By Candice Vieira, Biochemistry and Molecular Biology, ’17

Author’s Note:

After researching Fragile X-associated tremor/ataxia syndrome (FXTAS) treatment methods for a UWP 104F assignment, I learned that current FXTAS therapeutics is limited to symptomatic treatment. Most articles emphasized the need to better characterize the molecular mechanisms underlying FXTAS development to develop drugs specifically for FXTAS. Therefore, I questioned what researchers currently know regarding molecular events that lead to FXTAS signs and symptoms and how this knowledge can aid in drug therapies. This motivated me to prepare a literary review, intended to educate and inform practicing clinicians, especially neurologists and psychologists, about recent findings and the future directions for FXTAS research. For this assignment, we were expected to synthesize recent articles and provide relevant information for clinical practice. Specifically, I wanted clinicians to gain a better understanding of a primary focus within FXTAS research—molecular triggering events—and importantly, how this research relates to clinical treatment of FXTAS.

I. Introduction

Discovered in 2001, Fragile X-associated tremor/ataxia syndrome (FXTAS) was identified as a late-onset, progressive neurodegenerative disorder, typically affecting males over the age of 55 (1). FXTAS is linked to a dominant mutation in the FMR1 gene, located on the X chromosome (1). This leads to higher disease penetrance in males—approximately 45% in males compared to 16% in females. In the United States, more than 1 million individuals are impacted by FXTAS (2). Core clinical features of FXTAS include intention tremor, gait ataxia, cognitive decline and dementia, and these symptoms progressively become more debilitating throughout time. Unfortunately, a cure still remains unknown, and thus, advanced research continues, emphasizing the molecular processes that underlie FXTAS to pioneer drug development. Ideally, once molecular events are better characterized, therapies can be developed to specifically target those processes.

At the molecular level, FXTAS affects carriers of a small mutation, also called a premutation, in the FMR1 gene. Normally, individuals have less than 55 CGG repeats in the 5’ untranslated region (UTR) of the FMR1 gene, but premutation carriers have between 55-200 CGG repeats (1). This repeat expansion leads to increased levels of FMR1 mRNA, yet decreased levels of Fragile X mental retardation protein (FMRP) (3). Originally, studies suggested that the high levels of mRNA caused the symptoms of FXTAS. However, recently, a competing hypothesis has been proposed, involving a toxic protein produced by repeat-associated non-AUG translation (RAN). Therefore, this review will focus on describing these two leading toxic processes—RNA-mediated toxicity and RAN protein toxicity—to better characterize FXTAS pathogenesis and, ultimately, address therapeutic implications. This article will first discuss the molecular events involved in the disease, including the original hypothesis—RNA-mediated toxicity— and the recent hypothesis—RAN protein toxicity. Lastly, this review will examine therapeutic perspectives given our knowledge about these molecular processes.

II. Molecular Triggering Events

To advance therapeutics, FXTAS research capitalizes on understanding the underlying molecular processes that lead to FXTAS pathogenesis. Thus, this section discusses the two leading proposed molecular mechanisms that may lead to FXTAS signs and symptoms.

RNA-mediated toxicity

The earlier hypothesis investigates the unique molecular pathology of FXTAS. FXTAS patients and premutation carriers exhibit elevated FMR1 mRNA levels by as much as 10-fold, suggesting that regular transcriptional control is reduced (4-5). Importantly, the expanded CGG repeat in the mRNA transcripts excessively binds one or more proteins (6). However, by sequestering proteins, the proteins’ normal cellular function is blocked and prevented, leading to functional deficiencies within the cell (6). These proteins include DNA repair proteins, transcriptional control proteins, and proteins involved in processing microRNAs (miRNA) (6).  Several of these “RNA binding” proteins bound to FMR1 mRNA have been identified in the neuropathological hallmark of FXTAS—ubiquitin-positive intranuclear inclusions. Previous studies demonstrate that these inclusions may be positively correlated with clinical FXTAS symptoms (7). Therefore, identifying colocalized sequestered proteins attached to FMR1 mRNA with ubiquitin-positive intranuclear inclusions further validates and strengthens this model (5-6). Scientists first proposed that one or more of the aforementioned “RNA binding” proteins is/are acting to mediate the effect of abnormal mRNA levels, which cause FXTAS development. Sequestering these specific proteins leads to toxic RNA that may underlie the neurological impairment, cell loss, and brain atrophy in FXTAS patients (6-7).

RAN protein toxicity

Recently, a competing hypothesis has emerged, involving unconventional translation, referred to as RAN translation. In this second post-transcriptional mechanism,  the expanded CGG repeat is included in the peptide product, either FMRpolyG or FMRpolyA, and results in either a polyglycine or a polyalanine stretch, respectively. This occurs because translation of the FMR1 mRNA begins at a point upstream of the expanded CGG repeat instead of at the expected AUG start codon (7). Typically, standard translation machinery would bypass the CGG repeat region, as it is located in the 5’ UTR (1). However, RAN translation shifts the frame of the normal coding sequence. Interestingly, two possible shifts can occur during RAN translation and can recognize the expanded repeat sequence as either GCC, coding for glycine, or GCG, coding for alanine (7). Although both shifts occur, the shift resulting in a polyglycine-containing peptide, FMRpolyG, occurs at higher levels and has been found in intranuclear inclusions (7-8). Recent studies have demonstrated that the peptide product, FMRpolyG, is toxic to cells, causing proteins to misfold and aggregate (7-8). FMRpolyA products are also toxic, but the focus of research remains on FMRpolyG due its higher prevalence and co-localization with inclusions. Thus, it is more likely to participate in FXTAS pathogenesis. More importantly, however, the overall roles of RAN translation and these unexpected toxic peptides need to be further unraveled, as this research focusing on RAN translation is in its infancy. RAN translation adds another potential toxic mechanism, explaining how the expanded CGG repeat might manifest into the FXTAS clinical phenotype.

III.         Prospects for Drug Development

Though there is no cure for FXTAS, further elucidation of the RNA- and protein-mediated toxicity mechanisms have opened new perspectives for drug development. Therefore, this section will examine different therapeutic directions and targets, considering the information gleaned through FXTAS research at the molecular level as described above.

Gene-targeted Therapy

Because FXTAS is a genetic disorder, drug therapies ideally target genetic components contributing to the development of FXTAS, as employed through genome editing and gene silencing approaches. Genome editing explores the possibility of removing the expanded CGG trinulecotide repeat, which would address the core cause of FXTAS pathology. Essentially, it would correct the FMR1 gene. However, this is still a futuristic approach because currently, there is no technology available to accomplish this in non-dividing cells, like neurons (9).

Contrarily, gene silencing techniques are available for patients participating in a clinical trial and are more actively employed for other microsatellite diseases, mainly Huntington’s disease. Though gene silencing is currently being studied for patients with Huntington’s disease, it can easily be adapted for use in FXTAS patients, who also suffer from a trinucleotide repeat disorder (10). This approach aims to silence the FMR1 gene, eliminating toxic mRNA and FMRP. There is concern, though, because certain levels of FMRP need to be preserved for normal cognitive function. Similarly, therapy that targets the degradation of toxic FMR1 mRNA experiences this fundamental conflict between removing toxic mRNA and maintaining sufficient levels of FMRP. Both gene silencing and degradation of FMR1 mRNA approaches have the capacity to prevent both RNA-mediated and RAN protein-mediated toxicity, but if FMR1 mRNA is completely degraded, no FMRP can be produced (9). The absence of FMRP is known to cause FXS (Fragile-X Syndrome) and therefore, this approach may not be beneficial for FXTAS patients and premutation carriers, as it could lead to another Fragile-X disorder (1). Instead, a modest reduction in FMR1 mRNA levels may help the cell eliminate toxic RNA and RAN protein aggregates while preserving sufficient FMRP levels for cognitive activity (12).

Protein-targeted Therapy

Given recent findings, FMRpolyG is now also a therapeutic target due to its intracellular toxicity. In the cell, proteasomes mark FMRpolyG for degradation, yet simultaneously FMRpolyG inhibits this activity, blocking degradation and ultimately leading to its intracellular accumulation (7). Therefore, increased proteasome activity could prevent this toxic process (9). In fact, a recent study observed that increased proteasome activity led to degradation of toxic proteins, reduced aggregate formation and increased cell survival (11). This evidence suggests that elevated proteasome activity poses strong therapeutic potential. However, there are difficulties regarding the long-term toxicity of elevated proteasome activity and cellular adaptation leading to treatment resistance. Increased levels of protein degradation may be deleterious to cells. Therefore, it is critical to target a specific process in the protein degradation process, because this allows for higher regulation and control (9). Overall, this method may prove better suited to patients because it avoids the intrinsic FMR1 mRNA and FMRP problem, unlike gene-targeted therapy.

IV.  Conclusion

Primarily, there are two toxic mechanisms leading to FXTAS pathogenesis: RNA gain-of-function and protein gain-of-function. The RNA gain-of-function hypothesis consists of the sequestration of RNA-binding proteins specifically to the expanded CGG trinucleotide repeat region in the 5’ UTR. This results in the lack of required proteins for tasks such as gene expression and mRNA processing, which are essential for proper cellular function (6-7). The protein gain-of-function hypothesis was recently explored due to further understanding of non-conventional RAN translation mechanisms. RAN translation incorporates the expanded CGG trinucleotide repeat in the peptide product, which was previously thought to be untranslated (7). This leads to toxic peptide products that are prone to misfolding and aggregating within the cell (7-8). Though presented separately, the RNA-mediated and RAN protein-mediated toxicity mechanisms are not mutually exclusive and, in fact, may have synergistic effects (7, 9). Thus, FXTAS pathogenesis would best be described as a two-hit model—toxic RNA and toxic protein both may be critical for FXTAS development (7).

Although these two toxic mechanisms have been further characterized in research, there is no easy drug target for FXTAS treatment. Fundamentally, gene-targeted therapy—mainly genome editing and gene silencing—would prevent both RNA-mediated and RAN protein-mediated toxicity because it would target the root cause of FXTAS (i.e. expanded CGG trinucleotide repeat). However, this method is problematic. Both technologies remove or silence toxic FMR1 mRNA, which leads to diminished levels of FMRP. Therefore, drugs specifically targeting RAN protein toxicity may be more sensible, since they would avoid the fundamental conflict between promoting FMR1 degradation and preserving sufficient levels FMRP for cognitive function.

FXTAS research has made tremendous progress in recent years and has helped advance drug development possibilities and avenues. However, further insight is needed to explore the role of RAN translation and the resulting toxic peptide, FMRpolyG, in FXTAS pathology. RAN translation has important implications for FXTAS patients and premutation carriers and may be a promising target for the development of drug therapies. The reported findings included in this review excite researchers to continue investigation to ultimately discover a standardized cure for FXTAS.

Works Cited

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Edited by Nicole Strossman, Rachel Hull, and Carly Cheung