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Quantitative Evaluation of Toxic Polyglycine Biosynthesis and Aggregation in Cell Models Expressing Expanded CGG Repeats.

ABSTRACT: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by expanded CGG (CGG^exp) 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 CGG^exp 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 CGG^exp 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 CGG^exp 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.

SUBMITTER: Derbis M

PROVIDER: S-EPMC6018535 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Quantitative Evaluation of Toxic Polyglycine Biosynthesis and Aggregation in Cell Models Expressing Expanded CGG Repeats.

Derbis Magdalena M Konieczny Patryk P Walczak Agnieszka A Sekrecki Michal M Sobczak Krzysztof K

Frontiers in genetics 20180619

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by expanded CGG (CGG<sup>exp</sup>) trinucleotides in the 5'UTR of the <i>FMR1</i> 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-prod ...[more]

PMID: 29971092

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Project description:BackgroundNeuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disorder characterized by widespread intranuclear inclusions in the nervous system as well as multiple visceral organs. In 2019, expanded GGC repeats within the 5' untranslated region of the NOTCH2NLC gene was identified as the causative factor. NIID is a heterogeneous disorder with variable clinical manifestations including cognitive impairment, cerebellar ataxia, parkinsonism, paroxysmal symptoms, autonomic dysfunction, and muscle weakness. Although NIID primarily affects the central and peripheral nervous systems, growing evidence suggests potential cardiac abnormalities in NIID. However, the link between expanded GGC repeats within NOTCH2NLC and cardiac dysfunction remains uncertain.ResultsIn this study, we utilized two transgenic mouse models, expressing NOTCH2NLC-(GGC)98 ubiquitously or specifically in cardiomyocytes, and identified p62 (also known as sequestosome 1, SQSTM1)-positive intranuclear NOTCH2NLC-polyG inclusions in cardiomyocytes in two mouse models. We observed that both models exhibited cardiac-related pathological and echocardiographic changes, albeit exhibiting varying degrees of severity. Transcriptomic analysis revealed shared downregulation of genes related to ion channels and mitochondria in both models, with the cardiomyocyte-specific mice showing a more pronounced downregulation of mitochondria and energy metabolism-related pathways. Further investigations revealed decreased expression of mitochondria-related genes and electron transport chain activity. At last, we conducted a retrospective review of cardiac-related examination results from NIID patients at our hospital and also identified some cardiac abnormalities in NIID patients.ConclusionsOur study provided the first in vivo evidence linking GGC repeat expansions within NOTCH2NLC to cardiac abnormalities and highlighted the contribution of mitochondrial dysfunction in the development of cardiac abnormalities.

Dataset Information

Quantitative Evaluation of Toxic Polyglycine Biosynthesis and Aggregation in Cell Models Expressing Expanded CGG Repeats.

Publications

Quantitative Evaluation of Toxic Polyglycine Biosynthesis and Aggregation in Cell Models Expressing Expanded CGG Repeats.

Similar Datasets

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