Spinocerebellar ataxia type 12 (SCA12) is a neurodegenerative disorder recently described by the authors. SCA12 is linked to a CAG repeat expansion mutation in PPP2R2B, the gene that encodes PPP2R2B, as regulatory subunit of protein phosphatase 2A (PP2A). Goals are 1) to delineate the clinical phenotype of this new disease and 2) to gain insight into the molecular and cellular mechanisms mediating neuronal dysfunction and cell loss in SCA12. The main hypothesis to be tested is that the expansion mutation leads to increased expression of the regulatory subunit of PP2A, resulting in altered PP2A activity that contributes to neuronal toxicity.
Aim 1 is to delineate the clinical, MRI and neuropathologic features of SCA12. Neurological, neuropsychological and psychiatric evaluations will be performed on affected members of the first SCA12 kindred at two different times points to define the clinical features of SCA12 and their time course. These findings will be compared to changes seen on brain magnetic resonance images (MRIs). An autopsy brain from the proband in this first kindred will be examined for gross pathology and cytologic changes in specific brain regions.
Aim 2 will determine the mechanism by which the CAG repeat expansion in SCA12 alters gene expression. We will test the hypothesis that the SCA12 CAG repeat is within the promoter region of the phosphatase subunit gene PPP2R2B, and the expansion of the repeat causes increased PPP2R2B expression. Bioinformatic and experimental approaches will be used to confirm location of the CAG repeat in a 5' region of PPP2R2B outside of an open reading frame. Promoter assays will reveal if the region containing the repeat is a functional promoter, and if repeat expansion increases promoter activity. If so, the properties of the repeat expansion necessary for increased promoter activity will be determined.
Aim 3 will analyze neuronal injury in a cell culture model of SCA12 using over-expression of PPP2R2B in N2a neuroblastoma cells and primary neuronal cultures. This will test the hypothesis that over-expression of PPP2R2B is toxic to cells. Survival of cultured cells will be analyzed following over-expression of PPP2R2B under regulation of a CMV promoter, and then by over-expression of PPP2R2B under control of its own promoter. The relative toxicity of different levels of PPP2R2B expression will be tested by comparing loss of cells transfected with normal vs. expanded CAG repeats. A 2nd hypothesis is that the toxicity of over-expressed PPP2R2B is a consequence of altered protein phosphatase 2A function. To test function, we will assay enzyme activity, substrate specificity, and intracellular localization of PP2A following over-expression of PPP2R2B and determine the effect of PP2A modulators on cell survival. Investigation of SCA12 may provide insight into the mechanisms responsible for neuronal loss, and potential therapy, for this and other trinucleotide repeat diseases and SCAs, additional disorders due to mutations in gene promoters, and other conditions that result from dysfunctional regulation of phoshorylation.
| O'Hearn, Elizabeth; Holmes, Susan E; Margolis, Russell L (2012) Spinocerebellar ataxia type 12. Handb Clin Neurol 103:535-47 |
| Cagnoli, Claudia; Michielotto, Chiara; Matsuura, Tohru et al. (2004) Detection of large pathogenic expansions in FRDA1, SCA10, and SCA12 genes using a simple fluorescent repeat-primed PCR assay. J Mol Diagn 6:96-100 |
