The Molecular Basis of FXTAS

Although the precise molecular mechanism of FXTAS is not known, two observations led to the proposal of an RNA toxic gain-of-function model for disease pathogenesis in FXTAS (Hagerman et al. 2001; Greco et al. 2002; Jacquemont et al. 2003; Hagerman and Hagerman 2004). First, (expanded-repeat) FMR1 mRNA levels are elevated by up to eightfold in premutation carriers, even though FMRP levels are normal to slightly reduced in the premutation range (Tassone et al. 2000a, b; Kenneson et al. 2001). Second, FXTAS has not been reported in adults who harbor hypermethylated (silenced) full-mutation alleles, where little or no FMR1 mRNA is produced. In support of this RNA toxicity model, Jin et al. (2003) demonstrated that an expanded approximately 90 r(CGG) repeat, when expressed in the 5'-UTR context of an unrelated reporter gene, was still capable of inducing neuropathology (including inclusion formation) in the eye of the fly (Drosophila). This observation provided a direct demonstration that the expanded r(CGG) repeat in mRNA is capable of inducing neuropathology. The principal neuropathologic feature of FXTAS is the presence of ubiquitin-positive intranuclear inclusions, in both neurons and astrocytes, broadly distributed throughout the brain and spinal cord (Greco et al. 2002, 2006). The inclusions are immunohistochemi-cally negative for tau isoforms, a-synuclein, and polyglutamine peptides, and appear to reflect a new class of inclusion disorder (reviewed in Hagerman and Hagerman 2004). Much of the focus of recent research efforts have been on the composition of the inclusions, which should provide important clues as to the pathogenesis of the disease itself.

Myotonic dystrophy (DM) is another noncoding, trinucleotide d(CTG) repeat-expansion disorder that is thought to result from an RNA toxic gain of function (Finsterer 2002; Mankodi and Thornton 2002), where the expanded r(CUG) repeat, located in the 3'-UTR of the DM protein kinase (DMPK) mRNA, sequesters one or more protein mediators of the disease process. Thus, in DM, the RNA "toxicity" arises as a result of dysregulation of the function^) of those proteins owing to their excessive binding to the expanded r(CUG) repeat. One such mediator is believed to be MBNL1, the human homolog of Drosophila muscleblind (Miller et al. 2000; Fardaei et al. 2001; Ranum and Day 2004). Both DMPK mRNA and MBNL1 are found within intranuclear foci in DM, the sequestration of MBNL1 protein in turn leading to dysregulation of the splicing of several other mRNAs. Consistent with this RNA toxicity model, we have recently found FMR1 mRNA itself within the intranuclear inclusions of FXTAS patients (Tassone et al. 2004). This last observation gives added impetus to study the protein complement of the inclusions, since, by analogy with DM, one or more (potential) protein mediators should be present within the inclusions themselves.

An analysis of the protein composition of the inclusions in FXTAS, through a combination of fluorescence-based particle sorting of inclusions from postmortem tissue and mass spectroscopic and immunochemical approaches for protein identification, has revealed more than 20 protein species to date (Iwahashi et al. 2006). At least one of the identified proteins, hnRNP A2, is a well-known RNA binding protein (Dreyfuss et al. 2002) that could serve as a mediator of the expanded-repeat FMR1 mRNA in FXTAS. MBNL1 was also identified within the inclusions; however, the roles played by these two proteins await further investigation. Iwahashi et al. (2006) did not observe a dominant protein species within the inclusions, which argues against the simple accretion of specific, abnormal proteins that is thought to occur with many other inclusion disorders (Paulson 1999; Zoghbi and Orr 2000; Tarlac and Storey 2003; Taylor et al. 2002; Ross and Poirier 2004). No FMRP was detected within the inclusions. Interestingly, the inclusions do appear to contain several intermediate filament proteins, including lamin A/C. Although the lamin A/C isoforms are not believed to interact directly with FMR1 mRNA, they are believed to be involved with the regulation of RNA synthesis and processing (Hutchison and Worman 2004; Zastrow et al. 2004). Thus, the lamins could also be involved in mediating the effects of the expanded-repeat FMR1 mRNA (Arocena et al. 2006).

Acknowledgements This work was supported by the National Institutes of Child Health and Development, grant HD40661 (P.J.H.), by the National Institutes of Neurological Diseases and Stroke. grant NS43532 (P.J.H.), and by a National Fragile X Foundation Research Grant (F.T.).

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