Machado-Joseph disease (MJD) is a dominantly inherited ataxia caused by a polyglutamine-coding expansion in the ATXN3 gene. Suppressing expression of the toxic gene product represents a promising approach to therapy for MJD and other polyglutamine diseases. We performed an extended therapeutic trial of RNA interference (RNAi) targeting ATXN3 in a mouse model expressing the full human disease gene and recapitulating key disease features. Adeno-associated virus (AAV) encoding a microRNA (miRNA)-like molecule, miRATXN3, was delivered bilaterally into the cerebellum of 6- to 8-week-old MJD mice, which were then followed up to end-stage disease to assess the safety and efficacy of anti-ATXN3 RNAi. Despite effective, lifelong suppression of ATXN3 in the cerebellum and the apparent safety of miRATXN3, motor impairment was not ameliorated in treated MJD mice and survival was not prolonged. These results with an otherwise effective RNAi agent suggest that targeting a large extent of the cerebellum alone may not be sufficient for effective human therapy. Artificial miRNAs or other nucleotide-based suppression strategies targeting ATXN3 more widely in the brain should be considered in future preclinical tests.

Glycogen and lipids are major storage forms of energy that are tightly regulated by hormones and metabolic signals. We demonstrate that feeding mice a high-fat diet (HFD) increases hepatic glycogen due to increased expression of the glycogenic scaffolding protein PTG/R5. PTG promoter activity was increased and glycogen levels were augmented in mice and cells after activation of the mechanistic target of rapamycin complex 1 (mTORC1) and its downstream target SREBP1. Deletion of the PTG gene in mice prevented HFD-induced hepatic glycogen accumulation. Of note, PTG deletion also blocked hepatic steatosis in HFD-fed mice and reduced the expression of numerous lipogenic genes. Additionally, PTG deletion reduced fasting glucose and insulin levels in obese mice while improving insulin sensitivity, a result of reduced hepatic glucose output. This metabolic crosstalk was due to decreased mTORC1 and SREBP activity in PTG knockout mice or knockdown cells, suggesting a positive feedback loop in which once accumulated, glycogen stimulates the mTORC1/SREBP1 pathway to shift energy storage to lipogenesis. Together, these data reveal a previously unappreciated broad role for glycogen in the control of energy homeostasis.

No disease-modifying treatment exists for the fatal neurodegenerative polyglutamine disease known both as Machado-Joseph disease and spinocerebellar ataxia type 3. As a potential route to therapy, we identified small molecules that reduce levels of the mutant disease protein, ATXN3. Screens of a small molecule collection, including 1250 Food and Drug Administration-approved drugs, in a novel cell-based assay, followed by secondary screens in brain slice cultures from transgenic mice expressing the human disease gene, identified the atypical antipsychotic aripiprazole as one of the hits. Aripiprazole increased longevity in a Drosophila model of Machado-Joseph disease and effectively reduced aggregated ATXN3 species in flies and in brains of transgenic mice treated for 10 days. The aripiprazole-mediated decrease in ATXN3 abundance may reflect a complex response culminating in the modulation of specific components of cellular protein homeostasis. Aripiprazole represents a potentially promising therapeutic drug for Machado-Joseph disease and possibly other neurological proteinopathies.

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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...