Abstract
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder associated with a premutation repeat expansion (55–200 CGG repeats) in the 5′ noncoding region of the FMR1 gene. Solitary intranuclear inclusions within FXTAS neurons and astrocytes constitute a hallmark of the disorder, yet our understanding of how and why these bodies form is limited. Here, we have discovered that FXTAS inclusions emit a distinct autofluorescence spectrum, which forms the basis of a novel, unbiased method for isolating FXTAS inclusions by preparative fluorescence-activated cell sorting (FACS). Using a combination of autofluorescence-based FACS and liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based proteomics, we have identified more than two hundred proteins that are enriched within the inclusions relative to FXTAS whole nuclei. Whereas no single protein species dominates inclusion composition, highly enriched levels of conjugated small ubiquitin-related modifier 2 (SUMO 2) protein and p62/sequestosome-1 (p62/SQSTM1) protein were found within the inclusions. Many additional proteins involved with RNA binding, protein turnover, and DNA damage repair were enriched within inclusions relative to total nuclear protein. The current analysis has also allowed the first direct detection, through peptide sequencing, of endogenous FMRpolyG peptide, the product of repeat-associated non-ATG (RAN) translation of the FMR1 mRNA. However, this peptide was found only at extremely low levels and not within whole FXTAS nuclear preparations, raising the question whether endogenous RAN products exist at quantities sufficient to contribute to FXTAS pathogenesis. The abundance of the inclusion-associated ubiquitin- and SUMO-based modifiers supports a model for inclusion formation as the result of increased protein loads and elevated oxidative stress leading to maladaptive autophagy. These results highlight the need to further investigate FXTAS pathogenesis in the context of endogenous systems.
Highlights
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive X-linked neurodegenerative disorder that arises from premutation CGG-repeat expansions (55200 repeats) in the 5′ noncoding portion of the FMR1 gene [42]
Inclusion autofluorescence has previously been reported in neuronal intranuclear inclusion disease (NIID) [70, 110], but has not been reported previously for FXTAS inclusions
Stimulated emission depletion (STED) microscopy on a Leica SP8 STED 3x was used to measure the excitation/emission spectra of FXTAS inclusions, and the results corroborated what was seen on visual examination (Additional file 1: Figure S1a), with maximum emission intensity at 550 nm by STED
Summary
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive X-linked neurodegenerative disorder that arises from premutation CGG-repeat expansions (55200 repeats) in the 5′ noncoding portion of the FMR1 gene [42]. The mechanism(s) governing inclusion formation, and the nature of their composition, remain largely unknown; a better understanding of the properties of inclusions is likely to be key in understanding FXTAS pathogenesis. FXTAS is largely limited to the premutation range, where there is normal to increased transcription of the expanded CGG-repeat mRNA [42, 68, 134]. The absence of the neurodegenerative phenotype for alleles in the full mutation range (>200 CGG repeats), with rare exceptions among mosaics [55, 82, 114], is thought to be due to methylationcoupled transcriptional silencing of the FMR1 gene. The requirement for transcriptional activity supports an RNA gain-of-function toxicity model [43, 78], as described earlier for myotonic dystrophy (DM) [25, 30, 89, 136]
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