Date of Award
Doctor of Philosophy (PhD)
Nancy M. Bonini
Amyotrophic lateral sclerosis and frontotemporal dementia spectrum disorders (ALS/FTD) are characterized by the degeneration of motor and cortical neurons. Recently, a hexanucleotide-repeat expansion of >30 repeats was discovered within intron 1 of C9orf72, defining ~40% of familial and ~7% of sporadic ALS/FTD cases. Aberrant accumulation of G4C2-RNA and the aggregation of its repeat-associated non-AUG (RAN-) translation products occurs in diseased tissue. Herein, we developed two Drosophila models that overexpressed G4C2-repeats. Expression of >30 repeats causes degenerative effects supporting gain-of-function mechanisms are contributing to disease. To define disease mechanisms, we performed an unbiased, RNAi-based screen covering ~4000 genes (~25% of the fly genome) and identified the PAF1 complex (PAF1C), an RNAPII-transcription factor, as a suppressor of G4C2-toxicity. Loss of PAF1C reduced RNA and peptide expression from sense-G4C2 and antisense-G2C4 transgenes in Drosophila and in yeast. Importantly, components Paf1 and Leo1 were selective for expression from toxic (>30) G4C2-repeats versus inert (≤29) repeats, arguing selectivity to the disease-associated repeat expansion. Leo1 was also shown to interact with C9orf72-chromatin in C9+-patient cells. Expression of PAF1C components became upregulated in response to expression of >30 G4C2-repeats in flies, mice, patient cells, and FTD tissue, further supporting their importance in disease. Interestingly, expression from PAF1 and LEO1 in FTD tissue correlated with expression of the repeat-expansion within C9orf72, supporting that these components of PAF1C are important for expression of the repeat in disease. In addition to PAF1C, we also identified 11 translation factors that may be important for peptide production from G4C2-transcripts in a second, targeted screen. Follow-up studies revealed that eIF4B and eIF4H knockdown reduced GR-peptide levels produced from G4C2-transcripts and G4C2-toxicity in the fly, defining them as potential RAN-translation factors in C9+ disease. In summary, jumping from unbiased forward genetic screens in Drosophila, we took an interdisciplinary approach to uncover key players whose activity regulates expression of the unique G4C2-hexanucleotide repeat expansion found in C9orf72 in a subset of ALS/FTD cases. This work increases our understanding of disease mechanisms while highlighting potential therapeutic targets.
Goodman, Lindsey D., "Factors That Mediate Expression Of Unique Ggggcc Repeat Expansions In C9orf72-Associated Als/ftd." (2019). Publicly Accessible Penn Dissertations. 3329.