German Center for Neurodegenerative Diseases (DZNE) Tübingen, Germany; Hertie Institute for Clinical Brain Research, Laboratory of Functional Neurogenetics, Tübingen, Germany.
Hertie Institute for Clinical Brain Research, Laboratory of Functional Neurogenetics, Tübingen, Germany.
Neurobiol Dis. 2015 Feb;74:76-88. doi: 10.1016/j.nbd.2014.11.003. Epub 2014 Nov 8.
Inclusions containing Fused in Sarcoma (FUS) are found in familial and sporadic cases of the incurable progressive motor neuron disease amyotrophic lateral sclerosis and in a common form of dementia, frontotemporal dementia. Most disease-associated mutations are located in the C-terminal proline-tyrosine nuclear localization sequence (PY-NLS) of FUS and impair its nuclear import. It has been shown in cell culture that the nuclear import of FUS is mediated by transportin, which binds the PY-NLS and the last arginine/glycine/glycine-rich (RGG) domain of FUS. Methylation of this last RGG domain by protein arginine methyltransferases (PRMTs) weakens transportin binding and therefore impairs nuclear translocation of FUS. To investigate the requirements for the nuclear import of FUS in an in vivo model, we generated different transgenic Drosophila lines expressing human FUS wild type (hFUS wt) and two disease-related variants P525L and R495X, in which the NLS is mutated or completely absent, respectively. To rule out effects caused by heterologous hFUS expression, we analysed the corresponding variants for the Drosophila FUS orthologue Cabeza (Caz wt, P398L, Q349X). Expression of these variants in eyes and motor neurons confirmed the PY-NLS-dependent nuclear localization of FUS/Caz and caused neurodegenerative effects. Surprisingly, FUS/Caz toxicity was correlated to the degree of its nuclear localization in this overexpression model. High levels of nuclear FUS/Caz became insoluble and reduced the endogenous Caz levels, confirming FUS autoregulation in Drosophila. RNAi-mediated knockdown of the two transportin orthologues interfered with the nuclear import of FUS/Caz and also enhanced the eye phenotype. Finally, we screened the Drosophila PRMT proteins (DART1-9) and found that knockdown of Dart1 led to a reduction in methylation of hFUS P525L and aggravated its phenotype. These findings show that the molecular mechanisms controlling the nuclear import of FUS/Caz and FUS autoregulation are conserved between humans and Drosophila. In addition to the well-known neurodegenerative effects of FUS loss-of function, our data suggest toxic potential of overexpressed FUS in the nucleus and of insoluble FUS.
融合于肉瘤（FUS）的包含体存在于无法治愈的进行性运动神经元疾病肌萎缩性侧索硬化症的家族性和散发性病例中，以及一种常见的痴呆症额颞叶痴呆症中。大多数与疾病相关的突变位于 FUS 的 C 末端脯氨酸-酪氨酸核定位序列（PY-NLS）中，并损害其核输入。在细胞培养中已经表明，FUS 的核输入是由转运蛋白介导的，转运蛋白结合 PY-NLS 和 FUS 的最后一个精氨酸/甘氨酸/甘氨酸丰富（RGG）结构域。蛋白质精氨酸甲基转移酶（PRMTs）对最后一个 RGG 结构域的甲基化削弱了转运蛋白的结合，从而损害了 FUS 的核易位。为了研究 FUS 在体内模型中的核输入要求，我们生成了不同的转基因果蝇系，表达野生型人 FUS（hFUS wt）和两种与疾病相关的变体 P525L 和 R495X，其中 NLS 分别发生突变或完全缺失。为了排除异源 hFUS 表达引起的影响，我们分析了果蝇 FUS 同源物 Cabeza（Caz wt、P398L、Q349X）的相应变体。这些变体在眼睛和运动神经元中的表达证实了 FUS/Caz 的 PY-NLS 依赖性核定位，并导致神经退行性效应。令人惊讶的是，在这个过表达模型中，FUS/Caz 的毒性与它的核定位程度相关。高水平的核 FUS/Caz 变得不溶，并降低了内源性 Caz 水平，证实了 FUS 在果蝇中的自身调节。两种转运蛋白同源物的 RNAi 介导的敲低干扰了 FUS/Caz 的核输入，并也增强了眼睛表型。最后，我们筛选了果蝇 PRMT 蛋白（DART1-9），发现 Dart1 的敲低导致 hFUS P525L 的甲基化减少，并加重了其表型。这些发现表明，控制 FUS/Caz 的核输入和 FUS 自身调节的分子机制在人类和果蝇之间是保守的。除了 FUS 功能丧失的众所周知的神经退行性效应外，我们的数据还表明过表达核内 FUS 和不溶性 FUS 的潜在毒性。
