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Transcriptional phenotype in a novel ataxin-3 knock-in mouse model for spinocerebellar ataxia type 3

ABSTRACT: Spinocerebellar ataxia type 3 is the most common autosomal dominant inherited ataxia worldwide and caused by a CAG repeat expansion in the Ataxin-3 gene resulting in a polyQ expansion in the corresponding protein. The disease is characterized by neuropathological (aggregate formation, cell loss), phenotypical (gait instability, body weight reduction) and transcriptional changes. So far there is no mouse model available representing all the different aspects of the disease, but a representative model is highly needed to gain a better understanding of the disease pathomechanism. Here we characterized a novel Ataxin-3 knock-in mouse model, expressing an expansion of 304 CAG/CAAs either heterozygous or homozygous in the murine Ataxin-3 locus using biochemical, behavioral and transcriptomic approaches. Further, we correlated the transcriptional changes of the knock-in mice to those found in human SCA3 patients, to prove the comparability of our model. The novel Ataxin-3 knock-in mouse is characterized by the expression of polyQ-expanded Ataxin-3 in the murine protein, leading to massive aggregate formation, especially in brain regions known to be vulnerable in SCA3 patients, and impairment of Purkinje cells. Caused by these neuropathological changes, the mice demonstrated phenotypically reduction in body weight accompanied by gait and balance instability. Transcriptomic analysis of cerebellar tissue revealed downregulation of differentially expressed genes enriched in myelinating oligodendrocytes. Comparing these transcriptional changes with those found in cerebellar tissue of SCA3 patients, we discovered an overlap of differentially expressed genes pointing towards disturbances in the myelin sheaths and myelinating oligodendrocytes.

ORGANISM(S): Mus musculus

PROVIDER: GSE145613 | GEO | 2022/04/01

REPOSITORIES: GEO

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Project description:Spinocerebellar ataxia type 3 (SCA3/MJD) has a polyQ etiology but, the current knowledge on molecular processes and proteins involved in pathogenesis is insufficient. Due to its proteolytic function, Ataxin-3 can influence other proteins, yet the global picture of crucial proteins and pathways in SCA3 was not investigated previously. Here, we explored molecular SCA3 mechanism by interdisciplinary research paradigm combining SCA3 knock-in model, behavior, MRI, brain proteomics, precise axonal proteomics, neuronal energy recordings, labeling of vesicles, and inclusions and focusing in axonal compartment. Using the global proteomics, we found dysregulation of protein homeostasis over the entire disease progression. The early SCA3 phase was associated with reduced body weight gain, the presence of the number of dysregulated proteins related to metabolism and mitochondria, and an altered profile of oxygen consumption rate in neurons. Moreover, the early phase presented alterations of protein metabolism, cytoskeletal architecture, axonal and synaptic proteins. Protein dysregulations indicated that the compartment involved in SCA3 pathogenesis next to the mitochondria are also neuronal processes and axons in particular. To confirm that the axon is one of the central compartments in SCA3 pathogenesis, we performed targeted proteomics on axons and somatodendritic compartments. We revealed highly increased axonal localization of protein synthesis machinery, including ribosomes, translation factors, and RNA binding proteins, while the level of proteins responsible for cellular transport and mitochondria was decreased. In summary, the SCA3 disease mechanism is based on the broad influence of mutant ataxin-3 on the neuronal proteome. Processes central in our SCA3 model include disturbed localization of proteins between axonal and somatodendritic compartment, early neuronal energy deficit, altered neuronal cytoskeletal structure, an overabundance of protein synthetic machinery in axons.

Project description:Spinocerebellar ataxia type 3 (SCA3) is one of the polyglutamine (polyQ) diseases, which are caused by a CAG repeat expansion within the coding region of the associated genes. The CAG repeat specifies glutamine, and the expanded polyQ domain with mutation confers dominant toxicity on the protein. Traditionally, studies have focused on protein toxicity in polyQ disease mechanisms. Recent findings, however, demonstrate that the CAG repeat RNA, which encodes the toxic polyQ protein, also contributes to the disease in Drosophila. To provide insight into the nature of the RNA toxicity, we extracted brain-enriched RNA from flies expressing a toxic CAG repeat mRNA (CAG100) and a non-toxic interrupted CAA/G mRNA repeat (CAA/G105) for microarray analysis. This approach identified a set of genes that are differentially expressed specifically in CAG100 flies. Four independent replicates of flies expressing CAG0, CAG100, or CAA/G105 by elav-GAL4 were collected at 3 days. The transgenes are DsRed with either (CAG0) no CAG repeat in the 3'UTR, (CAG100) a CAG repeat of 100 CAGs in the 3'UTR, or (CAA/G105) an interrupted CAA CAG repeat in the 3'UTR (ref: Li et al., Nature 453:1107) The transgenes were adjusted to match in mRNA expression such that CAG0 flies had one copy of the transgene, CAG100 flies had 5 copies, and CAA/G105 had two copies. Fly brain tissue (about 20 brains per sample, dissected from head capsule, eyes, lamina and outer medulla removed) was dissected in cold phosphate buffered saline (PBS) and stored in Trizol reagent (Invitrogen Corporation, Carlsbad, CA) at -80Ë?C. Total brain RNA was extracted and purified using TRIzol reagent (Invitrogen) and the RNeasy Mini system (Qiagen), and treated with RNase-free DNase I (Qiagen). To define genes whose expression is altered in response to a toxic CAG repeat RNA, we compared CAG100 flies with age-matched flies expressing CAG0. To exclude transcriptional changes in response to a non-toxic trinucleotide repeat, a second gene list was generated by comparing CAA/G105 flies with age-matched CAG0 flies.

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Transcriptional phenotype in a novel ataxin-3 knock-in mouse model for spinocerebellar ataxia type 3

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