Studying C9orf72 dipeptide repeat polypeptide aggregation using an analytical ultracentrifuge equipped with...

Area of Science:

• Biochemistry and Molecular Biology
• Neuroscience
• Genetics

Background:

• Neurodegenerative diseases like ALS and FTD are linked to repeat expansions in genes such as c9orf72.
• Aberrant translation of these repeats produces dipeptide repeats (DPRs) that aggregate, contributing to disease pathology.
• Understanding DPR aggregation in vivo is crucial for developing therapeutic strategies.

Purpose of the Study:

• To apply sedimentation velocity and electrophoresis methods to study DPR aggregation in animal models.
• To analyze the aggregation patterns of specific DPRs, including (Gly-Pro)47, (Gly-Ala)50, and (Gly-Arg)50.
• To investigate DPR aggregation in transgenic *Drosophila melanogaster* and *C. elegans* models.

Main Methods:

• Utilized analytical ultracentrifugation with fluorescence detection (AU-FDS) to assess protein aggregation.
• Employed sedimentation, dimensionally-defined, size-exclusion chromatography (SDD-AGE) for aggregate analysis.
• Analyzed DPRs fused to fluorescent proteins in samples from transgenic *D. melanogaster* and *C. elegans*.

Main Results:

• (Gly-Pro)47 largely remained monomeric.
• (Gly-Ala)50 formed both intermediate and large-scale aggregates.
• (Gly-Arg)50 showed partial monomeric forms with aggregation potentially involving DNA/RNA co-aggregation.

Conclusions:

• AU-FDS and SDD-AGE are powerful tools for characterizing protein aggregation in animal models of neurodegenerative diseases.
• Different DPRs exhibit distinct aggregation behaviors, influencing disease mechanisms.
• These findings provide a foundation for evaluating therapeutic interventions targeting DPR aggregation.