Table1_Codon-optimized TDP-43 mediates neurodegeneration in a Drosophila model of ALS/FTLD.XLSX

Transactive response DNA binding protein-43 (TDP-43) is known to mediate neurodegeneration associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The exact mechanism by which TDP-43 exerts toxicity in the brains, spinal cord, and lower motor neurons of affected patients remains unclear. In a novel Drosophila melanogaster model, we report gain-of-function phenotypes due to misexpression of insect codon-optimized version of human wild-type TDP-43 (CO-TDP-43) using both the binary GAL4/UAS system and direct promoter fusion constructs. The CO-TDP-43 model showed robust tissue specific phenotypes in the adult eye, wing, and bristles in the notum. Compared to non-codon optimized transgenic flies, the CO-TDP-43 flies produced increased amount of high molecular weight protein, exhibited pathogenic phenotypes, and showed cytoplasmic aggregation with both nuclear and cytoplasmic expression of TDP-43. Further characterization of the adult retina showed a disruption in the morphology and function of the photoreceptor neurons with the presence of acidic vacuoles that are characteristic of autophagy. Based on our observations, we propose that TDP-43 has the propensity to form toxic protein aggregates via a gain-of-function mechanism, and such toxic overload leads to activation of protein degradation pathways such as autophagy. The novel codon optimized TDP-43 model is an excellent resource that could be used in genetic screens to identify and better understand the exact disease mechanism of TDP-43 proteinopathies and find potential therapeutic targets.

反式激活应答DNA结合蛋白43（Transactive response DNA binding protein-43，TDP-43）已被证实可介导与肌萎缩侧索硬化症（ALS）及额颞叶变性（FTLD）相关的神经退行性病变。目前，TDP-43在受累患者的大脑、脊髓及下运动神经元中产生毒性的确切机制仍不明确。本研究通过一款新型黑腹果蝇（Drosophila melanogaster）模型，报道了借助二元GAL4/UAS系统与直接启动子融合构建体，对人类野生型TDP-43的昆虫密码子优化版本（CO-TDP-43）进行异位表达所引发的功能获得性表型。该CO-TDP-43模型在成虫眼、翅及背板刚毛中展现出稳健的组织特异性表型。与非密码子优化的转基因果蝇相比，CO-TDP-43果蝇的高分子量蛋白表达量显著升高，呈现致病表型，且在TDP-43同时存在核表达与胞质表达的情况下出现胞质聚集。对成虫视网膜的进一步表征显示，感光神经元（photoreceptor neurons）的形态与功能遭到破坏，同时出现了自噬（autophagy）特征性的酸性液泡。基于上述观察结果，我们提出TDP-43倾向于通过功能获得性机制形成毒性蛋白聚集，而此类毒性负荷会激活自噬等蛋白质降解通路。这款新型密码子优化的TDP-43模型是一项优质研究资源，可用于遗传筛选，以明确并深入阐释TDP-43蛋白病的确切发病机制，并筛选潜在的治疗靶点。