A mutation in the C9ORF72 gene is the most common genetic cause of the devastating disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Currently, no effective treatments for these disorders exist. The C9ORF72 mutation results in the production of neurotoxic dipeptide repeat proteins (DPRs). Modifier and interactome studies suggest that DPRs affect nucleocytoplasmic transport. Altered nucleocytoplasmic transport may contribute to disease pathology as this vital mechanism is the main route of information exchange between the cytoplasmic and nuclear compartments. Smaller molecules can passively diffuse through the nuclear pore, the protein complex through which transport occurs. Conversely, larger molecules need to be actively transported by receptors. We aimed to specifically study passive nucleocytoplasmic transport in the context of C9ORF72 ALS/FTD as this yields information about nuclear pore integrity and function. In addition, passive diffusion is thought to be the main route of nuclear export of key ALS/FTD proteins TDP43 and FUS. An assay was optimised to specifically investigate passive nucleocytoplasmic transport monitoring inert fluorescent cargo of different sizes using live confocal microscopy. The influence of C9ORF72 DPRs on passive nuclear import of reporter cargo and on passive export of TDP43 and other nuclear proteins was studied using this assay in HeLa cells. Arginine containing DPRs were shown to enhance passive nuclear translocation of reporter cargo and passive nuclear export of TDP43 and other nuclear proteins. To study potential mechanisms of action of arginine containing DPRs the localisation of various transport receptors upon DPR treatment was studied using immunofluorescence. Levels of import receptors, nucleoporins and Ran regulating factors were reduced within the nucleus and the nuclear pore in the presence of arginine containing DPRs. This might contribute to the enhancement of passive nucleocytoplasmic transport. Findings of increased passive nucleocytoplasmic transport could be recapitulated in C9ORF72 ALS iPSC-derived neurons highlighting the disease relevance of this result. A trend of nucleocytoplasmic transport factor mislocalisation was also observed in C9ORF72 ALS iPSC-derived neurons. In this PhD project a potential pathology mechanism of C9ORF72 arginine containing DPRs was characterised which might contribute to cytoplasmic TDP43 mislocalisation, a pathological hallmark of ALS/FTD.
Dysfunctional nucleocytoplasmic transport dynamics in amyotrophic lateral sclerosis and frontotemporal dementia caused by mutation in C9ORF72
Salcher-Konrad, M. (Author). 1 Apr 2023
Student thesis: Doctoral Thesis › Doctor of Philosophy