Fig 2: Expression of human ATXN7 (wildtype and mutant variants) and human TOP1MT (wildtype and mutant variants) in the Drosophila central nervous system has variable effects on locomotor activity and lifespan.A Schematic representation outlining the development of transgenic flies prior to experimentation. Briefly, we used the pUASTattb vector system which allows for PhiC31-mediated transgenesis. We crossed flies expressing ataxin-7 Q35P and ataxin-7 N556K together with elav-GAL4 with those expressing top1mt R111W to obtain relevant genotypes. B Climbing indexes for all control and experimental genotypes described were obtained at one, three, and five weeks of age, with at least six trials per genotype per time point. Experiment was repeated three times. Error bars represent standard error. C Kaplan–Meier survival curves for lifespan. Experiment was repeated three times and results combined in the depicted graph. N = 200 flies per genotype. Figure created with BioRender.com.

Fig 3: Top1mt R111W is associated with bioenergetic deficits.A Immunofluorescence of primary human fibroblasts isolated from a healthy individual (a), a SCA7 patient (b), our proband (c), and our proband’s mother (d) and father (e) using antibodies against top1mt. No differences in subcellular localization were directly appreciable by eye. Secondary antibodies were conjugated to Alexa488. All representative images are post-deconvolution. All scale bars: 10 µm. B PhenoRipper multidimensional scaling plot of sorting of top1mt stained images. No discernable groups were observed on sorting. Every point represents one image of 20–25 cells, with 20 images per cell type for N = 400 cells per type. C Measurement of oxidative phosphorylation across skin fibroblasts obtained from three healthy individuals, our patient, a SCA7 patient, and our patient’s parents using the Seahorse XF 24 Mito Stress Test. Arrows indicate points at which pre-loaded mitochondrial poisons were applied to test different segments of the energetic system. Points are means for triplicates with error bars indicating the standard deviation. Oxygen consumption rate was measured in picomoles of O2/min and then normalized to protein per well by bicinchoninic acid assay. Clear defects are noted in cells from our patient, as well as cells from his father, both carrying the top1mt R111W mutation. D–I Analysis of specific oxidative phosphorylation components including basal respiration (D), proton leak (E), maximal respiration (F), spare respiratory capacity (G), non-mitochondrial respiration (H), and ATP production (I). Notably, for all electron transport chain-dependent components, cells from our patient and his father performed the poorest. All columns represent means with error bars indicating standard deviation. Values were normalized to non-mitochondrial respiration. J Southern blot analysis and qPCR analysis (K) of relative mitochondrial DNA content from patient skeletal muscle biopsy samples compared to healthy controls, with patient samples having substantially less mitochondrial DNA content. L Two-dimensional intact mitochondrial DNA agarose gel electrophoresis (2D-IMAGE) analysis of patient mitochondrial DNA compared to healthy controls. Patient samples were noted to have decreased covalently closed circular DNA levels (arrow) as well as increased linearity.