泛素结合酶UBE2N对肌萎缩侧索硬化症中变异蛋白TDP-43累积作用的研究

Amyotrophic Lateral Sclerosis (ALS) and Huntington disease (HD) are the important neurodegenerative diseases, which share the important pathological feature of the age-dependent accumulation of misfolded proteins in brain. Ubiquitination of misfolded proteins can lead to their aggregation or degradation, which involves different types of ubiquitin-conjugating enzymes (UBE2) and other molecules. By utilizing iTRAQ/proteomics approaches, our recent studies found that UBE2N showed the increased expression levels with age in old rhesus monkey brains. Inhibiting UBE2N expression in HD mice can reduce the accumulation of the HD protein. It remains unclear whether UBE2N has similar effect on other disease proteins that also accumulate in aged neurons. In this project, we will over-express or inhibit UBE2N in cellular and mouse models of ALS to examine whether UBE2N levels influence the accumulation and toxicity of the ALS mutant protein TDP-43. Using an in vitro assay, we will also examine whether mouse brain tissues containing UBE2N at different ages have differential effects on the ubiquitination of mutant TDP-43. This study aims to investigate whether UBE2N can selectively or broadly regulate the accumulation of misfolded proteins that can cause age-dependent neurodegeneration.

肌萎缩侧索硬化症与亨廷顿症均为典型的神经退行性疾病，与衰老密切相关，表现为变异蛋白在衰老的神经元中累积而引发神经细胞毒性。变异蛋白的累积和清除与泛素化过程密切相关。在前期工作中我们采用iTRAQ/蛋白组学技术发现在猕猴神经细胞中,泛素结合酶UBE2N的表达水平随衰老逐渐升高，导致亨廷顿症致病蛋白mutant Htt的聚合与累积而不易被清除。本项目将探讨UBE2N是否对肌萎缩侧索硬化症的致病蛋白TDP-43的累积亦有重要影响作用。我们将分别在体外培养的神经细胞和小鼠脑内，利用病毒载体过表达UBE2N或用siRNA抑制内源性UBE2N来观察UBE2N水平对突变蛋白TDP-43累积的作用。在分子机制上，我们将研究衰老过程中UBE2N的增加是否影响TDP-43泛素化水平及位点的选择。本项目旨在进一步论证UBE2N是否对不同变异蛋白的累积与毒性有广泛的调节功能。