Induction of inclusion formation and disruption of lamin A/C structure by premutation CGG-repeat RNA in human cultured neural cells

Abstract

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.

Expanded clinical phenotype of women with the FMR1 premutation

“…More than 20 proteins have been identified within the inclusions, including the small stress-response protein, aB-crystallin, the nuclear intermediate filament protein, lamin A/C, the RNA binding protein, hnRNP A2, and myelin basic protein Iwahashi et al, 2006]. Dysregulation of at least two of these proteins, aB-crystallin and lamin A/C, is clearly related to disease pathogenesis [Arocena et al, 2005].…”

ElevatedFMR1mRNA in premutation carriers is due to increased transcription

“…Our model is analogous to the RNA gain-offunction model proposed for myotonic dystrophy, where CUG or CCUG repeat expansions (DMPK or ZNF9 genes, respectively), sequestered in the nuclei of affected cells, are believed to sequester one or more proteins to the expanded RNA repeat element (Finsterer 2002;Mankodi and Thornton 2002;Kanadia et al 2003;Ho et al 2004;Ranum and Day 2004). More recent studies of the expanded repeats in Drosophila (Jin et al 2003), identification of the FMR1 mRNA in the intranuclear inclusions of FXTAS patients (Tassone et al 2004b), and the recapitulation of CGG repeat-induced neural cell toxicity and inclusion formation in neural cell culture (Arocena et al 2005) have provided additional support for an RNA-based pathogenesis of FXTAS .…”

The primary cognitive deficit among males with fragile X-associated tremor/ataxia syndrome (FXTAS) is a dysexecutive syndrome

“…Intranuclear eosinophilic astrocytic and neuronal inclusion bodies are found throughout the cortex and in many subcortical areas, and are especially prevalent in the hippocampus (Arocena et al, 2005;Greco et al, 2002;Greco et al, 2006;Iwahashi et al, 2006;Louis, Moskowitz, Friez, Amaya, & Vonsattel, 2006). Inclusions are rare in the cerebellum, which nevertheless shows considerable atrophy associated with large-scale dropout of Purkinje cells.…”