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Title: Nuclear Accumulation of Stress Response mRNAs Contributes to the Neurodegeneration Caused by Fragile X Premutation rCGG Repeats}
Article:
Abstract:
Fragile X–associated tremor/ataxia syndrome( FXTAS) is a neurodegenerative disorder seen in Fragile X premutation carriers. Previous studies found that Fragile X rCGG repeats are sufficient to cause neurodegeneration and that the rCGG repeat-binding proteins Pur α and hnRNP A2/B1 can modulate rCGG–mediated neuronal toxicity. To explore the role of Pur α in rCGG–mediated neurodegeneration further, we took a proteomic approach and identified more than 100 proteins that interact with Pur α. Of particular interest is Rm62, the Drosophila ortholog of p68 RNA helicase, which could modulate rCGG–mediated neurodegeneration. Here we show that rCGG repeats decreased the expression of Rm62 posttranscriptionally, leading to the nuclear accumulation of Hsp70 transcript, as well as additional mRNAs involved in stress and immune responses. Together these findings suggest that abnormal nuclear accumulation of these mRNAs, likely as a result of impaired nuclear export, could contribute to FXTAS pathogenesis.
Introduction:
Fragile X syndrome( FXS) is usually caused by expansion of the CGG trinucleotide repeat in the 5′ untranslated region( 5′ UTR) of the Fragile X Mental Retardation 1( FMR1) gene[1]. Whereas normal individuals generally possess between five and 54 repeats, fully affected individuals have more than 200 CGG repeats on what are referred to as full mutation alleles[2]. Premutation alleles( 55–200 CGG repeats) of the FMR1 gene are known to contribute to the Fragile X phenotype through genetic instability, and they can expand into the full mutation during germline transmission[3]. Within the last decade, Fragile X-associated Tremor/Ataxia Syndrome( FXTAS), a late-onset neurodegenerative disorder, has been recognized mainly among many male premutation carriers in or beyond their fifth decade of life[4], and FXTAS is distinct from the neurodevelopmental disorder, FXS. The most common clinical feature of FXTAS is a progressive action tremor with ataxia. More advanced or severe cases may show a progressive cognitive decline that ranges from executive and memory deficits to dementia[5]. Patients may also present with common psychiatric symptoms, such as increased anxiety, mood liability, and depression[6],[7]. Magnetic resonance imaging( MRI) of adult male patients affected with FXTAS demonstrated mild to moderate global brain atrophy, most common in the fontal and parietal regions, as well as the pons and the cerebellum[8]. Nearly all autopsy studies on the brains of symptomatic premutation carriers show degeneration in the cerebellum, which includes Purkinje neuronal cell loss, Bergman gliosis, spongiosis of the deep cerebellar white matter, and swollen axons[9],[10]. The major neuropathological hallmark and postmortem criterion for definitive FXTAS is eosinophilic, ubiquitin-positive intranuclear inclusions broadly distributed throughout the brain in neurons, astrocytes, and in the spinal column[9]. For the past several years, the hunt has been on to uncover the molecular basis of FXTAS. One unique molecular signature of the fragile X premutation allele is that the level of FMR1 mRNA is significantly elevated, while the FMR1 protein( FMRP) remains relatively unchanged in cells from premutation carriers[11],[12]. Given that the neurodegenerative phenotype of FXTAS is associated specifically with premutation carriers, but not with the full mutation, FMRP deficiency per se is likely not the culprit behind FXTAS[4]. Instead, the neurodegenerative phenotypes associated with FXTAS are suspected of being caused by a gain of function in fragile X premutation rCGG repeat RNAs[3],[13]. The hypothesis is that overproduced rCGG repeats in FXTAS sequester specific RNA-binding proteins and reduce their ability to perform their normal cellular functions, thereby contributing significantly to the pathology of this disorder. The presence of FMR1 mRNA in inclusions found in the brains of FXTAS patients, as well as the formation of similar inclusions upon ectopic expression of rCGG repeats in model systems, have provided strong support for this hypothesis[13]–[16]. Two RNA-binding proteins, Pur α and hnRNP A2/B1, are known to bind rCGG repeats specifically in both mammalian and Drosophila brains[17],[18]. Both Pur α and hnRNP A2/B1 are found to be present in the inclusions of FXTAS brain tissues. Furthermore, overexpression of either Pur α or hnRNP A2/B1 can alleviate neurodegeneration in the fly model of FXTAS[17],[18]. In particular, Pur α knock-out mice appeared normal at birth, but developed severe tremor and spontaneous seizures at two weeks of age due to drastically reduced numbers of neurons in regions of the hippocampus and cerebellum, suggesting that the depletion of Pur α alone could lead to ataxia[19]. In this study, to further investigate the role of Pur α in rCGG-mediated neurodegeneration, we took a proteomic approach to identify the proteins that interact with Pur α. Over 100 proteins, including several known interactors, such as Fmrp, were found to interact with Pur α in vitro. To evaluate the physiological role( s) of Pur α-interacting proteins in rCGG-mediated neuronal toxicity, we further tested their genetic interactions with rCGG repeats using our FXTAS fly model and identified several interactors of Pur α that could genetically modulate the toxicity caused by rCGG repeats. Among these, Rm62, the Drosophila ortholog of the p68 RNA helicase, was of particular interest. Rm62, physically interacting with Pur α, could modulate rCGG-mediated neurodegeneration, and biochemically, fragile X rCGG repeats could decrease the expression of Rm62 posttranscriptionally, leading to the accumulation of Hsp70 transcript, a previously identified target of Rm62, in the nucleus. Further microarray analyses revealed the nuclear accumulation of additional mRNAs involved in stress and immune responses in fragile X premutation flies. These findings suggest an unexpected nuclear accumulation of specific mRNAs caused by fragile X premutation rCGG repeats and point to likely deficits in the nuclear export of specific mRNA as a possible cause of the compromised stress response in neurons expressing rCGG repeats, which would predispose them to neuronal apoptosis.
Discussion:
Fragile X-associated tremor/ataxia syndrome is a neurodegenerative disorder seen in adult fragile X syndrome premutation carriers. Although several studies, including our own, have proposed a model of RNA-mediated sequestration, in which the sequestration of several RNA-binding proteins( RBP) by rCGG repeat-binding causes cell death and neurodegeneration, the precise mechanisms by which expanded rCGG repeats exert their toxic effects are less understood. Here we report an unexpected nuclear accumulation of specific mRNAs caused by fragile X premutation rCGG repeats, which indicates that a potential deficit in the nuclear export of these mRNAs could contribute to the pathogenesis of FXTAS. Our earlier studies demonstrated that Pur α and hnRNP A2/B1 are rCGG-repeat-binding proteins and that they could modulate rCGG-mediated neuronal toxicity[17],[18]. In particular, the loss of Pur α in mice is known to cause neurological phenotypes reminiscent of those observed in FXTAS patients[19]. Therefore, to better understand the set of molecular interactions that are dysregulated as a consequence of the sequestration of rCGG repeat-binding proteins( RBPs), we used proteomic analyses to identify the Pur α interactome, which includes proteins involved in different biological pathways. However, whether these interacting proteins could be involved in rCGG repeat-mediated neuronal toxicity was unclear. To address this question, we further tested the roles of Pur α-interacting proteins with the existing fly mutants in rCGG-mediated neurodegeneration. Interestingly, two proteins, Rm62 and Hts, the Drosophila orthologs of the p68 RNA helicase and Adducin, respectively, were found to prominently modulate rCGG-mediated neuronal toxicity. These findings suggest that, despite Pur α being involved in multiple biological pathways, only selective pathway( s) altered by fragile X premutation rCGG repeats and the sequestration of Pur α are involved in rCGG-mediated neurodegeneration. Our methods demonstrate the utility and power of combining a proteomic approach with genetic-interaction tests to identify key biological pathway( s) relevant to human diseases. Rm62/Dmp68 is a member of the DEAD-box family, which includes a large number of proteins that play important roles in almost all aspects of RNA metabolism[28],[29]. Specifically, Rm62/Dmp68 is found to be a multifunctional protein involved in gene transcription, pre-mRNA, pre-rRNA, and pre-miRNA processing, RNAi, and RNA export[21]-[23],[25],[26],[30],[31]. The belief now is that Rm62/Dmp68 may be associated with mRNP export, promoting gene silencing by removing transcripts from their active site of synthesis to promote an inactive heterochromatin configuration[31]. The mammalian ortholog of Rm62/Dmp68 is found to shuttle between cytoplasm and the nucleus[30]. In flies, disruption of its action alters the nuclear distribution of known RNA export components, producing defects similar to those seen in sbr mutants, a known component of the RNA export machinery[31]. Given the importance of Rm62/Dmp68 as part of an mRNP complex involved in mRNA transport, it is likely that its depletion or sequestration caused by fragile X premutation rCGG repeats could lead to dysfunction of mRNA transport in the cell and eventual cell death and neuronal toxicity. In line with our observation, other studies have shown that premutation rCGG repeats can biochemically associate with several other DEAD-box RNA helicases, like DDX3, DDX5/p68, and DDX17, as well as colocalize with CGG60 RNA aggregates, resulting in the formation of inclusions in cells[40]; this suggests sequestration as a potential mechanism for the Rm62 posttranscriptional reduction. In our FXTAS fly model, total steady-state levels of Hsp70 transcripts are increased, which could reflect an initial cellular response to the stress caused by fragile X premutation rCGG repeats. However, we found that most of the increased Hsp70 mRNAs are trapped in the nucleus, instead of being transported out of the nucleus to produce Hsp70 protein and respond to cell stress. Interestingly, this altered distribution of Hsp70 transcripts and their delay in turnover is similar to what was seen with Hsp70 transcripts in the Rm62/p68 mutation. Therefore, fragile X premutation rCGG repeats could likely produce deficits in the nuclear export of selective transcripts mediated by Rm62, such as Hsp70 mRNA. This nuclear accumulation will interfere with the neurons' ability to produce sufficient proteins to respond to cellular stress( Figure 5C). To identify additional accumulated mRNAs in the nucleus of neurons expressing fragile X premutation rCGG repeats, we performed gene expression profiling using total RNAs and RNAs from both nuclear and cytoplasmic fractions. Through these analyses, we identified transcripts that were uniquely enriched/restricted in the nuclear fraction. The gene functions of these nuclear-accumulated transcripts cover a variety of biological processes. For example, gene functions like oxidoreductases indicate the involvement of oxidative stress. Likewise, chaperones indicate maintenance of the conformational homeostasis of cellular proteins and RNAs. The cytokine-like interleukin-6( IL-6) and immune-induced molecules( IM2) regulate part of the immune system. Discovering that these nuclear-enriched transcripts fell into stress, apoptotic, and immunity/defense pathways rather than other biological processes implies that they are either a causative event, or are a consequence of FXTAS pathogenesis. There is a growing body of evidence from both animal and human studies to suggest that stress can intensify inflammation and increase a person's risk for developing various neurological disorders, like Parkinson's disease, multiple sclerosis( MS), and other inflammatory diseases[41],[42]. These studies, along with our findings presented here, suggest that in the presence of toxic RNAs, such as fragile X premutation rCGG repeats, a continuous build up of stress might allow the inflammatory process to run amok and may compromise homeostasis integrity, triggering neuronal cell death/neurodegeneration. Significantly with regard to the data provided above, overexpression of Hsp70 suppresses toxicity of premutation rCGG repeats( Figure 5B). This paradox could be explained by that the nucleus becomes saturated due to induced Hsp70 transcripts that are forced out of the nucleus to respond to cell stress. While our study provides insights into potential roles of stress response pathways in FXTAS neurodegeneration, in the future, a detailed analysis of accumulated mRNAs will be important to understand their implications in the pathogenesis of FXTAS. In summary, through systematic proteomic, genetic, and microarray analyses, here we show that the nuclear accumulation of select mRNAs caused by fragile X premutation rCGG repeats may contribute to FXTAS pathogenesis, and the mechanism could be via impaired nuclear export due to the decreased levels of Rm62 seen upon fragile X premutation rCGG expression.
Fragile X–associated tremor/ataxia syndrome ( FXTAS ) is an adult-onset neurodegenerative disorder that usually affects males over 50 years of age , and FXTAS patients are carriers of fragile X premutation alleles . Using a FXTAS Drosophila model , we previously showed that fragile X premutation rCGG repeats alone could cause neurodegeneration . Pur α and hnRNP A2/B1 were identified as specific premutation rCGG repeat-binding proteins ( RBPs ) that could bind and modulate fragile X premutation rCGG–mediated neuronal degeneration . Here , through systematic proteomic , genetic , and microarray analyses , we show that the nuclear accumulation of select mRNAs caused by fragile X premutation rCGG repeats may contribute to FXTAS pathogenesis , and the mechanism could be via impaired nuclear export due to the decreased levels of Rm62 seen upon fragile X premutation rCGG expression .