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      Autofluorescence-based analyses of intranuclear inclusions of Fragile X-associated tremor/ataxia syndrome

      brief-report
      1 , * , 1 , 2 ,
      BioTechniques
      Future Science Ltd

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          Abstract

          Intranuclear inclusions present in the brains of patients with Fragile X-associated tremor/ataxia syndrome (FXTAS) have historically been difficult to study due to their location and scarcity. The recent finding that these particles autofluoresce has complicated the use of immunofluorescence techniques, but also offers new opportunities for purification. We have ascertained the features of the autofluorescence, including its excitation/emission spectrum, similarities and differences compared with lipofuscin autofluorescence, and its presence/absence under various fixation, mounting and UV light exposure conditions. Immunofluorescence at various wavelengths was conducted to determine which conditions are ideal for minimizing autofluorescence confounds. We also present a technique for autofluorescence-based sorting of FXTAS inclusions using flow cytometry, which will allow researchers in the field to purify inclusions more successfully for unbiased analyses.

          METHOD SUMMARY

          The intranuclear inclusions of FXTAS are autofluorescent across a broad range of wavelengths. We performed experiments using fluorescence microscopy to define the characteristics of this autofluorescence and to determine what immunofluorescence conditions might overcome interference from inclusion autofluorescence. The inclusion-associated autofluorescence pattern was also used to develop a method for purifying inclusions through nuclear isolation, sucrose fractionation and FACS.

          Most cited references16

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          Fragile X-associated tremor/ataxia syndrome - features, mechanisms and management.

          Many physicians are unaware of the many phenotypes associated with the fragile X premutation, an expansion in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene that consists of 55-200 CGG repeats. The most severe of these phenotypes is fragile X-associated tremor/ataxia syndrome (FXTAS), which occurs in the majority of ageing male premutation carriers but in fewer than 20% of ageing women with the premutation. The prevalence of the premutation is 1 in 150-300 females, and 1 in 400-850 males, so physicians are likely to see people affected by FXTAS. Fragile X DNA testing is broadly available in the Western world. The clinical phenotype of FXTAS at presentation can vary and includes intention tremor, cerebellar ataxia, neuropathic pain, memory and/or executive function deficits, parkinsonian features, and psychological disorders, such as depression, anxiety and/or apathy. FXTAS causes brain atrophy and white matter disease, usually in the middle cerebellar peduncles, the periventricular area, and the splenium and/or genu of the corpus callosum. Here, we review the complexities involved in the clinical management of FXTAS and consider how targeted treatment for these clinical features of FXTAS will result from advances in our understanding of the molecular mechanisms that underlie this neurodegenerative disorder. Such targeted approaches should also be more broadly applicable to earlier forms of clinical involvement among premutation carriers.
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            Translation of Expanded CGG Repeats into FMRpolyG Is Pathogenic and May Contribute to Fragile X Tremor Ataxia Syndrome

            Summary Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a limited expansion of CGG repeats in the 5′ UTR of FMR1. Two mechanisms are proposed to cause FXTAS: RNA gain-of-function, where CGG RNA sequesters specific proteins, and translation of CGG repeats into a polyglycine-containing protein, FMRpolyG. Here we developed transgenic mice expressing CGG repeat RNA with or without FMRpolyG. Expression of FMRpolyG is pathogenic, while the sole expression of CGG RNA is not. FMRpolyG interacts with the nuclear lamina protein LAP2β and disorganizes the nuclear lamina architecture in neurons differentiated from FXTAS iPS cells. Finally, expression of LAP2β rescues neuronal death induced by FMRpolyG. Overall, these results suggest that translation of expanded CGG repeats into FMRpolyG alters nuclear lamina architecture and drives pathogenesis in FXTAS.
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              FMRpolyG-positive inclusions in CNS and non-CNS organs of a fragile X premutation carrier with fragile X-associated tremor/ataxia syndrome

              para Electronic supplementary material The online version of this article (doi:10.1186/s40478-014-0162-2) contains supplementary material, which is available to authorized users.
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                Author and article information

                Journal
                BTN
                BioTechniques
                Future Science Ltd (London, UK )
                0736-6205
                1940-9818
                02 June 2020
                April 2020
                : 69
                : 1
                : 57-63
                Affiliations
                1Department of Biochemistry & Molecular Medicine, University of California Davis School of Medicine, Davis, CA 95616, USA
                2MIND Institute, University of California Davis Health, Sacramento, CA 95817, USA
                Author notes
                [* ]Author for correspondence: pjhagerman@ 123456ucdavis.edu
                Author information
                https://orcid.org/0000-0003-1142-5680
                Article
                10.2144/btn-2019-0144
                93e78e85-9720-468f-af6c-8a24075947ad
                © 2020 Paul Hagerman

                This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License

                History
                : 29 October 2019
                : 03 February 2020
                : 02 June 2020
                Page count
                Pages: 7
                Categories
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                General life sciences,Cell biology,Molecular biology,Biotechnology,Genetics,Life sciences

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