Repeat size dependent-toxicity of dipeptide repeat proteins in C9orf72 ALS/FTD Open Access

Davis, Emma (Spring 2021)

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Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Degeneration (FTD) are two devastating neurodegenerative diseases with clinical, pathological and genetic overlaps. These two diseases both can be caused by the same most common genetic mutation: a hexanucleotide GGGGCC (G4C2) repeat expansion in the C9orf72 intron region. Toxicity through the translation of certain dipeptide repeat proteins (DPRs), GR and PR, have been shown to likely lead to disease; however, literature has focused on short repeat lengths rather than the long repeat lengths that are seen in patients. Furthermore, the translational mechanism of the repeat translation is relatively unknown, particularly in the antisense direction. In order to gain a better understanding of the translational mechanism of DPRs in C9orf72 ALS/FTD, we first looked at what role the intron plays in the translation of antisense DPRs. We found that when the intron sequence is removed, there is a significant reduction in DPR production, indicating that the intron region is likely involved in antisense DPR translation. Preliminary data suggests that this translation within the intron region is driven by start codons in frame with various DPRs. Due to this start codon driven translation, longer repeats within patients are likely to be completely translated. Comparing the shorter repeats used in literature with longer repeats, we found a change in the localization of repeats and a change in stress granule dynamics, a downstream pathway. Longer GR repeats localized within the cytoplasm, as do patient samples, while shorter GR repeats localized within the nucleolus. Furthermore, shorter PR repeats lead to stress granule formation, while longer PR repeats do not. These discrepancies, along with the insight into the translational mechanism of disease, indicate that researchers may need to reevaluate the conclusions that have been made using short DPRs in their applicability to the actual disease. 

Table of Contents



Intron sequence implicated in antisense DPR translation--------------------7

Start codons in antisense intron sequence likely initiating translation-------8

Long GR DPRs form cytoplasmic aggregates as in observed in patients------10

Long PR DPRs do not trigger stress granule formation-----------------------11


Materials and Methods------------------------------------------------------14

Molecular Cloning Mutated Start Codons-----------------------------------14

Molecular Cloning Long Repeats--------------------------------------------15




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