Machado-Joseph disease (MJD, also known as spinocerebellar ataxia type 3) is a fatal neurodegenerative disease that impairs control and coordination of movement. Here we tested whether treatment with the histone deacetylase inhibitor sodium valproate (valproate) prevented a movement phenotype that develops in larvae of a transgenic zebrafish model of the disease. We found that treatment with valproate improved the swimming of the MJD zebrafish, affected levels of acetylated histones 3 and 4, but also increased expression of polyglutamine expanded human ataxin-3. Proteomic analysis of protein lysates generated from the treated and untreated MJD zebrafish also predicted that valproate treatment had activated the sirtuin longevity signaling pathway and this was confirmed by findings of increased SIRT1 protein levels and sirtuin activity in valproate treated MJD zebrafish and HEK293 cells expressing ataxin-3 84Q, respectively. Treatment with resveratrol (another compound known to activate the sirtuin pathway), also improved swimming in the MJD zebrafish. Co-treatment with valproate alongside EX527, a SIRT1 activity inhibitor, prevented induction of autophagy by valproate and the beneficial effects of valproate on the movement in the MJD zebrafish, supporting that they were both dependent on sirtuin activity. These findings provide the first evidence of sodium valproate inducing activation of the sirtuin pathway. Further, they indicate that drugs that target the sirtuin pathway, including sodium valproate and resveratrol, warrant further investigation for the treatment of MJD and related neurodegenerative diseases. Graphical abstract

Spinocerebellar ataxia-3 (SCA3, also Machado Joseph disease), is a neurodegenerative disease caused by inheritance of a CAG repeat expansion within the ATXN3 gene, resulting in polyglutamine (polyQ) repeat expansion within the ataxin-3 protein. In this study we have identified protein aggregates in both neuronal-like (SHSY5Y) cells and transgenic zebrafish expressing human ataxin-3 with expanded polyQ. We have adapted a previously reported flow cytometry methodology named flow cytometric analysis of inclusions and trafficking (FloIT), allowing rapid quantification of detergent insoluble forms of ataxin-3 fused to a green fluorescent protein in the SHSY5Y cells and cells dissociated from the zebrafish larvae. Flow cytometric analysis revealed an increased number of detergent-insoluble ataxin-3 particles per nuclei in the cells and zebrafish expressing polyQ expanded ataxin-3 compared to those expressing wildtype human ataxin-3. Treatment with compounds known to modulate autophagy activity was found to alter the number of detergent-insoluble ataxin-3 particles in cells and zebrafish. We conclude that flow cytometry can be harnessed to rapidly count ataxin-3 aggregates, both in vitro and in vivo, and can be utilised to compare potential therapies targeting protein aggregates.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. ALS can be modeled in zebrafish (Danio rerio) through the expression of human ALS-causing genes, such as superoxide dismutase 1 (SOD1). Overexpression of mutated human SOD1 protein causes aberrant branching and shortening of spinal motor axons. Despite this, the functional relevance of this axon morphology remains elusive. Our aim was to determine whether this motor axonopathy is correlated with impaired movement in mutant (MT) SOD1-expressing zebrafish. Transgenic zebrafish embryos that express blue fluorescent protein (mTagBFP) in motor neurons were injected with either wild-type (WT) or MT (A4V) human SOD1 messenger ribonucleic acid (mRNA). At 48 hours post-fertilization, larvae movement (distance traveled during behavioral testing) was examined, followed by quantification of motor axon length. Larvae injected with MT SOD1 mRNA had significantly shorter and more aberrantly branched motor axons (p < 0.002) and traveled a significantly shorter distance during behavioral testing (p < 0.001) when compared with WT SOD1 and noninjected larvae. Furthermore, there was a positive correlation between distance traveled and motor axon length (R2 = 0.357, p < 0.001). These data represent the first correlative investigation of motor axonopathies and impaired movement in SOD1-expressing zebrafish, confirming functional relevance and validating movement as a disease phenotype for the testing of disease treatments for ALS.

Spinocerebellar ataxia-3, (SCA3, also known as Machado Joseph Disease) is a neurodegenerative disease caused by inheritance of a ATXN3 gene containing a CAG repeat expansion, resulting in presence of a polyglutamine (polyQ) repeat expansion within the encoded human ataxin-3 protein. SCA3 is characterized by the formation of ataxin-3 protein aggregates within neurons, neurodegeneration, and impaired movement. In this study we have identified protein aggregates in both neuronal-like cell (SHSY5Y) cells and in vivo (transgenic zebrafish) models expressing human ataxin-3 protein containing polyQ expansion. We have adapted a flow cytometric methodology, allowing rapid quantification of detergent insoluble forms of ataxin-3 fused to a green fluorescent protein. Flow cytometric analysis revealed an increased number of detergent-insoluble ataxin-3 particles in cells and zebrafish expressing polyQ expanded ataxin-3 when compared to cells and zebrafish expressing wildtype ataxin-3. Interestingly, a protein aggregation phenotype could be detected as early as two days of age in transgenic zebrafish, prior to the onset of a detectable movement impairment at 6 days of age, suggesting protein aggregation may be an early disease phenotype in SCA3. Further, treatment of SCA3 cells and transgenic zebrafish with compounds known to modulate the activity of the autophagy protein quality control pathway altered the number of detergent-insoluble ataxin-3 particles detected by flow cytometry. We conclude that flow cytometry is a powerful tool that can be harnessed to rapidly quantify ataxin-3 aggregates, both in vitro and in vivo, and can be utilised to screen and compare potential protein aggregate targeting therapies.

Machado-Joseph disease (MJD, also known as spinocerebellar ataxia-3) is a fatal neurodegenerative disease that impairs control and coordination of movement. Here we tested whether treatment with the histone deacetylase inhibitor sodium valproate (SV) prevented a movement phenotype that develops in larvae of a transgenic zebrafish model of the disease. We found that treatment with SV improved the swimming of the MJD zebrafish, increased levels of acetylated histones 3 and 4, but also increased expression of polyglutamine expanded human ataxin-3. Proteomic analysis of protein lysates generated from the treated and untreated MJD zebrafish also predicted that SV treatment had activated the sirtuin longevity signaling pathway and this was confirmed by findings of increased SIRT1 protein levels and sirtuin activity in SV treated MJD zebrafish and HEK293 cells expressing ataxin-3-84Q, respectively. Treatment with resveratrol (another compound known to activate the sirtuin pathway), also improved swimming in the MJD zebrafish. Co-treatment with SV alongside EX527, a SIRT1 activity inhibitor, prevented induction of autophagy by SV and the beneficial effects of SV on the movement in the MJD zebrafish, indicating that they were both dependent on sirtuin activity. These findings provide the first evidence of sodium valproate inducing activation of the sirtuin pathway. Further, they indicate that drugs that target the sirtuin pathway, including sodium valproate and resveratrol, warrant further investigation for the treatment of MJD and related neurodegenerative diseases.