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Polyglutamine expansion accelerates the dynamics of ataxin-1 and does not result in aggregate formation.

Krol HA, Krawczyk PM, Bosch KS, Aten JA, Hol EM, Reits EA - PLoS ONE (2008)

Bottom Line: Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1.Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before.These results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT

Background: Polyglutamine expansion disorders are caused by an expansion of the polyglutamine (polyQ) tract in the disease related protein, leading to severe neurodegeneration. All polyQ disorders are hallmarked by the presence of intracellular aggregates containing the expanded protein in affected neurons. The polyQ disorder SpinoCerebellar Ataxia 1 (SCA1) is caused by a polyQ-expansion in the ataxin-1 protein, which is thought to lead to nuclear aggregates.

Methodology/principal findings: Using advanced live cell fluorescence microscopy and a filter retardation assay we show that nuclear accumulations formed by polyQ-expanded ataxin-1 do not resemble aggregates of other polyQ-expanded proteins. Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1. During mitosis, ataxin-1 accumulations redistributed equally among daughter cells, in contrast to polyQ aggregates. Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before.

Conclusions/significance: These results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1. The unexpected findings for a polyQ-expanded protein and their consequences for ongoing SCA1 research are discussed.

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Related in: MedlinePlus

Nuclear ataxin-1 accumulations do not resemble aggregates formed by other disease-related polyglutamine-expanded proteins.(A). Live cell images of wild-type and polyQ-expanded ataxin-1 (Atx1[Q2]GFP and Atx1[Q85]GFP) and polyglutamine-expanded proteins androgen receptor (AR[Q112]GFP), huntingtin exon-1 (Htt[Q103]GFP), NLS[Q64]GFP and Q65-GPF in Cos7 cells. (B). Filtertrap assay of celllysates of Cos7 cells expressing Atx1[Q2]GFP, Atx1[Q85]GFP and NLS[Q64]GFP, 24, 48 and 72 hr after transfection (upper panel) and of Cos7 cells expressing AR[Q112]GFP, Q65-GFP and Htt[Q103]GFP 24 hours after transfection (lower panel). Sizebar = 1 µm.
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pone-0001503-g001: Nuclear ataxin-1 accumulations do not resemble aggregates formed by other disease-related polyglutamine-expanded proteins.(A). Live cell images of wild-type and polyQ-expanded ataxin-1 (Atx1[Q2]GFP and Atx1[Q85]GFP) and polyglutamine-expanded proteins androgen receptor (AR[Q112]GFP), huntingtin exon-1 (Htt[Q103]GFP), NLS[Q64]GFP and Q65-GPF in Cos7 cells. (B). Filtertrap assay of celllysates of Cos7 cells expressing Atx1[Q2]GFP, Atx1[Q85]GFP and NLS[Q64]GFP, 24, 48 and 72 hr after transfection (upper panel) and of Cos7 cells expressing AR[Q112]GFP, Q65-GFP and Htt[Q103]GFP 24 hours after transfection (lower panel). Sizebar = 1 µm.

Mentions: PolyQ disorders show accumulation of polyQ-expanded proteins into a single cytoplasmic or nuclear aggregate. In agreement with data published previously [13], [21] our experiments demonstrated that ataxin-1 is mostly accumulating into multiple nuclear accumulations and this process is independent of the length of the polyQ expansion (Fig 1A). To compare the distribution and aggregate formation of ataxin-1 to a variety of different polyQ-expanded proteins we transfected Cos-7 cells with different polyQ proteins tagged with green fluorescent protein (GFP), to enable visualization in living cells. Cos-7 cells were chosen since they have a low expression level of endogenous ataxin-1 [8]. This minimizes interactions between the transfected ataxin-1 fusion proteins and the endogenous wild-type ataxin-1, thereby preventing any additional effect on the attaxin-1 aggregate formation. Next to the wildtype ataxin-1 (Atx1[2Q]GFP) and the polyQ-expanded ataxin-1 (Atx1[85Q]GFP), two disease-related polyQ-expanded fusion proteins were used, i.e. the truncated androgen receptor (AR[Q112]GFP) and huntingtin exon1 (Htt[Q103]GFP) which are both aggregation-prone (Fig 1A). We also expressed a pure polyQ-tract fused to GFP (Q65-GFP) and a polyQ-GFP fused to a nuclear localization signal (NLS[Q64]GFP). These fusion proteins are also aggregation-prone due to a similar polyQ-expansion. The NLS signal targets the protein to the nucleus, which mimics ataxin-1 polyQ localization. When cells were analyzed by Confocal Laser Scanning Microscopy, all polyQ-expanded proteins formed irregularly shaped intracellular aggregates within 24 hours after transfection, with exception of Atx1[85Q]GFP and Atx1[2Q]GFP which formed multiple nuclear accumulations (Fig 1A). The formation of multiple nuclear accumulations of Atx1[2Q]GFP has been described before [13], [21]. The distinct pattern of the formation of multiple nuclear accumulations is probably due to features of the ataxin-1 protein, since nuclear polyQ-expanded GFP (NLS[Q64]GFP) resulted in a single nuclear aggregate which increased in size in time due to sequestering of newly synthesized polyQ-GFP proteins, similar as observed with other polyQ-expanded proteins. This main difference between Atx1[85Q]GFP and NLS[Q64]GFP was maintained up to 72 hours after transfection.


Polyglutamine expansion accelerates the dynamics of ataxin-1 and does not result in aggregate formation.

Krol HA, Krawczyk PM, Bosch KS, Aten JA, Hol EM, Reits EA - PLoS ONE (2008)

Nuclear ataxin-1 accumulations do not resemble aggregates formed by other disease-related polyglutamine-expanded proteins.(A). Live cell images of wild-type and polyQ-expanded ataxin-1 (Atx1[Q2]GFP and Atx1[Q85]GFP) and polyglutamine-expanded proteins androgen receptor (AR[Q112]GFP), huntingtin exon-1 (Htt[Q103]GFP), NLS[Q64]GFP and Q65-GPF in Cos7 cells. (B). Filtertrap assay of celllysates of Cos7 cells expressing Atx1[Q2]GFP, Atx1[Q85]GFP and NLS[Q64]GFP, 24, 48 and 72 hr after transfection (upper panel) and of Cos7 cells expressing AR[Q112]GFP, Q65-GFP and Htt[Q103]GFP 24 hours after transfection (lower panel). Sizebar = 1 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2200796&req=5

pone-0001503-g001: Nuclear ataxin-1 accumulations do not resemble aggregates formed by other disease-related polyglutamine-expanded proteins.(A). Live cell images of wild-type and polyQ-expanded ataxin-1 (Atx1[Q2]GFP and Atx1[Q85]GFP) and polyglutamine-expanded proteins androgen receptor (AR[Q112]GFP), huntingtin exon-1 (Htt[Q103]GFP), NLS[Q64]GFP and Q65-GPF in Cos7 cells. (B). Filtertrap assay of celllysates of Cos7 cells expressing Atx1[Q2]GFP, Atx1[Q85]GFP and NLS[Q64]GFP, 24, 48 and 72 hr after transfection (upper panel) and of Cos7 cells expressing AR[Q112]GFP, Q65-GFP and Htt[Q103]GFP 24 hours after transfection (lower panel). Sizebar = 1 µm.
Mentions: PolyQ disorders show accumulation of polyQ-expanded proteins into a single cytoplasmic or nuclear aggregate. In agreement with data published previously [13], [21] our experiments demonstrated that ataxin-1 is mostly accumulating into multiple nuclear accumulations and this process is independent of the length of the polyQ expansion (Fig 1A). To compare the distribution and aggregate formation of ataxin-1 to a variety of different polyQ-expanded proteins we transfected Cos-7 cells with different polyQ proteins tagged with green fluorescent protein (GFP), to enable visualization in living cells. Cos-7 cells were chosen since they have a low expression level of endogenous ataxin-1 [8]. This minimizes interactions between the transfected ataxin-1 fusion proteins and the endogenous wild-type ataxin-1, thereby preventing any additional effect on the attaxin-1 aggregate formation. Next to the wildtype ataxin-1 (Atx1[2Q]GFP) and the polyQ-expanded ataxin-1 (Atx1[85Q]GFP), two disease-related polyQ-expanded fusion proteins were used, i.e. the truncated androgen receptor (AR[Q112]GFP) and huntingtin exon1 (Htt[Q103]GFP) which are both aggregation-prone (Fig 1A). We also expressed a pure polyQ-tract fused to GFP (Q65-GFP) and a polyQ-GFP fused to a nuclear localization signal (NLS[Q64]GFP). These fusion proteins are also aggregation-prone due to a similar polyQ-expansion. The NLS signal targets the protein to the nucleus, which mimics ataxin-1 polyQ localization. When cells were analyzed by Confocal Laser Scanning Microscopy, all polyQ-expanded proteins formed irregularly shaped intracellular aggregates within 24 hours after transfection, with exception of Atx1[85Q]GFP and Atx1[2Q]GFP which formed multiple nuclear accumulations (Fig 1A). The formation of multiple nuclear accumulations of Atx1[2Q]GFP has been described before [13], [21]. The distinct pattern of the formation of multiple nuclear accumulations is probably due to features of the ataxin-1 protein, since nuclear polyQ-expanded GFP (NLS[Q64]GFP) resulted in a single nuclear aggregate which increased in size in time due to sequestering of newly synthesized polyQ-GFP proteins, similar as observed with other polyQ-expanded proteins. This main difference between Atx1[85Q]GFP and NLS[Q64]GFP was maintained up to 72 hours after transfection.

Bottom Line: Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1.Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before.These results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT

Background: Polyglutamine expansion disorders are caused by an expansion of the polyglutamine (polyQ) tract in the disease related protein, leading to severe neurodegeneration. All polyQ disorders are hallmarked by the presence of intracellular aggregates containing the expanded protein in affected neurons. The polyQ disorder SpinoCerebellar Ataxia 1 (SCA1) is caused by a polyQ-expansion in the ataxin-1 protein, which is thought to lead to nuclear aggregates.

Methodology/principal findings: Using advanced live cell fluorescence microscopy and a filter retardation assay we show that nuclear accumulations formed by polyQ-expanded ataxin-1 do not resemble aggregates of other polyQ-expanded proteins. Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1. During mitosis, ataxin-1 accumulations redistributed equally among daughter cells, in contrast to polyQ aggregates. Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before.

Conclusions/significance: These results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1. The unexpected findings for a polyQ-expanded protein and their consequences for ongoing SCA1 research are discussed.

Show MeSH
Related in: MedlinePlus