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.

Spinocerebellar ataxia type 7 (SCA7) and other polyglutamine (polyQ) diseases are caused by expansions of polyQ repeats in disease-specific proteins. Aggregation of the polyQ proteins resulting in various forms of cellular stress, that could induce the stress granule (SG) response, is believed to be a common pathological mechanism in these disorders. SGs can contribute to cell survival but have also been suggested to exacerbate disease pathology by seeding protein aggregation. In this study, we show that two SG-related proteins, TDP-43 and TIA1, are sequestered into the aggregates formed by polyQ-expanded ATXN7 in SCA7 cells. Interestingly, mutant ATXN7 also localises to induced SGs, and this association altered the shape of the SGs. In spite of this, neither the ability to induce nor to disassemble SGs, in response to arsenite stress induction or relief, was affected in SCA7 cells. Moreover, we could not observe any change in the number of ATXN7 aggregates per cell following SG induction, although a small, non-significant, increase in total aggregated ATXN7 material could be detected using filter trap. However, mutant ATXN7 expression in itself increased the speckling of the SG-nucleating protein G3BP1 and the SG response. Taken together, our results indicate that the SG response is induced, and although some key modulators of SGs show altered behaviour, the dynamics of SGs appear normal in the presence of mutant ATXN7. 

The image analysis tool FRIC (Fluorescence Ratiometric Imaging of Chromatin) quantitatively monitors dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As a proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation as well as expression of the mutant lamin A protein “progerin,” which causes Hutchinson-Gilford Progeria Syndrome. In summary FRIC is versatile, unbiased, robust, requires a minimum of experimental steps and is suitable for screening purposes.

Polyglutamine (polyQ) diseases, such as Spinocerebellar ataxia type 7 (SCA7), are caused by expansions of polyQ repeats in disease specific proteins. The sequestration of vital proteins into aggregates formed by polyQ proteins is believed to be a common pathological mechanism in these disorders. The RNA-binding protein FUS has been observed in polyQ aggregates, though if disruption of this protein plays a role in the neuronal dysfunction in SCA7 or other polyQ diseases remains unclear. We therefore analysed FUS localisation and function in a stable inducible PC12 cell model expressing the SCA7 polyQ protein ATXN7. We found that there was a high degree of FUS sequestration, which was associated with a more cytoplasmic FUS localisation, as well as a decreased expression of FUS regulated mRNAs. In contrast, the role of FUS in the formation of gamma H2AX positive DNA damage foci was unaffected. In fact, a statistical increase in the number of gamma H2AX foci, as well as an increased trend of single and double strand DNA breaks, detected by comet assay, could be observed in mutant ATXN7 cells. These results were further corroborated by a clear trend towards increased DNA damage in SCA7 patient fibroblasts. Our findings suggest that both alterations in the RNA regulatory functions of FUS, and increased DNA damage, may contribute to the pathology of SCA7.

In most cells, transcriptionally inactive heterochromatin is preferentially localized in the nuclear periphery and transcriptionally active euchromatin is localized in the nuclear interior. Different cell types display characteristic chromatin distribution patterns, which change dramatically during cell differentiation, proliferation, senescence and different pathological conditions. Chromatin organization has been extensively studied on a cell population level, but there is a need to understand dynamic reorganization of chromatin at the single cell level, especially in live cells. We have developed a novel image analysis tool that we term Fluorescence Ratiometric Imaging of Chromatin (FRIC) to quantitatively monitor dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation. We also found that peripheral heterochromatin depended on the levels of the inner nuclear membrane protein Samp1, suggesting an important role in promoting peripheral heterochromatin. Taken together, FRIC is a powerful and robust new tool to study dynamic chromatin redistribution in live cells.

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.

Recently alterations to the nuclear lamina, nuclear pore complexes (NPCs) and nucleocytoplasmic transport have emerged as a pathological mechanism in Huntington’s disease (HD). In this study, we investigated whether such a phenotype could also be detected in models of the hereditary disease Spinocerebellar ataxia type 7 (SCA7). This as both HD and SCA7 are caused by expansion of a polyglutamine domain in the respective disease protein and a common gain of function mechanism has been proposed for these disorders. Using an inducible SCA7 PC12 cell model and human SCA7 fibroblasts, we could not detect any nuclear lamina alterations, such as invaginations or Lamin B1 mislocalization, previously reported in HD. Unlike in HD models, we could also not detect any sequestration of nuclear pore proteins Nup62 or Nup153 into the polyglutamine aggregates formed by mutant ATXN7 in SCA7 cells. However, we could observe sequestration of the nuclear import receptor Importin β and increased cytoplasmic Ran levels in SCA7 cells. Despite this, no gross change in nuclear protein import could be observed. Taken together our data suggest that nuclear lamina, NPC and nucleocytoplasmic transport defects might not play the same early and/or central role in SCA7 pathology, as suggested in HD.

Polyglutamine (polyQ) diseases, such as Spinocerebellar ataxia type 7, are caused by the expansion of a CAG/polyglutamine repeat in a disease specific gene/protein. Misfolding and aggregation of the expanded protein can be observed in all polyQ disorders and sequestration of vital proteins into the aggregates formed have been suggested as a common pathological mechanism. FUS, an RNA binding protein, is frequently observed in polyglutamine aggregates. However, whether or not FUS disruption contributes to polyQ pathology is not clear.

To address this question we used confocal microscopy, cell fractionation, filter traps and western blot, to study how FUS localization and function is affected by the SCA7 disease protein ataxin-7 (ATXN7). We found that aggregates formed by polyQ expanded ATXN7 were to a high degree also FUS positive and FUS re-distributed into the insoluble cell fraction together with mutant ATXN7. Moreover, a shift in abundance of FUS from the nucleus to the cytoplasm was observed and associated with altered levels of FUS regulated mRNAs in mutant ATXN7 expressing cells. However, some of the affected mRNAs are also regulated by the RNA binding protein TDP-43, which we could also show co-localized with ATXN7 aggregates using microscopy. Moreover, increased phosphorylation of serine 409/410 in TDP-43, which has been linked to TDP-43 neurotoxicity, could be observed in mutant ATXN7 expressing cells. Taken together, these findings lead us to conclude that disruption of FUS and also TDP-43 could potentially play a role in SCA7 pathology.