Elsevier

Brain Research

Volume 1318, 8 March 2010, Pages 155-166
Brain Research

Research Report
Ubiquitin-positive intranuclear inclusions in neuronal and glial cells in a mouse model of the fragile X premutation

https://doi.org/10.1016/j.brainres.2009.12.077Get rights and content

Abstract

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder caused by CGG trinucleotide repeat expansions in the fragile X mental retardation 1 (FMR1) gene. The neuropathological hallmark of the disease is the presence of ubiquitin-positive intranuclear inclusions in neurons and in astrocytes. Ubiquitin-positive intranuclear inclusions have also been found in the neurons of transgenic mice model carrying an expanded CGG(98) trinucleotide repeat of human origin but have not previously been described in glial cells. Therefore, we used immunocytochemical methods to determine the pathological features of nuclear and/or cytoplasmic inclusions in astrocytes, Bergmann glia, and neurons, as well as relationships between inclusion patterns, age, and repeat length in CGG knock-in (KI) mice in comparison with wild-type mice. In CGG KI mice, ubiquitin-positive intranuclear inclusions were found in neurons (e.g., pyramidal cells, GABAergic neurons) throughout the brain in cortical and subcortical brain regions; these inclusions increased in number and size with advanced age. Ubiquitin-positive intranuclear inclusions were also present in protoplasmic astrocytes, including Bergmann glia in the cerebellum. The morphology of intranuclear inclusions in CGG KI mice was compared to that of typical inclusions in human neurons and astrocytes in postmortem FXTAS brain tissue. This new finding of previously unreported pathology in astrocytes of CGG KI mice now provides an important mouse model to study astrocyte pathology in human FXTAS.

Introduction

The fragile X mental retardation gene (FMR1) is polymorphic for the length of CGG tandem trinucleotide repeat in the 5′-untranslated region (5′-UTR). Repeat lengths in the general population range from 5 to 55, while full mutations with repeat lengths above 200 result in hypermethylation of FMR1, transcriptional silencing, and the clinical syndrome fragile X mental retardation (FXS). Intermediate length CGG expansions between 55 and 200, referred to as the fragile X premutation, have now been identified in patient populations and are associated with a unique, progressive, late onset, neurodegenerative disorder called fragile X-associated tremor/ataxia syndrome (FXTAS). The neurological symptoms of FXTAS include intention tremor and ataxia, peripheral neuropathy, neuropsychological problems (anxiety, depression), and cognitive impairments including dementia at late stages of the disorder (Berry-Kravis et al., 2007, Hagerman and Hagerman, 2004, Hagerman et al., 2007). Radiologic changes seen on MRI include patchy T2 signal hyperintensities in cerebral white matter and in the middle cerebellar peduncle (“the MCP sign”), and overall brain atrophy (Brunberg et al., 2002). The neuropathological hallmark of FXTAS is the presence of eosinophilic intranuclear inclusions in neurons and astrocytes throughout the brain that stain positive for ubiquitin (Greco et al., 2006, Greco et al., 2002, Tassone et al., 2004). In contrast to FXS where gene silencing occurs, FXTAS is associated with increased transcription resulting in 3- to 8-fold elevations in levels of FMR1 mRNA in leukocytes. However, translation of the FMR1 mRNA with expanded CGG repeats in the premutation range is inefficient so that levels of the gene product, fragile X mental retardation protein (FMRP) are paradoxically low (Tassone et al., 2000). The disorder appears to be due to an “RNA toxic gain of function”, although the mechanisms for disease progression, including formation of intranuclear ubiquitin-positive inclusions in neurons and astrocytes are not well understood (Oostra and Willemsen, 2009).

Human carriers with the fragile X premutation underlying FXTAS have intranuclear inclusions in neurons and astrocytes throughout the brain. In humans, it is still unknown at what age inclusions form due to the progressive nature of the disorder and the advanced age at which FXTAS is typically diagnosed, as well as that the tissue diagnosis is made at autopsy. It has been shown that inclusions can form after as few as 8 days in vitro after an expanded CGG repeat in the premutation range is introduced into human primary neural progenitor cells and into established cell lines (Arocena et al., 2005). In order to better characterize FXTAS and to study its molecular mechanisms, knock-in (KI) mice bearing expanded CGG trinucleotide repeats in the 5′-UTR have been created. The CGG KI mouse model of the fragile X premutation was generated by replacing the endogenous CGG8 trinucleotide repeat with a CGG98 trinucleotide repeat of human origin via homologous recombination (Bontekoe et al., 1997, Willemsen et al., 2003). Similar to the human cases of FXTAS, neurons of these CGG KI mice show intranuclear inclusions that stain for ubiquitin in neurons in a number of brain regions (Bontekoe et al., 1997, Brouwer et al., 2008, Willemsen et al., 2003). It is not yet known if the inclusions contribute directly to the neuropathology seen in FXTAS. It has been suggested that intranuclear inclusions may not be pathological of themselves but may reflect pathology such as mRNA toxicity due to the increased gene transcription resulting from the premutation or perhaps due to the presence of the mutant FMR1 mRNA itself (Brouwer et al., 2008, Oostra and Willemsen, 2009, Willemsen et al., 2003).

Intranuclear inclusions in these CGG KI mice are common in neurons at 50–100 weeks of age and have been observed as early as 20 weeks of age (Bontekoe et al., 1997, Brouwer et al., 2008, Willemsen et al., 2003). However, unlike FXTAS, intranuclear inclusions have not been previously reported in astrocytes in the CGG KI mice. The present study replicates the previous findings of intranuclear inclusions in neurons in the CGG KI mouse and expands upon these findings by describing the presence and distribution of such inclusions in glial cells (i.e., astrocytes).

Section snippets

Nuclear pathology in neurons of CGG KI mice and FXTAS patients

The presence of intranuclear inclusions in neurons and astrocytes is a pathological hallmark of FXTAS. Thus far, similar intranuclear inclusions have not been reported in oligodendrocytes or microglia in human FXTAS or CGG KI mice. Figs. 1A and B shows typical intranuclear inclusions in neurons and astrocytes in the hippocampus of a patient who was diagnosed with FXTAS at 65 years of age and later died at 78 (case 2 from Greco et al., 2002). These inclusions in a human patient are shown for

Discussion

Intranuclear inclusions in neurons and astrocytes that stain for ubiquitin are the neuropathological hallmark of FXTAS. Similar appearing ubiquitin-positive inclusions are also found in neurons in the brains of CGG KI mice, but previous studies have not demonstrated such inclusions in mouse astrocytes. In this study, we extend the description of the cellular and brain regional distribution and appearance of intranuclear inclusions in a CGG KI mouse model of FXTAS to include astrocytes. New

Animals

The generation of an expanded CGG trinucleotide repeat knock-in (CGG KI) mouse model of the fragile X premutation has been described previously in detail (Bontekoe et al., 1997, Willemsen et al., 2003). Briefly, the native CGG8 trinucleotide repeat of human origin in the 5′-UTR of the mouse Fmr1 gene was replaced by a human CGG98 trinucleotide repeat via homologous recombination. Across breedings, the CGG repeats showed mild instability, both expanding and contracting in length within the

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgments

We are grateful to Dr. Mareike Wenzel and Lee Rognlie for their excellent technical support. This research was supported by grants NINDSRL1 NS 062411, NIH Roadmap Consortium NIDCR UL1 DE19583, and NIAAG032119.

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