Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects some adult carriers of pre-mutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. FXTAS is thought to be caused by a toxic 'gain-of-function' of the expanded CGG-repeat FMR1 mRNA, which is found in the neuronal and astrocytic intranuclear inclusions associated with the disorder. Using a reporter construct with a FMR1 5' untranslated region harboring an expanded (premutation) CGG repeat, we have demonstrated that intranuclear inclusions can be formed in both primary neural progenitor cells and established neural cell lines. As with the inclusions found in post-mortem tissue, the inclusions induced by the expanded CGG repeat are alphaB-crystallin-positive; however, inclusions in culture are not associated with ubiquitin, indicating that incorporation of ubiquitinated proteins is a later event in the disease process. The absence of ubiquitinated proteins also argues against a model in which inclusion formation is due to a failure of the proteasomal degradative machinery. The presence of the expanded CGG repeat, as RNA, results in reduced cell viability as well as the disruption of the normal architecture of lamin A/C within the nucleus. This last observation, and the findings that lamin A/C is present in both the inclusions of FXTAS patients and the inclusions in cell culture, suggests that lamin A/C dysregulation may be a component of the pathogenesis of FXTAS; in particular, the Charcot-Marie-Tooth-type neuropathy associated with FXTAS may represent a functional laminopathy.

Expansion of a (CGG)n sequence in the 5′-UTR of the FMR1 gene to >200–2000 repeats abolishes its transcription and initiates fragile X syndrome (FXS). By contrast, levels of FMR1 mRNA are 5–10-fold higher in FXS premutation carriers of >55–200 repeats than in normal subjects. Lack of a corresponding increase in the amount of the product FMRP protein in carrier cells suggest that (CGG)>55–200 tracts thwart translation. Here we report that a (CGG)99 sequence positioned upstream to reporter firefly (FL) gene selectively diminished mRNA translation in coupled and separate T7 promoter-driven in vitro transcription and translation systems. The (CGG)99 tract similarly depressed mRNA utilization in HEK293 human cells transfected with plasmids bearing FMR1 promoter-driven FL gene. A (CGG)33 RNA tract formed a largely RNase T1-resistant intramolecular secondary structure in the presence of K+ ions. Expression of the quadruplex (CGG)n disrupting proteins hnRNP A2 or CBF-A in HEK293 cells significantly elevated the efficacy of (CGG)99 FL mRNA translation whereas hnRNP A2 or CBF-A mutants lacking quadruplex (CGG)n disrupting activity did not. Taken together, our results suggest that secondary structures of (CGG)n in mRNA obstruct its translation and that quadruplex-disrupting proteins alleviate the translational block.

Large expansions of a CGG-repeat element (>200 repeats; full mutation) in the fragile X mental retardation 1 (FMR1) gene cause fragile X syndrome (FXS), the leading single-gene form of intellectual disability and of autism spectrum disorder. Smaller expansions (55-200 CGG repeats; premutation) result in the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Whereas FXS is caused by gene silencing and insufficient FMR1 protein (FMRP), FXTAS is thought to be caused by 'toxicity' of expanded-CGG-repeat mRNA. However, as FMRP expression levels decrease with increasing CGG-repeat length, lowered protein may contribute to premutation-associated clinical involvement. To address this issue, we measured brain Fmr1 mRNA and FMRP levels as a function of CGG-repeat length in a congenic (CGG-repeat knock-in) mouse model using 57 wild-type and 97 expanded-CGG-repeat mice carrying up to ~250 CGG repeats. While Fmr1 message levels increased with repeat length, FMRP levels trended downward over the same range, subject to significant inter-subject variation. Human comparisons of protein levels in the frontal cortex of 7 normal and 17 FXTAS individuals revealed that the mild FMRP decrease in mice mirrored the more limited data for FMRP expression in the human samples. In addition, FMRP expression levels varied in a subset of mice across the cerebellum, frontal cortex, and hippocampus, as well as at different ages. These results provide a foundation for understanding both the CGG-repeat-dependence of FMRP expression and for interpreting clinical phenotypes in premutation carriers in terms of the balance between elevated mRNA and lowered FMRP expression levels.

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