Quantification of Ataxin-3 and Ataxin-7 aggregates formed in vivo in Drosophila reveals a threshold of aggregated polyglutamine proteins associated with cellular toxicity

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Highlights

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    The level of aggregated forms of polyQ proteins associates with toxicity.

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    Suppressors of polyQ protein-induced toxicity lower the level of cellular aggregates.

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    PolyQ protein aggregates accumulate as flies age until cell death is detected in vivo.

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    A critical threshold of aggregated polyQ proteins is associated to degeneration.

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    Three models of polyglutamine diseases lead to similar results.

Abstract

Polyglutamine diseases are nine dominantly inherited neurodegenerative pathologies caused by the expansion of a polyglutamine domain in a protein responsible for the disease. This expansion leads to protein aggregation, inclusion formation and toxicity. Despite numerous studies focusing on the subject, whether soluble polyglutamine proteins are responsible for toxicity or not remains debated. To focus on this matter, we evaluated the level of soluble and insoluble truncated pathological Ataxin-3 in vivo in Drosophila, in presence or absence of two suppressors (i.e. Hsp70 and non-pathological Ataxin-3) and along aging. Suppressing truncated Ataxin-3-induced toxicity resulted in a lowered level of aggregated polyglutamine protein. Interestingly, aggregates accumulated as flies aged and reached a maximum level when cell death was detected. Our results were similar with two other pathological polyglutamine proteins, namely truncated Ataxin-7 and full-length Ataxin-3. Our data suggest that accumulation of insoluble aggregates beyond a critical threshold could be responsible for toxicity.

Introduction

Polyglutamine (PolyQ) diseases are fatal dominantly inherited neurodegenerative disorders provoked by the expansion of a polyQ domain in a disease-specific protein prone to aggregate. With Huntington's disease, Spinobulbar Muscular Atrophy, Dentatorubral Pallidoluysian Atrophy, Spinocerebellar Ataxia (SCA) type 1, 2, 6 and 17, SCA3 and SCA7 are two of the nine polyQ diseases identified to this date. When the size of their polyQ domain exceeds a threshold of 54 glutamines for the Atx3 protein or 36 for the Atx7 protein, patients respectively develop the SCA3 or SCA7 disease [1].

A common feature to polyQ diseases is the presence of inclusions detectable by microscopy on histological brain slices from patients or in cellular and animal models. Inclusions are mainly nuclear [2], [3] and contain numerous proteins, including the polyQ protein responsible for the pathology and chaperone proteins such as Hsp70 [4]. Neither the conformation nor the oligomerization states of the pathological polyQ proteins can be fully characterized in vivo whether inside or outside inclusions. Therefore the link between aggregates and inclusions remains unclear.

Recent studies have focused on oligomers that can be formed by elongated polyQ proteins. Oligomers have been detected both in vitro and in vivo as multiple different oligomeric conformations [5], [6], [7], [8]. They can be considered as soluble [6], [9] or insoluble [8] but the notion of solubility is not defined on common grounds. Depending on the study, soluble oligomers are either toxic [10], [11] or not [12], and reciprocally, insoluble oligomers can be either toxic [13] or not [8].

In the present work we sought to investigate the toxicity of the SDS-soluble or -insoluble species of Atx3 and Atx7 polyQ proteins formed in vivo.

Section snippets

Fly genetics and phenotype observation

All crosses were grown at 25 °C or 19 °C on standard medium, which was changed every other day. Glass-Mediated Response-GAL4 (GMR-gal4) was used as a GAL4 driver. Drosophila eyes were photographed with a Leica MZFL III microscope.

Western and dot blotting

Twenty male and twenty female flies were sacrificed in liquid nitrogen. Their heads were crushed in denaturing buffer (TE pH 8.5; 2% SDS, 0.05M DTT; 10 μM Protease Inhibitor Cocktail AEBSF, Roche) and incubated at 96 °C for 12 min. Part of the protein extracts was dot

Quantification of SDS-soluble and -insoluble Atx proteins by western and dot blotting

The definition of protein aggregates often varies. They are defined in our study as oligomers that resist boiling in a 2% SDS reducing solution. Aggregate quantities are often evaluated from the amount of proteins trapped at the top of western blots. However, only small aggregates can enter stacking gels, while large aggregates cannot. An alternative method to quantify aggregates is dot blotting. To compare the accuracy of western and dot blotting quantifications of protein aggregates, we first

Discussion

Characterizing the toxic species among the monomers, small or large oligomers, fibrils, inclusions, soluble and insoluble forms of polyQ proteins has been a major goal in the research on polyQ diseases but the means, models and definitions vary from a study to another. We chose to focus on solubility in a denaturing and reducing environment classically found in SDS-PAGE. To minimize SDS-insoluble aggregate loss, we quantified aggregated polyQ proteins by dot blotting and SDS-soluble polyQ

Acknowledgments

This article is dedicated to the memory of Didier Contamine who initiated this study and Evelyne Maillier who participated to some of these experiments. We would like to thank Hervé Tricoire and Nancy Bonini respectively for SCA7 and SCA3 transgenic models. Transgenic flies expressing hsp70, hsc4, hsc3, puc were provided by the Bloomington Drosophila Resource Center. The bsk5680R-2 RNAi line was supplied by the National Institute of Genetics. We are thankful to Pierre Gandille, Alyssa

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    • Mass spectrometry analyses of normal and polyglutamine expanded ataxin-3 reveal novel interaction partners involved in mitochondrial function

      2018, Neurochemistry International
      Citation Excerpt :

      Some of the other most abundant interactors included a number of molecular chaperones (HSPH1, HSPA1A, HSPA8, HSPA4L) and the co-chaperone DNAJA1. The association of chaperones of the Hsp70 and Hsp40 family with ataxin-3 has previously been observed both in vitro and in SCA3 patients tissue (Chai et al., 1999; Gao et al., 2011) and have also been shown to reduce SCA3 toxicity and aggregation (Bilen and Bonini, 2007; Ito et al., 2016; Vinatier et al., 2015). We observed that overexpression of Hsp70 reduced the steady state levels of both normal and polyQ expanded ataxin-3 variants.

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