Characterization of C9orf72 function in autophagy and RNA metabolism

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two devastating neurodegenerative disorders that share clinical, pathological, and genetic features. The most common inheritable cause of ALS and FTD disorders is a hexanucleotide G4C2 repeat expansion within the non-coding region of the C9orf72 gene. Pathogenesis of C9orf72-mediated ALS/FTD is associated with both loss- and gain-of-function mechanisms involving three distinct disease factors. First, bidirectional transcription of the repeat expansion leads to the generation of repeat-containing sense and antisense RNAs. These repeat-RNAs are suggested to form RNA foci and sequester RNA-binding proteins eventually contributing to neurotoxicity. Second, non-conventional translation of repeat-RNAs in all six reading frames generates five distinct dipeptide repeat (DPR) proteins. DPR proteins disturb cellular homeostasis by affecting multiple cellular processes such as nucleocytoplasmic transport and protein translation. Finally, the repeat expansion causes a decrease in C9orf72 transcript and protein levels. C9orf72 protein is an important regulator of the autophagy-related vesicular pathway and the inflammatory response pathway, and its loss leads to their dysregulation. Toxicity due to repeat-RNAs and DPR proteins points towards the presence of gain-of-function mechanisms while haploinsufficiency of C9orf72 protein suggests a loss-of-function disease mechanism. However, the precise molecular and cellular changes caused by the individual and combinatorial expression of these three disease factors and how they contribute to disease pathogenesis remain elusive.
This thesis is aimed to further the understanding of the biochemical and cellular functions of the C9orf72 protein and to disentangle the isolated and combinatorial effects on cellular physiology that is caused by the expression of repeat-RNAs, DPR proteins, and the loss of C9orf72 protein. Single-particle cryo-electron microscopy was used to determine the structure of the C9orf72-SMCR8 complex. The structure shed light on the multifaceted role of the C9orf72 complex and how it might act as a binding platform for protein-protein interactions. Furthermore, a cellular model system was generated that allows the controlled expression of repeat-RNAs and DPR proteins in order to study their spatiotemporal dynamics in the presence and absence of the C9orf72 protein. Moreover, this system is used to systematically characterize individual and combinatorial effects of the three disease factors on RNA and protein metabolism. Therefore, the model system presented in this study is a valuable addition to study the cellular alterations caused by disease factors implicated in C9orf72-mediated ALS/FTD disease.