong et al., 1993). Northern blot analysis has shown that SCA7 mRNA is expressed throughout the CNS, with higher levels found in the cerebellum than in other parts of the brain (David et al., 1997). The level of expression could therefore in part explain why neuronal cells are more vulnerable to polyglutamine toxicity than non-neuronal cells. It could further be speculated that subtie differences in expression leveis, smaller than which can be detected by in situ hybridisation techniques available today, designates which neurons will be affected and which ones will be spared. Another suggestion involves the cell type-specific interaction of the respective disease proteins and its polyglutamine domain with other proteins. A number of huntingtin interacting partners have been identified using two-hybrid screening, denoted Huntingtin Associated Protein (HAP-1), Huntingtin Interacting Protein (HIP-1) and HIP-2 respectively (Kalchman et al., 1996; Kalchman et al., 1997; Li et al., 1995; Wanker et al., 1997). However, HIP-1 and HAP-1 are both expressed in the cerebellum, and the cell type specific degeneration in HD must therefore include additional factors. HIP-2 encodes an ubiquitin-conjugating enzyme that is expressed predominantly in striatum and cortex, but has not been shown to interact with huntingtin in a length-dependent manner (Kalchman et al., 1996). Furthermore, the leucine-rich acidic nuclear protein (LANP), a protein highly abundant in Purkinje cells of the cerebellum, has been shown to interact and bind to ataxin-1 in a polyglutamine length-dependent manner. LANP has therefore been suggested as a good candidate for conferring the specificity of neurodegeneration in SCA1 (Matilla et al., 1997). It is likely that the mechanism behind the cell-type specific neurodegeneration in the different polyglutamine disorders is complex and involves a combination of different pathways, specific to each disease. It may thus involve different combinations specific interaction with other proteins, NIls may sequester

34

different proteins in different cell types and proteolytic processing and altered gene expression could differ between different cell types and brain regions. Additional studies are needed to investigate if altered gene expression is a common theme in polyglutamine disorders and if so, if this involves altered regulation of different genes, specific for the cell types primarily affected in the different disorders.

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RESULTS AND DISCUSSION

Genetic analysis of the SCA710cus Chromosomallocalisation of the SCA7 gene In order to determine the localisation of the SCA7 gene, a genome-wide screen using 270 polymorphic microsatellite markers was performed in a large Swedish SCA7/ADCAII kindred (figure 1, paper I). Allloci for ADCA, known at the time, including SCA1 (6p), SCA2 (12q), SCA3IMJD (14q), SCA4 (16q) and SCA5 (lIcen), could be excluded in the Swedish ADCA II family. Two-point lod scores were calculated between the SCA7 disease locus and all 270 markers used in the study. Significant lod scores, over 3, were obtained for markers D3S1600, D3S1296 and D3S1598, alllocalised on the short arm of chromosome 3 (table 2, paper I). Lod score calculations were performed using a model with age-dependent penetrance for the disorder. Seven additional markers in the region gave positive, but not significant, lod scores using the same model. Marker D3S1600 gave a maximum lod score of 4.53 at e = o using the above model, and a lod score of 3.03 using an affected-only model. The disease gene could be restricted to a 32 cM region between the markers D3S1547 (telomeric) and D3S1274 (centromeric) on chromosome 3p12-21.1 by recombination events. Multipoint analysis was performed using five microsatellite markers; markers D3S1547 and D3S1274 restricting the SCA7 disease gene region and the three most informative markers, D3S1600, D3S1296 and D3S1598, located within the SCA7 disease region. A maximum lod score of 4.31 was obtained, with the most likely position of the SCA7 gene close to marker D3S 1598 (figure 2, paper I).

36

Two other independent studies also mapped the SCA7 gene to the same region on chromosome 3p (Benomar et al., 1995; Gouw et al., 1995). Benomar et al. included four SCA7 families originating from Belgium, Morocco and France, and restricted the SCA7 disease gene to a region of 33 cM between markers D3S1300 and D3S1276. The candidate region was further refined to 8 cM surrounding marker D3S 1285, using the Zmax-l method of combined multipoint analysis (Benomar et al., 1995). In the study by Gouw et al. the SCA7 disease gene was located to a 24.8 cM interval restricted by markers D3S 1767 and D3S1261, in two African-American and two Caucasian SCA7 families. Multipoint analysis over this region gave a maximum lod score at the D3S1287 locus (Gouw et al., 1995). Fine-mapping reports were later published where David et al. restricted the SCA7 disease gene region to a 5 cM region between telomeric marker D3S1312 and centromeric marker D3S1600 (David et al., 1996). A second fine-mapping report from Krols and co-workers limited the disease region to 12 cM between markers D3S1300 and D3S1285 (Krols et al., 1997), the latter in agreement with cloning data localising the SCA7 gene to a 2.5 cM region between D3S1600 (telomeric) and D3S1287 (centromeric) (Del-Favero et al., 1998).

Isolation of SCA7 candidate genes The Swedish SCA7 family used for linkage analysis showed marked genetic anticipation. This phenomenon had been described for several other hereditary neurodegenerative disorders and shown to be correlated to the presence of expanded CAG repeats in the respective disease genes (Group, 1993; Imbert et al., 1996; Kawaguchi et al., 1994; Koide et al., 1994; La Spada et al., 1991; Orr et al., 1993; Pulst et al., 1996; Sanpei et al., 1996). In order to isolate the SCA7 disease gene we, therefore, screened a human infant brain stem cDNA library

37

(Stratagene) for CAG repeat containing clones and tested positive clones for localisation to chromosome 3. Approximately 1.9x106 clones were screened, using a (CTG)g probe, resulting in the identification of 718 CAG positive clones (frequency of 0.03%). 155 out of 718 CAG positive clones were isolated and tested for localisation on chromosome 3 by Southern blot hybridisation using DNA from a mouselhuman hybrid somatic cell line (GMl1713, Coriell Cell Repositories) containing human chromosome 3. A chromosome 4 hybrid (GMl1687, Coriell Cell Repositories) was used as a negative controi and human genomic DNA as a positive controI. Four CAG containing, chromosome 3 positive, clones were identified and denoted CAG28, CAG35, CAG117 and CAG164 respectively. The four clones were end-sequenced using vector primers and homology searches were performed using the GCG BLASTN program. Three clones showed homology to previously known sequences/genes; CAG28 to a sequence isolated from human pancreatic islets (EST U16738), CAG35 to the MAR/SAR DNA-binding protein SATB1 (NM002971) and CAG117 to clone CTG-B33 (EST L10376). CAG 164 did not show homology to any known sequence in the databases. Primers were constructed for amplification of the four CAG repeat regions in SCA7 patients and controis. None of the repeat regions were expanded in SCA7 patients, and therefore all 4 clones were excluded as candidate genes for the disease (unpublished data). The clones CAG28, CAG117 and CAG164 were also evaluated as candidate genes for a form of frontotemporal dementia with anticipation, shown to be localised on chromosome 3 (FTD-3), but could be excluded for this disorder also (Ashworth et al., 1999). In 1997, the successful cloning of the SCA7 gene was reported by three independent research groups, all using cloning approaches based on the

38

assumption of expanded CAG repeats as the disease causing mutation (David et al., 1997; Del-Favero et al., 1998; Koob et al., 1998). The SCA7 cDNA was shown to contain a CAG repeat in the first translated exon, and the repeat was, as expected, shown to be expanded in SCA7 patients (David et al., 1997; DelFavero et al., 1998; Koob et al., 1998).

Molecular analysis of the SCA7 gene Expanded CAG repeats in Swedish SCA7 patients Following the cloning of the SCA7 gene, we characterised the SCA7 mutation in Swedish SCA7 patients and controis. We amplified and analysed the CAG repeat region in four Swedish SCA7 families, A-D (figure 1, paper I and figure 4, paper II), three of which were novel (families B-D). A total of 16 clinically diagnosed SCA7 patients, three asymptomatic individuals carrying the disease haplotype, their unaffected relatives and 57 healthy controls were included in the study. Our analysis showed that normal chromosomes harboured a CAG repeat ranging from 7 to 15 repeat units in size (figure 1, paper II). These findings are in agreement with studies undertaken in other populations, where CAG repeats on normal chromosomes have been shown to range from 4-35 repeat units (DelFavero et al., 1998; Giunti et al., 1999). The most common allele in the Swedish population carried 10 CAG repeats and was found in 63% of the chromosomes in the controi material, similar to frequencies reported by others (Giunti et al., 1999). All clinically affected SCA7 patients showed CAG repeat expansions ranging from 40->200 CAG repeats (figures 1 and 2, paper II). The size of the CAG expansions in Swedish SCA7 patients mirrors weIl SCA7 mutation sizes reported in other populations (David et al., 1998; Giunti et al., 1999; Gouw et

39

al., 1998). The repeat region failed to amplify in two early onset cases (age at onset 1 and 4 years respectively), due to poor quaiity of DNA extracted from paraffin embedded tissue. However, an extreme expansion of >200 repeat units was detected in a Swedish infantile SCA7 case and constituted one of the largest CAG repeats ever reported in a polyglutamine disorder (figure 2, paper II). This child was severely affected and failed to gain weight, showed breathing problems and metabolic acidosis. A patent ductus arteriosus (PDA) was discovered and successfully closed. Age at onset was as earlyas 3 months of age in this patient, and the aggressive disease progression lead to death at 7 months (table 1, paper II). This particularly severe phenotype, including PDA, has been reported in other infantile-onset cases of SCA7, all resulting in early childhood death (Benton et al., 1998; Enevoldson et al., 1994). The smallest repeat size reported to lead to a SCA7 phenotype is 37 CAG repeats (David et al., 1998). We have identified one individual with an allele of 40 CAG, still healthy at age 85, indicating that there exists an intermediate repeat interval with reduced penetrance of the disease (paper III). Giunti et al. reported on CAG repeats of intermediate size, 28-35 repeats, in neurologically healthy individuals (Giunti et al., 1999). They could also demonstrate that these intermediate alleles (lA) were more prone to further expansion than CAG repeats in the normal range, when transmitted to the next generation. lAs in the 28-35 CAG range were however not detected in Swedish families A-D and none of the normally sized CAG repeats were expanded during transmission (paper II).

Effect of CAG repeat size on the SCA7 phenotype We next investigated the impact of repeat size on age at onset of disease phenotype. As the symptoms at disease onset are variable among patients and

40

gradual in nature, age at onset was determined as the first time the patient experienced any clinical sign of neurodegenerative disease, irrespective of whether medical contact was taken at that time or not. For the 15 Swedish SCA7 patients included in this study a strong negative correlation between repeat size and age at onset of disease was observed (figure 3, paper II). The infantile case A V: 16, carrying a very large CAG repeat, was excluded from this analysis due to difficulties to determine of the exact repeat size. Patients with a larger CAG repeat size had an earlier age of onset of disease as weIl as a more rapid progression of disease (figures 2 and 3, table 1, paper II). We also observed a tendency for the CAG repeat to further expand when transmitted from one generation to the next, thereby providing a molecular explanation for the genetic anticipation seen in the SCA7 families. Patients with smaller repeat expansions «59 CAG) usually presented with ataxia prior to subjective visual impairment, whereas in patients with larger CAG repeats (>59 CAG) the situation was reversed (table 1, paper II). Likewise, Giunti et al. reported that patients with onset of maculopathy preceding onset of ataxia showed significantly larger repeats, than patients where the mode of presentation was reversed (Giunti et al., 1999). It is possible that some clinical symptoms observed in SCA7 depend on additional genetic or environmental factors. David et al. described the frequent symptom of sphincter disturbances in 19 SCA7 families of various geographical origins. The authors also reported decreased hearing in 24% of these patients (David et al., 1998). These two symptoms are not found in Swedish SCA7 families A-D. In addition, a recent publication describing three Black SouthAfrican families, with genetically confirmed SCA7, reported that three out of four patients showed dementia, a symptom not described in SCA7 before (Modi et al., 2000).

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Although the expanded CAG repeats are believed to cause disease through a gain-of-function mechanism, it can not be ruled out that some of the symptoms are in fact due to loss-of-function of the normal gene product. It could be speculated that the symptoms of muscle hypotonia and PDA found in infantile cases of SCA7 represent a partialloss-of-function phenotype. Further studies of the normal function of the SCA7 gene as weIl as disease mechanisms leading to pathology in SCA7 will be needed to shed light on the answers to these questions.

Maternai versus paternal transmission: Effect on the CAG repeat expansion Genetic anticipation observed in SCA7 pedigrees has been reported to be particularly pronounced when the disease is inherited through the patemalline (Benomar et al., 1995; Enevoldson et al., 1994; Gouw et al., 1995; Martin et al., 1994). In the four Swedish families investigated, only two affected fathers transmitted the SCA7 mutation with a further expansion to an affected chiid, in both cases resulting in childhood onset of disease (3 months and 4 years, respectively). However, two affected fathers transmitted their mutated allele without further expansion to three as yet unaffected children. Furthermore, two out of four paternal transmissions, but none of the maternai transmissions, resulted in a decrease ofrepeat size (-2 and -4 repeats). The changes in repeat size upon patemal transmission ranged from -4 to +>150 and in maternai transmissions from ±O to +19. Although patemal bias of anticipation is not pronounced in our four SCA7 families, other studies have reported that SCA7 CAG expansions are greater in patemal transmissions (David et al., 1998; Gouw et al., 1998). The largest expansion observed in the four Swedish SCA7 families was that from apaternai transmission, where the CAG repeat expanded from 49 repeats to over 200

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repeat units. It has been speculated that the large paternal expansions observed are due to that the CAG repeats are particularly unstable in spermatogenesis. A comparison of somatic and gonadal mosaicism in a male SCA7 patient showed a predominant repeat size of 45 CAG with very small variations in blood, whereas sperm contained repeats from 49 CAG and up to more than twice that size, indicating that CAG repeats indeed are very unstable in sperm (David et al., 1998). Single sperm analysis in two affected SCA7 patients has shown these cells to have a mutation rate of nearly 100%, with a 99% bias towards further expansion (Monckton et al., 1999). In agreement with our findings in the Swedish SCA7 families, it has been shown that even though the ratio of affected females and males are 1: 1, a larger proportion of the patients have inherited the mutation from their mother (Gouw et al., 1998). These findings have been speculated to reflect a selective disadvantage for the unstable, very large paternal alleles during spermatogenesis, and/or at fertilization or in utero (Gouw et al., 1998). There are no publications showing that spontaneous abortions are more frequent in SCA7 families where the father is affected with SCA7. There is, however, one report on an ADCA II family with one severely affected child where the mother also had four early spontaneous abortions (Amit et al., 1986).

DNA-based diagnosis of SCA7

The retinal degeneration observed in SCA7 patients is a symptom that enables differentiation between SCA7 and other hereditary forms of SCA on a clinical basis. SCA1 patients that show progressive visual impairment, and one infantile case of SCA2 with retinitis pigmentosa, have however been reported (Abe et al., 1997; Babovic-Vuksanovic et al., 1998; Illarioshkin et al., 1996). Differential diagnoses can also occur in situations where individuals with ataxia show

43

retinal degeneration for reasons other than SCA7, such as diabetes, multiple sclerosis or age-related macular degeneration. In addition, ataxia in association with retinal degeneration in children can be confused with lipid storage disease such as neuronal ceroid lipofuchsinosis (CLN1). Children with CLN1 present with ataxia, muscular hypotonia, loss of speech and macular and retinal changes, similar to infantile SCA7. Although CLN1 is an autosomal recessive disease, the mode of inheritance is not always obvious in infantile SCA7, due to the strong genetic anticipation. The previous problem to diagnose SCA7 patients solely on a clinical basis can today be overcome by the use of a simple PCR-based DNA test. In our initial analysis of SCA7 CAG repeat size in the Swedish population, no intermediate sized alleles in the range 16-39 repeats were found. This indicated that a DNA based mutation test would be conclusive in molecular diagnosis of SCA7. The occurrence of SCA7 family members with intermediate sized repeats lacking disease phenotype have since been reported by us and others (fig 1, paper III and David et al., 1998; Giunti et al., 1999; Stevanin et al., 1998). Reduced penetrance due to intermediate sized alleles has also been reported in HD. Because of the instability of intermediate sized alleles and the reduced penetrance observed in individuals with CAG repeats in this size range, DNAbased testing of SCA7, including presymptomatic and prenatal testing, should follow the same guidelines as for Huntington~s disease.

A founder effect for SCA7 in Scandinavia Haplotype analysis in four Swedish SCA7 families

SCA7 has been reported in families of various ethnic background, including kindreds from Europe (Sweden, Finland, Belgium and France), Africa (Morocco, Libya and South Africa), North America (Caucasian-Americana and

44

Afro-Americans), South America (Brazil) and Asia (Korea) (Anttinen et al., 1986; David, et al., 1996; Enevoldson et. al, 1994; Gouw et al., 1994; Holmberg et al., 1995; Krols et al., 1997; Raitta et al., 1984; Stevanin et al., 1999). The presence of SCA7 in such a variety of ethnic groups suggests that the disease causing mutation has arisen several times, as opposed to all SCA7 families having a common founder. It is, however, likely that regional founders exist in populations such as Sweden and Finland, where the immigration has been limited. In paper II we investigated the possibility of a regional founder effect in four Swedish SCA7 families, by applying genetic as weIl as genealogical methods. A haplotype study Swedish SCA7 families A, B, C and D, including 16 affected individuals and their healthy relatives, was performed using a total of 10 microsatellites in a 12 cM region harbouring the SCA710cus on chromosome 3. Families A and B could, by genealogical investigations, be traced back to a common ancestor born in the mid 17th century, 6-8 generations ago. This kinship was further supported by haplotype analysis, which demonstrated that patients in families A and B shared a haplotype over the 12 cM analysed in the study (fig 4, paper II). Families C or D could not be genealogically traced back to any of the other Swedish families, they did however share a haplotype centromeric ofD3S1287, indicating that they could be distantly related (fig 4, paper II). SCA7 patients in all four families also shared alleles for two markers very close to the SCA7 gene, D3S1287 and D3S3635 (figure 4, paper II). The fact that the first reports of neurological disease in members of families A and B date back to the early 20th century, 6-8 generations from the common ancestor, indicates that a premutation may have ansen several generations before the occurrence of disease. It could be speculated that such a premutation,

45

perhaps in form of an intermediate sized CAG repeat, could be more susceptible for further expansion, resulting in pathology.

A/ounder mutation/or SCA7 in Scandinavia The development of a DNA-based diagnostic test for SCA7 allowed for the identification of three additional Swedish families, making SCA7the most common form of ADCA genetically verified in Sweden. This is in striking contrast to other populations, where SCA7 has been reported to constitute only 4.5 to 11.6% of the ADCAs diagnosed (Benton et al., 1998; Moseley et al., 1998). To investigate whether SCA7 was also common in other Scandinavian countries, we performed an inventory of genetically verified SCA7 patients in Sweden, Finland, Denmark and Norway (paper III). A total of 25 Swedish and 15 Finnish SCA7 patients from 8 Swedish and 7 Finnish families were found, including previously described Swedish families AlB, C and D. No SCA7 patients were identified in Norway or Denmark. Even though SCA7 was the most common genetically verified dominant ataxia in both Sweden and Finland, the frequencies of other ataxias differed. In Sweden, only two other forms of ADCA were reported, SCA2 and SCA3 (1 and 4 patients respectively). In contrast, in the Finnish population families with SCA1 (12 patients from 5 families), SCA2 (3 patients from one family) and SCA6 (1 patient) were found (paper III). We wanted to investigate if this overrepresentation of SCA7 among the genetically verified ataxias in Sweden and Finland was the result of a common founder mutationIpremutation. We, therefore, performed a haplotype analysis on all 15 Swedish and Finnish SCA7 families identified (paper III). Nine microsatellite markers and one intragenic polymorphism (G3145TGIA3145TG), covering a 10.2 cM region surrounding the SCA710cus, were analysed and

46

haplotypes were constructed. Ten families shared a haplotype over a region of 9.3 cM between markers D3S3631 and D3S1228. In addition, we identified a core haplotype A-3-10 that was shared by all 15 families, consisting of the intragenic polymorphism and markers D3S1287 and D3S1228. The presence of a common haplotype A-3-10 for the closely linked markers in all Scandinavian SCA7 families supports our hypothesis of a common ancestor. Furthermore, marker D3S1287 proved particularly informative as allele 3, which segregated with SCA7 in all patients, was only present in 7 out of 264 unrelated controi chromosomes (2.6%). Out of these seven individuals, only two carried allele A for the intragenic polymorphism. These individuals were both heterozygous for the two markers, resulting in a maximum frequency of 0.75% for the haplotype A-3 in the Swedish population. De novo expansions of lA to pathological SCA7 alleles are rare, but have been

demonstrated in four families of Tunisian, Jamaican and Italian origin, and support the hypothesis that founder premutations in the lA range may exist (Giunti et al., 1999). A de novo expansion from a CAG repeat of normal size to and lA or to a full sized mutation has so far not been reported. We therefore find it unlikely that the SCA7 mutation has occurred independently, 15 times, in the Swedish and Finnish populations and that these 15 independent mutations by chance also carry the same, rare haplotype for three closely linked markers. Instead, we suggest that all Scandinavian SCA7 patients share a common ancestor. We hypothesise that this ancestor carried a premutation in the form of an intermediate sized CAG repeat, more susceptible to expand to disease causing size in subsequent generations.

47

Expression of ataxin-7 in normal and pathological tissue Polyglutamine disorders have been found to show overlapping, yet distinct patterns of neurodegeneration. Still, the respective disease genes are reported to be widely expressed in both CNS and non-CNS tissues (Jou and Myers, 1995; Li et al., 1993; Nagafuchi et al., 1994; Servadio et al., 1995; Sharp et al., 1995; Strong et al., 1993; Trottier et al., 1995). Recent studies have implicated the importance of nuclear localisation of mutant protein for the development of neurological disease, but the reason for the discrepancies in cell specificity of neurodegeneration between the polyglutamine disorders is a question that remains to be answered (Klement et al., 1998; Saudou et al., 1998). To investigate the role of ataxin-7 expression in SCA7 pathology as weIl as to gain insight into the function of wild-type ataxin-7, the expression pattern of ataxin-7 was analysed in a number of CNS and non-CNS tissues from SCA7 patients and controls (paper IV). One adult onset SCA7 patient, age at onset 19 years, and one childhood case of SCA7, age at onset 4 years, as weIl as neurologically healthy controis, were investigated. Tissues analysed included those considered major sites of pathology in SCA7 (cerebellum, olivary nuclei of the pons, and retina), as weIl as other areas of the CNS (hippocampus and four different regions of cerebral cortex) and peripheral tissues (heart, lung, kidney, adrenal gland, liver, spleen, thyroid and pancreas). Expression of ataxin-7 was analysed on sections from cryo-preserved or paraffin-embedded tissue, using a polyclonal anti-ataxin-7 antibody, SCA7pepl-15, raised against a synthetic peptide corresponding to the 15 most N-terminal amino acids of the human protein.

48

Specificity of SCA7pepl-15 Immunohistochemical staining using the affinity-purified polyclonal antiataxin-7 antibody, SCA7pepl-15, revealed a distinct nuclear, mainly neuronal immunoreactivity in both patients and controis. In tissue from SCA7 patients the SCA7pepl-15 antibody also stained neuronal intranuclear inclusions, NIls, as previously reported in SCA7 brain (Holmberg et al., 1998). The specificity of SCA7pepl-15 was analysed on paraffin sections from cerebral cortex of the control, where the staining was most intense. Nuclear immunoreactivity was completely blocked by pre-incubation of SCA7pepl-15 with the peptide used to raise this antibody (fig la-b, paper IV). Staining of NIls in patient material was also completely abolished by competing with this peptide (data not shown). In contrast, pre-incubation with a non-related peptide had no effect on nuclear immunoreactivity (fig 1c, paper IV). No labelling was observed using preimmune serum, or when the primary antibody was omitted (data not shown).

Expression of ataxin-7 in cerebellum, olivary nuclei and retina Neurodegeneration has been reported to be pronounced in the cerebellum, the olivary nuclei of the pons, and the retina of SCA7 patients (Gouw et al., 1994; Martin et al., 1994). To investigate the distribution of ataxin-7 in these regions, we performed immunohistochemical analysis on the corresponding tissues from SCA7 patients and controis. Immunoreactivity was found to be exclusively nuclear and predominantly restricted to neurons. There was no observable difference in the staining pattem or frequency of immunopositve cells between patients and the controI. Neurons in the cerebellum of the control, including Purkinje cells, showed intense nuclear staining (figures 2a-c, paper IV). In patients, the few Purkinje cells remaining were dysmorphic and had migrated into the molecular layer

49

(figure 2b-c, paper IV). Heterotopic Purkinje cells have previously been reported as a prominent feature of SCA1 transgenic mice, and have been suggested to reflect an attempt by the cell to preserve synapses (Clark et al., 1997; Lin et al., 1999). NIls in cerebellum of SCA7 patients occurred at a very low frequency and rarely in Purkinje cells. The presence of NIls in Purkinje cells and other cerebellar neurons of a young onset SCA7 patient has previously been demonstrated using the 1C2 antibody. AIso in this study the frequency of NIls in Purkinje cells was low, likely due to the severe cerebellar degeneration (Holmberg et al., 1998). In olivary nuclei, ataxin-7 immunoreactivity was detected as an evenly distributed nuclear staining in neurons of the controi brain (figure 2d, paper IV). Analysis of SCA7 brains revealed a similar pattern (figure 2e, paper IV), accompanied by the occasional detection of NIls (data not shown). Again, the relatively low numbers of ataxin-7 positive cells in the patient are likely to be the result of neuronalloss. Neuropathological examination of the eye of SCA7 patients have reported atrophy of the choroid and degeneration of a number of cell types in the retina, including the pigment epithelial cells, both types of photoreceptor cells of the outer nuclear layer (ONL), bipolar cells of the inner nuclear layer (INL)/ bipolar celllayer (BCL), and ganglion cells (Gouw et al., 1994; Martin et al., 1994). Immunolocalisation of ataxin-7 in controi retina demonstrated nuclear staining of a subset of the ganglion cells and a low frequency of positive cells in the bipolar celllayer (BCL) and photoreceptors (PR) (figure 2f, paper IV). This is in contrast to the study by Mauger et al., where no nuclear staining in controi retina could be detected using a polyclonal anti-ataxin-7 peptide antibody, CM189, directed towards amino acids 2-14 of the human ataxin-7 (Mauger et al., 1999). However, Mauger et al. did detect NIls in both inner and outer

50

nuclear layers, as weIl as in ganglion cells of SCA7 retina. The NIls found in patient retina could also be confirmed using antibody 1C2 (Mauger et al., 1999). In our analysis, retina was only available from the adult onset SCA7 patient. This patient had suffered from visual impairment since age 19 and had been completely blind for many years at the time of her death. The retina therefore showed severe degeneration and no staining was observed in any of the tissue that remained (figure 2g, paper IV)

Ataxin-7 expression in cerebral cortex and hippocampus We further examined the expression of ataxin-7 in regions of the CNS not considered to represent major sites ofpathology, including frontal, parietal, temporal and occipitallobes of cerebral cortex, as weIl as hippocampus. In controi brain nuclear immunoreactivity was intense in neurons though out layers III-VI of cerebral cortex (figure 3a-d and data not shown, paper IV). The staining was similar in the different lobes examined, and there was no detectable difference in number of staining cells between patient and controI. Immunolocalisation appeared to be restricted to neurons, although the involvement of immunopositive astrocytes can not be ruled out. NIls were relatively frequent in all cortical regions analysed in SCA7 patients, as reported previously (Holmberg et al., 1998). The nuclear staining of neurons containing NIls appeared less intense than in nuclei that did not contain NIls, possibly due to recruitment of normal ataxin-7 to the aggregates (figure 3b and d, paper IV). In support of this hypothesis, Perez et al. have demonstrated that cells expressing ataxin-3 containing an expanded polyglutamine stretch form nuclear inclusions that sequester mutant as weIl as normal protein (Perez et al., 1998).

51

Intense nuclear immunoreactivity was also detected in neurons of both the dendate gyms and area CA3 of the hippocampus (figure 3e-h, paper IV). Nuclear inclusions in the hippocampus of the adult onset case studied here were rare, compared to the young onset case reported by Holmberg et al. (Holmberg et al., 1998). An unusually high frequency of nuclear inclusions was found in layers III-IV of cerebral cortex in the young onset patient (figure 4a, paper IV). This finding is consistent with the higher frequency of NIls observed in a juvenile-onset case of HD (Di Figlia et al., 1997). We estimate the frequency of NIs in cerebral cortex of the young onset SCA7 case to be in the order of 10 times that of the frequency in the adult onset case (figures 4a-b, paper IV).

Expression of ataxin-7 in peripheral tissue Apart from the hypotonia and PDA occasionally found in extreme infantile cases of SCA7, clinical symptoms other than those derived from the CNS are generally not present in patients. Expression of SCA7 mRNA has however been demonstrated by Northern blot analysis, in a number of peripheral tissues including skeletal muscle and heart, from non-affected individuals (David et al., 1998; David et al., 1997). In order to analyse if ataxin-7 is expressed outside the CNS, a number of non-neuronal tissues were analysed in patients and controis. No nuclear staining could be detected in heart, adrenal gland, liver, lung, thyroid or spleen of either the patient or the controI. In spite of the apparent lack of nuclear staining in the peripheral tissues examined, nuclear inclusions were observed in the kidney and pancreas of the adult-onset SCA7 patient (figure 5, paper IV). The number of nuclear inclusions was low in the kidney but more frequent in the pancreas (figure 5, paper IV).

52

The finding that ataxin-7 expression is as abundant in areas showing severe neurodegeneration in SCA7 patients, as weIl as in areas only mildly affected, suggests that expression pattem is not a major determinant of pathology in SCA7 brain. This is consistent with reports of protein expression in other polyglutamine disorders, including HD, SCA1 and SCA3/MJD (Paulson et al., 1997; Servadio et al., 1995). Our data also confirms previous conclusions that NIls are not the primary cause of disease pathology in SCA7 (Holmberg et al., 1998).

53

CONCLUSIONS

• The SCA7 disease gene is localised on chromosome 3p12-21.1

• Swedish SCA7 patients carry expansions of a CAG motif in the 5 ~region of the SCA7 gene, ranging from 40 to more than 200 CAG repeats.

• The CAG expansions are inversely correlated to the age at onset of disease and disease severity (genetic anticipation).

• There is a strong founder effect for SCA7 in the Scandinavian population.

• Ataxin-7 is a nuclear protein, widely expressed in brain, but also detected in pancreas and kidney.

• Expression pattem of ataxin-7, subcellular localisation or formations of NIls in SCA7 patients are not the main determinants of cell-specific neuronal dysfunction and degeneration in this disease.

54

ACKNOWLEDGEMENTS

• I am most grateful to all the SCA7 family members for their kind co-operation and participation, without whom these projects would not have been possible. I truly hope that they will benefit from our findings in some way.

• My gratitude to Oskarfonden, Neurologiskt Handikappades Riksförbund and Torsten

och Ragnar Söderbergs Stiftelser for financially supporting my research.

• Naturally, I want to thank my supervisor Dr. Monica Holmberg for her true devotion to the project, for support and criticism of my work. You made me promise only to write nice things about you- if's really not that difficult...1 can always save the rest for aspeech!

• A number of people have contributed their crucial skills, enthusiasm and support for my work, of whom I would especially like to mention professor Lars Forsgren, professor

Gösta Holmgren, associate professor Jaakko Ignatius and associate professor Ola Sandgren. I have also had the opportunity to be involved in collaborations with Dr Erik Björck, professor Alexis Brice, associate professor Thomas Brännström, Vesa Juvonen, associate professor Atle Melberg, Dr. Pertti Sistonen and professor Jan Wahlström, who are all acknowledged.

• All the people who make life in the lab easier: the technical staff at the Media

departments of CMB and Microbiology, P-A Lundström and Johnny Stenman, EvaChristine Lundström, Ethel Strömdahl, Berit Nordh and Marita Waern. May the sun always shine on you!

• All the people who guided me through the year Monica was in Paris- especially associate professors Kristina Forsman-Semb and Lisbet Lind for co-supervising.

• Dr. Patricia Hart for correcting my Swenglish and for other friendly deeds!

55

Then, of course, as you all know:

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56

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