Polyglutamine and polyalanine expansions in ataxin7 result in different types of aggregation and levels of toxicity

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Abstract

Spinocerebellar ataxia type 7 (SCA7) is caused by expansion of a (CAG)n repeat in the ataxin7 gene, resulting in an abnormally long polyglutamine polyQ tract in the translated protein that aggregates in the form of neuronal intranuclear inclusions. Polyalanine (polyA) stretches, implicated in several genetic disorders, also appear to aggregate. To investigate the role of the aggregates in the pathologies, we compared the effects of ataxin7 containing a polyA (ataxin7–90A) or polyQ (ataxin7–100Q) expansion in HEK 293 cells and in primary cultures of rat mesencephalon. Both proteins formed nuclear and perinuclear aggregates that contained molecular chaperones and components of the ubiquitin–proteasome system, suggesting that they were abnormally folded. Ataxin-90A aggregates differed morphologically from ataxin7–100Q aggregates, consisted of small and amorphous rather than fibrillar inclusions and were more toxic to mesencephalic neurons, suggesting that toxicity was determined by the type of aggregate rather than the cellular misfolding response.

Introduction

Protein misfolding, often leading to the formation of aggregates (Temussi et al., 2003), is observed in many late-onset neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, prion encephalopathies and other amyloidopathies. Nine of these disorders, Huntington's disease, spinobulbar muscular atrophy, dentatorubral–pallidoluysian atrophy and six types of spinocerebellar ataxia (SCA1, 2, 3, 6, 7 and 17), are known as polyglutamine (polyQ) diseases. They all lead to dramatic neurological dysfunction and ultimately to death, and no treatment is as yet available that can stop or even slow disease progression.

PolyQ diseases are all caused by the same kind of mutation: an abnormally high number of coding CAG repeats translated into expanded polyQ sequences in the corresponding protein. The number of glutamine repeats in the proteins varies from 21 to >306 in the nine disorders, but, in most cases, the phenotype becomes apparent at a threshold of 30–40 repeats. Another characteristic of polyQ diseases is the presence of neuronal intranuclear inclusions (NIIs) or aggregates of the pathological proteins in the brains of patients (Paulson, 1999, Tran and Miller, 1999). There is growing evidence that NIIs reflect the tendency of proteins containing large polyQ stretches to assume abnormal β-sheet conformations that assemble into fibrillary aggregates (Perutz, 1996), which also sequester other proteins with specific functions (e.g. ubiquitin, proteasome subunits, transcription factors, heat shock proteins) or structures (polyQ-rich proteins) (Chai et al., 1999a, Chai et al., 1999b, Ferrigno and Silver, 2000). Although protein aggregates are the major pathological hallmark of these diseases, their contribution to the pathological process remains unclear. Experimental studies have concluded diversely that NIIs may be toxic (Becher et al., 1998, Davies et al., 1997, Gusella and MacDonald, 2000, Scherzinger et al., 1997, Scherzinger et al., 1999, Skinner et al., 1997), non-toxic (Holmberg et al., 1998, Klement et al., 1998, Sathasivam et al., 1999, Saudou et al., 1998) or even protective (Saudou et al., 1998).

Interestingly, there is also a family of nine coding GCN repeat disorders, the polyalanine (polyA) diseases (Brown and Brown, 2004, Amiel et al., 2004). These diseases do not have a neurodegenerative phenotype, but oculopharyngeal muscular dystrophy (OPMD), which is caused by a coding (GCG)7–13 repeat instead of a (GCG)6 repeat in the PABPN1 gene (Brais et al., 1999), is associated with the formation of fibrillary intranuclear inclusions in muscle. These inclusions have some of the same components as polyglutamine NIIs (Askanas et al., 1991, Calado et al., 2000). PolyA expansions in the disease-related FOXL2 and ARX genes also induce protein aggregation in vitro (Caburet et al., 2004, Nasrallah et al., 2004). Furthermore, when polyA tracts were expressed in COS7 cells, they formed fibrillary inclusions and induced cell death (Rankin et al., 2000).

These observations suggest that polyQ and polyA expansions may have the same mechanism of toxicity. To address this issue, we compared protein aggregation and toxicity resulting from the transient expression, in HEK 293 cells and primary cultures of rat embryonic mesencephalic neurons, of ataxin7, the protein encoded by the SCA7 gene responsible for SCA7 (Stevanin et al., 2000, Stevanin et al., 2002) carrying either a polyQ or a polyA expansion. The disease develops when ataxin7 carries a stretch of at least 36 glutamines. This protein also normally contains an imperfect polyA sequence upstream of the polyQ stretch. For the purposes of our study, we expressed ataxin7 with either a 90-repeat polyA or a 100-repeat polyQ expansion.

Section snippets

Ataxin7 with polyalanine or polyglutamine expansions forms aggregates

Vectors allowing the expression of wild-type ataxin7 (ataxin7–10Q), ataxin7 with a polyA expansion (ataxin7–90A) and ataxin7 with a polyQ expansion (ataxin7–100Q) fused with EGFP (Fig. 1) were transiently expressed in human embryonic kidney cells (HEK 293). Western blot analysis of cell extracts using an antibody targeting the N-terminal region of ataxin7 (1C1, kind gift from J. L. Mandel, IGBMC, Illkirch, France) detected proteins of appropriate apparent molecular weights, except for

Discussion

We have shown that polyA and polyQ expansions produced at similar levels in the same protein differ both in the way they accumulate and the toxicity of aggregates. However, as truncated forms of these proteins were also observed, we cannot exclude that aggregation was mainly due to these forms that are well known to be more prone to aggregate. Both expansions evoked cellular responses to protein misfolding, as shown by the presence in the aggregates of the molecular chaperones Hsp40 and Hsp70

SCA7 constructs

All SCA7 constructs (Fig. 1) were cloned in pEGFP-N1 vectors (Clontech). The wild-type SCA7 (10Q) and SCA7–100Q constructs have previously been described (Zander et al., 2001). SCA7–90A was generated by inserting a 90GCA cassette into the wild-type SCA7 construct. This cassette was created by generating a frameshift in the SCA7–100Q construct by PCR. All constructs were verified by DNA sequencing. The sizes of the proteins were confirmed by Western blot.

Cell cultures/transfections

Human embryonic kidney (HEK) 293 cells

Acknowledgments

The authors are indebted to Drs. Eric Leguern, Erich Wanker, Gael Yvert and Didier Devys for helpful discussions during the design stage, to Drs. Nacer Edine Abbas, Benedicte Salthun-Lassalle, Cornelia Hampe and Anne Sophie Lebre for their contribution to some aspects of this work and to Dr. Olga Corti for invaluable advice, support and critical reading of the manuscript. We also thank Dr. Jean-Louis Mandel for providing us with the 1C1 antibody. M.L. was the recipient of a fellowship from the

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    Present address: CECS/I-STEM, Evry, France.

    2

    Present address: Department of Genetics and Pathology, University Hospital, 70185 Uppsala, Sweden.

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