This application addresses broad Challenge Area (14): Stem Cells and specific Challenge Topic, 14-AG-101, Induced Pluripotent Stem (iPS) Cells for Aging and Neurodegeneration Research. The main objective of the proposed research is the elucidation of the pathogenic mechanism of a leading single-gene neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS), using human neuronal cells reprogrammed from patient fibroblasts through induced pluripotent stem cell (iPSC) technology. FXTAS involves both progressive motor dysfunction (tremor, gait ataxia, parkinsonism) and cognitive impairment/dementia. The disorder is caused by non-coding CGG-repeat expansions (premutation range;55- 200 repeats) in the fragile X mental retardation 1 (FMR1) gene, specifically by toxicity of the expanded CGG- repeat FMR1 mRNA. Our recent work with pure neuronal cultures from premutation CGG-repeat Fmr1 knock- in mice has revealed that (mouse) CGG-repeat-induced pathogenesis involves altered dendritic growth, altered pre- and post-synaptic volumes, and decreased neuronal cell longevity. These altered functions appear within one to three weeks in neurons cultured from one-day old mice, raising the possibility of a developmental component that precedes the neurodegenerative phenotype in humans. To investigate the basis of neuronal dysfunction in FXTAS, we propose to use iPSC technology to reprogram fibroblasts from individuals with FXTAS, along with controls, to produce cultured human neurons and astrocytes. This effort will be followed by parallel studies to those performed in the KI mouse neurons. A highly innovative feature of our proposed studies is the use of single individuals to produce both normal and premutation FMR1 expression phenotypes, either by sub-cloning fibroblasts with various allele sizes from mosaic individuals, or by sub-cloning iPSC-derived neural progenitor cells and selecting sub-clones based on which FMR1 allele (normal or premutation) is active. In the former case, the normal alleles are compared to expanded alleles from single (mosaic) individuals, thus providing an isogenic background;in the latter case, alleles are compared on an isoautosomal background. This approach exploits both the power of our clinical resources (over 100 fibroblast lines to date), the unique features of the FMR1 gene in providing the fibroblast substrates for isogenic comparison, and the fact that the trigger for neurodegeneration is known and is a function of the CGG repeat size. To our knowledge the proposed intra-individual (isogenic, isoautosomal) comparisons are unique among studies of neurodegenerative disorders. Based on current operation of the UC Davis Stem Cell Core, production and characterization of the iPSCs, and differentiation to neural stem cells, is expected to take approximately three to six months, with up to eighteen months afforded for the characterization of the trajectory for neural stem cell maturation and degeneration. Thus, the stated aims of the proposed research should be realized well within the two-year window of the proposed project. Consistent with the objective of the Challenge Grant Initiative, much of the requested funding is for purpose of retention and hiring of research staff.
FXTAS is a leading single-gene neurodegenerative disorder, with features of parkinsonism and dementia in addition to its core motor dysfunction (tremor and gait ataxia). Thus, the disorder represents a paradigm for studying late-onset neurodegeneration, since the gene (FMR1) and the pathogenic trigger (mRNA) are known and can be manipulated. Approximately 1/130 women and 1/300 men in the general population are carriers of premutation alleles, and a significant fraction of these will develop some features of FXTAS, underscoring the societal impact of this disorder.
