Investigating disease mechanisms and potential drug treatments in a transgenic zebrafish model of Machado-Joseph disease

Machado-Joseph disease (MJD) is a neurodegenerative disease resulting in the loss of muscle control and coordination. This disease is caused by inheritance of the ATXN3 gene containing an expanded CAG trinucleotide repeat region encoding for the polyglutamine (polyQ) tract in the ataxin-3 protein. Normally, the length of the CAG repeat region is between 12-44 repeats whilst MJD patients harbour >44 repeats. There is no known treatment or cure to prevent disease progression and understanding the mechanisms causing MJD neuropathology are limited. Thus, there are various cell and animal models exploring potential mechanisms of disease and investigating which treatments could ameliorate disease phenotypes. Our team has generated the first transgenic zebrafish model of MJD. Zebrafish are a popular animal model to investigate neurodegeneration due to external development of the embryos, for easy genetic manipulation and observation. The main advantage is the permeability of embryos, allowing for easy absorption of compounds dissolved in their environment. In combination with this, zebrafish embryos develop rapidly, allowing for high throughput drug testing. This thesis aimed to characterise disease phenotypes that develop in this zebrafish model and study these phenotypes to investigate disease mechanisms and potential treatments. Disease phenotypes identified within the zebrafish expressing human polyQ expanded ataxin-3 protein were motor impairment and ataxin-3 positive cleavage fragments. We monitored these phenotypes to explore a number of related pathways, including autophagy and transcription regulation, to understand how these pathways may relate to the development of the disease phenotypes. Finally, we tested whether a variety of small compounds/drugs are protective. The findings of this drug testing provided valuable insight towards the development of a treatment for MJD

A Novel Calpain Inhibitor Compound Has Protective Effects on a Zebrafish Model of Spinocerebellar Ataxia Type 3

Spinocerebellar ataxia type 3 (SCA3) is a hereditary ataxia caused by inheritance of a mutated form of the human ATXN3 gene containing an expanded CAG repeat region, encoding a human ataxin-3 protein with a long polyglutamine (polyQ) repeat region. Previous studies have demonstrated that ataxin-3 containing a long polyQ length is highly aggregation prone. Cleavage of the ataxin-3 protein by calpain proteases has been demonstrated to be enhanced in SCA3 models, leading to an increase in the aggregation propensity of the protein. Here, we tested the therapeutic potential of a novel calpain inhibitor BLD-2736 for the treatment of SCA3 by testing its efficacy on a transgenic zebrafish model of SCA3. We found that treatment with BLD-2736 from 1 to 6 days post-fertilisation (dpf) improves the swimming of SCA3 zebrafish larvae and decreases the presence of insoluble protein aggregates. Furthermore, delaying the commencement of treatment with BLD-2736, until a timepoint when protein aggregates were already known to be present in the zebrafish larvae, was still successful at removing enhanced green fluorescent protein (EGFP) fused-ataxin-3 aggregates and improving the zebrafish swimming. Finally, we demonstrate that treatment with BLD-2736 increased the synthesis of LC3II, increasing the activity of the autophagy protein quality control pathway. Together, these findings suggest that BLD-2736 warrants further investigation as a treatment for SCA3 and related neurodegenerative diseases.</jats:p

Autophagy Function and Benefits of Autophagy Induction in Models of Spinocerebellar Ataxia Type 3

Background: Spinocerebellar ataxia 3 (SCA3, also known as Machado Joseph disease) is a fatal neurodegenerative disease caused by the expansion of the trinucleotide repeat region within the ATXN3/MJD gene. The presence of this genetic expansion results in an ataxin-3 protein containing a polyglutamine repeat region, which renders the ataxin-3 protein aggregation prone. Formation of ataxin-3 protein aggregates is linked with neuronal loss and, therefore, the development of motor deficits. Methods: Here, we investigated whether the autophagy protein quality control pathway, which is important in the process of protein aggregate removal, is impaired in a cell culture and zebrafish model of SCA3. Results: We found that SH-SY5Y cells expressing human ataxin-3 containing polyglutamine expansion exhibited aberrant levels of autophagy substrates, including increased p62 and decreased LC3II (following bafilomycin treatment), compared to the controls. Similarly, transgenic SCA3 zebrafish showed signs of autophagy impairment at early disease stages (larval), as well as p62 accumulation at advanced age stages (18 months old). We then examined whether treating with compounds known to induce autophagy activity, would aid removal of human ataxin-3 84Q and improve the swimming of the SCA3 zebrafish larvae. We found that treatment with loperamide, trehalose, rapamycin, and MG132 each improved the swimming of the SCA3 zebrafish compared to the vehicle-treated controls. Conclusion: We propose that signs of autophagy impairment occur in the SH-SY5Y model of SCA3 and SCA3 zebrafish at larval and advanced age stages. Treatment of the larval SCA3 zebrafish with various compounds with autophagy induction capacity was able to produce the improved swimming of the zebrafish, suggesting the potential benefit of autophagy-inducing compounds for the treatment of SCA3

Flow cytometry allows rapid detection of protein aggregates in cellular and zebrafish models of spinocerebellar ataxia 3

ABSTRACT Spinocerebellar ataxia 3 (SCA3, also known as 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, allowing rapid quantification of detergent insoluble forms of ataxin-3 fused to a GFP in 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 cells and in zebrafish expressing polyQ-expanded ataxin-3 compared to those expressing wild-type human ataxin-3. Treatment with compounds known to modulate autophagic activity altered the number of detergent-insoluble ataxin-3 particles in cells and zebrafish expressing mutant human ataxin-3. We conclude that flow cytometry can be harnessed to rapidly count ataxin-3 aggregates, both in vitro and in vivo, and can be used to compare potential therapies targeting protein aggregates. This article has an associated First Person interview with the first author of the paper.</jats:p

Flow cytometry allows rapid detection of protein aggregates in cell culture and zebrafish models of spinocerebellar ataxia-3

AbstractSpinocerebellar ataxia-3 (SCA3, also known as Machado Joseph Disease), is a neurodegenerative disease caused by inheritance of aATXN3gene 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 (SHSY5Y) cells andin 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 human 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, bothin vitroandin vivo, and can be utilised to screen and compare potential protein aggregate targeting therapies.</jats:p

Spermidine treatment: induction of autophagy but also apoptosis?

Abstract Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is a fatal neurodegenerative disease that causes loss of balance and motor co-ordination, eventually leading to paralysis. It is caused by the autosomal dominant inheritance of a long CAG trinucleotide repeat sequence within the ATXN3 gene, encoding for an expanded polyglutamine (polyQ) repeat sequence within the ataxin-3 protein. Ataxin-3 containing an expanded polyQ repeat is known to be highly prone to intraneuronal aggregation, and previous studies have demonstrated that protein quality control pathways, such as autophagy, are impaired in MJD patients and animal models of the disease. In this study, we tested the therapeutic potential of spermidine on zebrafish and rodent models of MJD to determine its capacity to induce autophagy and improve functional output. Spermidine treatment of transgenic MJD zebrafish induced autophagy and resulted in increased distances swum by the MJD zebrafish. Interestingly, treatment of the CMVMJD135 mouse model of MJD with spermidine added to drinking water did not produce any improvement in motor behaviour assays, neurological testing or neuropathology. In fact, wild type mice treated with spermidine were found to have decreased rotarod performance when compared to control animals. Immunoblot analysis of protein lysates extracted from mouse cerebellar tissue found little differences between the groups, except for an increased level of phospho-ULK1 in spermidine treated animals, suggesting that autophagy was indeed induced. As we detected decreased motor performance in wild type mice following treatment with spermidine, we conducted follow up studies into the effects of spermidine treatment in zebrafish. Interestingly, we found that in addition to inducing autophagy, spermidine treatment also induced apoptosis, particularly in wild type zebrafish. These findings suggest that spermidine treatment may not be therapeutically beneficial for the treatment of MJD, and in fact warrants caution due to the potential negative side effects caused by induction of apoptosis

Treatment with sodium butyrate has therapeutic benefits for Machado-Joseph disease through the induction of autophagy

AbstractMachado-Joseph disease (MJD) is a fatal neurodegenerative disease caused by expansion of the trinucleotide repeat region within the ATXN3/MJD gene. Mutation of ATXN3 causes formation of neurotoxic ataxin-3 protein aggregates, neurodegeneration and motor deficits. Here we investigated the therapeutic potential of sodium butyrate (SB), the sodium salt of butyric acid, a metabolite naturally produced by gut microbiota, on cultured SH-SY5Y cells and transgenic zebrafish expressing human ataxin-3 containing 84 glutamine (Q) residues to model MJD. MJD SH-SY5Y cells were found to contain ataxin-3 oligomeric species and protein aggregates. Interestingly, treatment with SB decreased the size of detergentinsoluble ataxin-3 aggregates in vitro. Further investigation revealed that SB treatment increased activity of the autophagy protein quality control pathway in the MJD cells and decreased presence of ataxin-3 oligomers in an autophagy-dependent manner. Treatment with SB was also beneficial in vivo through induction of autophagy and improving swimming performance in transgenic MJD zebrafish. Co-treating the MJD zebrafish with SB and chloroquine, an autophagy inhibitor, prevented the beneficial effects of SB, suggesting that the improved swimming performance was autophagy-dependent. Furthermore, intraperitoneal injection of SB to wild type mice resulted in increased levels of neuronal LC3B levels, indicating induction of autophagy within the brain. Collectively, our findings suggest that SB can induce activity of the autophagy pathway and can produce beneficial effects in vitro and in vivo. We propose that treatment with sodium butyrate warrants further investigation for the treatment of neurodegenerative diseases underpinned by proteinopathy mechanisms, including MJD.</jats:p

Additional file 1 of Spermidine treatment: induction of autophagy but also apoptosis?

Supplementary Material