Spermidine treatment: induction of autophagy but also apoptosis?

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 induces autophagy resulting in therapeutic benefits for spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 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. Mutation of ATXN3 causes formation of ataxin-3 protein aggregates, neurodegeneration, and motor deficits. Here we investigated the therapeutic potential and mechanistic activity 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 SCA3. SCA3 SH-SY5Y cells were found to contain high molecular weight ataxin-3 species and detergent-insoluble protein aggregates. Treatment with SB increased the activity of the autophagy protein quality control pathway in the SCA3 cells, decreased the presence of ataxin-3 aggregates and presence of high molecular weight ataxin-3 in an autophagy-dependent manner. Treatment with SB was also beneficial in vivo, improving swimming performance, increasing activity of the autophagy pathway, and decreasing the presence of insoluble ataxin-3 protein species in the transgenic SCA3 zebrafish. Co-treating the SCA3 zebrafish with SB and chloroquine, an autophagy inhibitor, prevented the beneficial effects of SB on zebrafish swimming, indicating that the improved swimming performance was autophagy-dependent. To understand the mechanism by which SB induces autophagy we performed proteomic analysis of protein lysates from the SB-treated and untreated SCA3 SH-SY5Y cells. We found that SB treatment had increased activity of Protein Kinase A and AMPK signaling, with immunoblot analysis confirming that SB treatment had increased levels of AMPK protein and its substrates. Together our findings indicate that treatment with SB can increase activity of the autophagy pathway process and that this has beneficial effects in vitro and in vivo. While our results suggested that this activity may involve activity of a PKA/AMPK-dependent process, this requires further confirmation. We propose that treatment with sodium butyrate warrants further investigation as a potential treatment for neurodegenerative diseases underpinned by mechanisms relating to protein aggregation including SCA3.

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.

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.

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

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.

Dystrophin deficiency disrupts muscle clock expression and mitochondrial quality control in mdx mice.

Impaired oxidative capacity and mitochondrial function contribute to the dystrophic pathology in muscles of patients with Duchenne muscular dystrophy (DMD) and in relevant mouse models of the disease. Emerging evidence suggests an association between disrupted core clock expression and mitochondrial quality control, but this has not been established in muscles lacking dystrophin. We examined the diurnal regulation of muscle core clock and mitochondrial quality control expression in dystrophin-deficient C57BL/10ScSn-Dmdmdx (mdx) mice, an established model of DMD. Male C57BL/10 (BL/10; n = 18) and mdx mice (n = 18) were examined every 4 h beginning at the dark cycle. Throughout the entire light-dark cycle, extensor digitorum longus (EDL) muscles from mdx mice had decreased core clock mRNA expression (Arntl, Cry1, Cry2, Nr1d2; P < 0.05) and disrupted mitochondrial quality control mRNA expression related to biogenesis (decreased; Ppargc1a, Esrra; P < 0.05), fission (increased; Dnm1l; P < 0.01), fusion (decreased; Opa1, Mfn1; P < 0.05), and autophagy/mitophagy (decreased: Bnip3; P < 0.05; increased: Becn1; P < 0.05). Cosinor analysis revealed a decrease in the rhythmicity parameters mesor and amplitude for Arntl, Cry1, Cry2, Per2, and Nr1d1 (P < 0.001) in mdx mice. Diurnal oscillations in Esrra, Sirt1, Map1lc3b, and Sqstm1 were absent in mdx mice, along with decreased mesor and amplitude of Ppargc1a mRNA expression (P < 0.01). The expression of proteins involved in mitochondrial biogenesis (decreased: PPARGC1A, P < 0.05) and autophagy/mitophagy (increased: MAP1LC3BII, SQSTM1, BNIP3; P < 0.05) were also dysregulated in tibialis anterior muscles of mdx mice. These findings suggest that dystrophin deficiency in mdx mice impairs the regulation of the core clock and mitochondrial quality control, with relevance to DMD and related disorders.