Higelin Julia, Demestre Maria, Putz Stefan, Delling Jan P, Jacob Christian, Lutz Anne-Kathrin, Bausinger Julia, Huber Anne-Kathrin, Klingenstein Moritz, Barbi Gotthold, Speit Günter, Huebers Annemarie, Weishaupt Jochen H, Hermann Andreas, Liebau Stefan, Ludolph Albert C, Boeckers Tobias M
Institute for Anatomy and Cell Biology, Ulm University Ulm, Germany.
Institute for Anatomy and Cell Biology, Ulm UniversityUlm, Germany; Department of Neurology, Ulm UniversityUlm, Germany.
Front Cell Neurosci. 2016 Dec 26;10:290. doi: 10.3389/fncel.2016.00290. eCollection 2016.
Mutations within the gene (Fused in Sarcoma) are known to cause Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease affecting upper and lower motoneurons. The gene codes for a multifunctional RNA/DNA-binding protein that is primarily localized in the nucleus and is involved in cellular processes such as splicing, translation, mRNA transport and DNA damage response. In this study, we analyzed pathophysiological alterations associated with ALS related FUS mutations (mFUS) in human induced pluripotent stem cells (hiPSCs) and hiPSC derived motoneurons. To that end, we compared cells carrying a mild or severe mFUS in physiological- and/or stress conditions as well as after induced DNA damage. Following hyperosmolar stress or irradiation, mFUS hiPS cells recruited significantly more cytoplasmatic FUS into stress granules accompanied by impaired DNA-damage repair. In motoneurons wild-type FUS was localized in the nucleus but also deposited as small punctae within neurites. In motoneurons expressing mFUS the protein was additionally detected in the cytoplasm and a significantly increased number of large, densely packed FUS positive stress granules were seen along neurites. The amount of FUS mislocalization correlated positively with both the onset of the human disease (the earlier the onset the higher the FUS mislocalization) and the maturation status of the motoneurons. Moreover, even in non-stressed post-mitotic mFUS motoneurons clear signs of DNA-damage could be detected. In summary, we found that the susceptibility to cell stress was higher in mFUS hiPSCs and hiPSC derived motoneurons than in controls and the degree of FUS mislocalization correlated well with the clinical severity of the underlying ALS related mFUS. The accumulation of DNA damage and the cellular response to DNA damage stressors was more pronounced in post-mitotic mFUS motoneurons than in dividing hiPSCs suggesting that mFUS motoneurons accumulate foci of DNA damage, which in turn might be directly linked to neurodegeneration.
已知肉瘤融合基因（Fused in Sarcoma）内的突变会导致肌萎缩侧索硬化症（ALS），这是一种影响上下运动神经元的神经退行性疾病。肉瘤融合基因编码一种多功能RNA/DNA结合蛋白，该蛋白主要定位于细胞核，并参与剪接、翻译、mRNA运输和DNA损伤反应等细胞过程。在本研究中，我们分析了人类诱导多能干细胞（hiPSC）和hiPSC衍生的运动神经元中与ALS相关的FUS突变（mFUS）相关的病理生理改变。为此，我们比较了在生理和/或应激条件下以及诱导DNA损伤后携带轻度或重度mFUS的细胞。在高渗应激或辐射后，mFUS hiPS细胞将显著更多的细胞质FUS募集到应激颗粒中，同时DNA损伤修复受损。在运动神经元中，野生型FUS定位于细胞核，但也以小斑点的形式沉积在神经突内。在表达mFUS的运动神经元中，该蛋白在细胞质中也有检测到，并且在神经突上可见大量密集堆积的FUS阳性应激颗粒显著增加。FUS定位错误的程度与人类疾病的发病时间（发病越早，FUS定位错误越高）和运动神经元的成熟状态呈正相关。此外，即使在非应激的有丝分裂后mFUS运动神经元中，也能检测到明显的DNA损伤迹象。总之，我们发现mFUS hiPSC和hiPSC衍生的运动神经元比对照细胞对细胞应激更敏感，FUS定位错误的程度与潜在的ALS相关mFUS的临床严重程度密切相关。有丝分裂后mFUS运动神经元中DNA损伤的积累和细胞对DNA损伤应激源的反应比分裂的hiPSC更明显，这表明mFUS运动神经元积累了DNA损伤灶，这反过来可能与神经退行性变直接相关。
