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

Robinson, Katherine J.; Tym, Madelaine C.; Hogan, Alison; Watchon, Maxinne; Yuan, Kristy C.; Plenderleith, Stuart K.; Don, Emily K.; Laird, Angela S.

doi:10.1242/dmm.049023

Cited by 8 publications

(19 citation statements)

References 63 publications

(143 reference statements)

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“…Nuclei were identified and gated based on the intensity of UV fluorescence and relative size (forward scatter). The number of insoluble GFP particles, indicating insoluble ataxin‐3 particles, was analyzed based on GFP fluorescent intensity and forward scatter 47 . Gating of Triton‐X insoluble GFP + positive particles was performed by comparing populations to an un‐transfected control sample.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we demonstrated that SH-SY5Y cells transiently transfected with EGFPfused human ataxin-3-84Q carry Triton-X insoluble protein aggregates that can be detected by a flow cytometry protocol called FloIT. 47 Upon immunostaining the stably expressing ataxin-3 cells for ataxin-3, the presence of cytoplasmic ataxin-3-positive puncta was revealed in cells expressing ataxin-3-28Q and -84Q (Figure 1D; white arrows). Automated quantification of the number of potential ataxin-3 protein aggregates (puncta with a diameter of 2.25-6 μm) within the images revealed that cells stably expressing ataxin-3-84Q had approximately double the number of puncta than cells expressing ataxin-3-28Q or an empty vector control (Figure 1E).…”

Section: A Sca3 Sh-sy5y Model Recapitulates Phenotypes Found In Human...mentioning

confidence: 98%

“…Whole zebrafish larvae (2 or 6 dpf) were euthanized and larvae were digested and dissociated into a single-cell solution, as previously described. 47 In brief, following euthanasia, whole larvae were dissected into smaller pieces using a scalpel and forceps and enzymatically digested using 0.5% Trypsin/EDTA. Dissociated cells were then lysed using PBS containing 0.5% Triton-X 100 and a marker of nuclei, either DAPI (final concentration 5 μM) or 1 × Red Dot (Gene Target Solutions, catalog # 40060).…”

Section: Dissociation Of Zebrafish For Flow Cytometrymentioning

confidence: 99%

“…The number of insoluble GFP particles, indicating insoluble ataxin-3 particles, was analyzed based on GFP fluorescent intensity and forward scatter. 47 Gating of Triton-X insoluble GFP + positive particles was performed by comparing populations to an un-transfected control sample. For analysis comparing the effect of different drug treatments on SCA3 cells and zebrafish expressing ataxin-3-84Q, data were analyzed as the number of insoluble GFP + particles divided by the number of detected nuclei.…”

Section: Flow Cytometric Analysis Of Insoluble Ataxin-3mentioning

confidence: 99%

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Treatment with sodium butyrate induces autophagy resulting in therapeutic benefits for spinocerebellar ataxia type 3

Watchon,

Robinson,

Luu

et al. 2024

The FASEB Journal

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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.

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Section: Methodsmentioning

confidence: 99%

Section: A Sca3 Sh-sy5y Model Recapitulates Phenotypes Found In Human...mentioning

confidence: 98%

Section: Dissociation Of Zebrafish For Flow Cytometrymentioning

confidence: 99%

Section: Flow Cytometric Analysis Of Insoluble Ataxin-3mentioning

confidence: 99%

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Treatment with sodium butyrate induces autophagy resulting in therapeutic benefits for spinocerebellar ataxia type 3

Watchon,

Robinson,

Luu

et al. 2024

The FASEB Journal

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“…These models, with the full gene [95,102] or with a truncated gene, were used with the expanded CAG repeat tract [96][97][98]. They convincingly showed pathogenic features of polyQ diseases including aggregate formation, the toxicity of the mutant proteins, neurotransmission defects and progressive neuronal degeneration [92,[96][97][98]102,[165][166][167]. Additionally, they are excellent models with which to study the mechanisms underlying polyQ disease symptoms, find potential targets for therapeutic interventions, search for new interactomes and verify findings from other models or patients [95][96][97]101,167].…”

Section: Simple Model Organismsmentioning

confidence: 99%

Advances in Modeling Polyglutamine Diseases Using Genome Editing Tools

Karwacka

Olejniczak

2022

Cells

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Polyglutamine (polyQ) diseases, including Huntington’s disease, are a group of late-onset progressive neurological disorders caused by CAG repeat expansions. Although recently, many studies have investigated the pathological features and development of polyQ diseases, many questions remain unanswered. The advancement of new gene-editing technologies, especially the CRISPR-Cas9 technique, has undeniable value for the generation of relevant polyQ models, which substantially support the research process. Here, we review how these tools have been used to correct disease-causing mutations or create isogenic cell lines with different numbers of CAG repeats. We characterize various cellular models such as HEK 293 cells, patient-derived fibroblasts, human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs) and animal models generated with the use of genome-editing technology.

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The deubiquitinase function of ataxin-3 and its role in the pathogenesis of Machado-Joseph disease and other diseases

Potapenko,

Davidson,

Lee

et al. 2024

Biochemical Journal

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Machado-Joseph disease (MJD) is a devastating and incurable neurodegenerative disease characterised by progressive ataxia, difficulty speaking and swallowing. Consequently, affected individuals ultimately become wheelchair dependent, require constant care, and face a shortened life expectancy. The monogenic cause of MJD is expansion of a trinucleotide (CAG) repeat region within the ATXN3 gene, which results in polyglutamine (polyQ) expansion within the resultant ataxin-3 protein. While it is well established that the ataxin-3 protein functions as a deubiquitinating (DUB) enzyme and is therefore critically involved in proteostasis, several unanswered questions remain regarding the impact of polyQ expansion in ataxin-3 on its DUB function. Here we review the current literature surrounding ataxin-3's DUB function, its DUB targets, and what is known regarding the impact of polyQ expansion on ataxin-3's DUB function. We also consider the potential neuroprotective effects of ataxin-3's DUB function, and the intersection of ataxin-3's role as a DUB enzyme and regulator of gene transcription. Ataxin-3 is the principal pathogenic protein in MJD and also appears to be involved in cancer. As aberrant deubiquitination has been linked to both neurodegeneration and cancer, a comprehensive understanding of ataxin-3's DUB function is important for elucidating potential therapeutic targets in these complex conditions. In this review, we aim to consolidate knowledge of ataxin-3 as a DUB and unveil areas for future research to aid therapeutic targeting of ataxin-3's DUB function for the treatment of MJD and other diseases.

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

Cited by 8 publications

References 63 publications

Treatment with sodium butyrate induces autophagy resulting in therapeutic benefits for spinocerebellar ataxia type 3

Treatment with sodium butyrate induces autophagy resulting in therapeutic benefits for spinocerebellar ataxia type 3

Advances in Modeling Polyglutamine Diseases Using Genome Editing Tools

The deubiquitinase function of ataxin-3 and its role in the pathogenesis of Machado-Joseph disease and other diseases

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