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Novel polyglutamine model uncouples proteotoxicity from aging.

Christie NT, Lee AL, Fay HG, Gray AA, Kikis EA - PLoS ONE (2014)

Bottom Line: Polyglutamine expansions in certain proteins are the genetic determinants for nine distinct progressive neurodegenerative disorders and resultant age-related dementia.In these cases, neurodegeneration is due to the aggregation propensity and resultant toxic properties of the polyglutamine-containing proteins.However, surprisingly, this aggregation and resultant toxicity was not influenced by aging.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, The University of the South, Sewanee, Tennessee, United States of America.

ABSTRACT
Polyglutamine expansions in certain proteins are the genetic determinants for nine distinct progressive neurodegenerative disorders and resultant age-related dementia. In these cases, neurodegeneration is due to the aggregation propensity and resultant toxic properties of the polyglutamine-containing proteins. We are interested in elucidating the underlying mechanisms of toxicity of the protein ataxin-3, in which a polyglutamine expansion is the genetic determinant for Machado-Joseph Disease (MJD), also referred to as spinocerebellar ataxia 3 (SCA3). To this end, we have developed a novel model for ataxin-3 protein aggregation, by expressing a disease-related polyglutamine-containing fragment of ataxin-3 in the genetically tractable body wall muscle cells of the model system C. elegans. Here, we demonstrate that this ataxin-3 fragment aggregates in a polyQ length-dependent manner in C. elegans muscle cells and that this aggregation is associated with cellular dysfunction. However, surprisingly, this aggregation and resultant toxicity was not influenced by aging. This is in contrast to polyglutamine peptides alone whose aggregation/toxicity is highly dependent on age. Thus, the data presented here not only describe a new polyglutamine model, but also suggest that protein context likely influences the cellular interactions of the polyglutamine-containing protein and thereby modulates its toxic properties.

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Expression of polyQ-expanded AT3CT in C. elegans body wall muscle cells.A. Schematic representations of gene constructs. The polyQ-containing C-terminal domain (lacking the N-terminal 257 amino acids) of AT3 was fused to YFP and expressed in body wall muscle cells under the control of the unc-54 promoter to generate Punc-54257cAT3(Qn)::YFP, herein referred to as AT3CT(Qn) B. Fluorescent micrographs showing fixed N2 (wild type), AT3CT(Q45), and AT3CT(Q63) animals imaged for YFP fluorescence (green) or phalloidin-stained actin filaments (red). C. Western blot with an anti-GFP antibody showing relative protein levels for C. elegans expressing YFP alone or AT3CT(Q45) or AT3CT(Q63). The asterisk (*) represents protein running as a GFP monomer. In the AT3CT (Q45) and AT3CT (Q63) lanes, these bands seem to represent cleavage fragments that are likely artifacts of protein extraction.
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pone-0096835-g001: Expression of polyQ-expanded AT3CT in C. elegans body wall muscle cells.A. Schematic representations of gene constructs. The polyQ-containing C-terminal domain (lacking the N-terminal 257 amino acids) of AT3 was fused to YFP and expressed in body wall muscle cells under the control of the unc-54 promoter to generate Punc-54257cAT3(Qn)::YFP, herein referred to as AT3CT(Qn) B. Fluorescent micrographs showing fixed N2 (wild type), AT3CT(Q45), and AT3CT(Q63) animals imaged for YFP fluorescence (green) or phalloidin-stained actin filaments (red). C. Western blot with an anti-GFP antibody showing relative protein levels for C. elegans expressing YFP alone or AT3CT(Q45) or AT3CT(Q63). The asterisk (*) represents protein running as a GFP monomer. In the AT3CT (Q45) and AT3CT (Q63) lanes, these bands seem to represent cleavage fragments that are likely artifacts of protein extraction.

Mentions: To express the AT3 C-terminal cleavage fragment (257cAT3) in body wall muscle cells, we expressed it under the control of the myosin heavy chain promoter, unc-54. 257cAT3 was C-terminally fused to YFP for visualization, resulting in 257cAT3(Qn)::YFP, herein referred to as AT3CT(Qn) for the sake of simplicity. Because polyQ-length is known to affect the aggregation and toxicity of polyQ-containing disease-associated proteins [33]–[35], we generated two lines, one with an intermediate polyQ length that we predicted to be close to the threshold for aggregation (Q45), and a longer, likely pathogenic length (Q63) (Fig. 1A).


Novel polyglutamine model uncouples proteotoxicity from aging.

Christie NT, Lee AL, Fay HG, Gray AA, Kikis EA - PLoS ONE (2014)

Expression of polyQ-expanded AT3CT in C. elegans body wall muscle cells.A. Schematic representations of gene constructs. The polyQ-containing C-terminal domain (lacking the N-terminal 257 amino acids) of AT3 was fused to YFP and expressed in body wall muscle cells under the control of the unc-54 promoter to generate Punc-54257cAT3(Qn)::YFP, herein referred to as AT3CT(Qn) B. Fluorescent micrographs showing fixed N2 (wild type), AT3CT(Q45), and AT3CT(Q63) animals imaged for YFP fluorescence (green) or phalloidin-stained actin filaments (red). C. Western blot with an anti-GFP antibody showing relative protein levels for C. elegans expressing YFP alone or AT3CT(Q45) or AT3CT(Q63). The asterisk (*) represents protein running as a GFP monomer. In the AT3CT (Q45) and AT3CT (Q63) lanes, these bands seem to represent cleavage fragments that are likely artifacts of protein extraction.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016013&req=5

pone-0096835-g001: Expression of polyQ-expanded AT3CT in C. elegans body wall muscle cells.A. Schematic representations of gene constructs. The polyQ-containing C-terminal domain (lacking the N-terminal 257 amino acids) of AT3 was fused to YFP and expressed in body wall muscle cells under the control of the unc-54 promoter to generate Punc-54257cAT3(Qn)::YFP, herein referred to as AT3CT(Qn) B. Fluorescent micrographs showing fixed N2 (wild type), AT3CT(Q45), and AT3CT(Q63) animals imaged for YFP fluorescence (green) or phalloidin-stained actin filaments (red). C. Western blot with an anti-GFP antibody showing relative protein levels for C. elegans expressing YFP alone or AT3CT(Q45) or AT3CT(Q63). The asterisk (*) represents protein running as a GFP monomer. In the AT3CT (Q45) and AT3CT (Q63) lanes, these bands seem to represent cleavage fragments that are likely artifacts of protein extraction.
Mentions: To express the AT3 C-terminal cleavage fragment (257cAT3) in body wall muscle cells, we expressed it under the control of the myosin heavy chain promoter, unc-54. 257cAT3 was C-terminally fused to YFP for visualization, resulting in 257cAT3(Qn)::YFP, herein referred to as AT3CT(Qn) for the sake of simplicity. Because polyQ-length is known to affect the aggregation and toxicity of polyQ-containing disease-associated proteins [33]–[35], we generated two lines, one with an intermediate polyQ length that we predicted to be close to the threshold for aggregation (Q45), and a longer, likely pathogenic length (Q63) (Fig. 1A).

Bottom Line: Polyglutamine expansions in certain proteins are the genetic determinants for nine distinct progressive neurodegenerative disorders and resultant age-related dementia.In these cases, neurodegeneration is due to the aggregation propensity and resultant toxic properties of the polyglutamine-containing proteins.However, surprisingly, this aggregation and resultant toxicity was not influenced by aging.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, The University of the South, Sewanee, Tennessee, United States of America.

ABSTRACT
Polyglutamine expansions in certain proteins are the genetic determinants for nine distinct progressive neurodegenerative disorders and resultant age-related dementia. In these cases, neurodegeneration is due to the aggregation propensity and resultant toxic properties of the polyglutamine-containing proteins. We are interested in elucidating the underlying mechanisms of toxicity of the protein ataxin-3, in which a polyglutamine expansion is the genetic determinant for Machado-Joseph Disease (MJD), also referred to as spinocerebellar ataxia 3 (SCA3). To this end, we have developed a novel model for ataxin-3 protein aggregation, by expressing a disease-related polyglutamine-containing fragment of ataxin-3 in the genetically tractable body wall muscle cells of the model system C. elegans. Here, we demonstrate that this ataxin-3 fragment aggregates in a polyQ length-dependent manner in C. elegans muscle cells and that this aggregation is associated with cellular dysfunction. However, surprisingly, this aggregation and resultant toxicity was not influenced by aging. This is in contrast to polyglutamine peptides alone whose aggregation/toxicity is highly dependent on age. Thus, the data presented here not only describe a new polyglutamine model, but also suggest that protein context likely influences the cellular interactions of the polyglutamine-containing protein and thereby modulates its toxic properties.

Show MeSH
Related in: MedlinePlus