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Intracellular green fluorescent protein-polyalanine aggregates are associated with cell death
J Rankin1, A Wyttenbach, D C Rubinsztein
1Department of Medical Genetics, Wellcome Trust Centre for Molecular Mechanisms in Disease, Cambridge Institute for Medical Research, Cambridge CB2 2XY, U.K.
Abstract:
Eight diseases, exemplified by Huntington's disease and spinocerebellar ataxia type 1, are caused by CAG-repeat expansion mutations. The CAG repeats are translated into expanded polyglutamine tracts, which are associated with deleterious novel functions. While these diseases are characterized by intraneuronal aggregate formation, it is unclear whether the aggregates cause disease. We have addressed this debate by generating intracellular aggregates with green fluorescent protein (GFP) fused to 19-37 alanines. No aggregates were seen in cells expressing native GFP or GFP fused to seven alanines. Aggregate-containing cells expressing GFP fused to 19-37 polyalanines show high rates of nuclear fragmentation compared with cells expressing the same constructs without aggregates, or cells expressing GFP fused to seven alanines. This suggests an association between aggregate formation and cell death.
Insights
CAG-repeat expansion mutations cause neurodegenerative diseases. Researchers created intracellular aggregates, finding that aggregate formation is associated with nuclear fragmentation and cell death, suggesting a role in disease pathogenesis.
Area of Science:
- Neuroscience
- Genetics
- Cell Biology
Background:
- Eight diseases, including Huntington's disease, stem from CAG-repeat expansion mutations.
- These mutations lead to expanded polyglutamine tracts, associated with harmful new functions.
- The role of intraneuronal aggregate formation in causing these diseases remains unclear.
Purpose of the Study:
- To investigate the causal relationship between intracellular aggregate formation and cell death in polyglutamine diseases.
- To determine if experimentally generated aggregates contribute to cellular pathology.
Main Methods:
- Generated intracellular aggregates using green fluorescent protein (GFP) fused to polyalanine tracts of varying lengths (7, 19-37 alanines).
- Compared aggregate formation and cellular phenotypes (nuclear fragmentation) in cells expressing different GFP fusion constructs.
- Assessed cell death rates in relation to the presence or absence of aggregates.
Main Results:
- No aggregates formed with native GFP or GFP fused to seven alanines.
- Cells expressing GFP fused to 19-37 polyalanines formed intracellular aggregates.
- Aggregate-containing cells exhibited significantly higher rates of nuclear fragmentation compared to control cells.
Conclusions:
- Intracellular aggregate formation is associated with nuclear fragmentation and increased cell death.
- These findings suggest that aggregate formation may play a causal role in the pathogenesis of polyglutamine diseases.