Astroglial-targeted expression of the fragile X CGG repeat premutation in mice yields RAN translation, motor deficits and possible evidence for cell-to-cell propagation of FXTAS pathology

Abstract

The fragile X premutation is a CGG trinucleotide repeat expansion between 55 and 200 repeats in the 5′-untranslated region of the fragile X mental retardation 1 ( FMR1 ) gene. Human carriers of the premutation allele are at risk of developing the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Characteristic neuropathology associated with FXTAS includes intranuclear inclusions in neurons and astroglia. Previous studies recapitulated these histopathological features in neurons in a knock-in mouse model, but without significant astroglial pathology. To determine the role of astroglia in FXTAS, we generated a transgenic mouse line (Gfa2-CGG99-eGFP) that selectively expresses a 99-CGG repeat expansion linked to an enhanced green fluorescent protein (eGFP) reporter in astroglia throughout the brain, including cerebellar Bergmann glia. Behaviorally these mice displayed impaired motor performance on the ladder-rung test, but paradoxically better performance on the rotarod. Immunocytochemical analysis revealed that CGG99-eGFP co-localized with GFAP and S-100ß, but not with NeuN, Iba1, or MBP, indicating that CGG99-eGFP expression is specific to astroglia. Ubiquitin-positive intranuclear inclusions were found in eGFP-expressing glia throughout the brain. In addition, intracytoplasmic ubiquitin-positive inclusions were found outside the nucleus in distal astrocyte processes. Intriguingly, intranuclear inclusions, in the absence of eGFP mRNA and eGFP fluorescence, were present in neurons of the hypothalamus and neocortex. Furthermore, intranuclear inclusions in both neurons and astrocytes displayed immunofluorescent labeling for the polyglycine peptide FMRpolyG, implicating FMRpolyG in the pathology found in Gfa2-CGG99 mice. Considered together, these results show that Gfa2-CGG99 expression in mice is sufficient to induce key features of FXTAS pathology, including formation of intranuclear inclusions, translation of FMRpolyG, and deficits in motor function. Electronic supplementary material The online version of this article (10.1186/s40478-019-0677-7) contains supplementary material, which is available to authorized users.

In silico, in vitro, and in vivo Approaches to Identify Molecular Players in Fragile X Tremor and Ataxia Syndrome

“…Although astrocytes with inclusion bodies were low in number, they were widely distributed in the brain such as in the neocortex, cerebellum and occasionally in subcortical regions in the hypothalamus and some brain stem nuclei. These mice had no sign of Purkinje neuronal dropout as well as no ubiquitinpositive inclusions in Purkinje cells could be found (Sellier et al, 2017;Wenzel et al, 2019). This study was the first of its kind to provide evidence of FXTAS related RAN-translation products being present in mouse astroglia.…”

“…Therefore an astroglialspecific mouse model was necessary. This transgenic mouse line with a C57BL/6j background was generated through pronuclear injection using a astrocyte-specific Gfa2 promoter to induce expression of an expanded 99CGG repeat fused to an eGFP marker gene only in astrocytes (Figure 7) (Wenzel et al, 2019). Immunocytochemical analysis of eGFP expression patterns show expression of 99CGG RNA was restricted to astroglia and Bergmann glia only and was not present in neurons, microglia or oligodendrocytes.…”

Drosophila Glia: Models for Human Neurodevelopmental and Neurodegenerative Disorders

“…Although a FXTAS Drosophila model expressing a human FMR1 premutation allele produces neuron-specific degeneration as well as inclusion bodies similar to those seen in FXTAS patients, the effects of its glial expression have not been reported. It was recently shown that astroglial-targeted expression of the FMRpolyG repeat expansion in mice induces key features of FXTAS pathology, including the formation of intranuclear inclusions, RAN translation, and deficits in motor function [323]. Therefore, an investigation into the response of Drosophila glial subtypes to the ectopic expression of human FMR1 premutation alleles seems intriguing.…”

Psychosis and Catatonia in Fragile X Syndrome

“…The syndrome has an X-linked dominant inheritance pattern and results from a full mutation (>200 CGC repeats) in the fragile X mental retardation 1 (FMR1) gene located at Xq27.3.2 The FMR1 gene encodes a protein named the fragile X mental retardation protein (FMRP), which is essential for normal brain development. Healthy individuals have 6-54 repeats of the CGG sequence in the 5’ untranslated region of the FMR1 gene [ 4 ]. When an individual carries an intermediate number of CGG repeats (55-200) excess FMR1 mRNA is produced and this is considered a pre-mutation allele.…”