Abstract
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by expanded CGG (CGGexp) trinucleotides in the 5′UTR of the FMR1 gene encoding fragile X mental retardation protein (FMRP). The patients, with the number of the repeats ranging from 55 to 200, show specific manifestation of clinical symptoms that include intention tremor, gait ataxia, cognitive deficits, and brain atrophy. Accumulation of toxic polyglycine (FMRpolyG), a by-product of the CGGexp repeat-associated non-ATG (RAN) translation, is considered to be one of the main factors triggering neurodegenerative processes in FXTAS patients. Nevertheless, the nature of the FMRpolyG-induced cell damage, especially in the context of its soluble and inclusion-associated forms, is still elusive. Targeting either biosynthesis, cellular stability or aggregation capacity of toxic FMRpolyG could be considered as a potential therapeutic strategy for FXTAS. Therefore, we tested a variety of quantitative methods based on forced expression of genetic constructs carrying CGGexp repeats in the context of the FMR1 5′UTR fused to GFP, mCherry or Firefly luciferase gene in or out of frame to the polyglycine encoding sequence. We show that FMRpolyG translation either from native or an AUG-induced start codon as well as the translation yield of the FMRP open reading frame equivalent located downstream of the CGGexp element can be effectively estimated using fluorescence microscopy, flow cytometry or luciferase assay. We also quantitatively estimated soluble fraction and insoluble form of FMRpolyG aggregated in foci using an electrophoretic separation of cell lysates and fluorescence microscopy, respectively. Importantly, we show that dependent on a fusion tag, FMRpolyG has a different potential for aggregate formation. Our established protocols enable sensitive tracking of FMRP and FMRpolyG quantitative and qualitative changes after treatment with potential therapeutic agents for FXTAS. Furthermore, they can be modified for application to other RAN translation- and aggregation-related diseases.
Highlights
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a lateonset neurodegenerative disorder caused by expanded CGG (CGGexp) trinucleotides in the 5 -untranslated region (5 UTR) of the FMR1 gene (Hagerman and Hagerman, 2016; Boivin et al, 2017)
Fluorescence-Based Systems to Monitor Biosynthesis and Aggregation of FMRpolyG. To evaluate both biosynthesis and aggregation of FMRpolyG we force expressed its fluorescent fusion version (FMRpolyGGFP) in COS7 cells by delivering one of the two plasmids, differing in its expression rate (Figure 1A). 5 (CGGexp)GFP(+1) contains the native 5 untranslated region (5 UTR) of the FMR1 gene with CGGexp repeats preceded by the near cognate ACG triplet embedded in the Kozak sequence context
ATG(CGGexp)-GFP(+1) lacks the upstream FMR1 5 UTR sequence from the start codon and the ACG is replaced for ATG, which results in a robust FMRpolyG-GFP production (Figure 1B)
Summary
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a lateonset neurodegenerative disorder caused by expanded CGG (CGGexp) trinucleotides in the 5 -untranslated region (5 UTR) of the FMR1 gene (Hagerman and Hagerman, 2016; Boivin et al, 2017). As the FMR1 gene is located on the X chromosome, the male carriers are mostly affected, the predominance is incomplete, with the current estimate of 16–20% females and 40–75% males to develop FXTAS (Seltzer et al, 2012; Tassone et al, 2012; Hunter et al, 2014). Despite the late onset of the full blown FXTAS symptoms, the carriers show manifestation of a milder phenotype earlier (Hagerman and Hagerman, 2016), including fragile X-associated primary ovarian insufficiency in females (FXPOI, Sullivan et al, 2005)
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