Protein composition of the intranuclear inclusions of FXTAS

TLDR

FXTAS is a late‑onset neurodegenerative disorder caused by premutation CGG repeats in FMR1, characterized by ubiquitin‑positive intranuclear inclusions and likely driven by RNA toxicity, yet the proteins mediating this response remain largely unknown. The study aimed to define the protein complement of the intranuclear inclusions to identify potential mediators. Inclusion bodies were purified from post‑mortem FXTAS brain tissue using fluorescence‑activated flow‑based methods, followed by mass spectrometry and immunohistochemistry to profile their protein content. Analysis identified more than 20 inclusion‑associated proteins—including neurofilaments, lamin A/C, and the RNA‑binding proteins hnRNP A2 and MBL1—while ubiquitinated proteins were minor, indicating inclusion formation is not due to proteasomal degradation.

Abstract

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by premutation expansions (55–200 CGG repeats) in the fragile X mental retardation 1 (FMR1) gene. The pathologic hallmark of FXTAS is the ubiquitin-positive intranuclear inclusion found in neurons and astrocytes in broad distribution throughout the brain. The pathogenesis of FXTAS is likely to involve an RNA toxic gain-of-function mechanism, and the FMR1 mRNA has recently been identified within the inclusions. However, little is known about the proteins that mediate the abnormal cellular response to the expanded CGG repeat allele. As one approach to identify the protein mediators, we have endeavoured to define the protein complement of the inclusion itself. Fluorescence-activated flow-based methods have been developed for the efficient purification of inclusions from the post-mortem brain tissue of FXTAS patients. Mass spectrometric analysis of the entire protein complement of the isolated inclusions, combined with immunohistochemical analysis of both isolated nuclei and tissue sections, has been used to identify inclusion-associated proteins. More than 20 inclusion-associated proteins have been identified on the basis of combined immunohistochemical and mass spectrometric analysis, including a number of neurofilaments and lamin A/C. There is no dominant protein species in the inclusions, and ubiquitinated proteins represent only a minor component; thus, inclusion formation is not likely to reflect a breakdown in proteasomal degradation of nuclear proteins. The list of proteins includes at least two RNA binding proteins, heterogeneous nuclear ribonucleoprotein A2 and muscle blind-like protein 1, which are possible mediators of the RNA gain-of-function in FXTAS.