Machado-Joseph disease, also known as spinocerebellar ataxia type 3, is a fatal polyglutamine disease with no disease-modifying treatment. The selective serotonin reuptake inhibitor citalopram was shown in nematode and mouse models to be a compelling repurposing candidate for Machado-Joseph disease therapeutics. We sought to confirm the efficacy of citalopram to decrease ATXN3 aggregation in an unrelated mouse model of Machado-Joseph disease. Four-week-old YACMJD84.2 mice and non-transgenic littermates were given citalopram 8 mg/kg in drinking water or water for 10 weeks. At the end of treatment, brains were collected for biochemical and pathological analyses. Brains of citalopram-treated YACMJD84.2 mice showed an approximate 50% decrease in the percentage of cells containing ATXN3-positive inclusions in the substantia nigra and three examined brainstem nuclei compared to controls. No differences in ATXN3 inclusion load were observed in deep cerebellar nuclei of mice. Citalopram effect on ATXN3 aggregate burden was corroborated by immunoblotting analysis. While lysates from the brainstem and cervical spinal cord of citalopram-treated mice showed a decrease in all soluble forms of ATXN3 and a trend toward reduction of insoluble ATXN3, no differences in ATXN3 levels were found between cerebella of citalopram-treated and vehicle-treated mice. Citalopram treatment altered levels of select components of the cellular protein homeostatic machinery that may be expected to enhance the capacity to refold and/or degrade mutant ATXN3. The results here obtained in a second independent mouse model of Machado-Joseph disease further support citalopram as a potential drug to be repurposed for this fatal disorder.

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Machado-Joseph disease (MJD) is a dominant neurodegenerative disease caused by an expanded CAG repeat in the ATXN3 gene encoding the ataxin-3 protein. Several cellular processes, including transcription and apoptosis, are disrupted in MJD. To gain further insights into the extent of dysregulation of mitochondrial apoptosis in MJD and to evaluate if expression alterations of specific apoptosis genes/proteins can be used as transcriptional biomarkers of disease, the expression levels of BCL2, BAX and TP53 and the BCL2/BAX ratio (an indicator of susceptibility to apoptosis) were assessed in blood and post-mortem brain samples from MJD subjects and MJD transgenic mice and controls. While patients show reduced levels of blood BCL2 transcripts, this measurement displays low accuracy to discriminate patients from matched controls. However, increased levels of blood BAX transcripts and decreased BCL2/BAX ratio are associated with earlier onset of disease, indicating a possible association with MJD pathogenesis. Post-mortem MJD brains show increased BCL2/BAX transcript ratio in the dentate cerebellar nucleus (DCN) and increased BCL2/BAX insoluble protein ratio in the DCN and pons, suggesting that in these regions, severely affected by degeneration in MJD, cells show signs of apoptosis resistance. Interestingly, a follow-up study of 18 patients further shows that blood BCL2 and TP53 transcript levels increase over time in MJD patients. Furthermore, while the similar levels of blood BCL2, BAX, and TP53 transcripts observed in preclinical subjects and controls is mimicked by pre-symptomatic MJD mice, the expression profile of these genes in patient brains is partially replicated by symptomatic MJD mice. Globally, our findings indicate that there is tissue-specific vulnerability to apoptosis in MJD subjects and that this tissue-dependent behavior is partially replicated in a MJD mouse model.

27Background: Spinocerebellar Ataxia type 3 (SCA3, also known as Machado-Joseph disease) 28 is a neurodegenerative disorder caused by a CAG repeat expansion encoding an abnormally 29 long polyglutamine (polyQ) tract in the disease protein, ataxin-3 (ATXN3). No preventive 30treatment is yet available for SCA3. Because SCA3 is likely caused by a toxic gain of ATXN3 31 function, a rational therapeutic strategy is to reduce mutant ATXN3 levels by targeting pathways 32 that control its production or stability. Here, we sought to identify genes that modulate ATXN3 33 levels as potential therapeutic targets in this fatal disorder. 34 Methods:We screened a collection of siRNAs targeting 2742 druggable human genes using a 35 cell-based assay based on luminescence readout of polyQ-expanded ATXN3. From 317 36 candidate genes identified in the primary screen, 100 genes were selected for validation. 37Among the 33 genes confirmed in secondary assays, 15 were validated in an independent cell 38 model as modulators of pathogenic ATXN3 protein levels. Ten of these genes were then 39 assessed in a Drosophila model of SCA3, and one was confirmed as a key modulator of 40 physiological ATXN3 abundance in SCA3 neuronal progenitor cells. 41 Results: Among the 15 genes shown to modulate ATXN3 in mammalian cells, orthologs of 42 CHD4, FBXL3, HR and MC3R regulate mutant ATXN3-mediated toxicity in fly eyes. Further 43 mechanistic studies of one of these genes, FBXL3, encoding a F-box protein that is a 44 component of the SKP1-Cullin-F-box (SCF) ubiquitin ligase complex, showed that it reduces 45 levels of normal and pathogenic ATXN3 in SCA3 neuronal progenitor cells, primarily via a SCF 46 complex-dependent manner. Bioinformatic analysis of the 15 genes revealed a potential 47 molecular network with connections to tumor necrosis factor-a/nuclear factor-kappa B (TNF/NF-48 kB) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways. 49 Conclusions:We identified 15 druggable genes with diverse functions to be suppressors or 50 enhancers of pathogenic ATXN3 abundance. Among identified pathways highlighted by this 51 screen, the FBXL3/SCF axis represents a novel molecular pathway that regulates physiological 52 levels of ATXN3 protein. 53 54 Keywords: polyglutamine, spinocerebellar ataxia, Machado-Joseph disease, 55 neurodegeneration, high-throughput screen, human embryonic stem cells, Drosophila 56 57 Introduction 58The polyglutamine (polyQ) diseases are inherited neurodegenerative diseases caused by 59 expanded CAG repeats that encode abnormally long glutamine repeats in the disease proteins 60[1, 2]. Spinocerebellar Ataxia type 3 (SCA3) is one of nine known polyQ disorders and the most 61 common dominant ataxia, primarily manifesting with degeneration of the cerebellum, brainstem, 62spinal cord, and basal ganglia [3][4][5][6][7]. The CAG repeat in the ATXN3 gene, which normally is 12 63 to 44 triplets, becomes expanded to ~60 to 87 repeats in SCA3 [8, 9]. Despite sharing a 64 propensity to misfold and aggregate, polyQ disease p...

Machado-Joseph disease (MJD) is a dominant neurodegenerative disease caused by an expanded CAG repeat in the ATXN3 gene encoding the ataxin-3 protein. Several cellular processes, including transcription and apoptosis, are disrupted in MJD. To gain further insights into the extent of dysregulation of mitochondrial apoptosis in MJD, and to evaluate if expression alterations of specific apoptosis genes/proteins could be used as transcriptional biomarkers of disease, the levels of BCL2, BAX and TP53 and the BCL2/BAX ratio, an indicator of susceptibility to apoptosis, were assessed in blood and post-mortem brain samples from MJD subjects and MJD transgenic mice and controls. While patients show reduced levels of blood BCL2 transcripts, this measurement displays low accuracy to discriminate patients from matched controls. However, increased levels of blood BAX transcripts and decreased BCL2/BAX ratio are associated with earlier onset, indicating a possible association with MJD pathogenesis. Post-mortem MJD brains show increased BCL2/BAX transcript ratio in the dentate cerebellar nucleus (DCN) and increased BCL2/BAX insoluble protein ratio in the DCN and pons, suggesting that in these regions, severely affected by degeneration in MJD, cells show signs of apoptosis resistance. Interestingly, a follow-up study of 18 patients further shows that blood BCL2 and TP53 transcript levels increase over time in MJD patients. Furthermore, while the similar levels of blood BCL2, BAX, and TP53 transcripts observed in preclinical subjects and controls is mimicked by pre-symptomatic MJD mice, the expression profile of these genes in patient brains is partially replicated by symptomatic MJD mice. Globally, our findings indicate that there is tissue-specific vulnerability to apoptosis in MJD subjects and that this tissue dependent behavior is partially replicated in a MJD mouse model.