RESUMO
Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant cerebellar ataxia caused by the expansion of polyglutamine (polyQ) within the TATA box-binding protein (TBP). Previous studies have shown that polyQ-expanded TBP forms neurotoxic aggregates and alters downstream genes. However, how expanded polyQ tracts affect the function of TBP and the link between dysfunctional TBP and SCA17 is not clearly understood. In this study, we generated novel Drosophila models for SCA17 that recapitulate pathological features such as aggregate formation, mobility defects and premature death. In addition to forming neurotoxic aggregates, we determined that polyQ-expanded TBP reduces its own intrinsic DNA-binding and transcription abilities. Dysfunctional TBP also disrupts normal TBP function. Furthermore, heterozygous dTbp amorph mutant flies exhibited SCA17-like phenotypes and flies expressing polyQ-expanded TBP exhibited enhanced retinal degeneration, suggesting that loss of TBP function may contribute to SCA17 pathogenesis. We further determined that the downregulation of TBP activity enhances retinal degeneration in SCA3 and Huntington's disease fly models, indicating that the deactivation of TBP is likely to play a common role in polyQ-induced neurodegeneration.
Assuntos
Drosophila melanogaster/genética , Doença de Huntington/genética , Doença de Machado-Joseph/genética , Degeneração Retiniana/genética , Ataxias Espinocerebelares/genética , Proteína de Ligação a TATA-Box/genética , Animais , Modelos Animais de Doenças , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Heterozigoto , Humanos , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Longevidade/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Peptídeos/química , Fenótipo , Agregados Proteicos , Ligação Proteica , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Transdução de Sinais , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/metabolismoRESUMO
We have developed a sensitive gold-nanoparticle-based (AuNP-based) graphite furnace atomic absorption spectrometry (GFAAS) amplification and magnetic separation method for the detection of mercuric ions (Hg²âº). The assay relies on (i) a sandwich-type structure containing two thymine-thymine (T-T) mismatches for selectively recognizing Hg²âº ions; (ii) magnetic beads for homogeneous separation; and (iii) AuNP-based GFAAS amplification detection. The limit of detection (LOD) of this assay is 0.45 nM (0.09 µg L⻹)--one order of magnitude lower than the United States Environmental Protection Agency (US EPA) limit for Hg²âº in drinking water. Furthermore, because a shorter hybridization step and a simpler AuNP-based GFAAS amplification detection were employed, a faster analytical run time allowing us to analyze a batch of 24 samples within 0.5 h. We demonstrated the feasibility of the developed approach for the determination of Hg²âº in urine and aqueous environmental samples.