Polyglutamine diseases are a group of nine disorders that includes, among others SCA7. The common denominator is an expanded glutamine tract in the respective disease protein caused by unstable replication during meiosis. Most research within this field points to a combination of gain-of-function and loss-of-function mechanisms causing all polyglutamine diseases. Using a SCA7 model we are thus attempting to study both of these mechanisms. The glutamine tract expansion responsible for SCA7 is located in the protein Ataxin-7, which like the other polyglutamine proteins aggregates into large inclusions in patient cells. In a gain-of-function mechanism, the aggregates are suggested to cause stress to the cell by e.g. sequestering vital proteins into the aggregates, which could disrupt their function. RNA-binding proteins such as FUS and TDP-43 are often found in aggregates in neurodegenerative diseases, and have been observed in SCA7 aggregates as well. However, if disruption of FUS and TDP-43 function occurs, or if it plays a role in SCA7 pathology is unclear. We found a high rate of co-aggregation of FUS with Ataxin-7 using immunofluorescence and filter trap assays. Furthermore, we found that both the localization and function of FUS was altered in a SCA7 cell model using cell fractionations and RT-PCR. Additionally, we found that TDP-43 also co-aggregated with Ataxin-7 and phosphorylation of TDP-43 was increased during the disease phenotype.

Wild-type Ataxin-7 normally functions within chromatin regulation processes, and loss-of-function pathology in SCA7 could therefore involve a disruption of these processes. We have developed a method, FRIC, that enables us to study chromatin organization in live cells using confocal microscopy and fluorescently tagged histones. Using inhibitors of HATs and HDACs, as well as a previously known protein that regulates chromatin structure, we were able to observe changes in chromatin structure in the nuclear periphery, confirming the usefulness of FRIC. Additionally, we investigated the involvement of an inner nuclear membrane protein, Samp1, in chromatin organization and found Samp1 to be instrumental in organizing peripheral chromatin.

Taken together, the results from these two studies indicate that SCA7 pathology disturbs RNA-binding protein mediated transcriptional regulation in a gain-of-function mechanism, and that FRIC is a powerful new tool for examining chromatin regulation in diseases with disrupted transcription, like SCA7.

In eukaryotes the genetic material is separated from the cytoplasm by the nuclear envelope (NE), consisting of the outer and inner nuclear membrane, the nuclear lamina and the nuclear pores. The genetic material is highly structured with transcriptionally inactive heterochromatin enriched at the nuclear periphery and transcriptionally active euchromatin in the nuclear interior. Underlying the inner nuclear membrane is the nuclear lamina (nucleoskeleton) that together with several hundred nuclear envelope transmembrane proteins (NETs) connect chromatin to the nuclear periphery. Most NETs are uncharacterized and expressed in a tissue-specific manner. Mutations in NE proteins are linked to distinct degenerative disorders, referred to as envelopathies or laminopathies. The NET primarily studied in this thesis is called Spindle-Associated Membrane Protein 1 (Samp1). We showed that overexpression of Samp1 induced a fast differentiation of human induced pluripotent stem cells and that the binding between two NETs, Samp1 and Emerin, is regulated by RanGTP. Another focus of this thesis was the development and use of a novel method called Fluorescent Ratiometric Imaging of Chromatin (FRIC). FRIC quantitatively monitors the epigenetic state of chromatin in live cells. Using FRIC, we were able to show that Samp1 promotes peripheral heterochromatin organization. FRIC also detected an increased distribution of heterochromatin at the nuclear periphery during neuronal differentiation. In conclusion, FRIC is a useful tool that could serve medical research in elucidating the effects of different chemical agents and the roles of NE proteins in chromatin organization.

Spinocerebellar Ataxia type 7 (SCA7) is a genetic neurodegenerative disease with lethal outcome that affects the cerebellum and retina of patients. This thesis focuses on characterising molecular pathological pathways that cause toxicity and cell death in SCA7. Using primarily an inducible cell model and patient fibroblasts I found that the three RNA binding proteins FUS, TDP-43 and TIA1 are co-sequestered into aggregates formed by the SCA7 causing protein, ATXN7. Consequently I investigated the cellular functions in which these proteins have important roles. I found that FUS’ ability to regulate mRNAs is altered due to mislocalisation, DNA damage is increased, and that stress granules (SGs) are induced in a SCA7 cell model and in patient fibroblasts. Surprisingly, I also found that ATXN7 was present within SGs, and that SGs exhibited an altered shape upon induction of mutant ATXN7. I also participated in developing a microscopy-based method for monitoring chromatin organisation in live cells called FRIC. FRIC is able to detect even subtle changes to peripheral chromatin organisation, and since ATXN7 is a subunit of the transcription regulational complex SAGA, we used FRIC to investigate the effect of mutant ATXN7 on peripheral chromatin organisation. While we found no evidence that mutant ATXN7 affected peripheral chromatin organisation, the inner nuclear membrane protein Samp1 was found to be important for normal chromatin organisation in the nuclear periphery. Finally, I characterised the effect of mutant ATXN7 expression on the nuclear lamina, nuclear pore complexes, and nucleocytoplasmic transport. I found that although key transport factors such as Ran and Importin ß intermittently co-localised with ATXN7 aggregates, there were no apparent defects in nucleocytoplasmic protein import or nuclear envelope integrity. 

In summation, my investigations resulted in new findings that may be built upon to find key targets for treating SCA7 patients.