Quantification of the Relative Contributions of Loss-of-function and Gain-of-function Mechanisms in TAR DNA-binding Protein 43 (TDP-43) Proteinopathies
Quantification of the Relative Contributions of Loss-of-function and Gain-of-function Mechanisms in TAR DNA-binding Protein 43 (TDP-43) Proteinopathies
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DOI：
10.1074/jbc.m116.737726
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发表时间：
2016-09-09
影响因子：
4.8
通讯作者：
Chiti, Fabrizio
中科院分区：
文献类型：
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作者：
Cascella, Roberta;Capitini, Claudia;Chiti, Fabrizio
肌萎缩侧索硬化症（ALS）和额颞叶变性伴泛素阳性包涵体（FTLD-U）是两种临床上不同的神经退行性疾病，具有相似的组织病理学特征，其特征在于TDP-43的核清除及其相关沉积到中枢神经系统不同区域的细胞质包涵体中。鉴于TDP-43核耗竭和细胞质蓄积的同时发生，已经提出TDP-43蛋白病起源于功能丧失（LOF）机制、功能获得（GOF）过程或两者。我们已经通过用内源性鼠TDP-43的siRNA和用人重组TDP-43包涵体（IB）转染鼠NSC 34和N2 a细胞解决了这个问题。这两种策略允许细胞核TDP-43的消耗和细胞质TDP-43聚集体的积累分别和独立地发生。内源性和外源性TDP-43的监测和定量使用免疫荧光和蛋白质印迹分析，和核功能TDP-43通过监测分拣蛋白1 mRNA剪接活性测量。实现了不同程度的TDP-43细胞质积累和细胞核TDP-43消耗，并评价了所得细胞活力，从而对LOF和GOF对总体细胞毒性的相对影响进行了定量全局分析。在两种细胞系中，并使用两种细胞毒性读数，发现这些分别约为55%和45%，表明这两种机制可能对ALS和FTLD-U的病理学有明显相同的贡献。
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, it has been proposed that TDP-43 proteinopathies originate from either a loss-of-function (LOF) mechanism, a gain-of-function (GOF) process, or both. We have addressed this issue by transfecting murine NSC34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs). These two strategies allowed the depletion of nuclear TDP-43 and the accumulation of cytoplasmic TDP-43 aggregates to occur separately and independently. Endogenous and exogenous TDP-43 were monitored and quantified using both immunofluorescence and Western blotting analysis, and nuclear functional TDP-43 was measured by monitoring the sortilin 1 mRNA splicing activity. Various degrees of TDP-43 cytoplasmic accumulation and nuclear TDP-43 depletion were achieved and the resulting cellular viability was evaluated, leading to a quantitative global analysis on the relative effects of LOF and GOF on the overall cytotoxicity. These were found to be approximate to 55% and 45%, respectively, in both cell lines and using both readouts of cell toxicity, showing that these two mechanisms are likely to contribute apparently equally to the pathologies of ALS and FTLD-U.