FUS/TLS acts as an aggregation-dependent modifier of polyglutamine disease model mice (I)

FUS/TLS is an RNA/DNA-binding protein associated with neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Previously, we found that a prion-like domain in the N-terminus of FUS/TLS mediates co-aggregation between FUS/TLS and mutant huntingtin, the gene product of Huntington's disease (HD). Here, we show that heterozygous knockout of FUS/TLS worsened the phenotypes of model mice of Huntington's disease (HD) but not spinal and bulbar muscular atrophy (SBMA). This difference was correlated with the degree of pathological association between disease proteins and FUS/TLS. Co-aggregation between FUS/TLS and mutant huntingtin resulted in the depletion of free FUS/TLS protein in HD mice that was detected as a monomer in SDS-PAGE analysis. Recently, we found that FUS/TLS paralogs, TAF15 and EWS, were up-regulated in homozygous FUS/TLS knockout mice. These two proteins were up-regulated in both HD and FUS/TLS heterozygote mice, and were further elevated in HD-TLS+/- double mutant mice, consistent with the functional impairment of FUS/TLS. These results suggest that FUS/TLS sequestration by co-aggregation is a rate-limiting factor of disease phenotypes of HD and that inclusions may have an adverse aspect, rather than being simply benign or protective. In addition, our results highlight inclusions as repositories of potential modifiers of neurodegeneration. Gene expression profiles were analyzed to examine the effects of FUS/TLS heterozygosity in mouse with or without the transgene of mutant huntingtin exon 1. To examine the effect of FUS/TLS heterozygosity on the transcriptome of Huntington's disease model mice, we crossed FUS/TLS heterozygous knockout mice with HD model mice. We extracted total RNA from the striatum of these mice at 8 weeks and used for a microaaray analysis. The samples are HD transgenic mice with FUS/TLS heterozygosity (HD_TLS+/-), HD mice with normal FUS/TLS (HD_TLS+/+), non-HD-transgenic mice with FUS/TLS heterozygosity (NT_TLS+/-), and non-HD-transgenic mice with normal FUS/TLS (NT_TLS+/+).

FUS/TLS是一种RNA/DNA结合蛋白，与肌萎缩侧索硬化（amyotrophic lateral sclerosis）、额颞叶变性（frontotemporal lobar degeneration）等神经退行性疾病相关。既往研究发现，FUS/TLS N端的朊蛋白样结构域（prion-like domain）可介导FUS/TLS与突变亨廷顿蛋白——亨廷顿病（Huntington's disease, HD）的基因产物——之间的共聚集（co-aggregation）。本研究证实，FUS/TLS的杂合敲除（heterozygous knockout）会恶化亨廷顿病模型小鼠的表型，但对脊髓延髓肌萎缩症（spinal and bulbar muscular atrophy, SBMA）模型小鼠无此影响。该差异与疾病蛋白和FUS/TLS的病理结合程度密切相关。FUS/TLS与突变亨廷顿蛋白的共聚集会导致HD小鼠体内游离FUS/TLS蛋白耗竭，这一现象可通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析中的单体条带检测到。近期研究表明，FUS/TLS的旁系同源物（paralogs）TAF15与EWS在纯合FUS/TLS敲除小鼠中表达上调。这两种蛋白在HD小鼠及FUS/TLS杂合小鼠中均出现表达升高，且在HD-TLS+/-双突变小鼠中上调幅度进一步增加，这与FUS/TLS的功能受损状态一致。上述结果提示，通过共聚集被螯合的FUS/TLS是HD疾病表型的限速因子，而神经元包涵体可能具有有害的病理效应，而非单纯的良性或保护性结构。此外，本研究将包涵体定位为神经退行性病变潜在修饰因子的储存库。为探究FUS/TLS杂合性对携带或不携带突变亨廷顿蛋白外显子1转基因小鼠的影响，我们对其基因表达谱进行了分析。为研究FUS/TLS杂合性对HD模型小鼠转录组（transcriptome）的调控作用，我们将FUS/TLS杂合敲除小鼠与HD模型小鼠进行杂交繁育。于小鼠8周龄时，从其纹状体中提取总RNA，用于微阵列（microarray）分析。本次实验的样本共分为四组：携带FUS/TLS杂合性的HD转基因小鼠（HD_TLS+/-）、携带正常FUS/TLS的HD小鼠（HD_TLS+/+）、携带FUS/TLS杂合性的非HD转基因小鼠（NT_TLS+/-），以及携带正常FUS/TLS的非HD转基因小鼠（NT_TLS+/+）。