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Rapid aggregate formation of the huntingtin N-terminal fragment carrying an expanded polyglutamine tract
1CREST, Japan Science and Technology Corporation, Tokyo, Japan.
Insights
Huntington's disease (HD) involves CAG repeat expansion, leading to huntingtin protein aggregation. This study shows mutated huntingtin aggregates rapidly in cells, suggesting a nucleation-dependent mechanism for HD pathogenesis.
Area of Science:
- Molecular Biology
- Neuroscience
- Genetics
Background:
- Huntington's disease (HD) is a neurodegenerative disorder caused by CAG repeat expansion in the huntingtin (HD) gene.
- Expanded polyglutamine tracts in huntingtin lead to protein aggregation in vitro and in vivo.
Purpose of the Study:
- To investigate the molecular mechanism of huntingtin aggregate formation.
- To analyze the time course and characteristics of aggregate formation using fusion proteins.
Main Methods:
- Transient expression of HD exon 1-GFP fusion proteins with varying glutamine repeat lengths (25 and 77 repeats) in COS-7 cells.
- Time-lapse microscopy to observe protein aggregation.
- Treatment with cycloheximide to assess the role of protein synthesis.
Main Results:
- Fusion proteins with 77 glutamine repeats formed aggregates rapidly and time-dependently, unlike those with 25 repeats.
- Aggregate formation initiated approximately 40 hours post-transfection, with rapid growth and subsequent loss of diffuse fluorescence.
- Cycloheximide treatment decreased the frequency of aggregate formation, suggesting a role for de novo protein synthesis.
Conclusions:
- Huntingtin aggregate formation is a time-dependent process, potentially involving nucleation.
- Focal concentration of mutant huntingtin in neurons may initiate aggregation.
- Understanding this mechanism is crucial for developing therapeutic strategies for Huntington's disease.
Abstract:
Huntington's disease (HD) is caused by an expansion of the CAG repeat in the HD gene. The repeat is translated to the polyglutamine tract as huntingtin, the product of HD gene. Several studies showed that the expansion of polyglutamine tract leads to formation of cytoplasminc and/or intranuclear aggregates in vivo or in vitro. To understand the molecular mechanism of the aggregate formation, we studied the transient expression of HD exon 1-GFP fusion proteins in COS-7 cells. The fusion protein carrying 77 glutamine repeats aggregated in a time-dependent manner, while the fusion protein carrying 25 glutamine tract remained to be distributed diffusely in the cytoplasm even 72 hours after transfection. Initially, fluorescent signals were diffusely distributed in the COS-7 cells that were transfected with the construct containing the 77 CAG repeats. Approximately 40 hours later after the transfection, large aggregates grew very rapidly in those cells and the diffuse cytoplasmic fluorescence faded out. This process was completed within 40 minutes from the appearance of small aggregates in the perinuclear regions. The addition of cycloheximide reduced the frequencies of aggregate formation. A possibility was discussed that the aggregate formation was via nucleation. The focal concentration of mutated proteins in neurons may trigger the aggregate formation.