Structural Analysis and Spatiotemporal Expression of Atxn1 Genes in Zebrafish Embryos and Larvae.

Zebrafish have come into focus to model cerebellar diseases such as spinocerebellar ataxias (SCAs), which is caused by an expansion of translated CAG repeats in several unrelated genes. In spinocerebellar ataxia type 1 (SCA1), gain-of-function in the mutant ATXN1 contributes to SCA1's neuropathy. Human ATXN1 and its paralog ATXN1L are chromatin-binding factors, act as transcriptional repressors, and have similar expression patterns. However, little is known about atxn1 genes in zebrafish. Recently, two family members, atxn1a and atxn1b, were identified as duplicate orthologs of ATXN1, as was atxn1l, the ortholog of ATXN1L. In this study, we analyzed the phylogenetic relationship of the atxn1 family members in zebrafish, compared their genetic structures, and verified the predicted transcripts by both RT-PCR and whole-mount in situ hybridization. All three genes, atxn1a, atxn1b, and atxn1l, show overlapping, but also distinct, expression domains during embryonic and larval development. While atxn1a and atxn1l display similar spatiotemporal embryonic expression, atxn1b expression is initiated during the onset of brain development and is predominantly expressed in the cerebellum throughout zebrafish development. These results provide new insights into atxn1 genes and their expression patterns in zebrafish during embryonic and late-larval development and may contribute importantly to future experiments in disease modeling of SCAs.

Genetic Modeling of the Neurodegenerative Disease Spinocerebellar Ataxia Type 1 in Zebrafish.

Dominant spinocerebellar ataxias (SCAs) are progredient neurodegenerative diseases commonly affecting the survival of Purkinje cells (PCs) in the human cerebellum. Spinocerebellar ataxia type 1 (SCA1) is caused by the mutated ataxin1 (Atx1) gene product, in which a polyglutamine stretch encoded by CAG repeats is extended in affected SCA1 patients. As a monogenetic disease with the Atx1-polyQ protein exerting a gain of function, SCA1 can be genetically modelled in animals by cell type-specific overexpression. We have established a transgenic PC-specific SCA1 model in zebrafish coexpressing the fluorescent reporter protein mScarlet together with either human wild type Atx1[30Q] as control or SCA1 patient-derived Atx1[82Q]. SCA1 zebrafish display an age-dependent PC degeneration starting at larval stages around six weeks postfertilization, which continuously progresses during further juvenile and young adult stages. Interestingly, PC degeneration is observed more severely in rostral than in caudal regions of the PC population. Although such a neuropathology resulted in no gross locomotor control deficits, SCA1-fish with advanced PC loss display a reduced exploratory behaviour. In vivo imaging in this SCA1 model may help to better understand such patterned PC death known from PC neurodegeneration diseases, to elucidate disease mechanisms and to provide access to neuroprotective compound characterization in vivo.

Mood alterations in mouse models of Spinocerebellar Ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by abnormal expansion of glutamine-encoding CAG repeats in the Ataxin-1 (ATXN1) gene. SCA1 is characterized by progressive motor deficits, cognitive decline, and mood changes including anxiety and depression, with longer number of repeats correlating with worse disease outcomes. While mouse models have been very useful in understanding etiology of ataxia and cognitive decline, our understanding of mood symptoms in SCA1 has lagged. It remains unclear whether anxiety or depression stem from an underlying brain pathology or as a consequence of living with an untreatable and lethal disease. To increase our understanding of the etiology of SCA1 mood alterations, we used the elevated-plus maze, sucrose preference and forced swim tests to assess mood in four different mouse lines. We found that SCA1 knock-in mice exhibit increased anxiety that correlated with the length of CAG repeats, supporting the idea that underlying brain pathology contributes to SCA1-like anxiety. Additionally, our results support the concept that increased anxiety is caused by non-cerebellar pathology, as Purkinje cell specific SCA1 transgenic mice exhibit decreased anxiety-like behavior. Regarding the molecular mechanism, partial loss of ATXN1 may play a role in anxiety, based on our results for Atxn1 haploinsufficient and null mice.

Complementary proteomics strategies capture an ataxin-1 interactome in Neuro-2a cells.

Ataxin-1 mutation, arising from a polyglutamine (polyQ) tract expansion, is the underlying genetic cause of the late-onset neurodegenerative disease Spinocerebellar ataxia type 1 (SCA1). To identify protein partners of polyQ-ataxin-1 in neuronal cells under control or stress conditions, here we report our complementary proteomics strategies of proximity-dependent biotin identification (BioID) and affinity purification (via GFP-Trap pulldown) in Neuro-2a cells expressing epitope-tagged forms of ataxin-1[85Q]. These approaches allowed our enrichment of proximal proteins and interacting partners, respectively, with the subsequent protein identification performed by liquid chromatography-MS/MS. Background proteins, not dependent on the presence of the polyQ-ataxin-1 protein, were additionally defined by their endogenous biotinylation (for the BioID protocol) or by their non-specific interaction with GFP only (in the GFP-Trap protocol). All datasets were generated from biological replicates. Following the removal of the identified background proteins from the acquired protein lists, our experimental design has captured a comprehensive polyQ-ataxin-1 proximal and direct protein partners under normal and stress conditions. Data are available via ProteomeXchange, with identifier PXD010352.

Proliferation, differentiation and migration of SCA1-/CD31- cardiac side population cells in vitro and in vivo.

BACKGROUND: Side-population (SP) cells, identified by their capacity to efflux Hoechst dye, are highly enriched for stem/progenitor cell activity. They are found in many mammalian tissues, including mouse heart. Studies suggest that cardiac SP (CSP) cells can be divided into SCA1+/CD31-, SCA1+/CD31+ and SCA1-/CD31- CSP subpopulations. SCA1+/CD31- were shown to be cardiac and endothelial stem/progenitors while SCA1+/CD31+ CSP cells are endothelial progenitors. SCA1-/CD31- CSP cells remain to be fully characterized. In this study, we characterized SCA1-/CD31- CSP cells in the adult mouse heart, and investigated their abilities to proliferate, differentiate and migrate in vitro and in vivo. METHODS AND RESULTS: Using fluorescence-activated cell sorting, reverse transcriptase/polymerase chain reaction, assays of cell proliferation, differentiation and migration, and a murine model of myocardial infarction we show that SCA1-/CD31- CSP cells are located in the heart mesenchyme and express genes characteristic of stem cells and endothelial progenitors. These cells were capable of proliferation, differentiation, migration and vascularization in vitro and in vivo. Following experimental myocardial infarction, the SCA1-/CD31- CSP cells migrated from non-infarcted areas to the infarcted region within the myocardium where they differentiated into endothelial cells forming vascular (tube-like) structures. We further demonstrated that the SDF-1α/CXCR4 pathway may play an important role in migration of these cells after myocardial infarction. CONCLUSIONS: Based on their gene expression profile, localization and ability to proliferate, differentiate, migrate and vascularize in vitro and in vivo, we conclude that SCA1-/CD31- CSP cells may serve as endothelial progenitor cells in the adult mouse heart.

Systematic analysis of fly models with multiple drivers reveals different effects of ataxin-1 and huntingtin in neuron subtype-specific expression.

The fruit fly, Drosophila melanogaster, is a commonly used model organism for neurodegenerative diseases. Its major advantages include a short lifespan and its susceptibility to manipulation using sophisticated genetic techniques. Here, we report the systematic comparison of fly models of two polyglutamine (polyQ) diseases. We induced expression of the normal and mutant forms of full-length Ataxin-1 and Huntingtin exon 1 in cholinergic, dopaminergic, and motor neurons, and glial cells using cell type-specific drivers. We systematically analyzed their effects based on multiple phenotypes: eclosion rate, lifespan, motor performance, and circadian rhythms of spontaneous activity. This systematic assay system enabled us to quantitatively evaluate and compare the functional disabilities of different genotypes. The results suggest different effects of Ataxin-1 and Huntingtin on specific types of neural cells during development and in adulthood. In addition, we confirmed the therapeutic effects of LiCl and butyrate using representative models. These results support the usefulness of this assay system for screening candidate chemical compounds that modify the pathologies of polyQ diseases.