Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder associated with a premutation repeat expansion (55-200 CGG repeats) in the 5' noncoding region of the FMR1 gene. Solitary intranuclear inclusions within FXTAS neurons and astrocytes constitute a hallmark of the disorder, yet our understanding of how and why these bodies form is limited. Here, we have discovered that FXTAS inclusions emit a distinct autofluorescence spectrum, which forms the basis of a novel, unbiased method for isolating FXTAS inclusions by preparative fluorescence-activated cell sorting (FACS). Using a combination of autofluorescence-based FACS and liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based proteomics, we have identified more than two hundred proteins that are enriched within the inclusions relative to FXTAS whole nuclei. Whereas no single protein species dominates inclusion composition, highly enriched levels of conjugated small ubiquitin-related modifier 2 (SUMO 2) protein and p62/sequestosome-1 (p62/SQSTM1) protein were found within the inclusions. Many additional proteins involved with RNA binding, protein turnover, and DNA damage repair were enriched within inclusions relative to total nuclear protein. The current analysis has also allowed the first direct detection, through peptide sequencing, of endogenous FMRpolyG peptide, the product of repeat-associated non-ATG (RAN) translation of the FMR1 mRNA. However, this peptide was found only at extremely low levels and not within whole FXTAS nuclear preparations, raising the question whether endogenous RAN products exist at quantities sufficient to contribute to FXTAS pathogenesis. The abundance of the inclusion-associated ubiquitin- and SUMO-based modifiers supports a model for inclusion formation as the result of increased protein loads and elevated oxidative stress leading to maladaptive autophagy. These results highlight the need to further investigate FXTAS pathogenesis in the context of endogenous systems.

中文翻译：

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脆性 X 相关震颤/共济失调综合征的核内包涵体的组成。


脆性 X 相关震颤/共济失调综合征 (FXTAS) 是一种与 FMR1 基因 5' 非编码区的前突变重复扩展（55-200 个 CGG 重复）相关的神经退行性疾病。 FXTAS 神经元和星形胶质细胞内的孤立核内包涵体构成了该疾病的标志，但我们对这些小体如何以及为何形成的理解有限。在这里，我们发现 FXTAS 内含物发出独特的自发荧光光谱，这构成了通过制备型荧光激活细胞分选 (FACS) 分离 FXTAS 内含物的新颖、无偏见方法的基础。通过结合基于自发荧光的 FACS 和基于液相色谱/串联质谱 (LC-MS/MS) 的蛋白质组学，我们已经鉴定出 200 多种蛋白质，这些蛋白质相对于 FXTAS 全核在包涵体中富集。虽然没有单一蛋白质种类主导内含物组成，但在内含物中发现了高度富集水平的缀合小泛素相关修饰剂 2 (SUMO 2) 蛋白和 p62/sequestosome-1 (p62/SQSTM1) 蛋白。相对于总核蛋白，许多与 RNA 结合、蛋白质周转和 DNA 损伤修复有关的其他蛋白质在内含物中富集。目前的分析还首次通过肽测序直接检测内源性 FMRpolyG 肽，即 FMR1 mRNA 重复相关非 ATG (RAN) 翻译的产物。然而，这种肽的含量极低，并且不在整个 FXTAS 核制剂中，这就提出了内源性 RAN 产物的存在量是否足以促进 FXTAS 发病机制的问题。 与包涵体相关的泛素和 SUMO 修饰剂的丰富性支持了包涵体形成的模型，该模型是由于蛋白质负荷增加和氧化应激升高导致适应不良自噬的结果。这些结果强调需要在内源系统的背景下进一步研究 FXTAS 发病机制。