Spinocerebellar ataxia type 6 (SCA6) is a dominantly-inherited, untreatable neurodegenerative disease characterized by progressive ataxia and Purkinje cell degeneration associated with CAG repeat expansions in the gene, CACNA1A. Our recent evidence suggests that the disease is attributable to expression of a polyQ repeat expansion within a second CACNA1A gene product, a1ACT, that normally serves as a transcription factor (TF) critical for cerebellar cortical development, and that arises through the action of a cryptic cellular internal ribosomal entry site (IRES) within the coding region. SCA6-sized polyQ expansions in the ?1ACT TF interrupt its cellular and molecular function, cause cell death in vitro, ataxia and cortical thinning. The long-term goal of this project is to understand te pathogenesis of SCA6 by characterizing how a1ACTSCA6 alters gene expression, by confirming the role of the CACNA1A IRES in vivo and by developing a screening strategy for potential IRES-inhibiting compounds as potential therapies. Specifically we propose to ask: 1. Does the SCA6 polyQ expansion in a1ACT in the transcription factor (a1ACTSCA6) changes the gene binding patterns and the expression patterns of Purkinje cell genes? We will utilize chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) to analyze in detail binding profiles of a1ACTWT and a1ACTSCA6 expressed in PC12 cells and isolate RNA from Purkinje cells of mice expressing a1ACTWT or a1ACTSCA6 to generate global gene expression profiles by RNA-seq to correlate a1ACTWT-DNA binding with transcriptional activity and to identify allele-specific changes in gene expression patterns. 2. Does a1ACT expression by CACNA1A IRES and promoter reproduce the normal and pathological functions of ?1ACT? We will generate new lines of a1ACT transgenic mice that will only yield a1ACTWT or a1ACTSCA6 protein by CAP-independent translation, and use the tet-off expression system to drive conditional expression and use a CACNA1A-tTA transgene to generate an endogenous pattern of expression. 3. Can we identify IRES-directed molecules that selectively suppress ?1ACT translation, but not a1A subunit expression? We will use our dual-luciferase bicistronic reporter to determine the optimal RNA sequences involved in IRES function, identify the ITAFs by EMSA and predict the secondary structure of CACNA1A IRES. We will carry out a high throughput screening assay to identify compounds that interfere with ?1ACT IRES function.

Public Health Relevance

We have discovered that the calcium channel gene responsible for spinocerebellar ataxia type 6 encodes a second transcription factor protein, ?1ACT made via a rare genetic mechanism, that bears the mutation responsible for SCA6. The control sequence leading to expression of ?1ACT, an internal ribosomal entry site (IRES) may be a highly amenable target for drug development. In this study we will better characterize the role of the a1ACT in health and disease, characterize the structure and binding proteins of the IRES, and carry out a high throughput screen to identify small molecules that interfere with IRES function.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Molecular Neurogenetics Study Section (MNG)
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Miller, Daniel L
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University of Chicago
Schools of Medicine
United States
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Pastor, Parviz Daniel Hejazi; Du, Xiaofei; Fazal, Sarah et al. (2018) Targeting the CACNA1A IRES as a Treatment for Spinocerebellar Ataxia Type 6. Cerebellum 17:72-77
Karginov, Timofey A; Pastor, Daniel Parviz Hejazi; Semler, Bert L et al. (2017) Mammalian Polycistronic mRNAs and Disease. Trends Genet 33:129-142
Miyazaki, Yu; Du, Xiaofei; Muramatsu, Shin-Ichi et al. (2016) An miRNA-mediated therapy for SCA6 blocks IRES-driven translation of the CACNA1A second cistron. Sci Transl Med 8:347ra94
Tsou, Wei-Ling; Hosking, Ryan R; Burr, Aaron A et al. (2015) DnaJ-1 and karyopherin ?3 suppress degeneration in a new Drosophila model of Spinocerebellar Ataxia Type 6. Hum Mol Genet 24:4385-96
Du, Xiaofei; Semler, Bert L; Gomez, Christopher M (2014) Revelations from a bicistronic calcium channel gene. Cell Cycle 13:875-6