神经退行性疾病相关的RNA结合蛋白TDP-43引起线粒体损伤的机制研究

TAR-DNA binding protein 43 (TDP-43), encoding a multi-functional DNA/RNA binding protein, plays an important role in RNA metabolism, from transcriptional to posttranscriptional gene regulation. Recent studies have discovered mutations in the human TDP-43 gene among patients with TDP-43 proteinopathy. This group of common and fatal neurodegenerative diseases includes motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), the second common form of early-onset dementia after Alzheimer’s diseases (AD). ALS and FTLD can cause movement defects and cognitive impairments, representing a huge socio-economical burden for countries including China. In our pilot study, immuno-electronic microscopy (immuno-EM) analyses of FTLP-TDP patient brain samples reveal that TDP-43 positive fibril-containing neurons exhibit severe mitochondrial damage. In this project, we propose to use multi-disciplinary approaches to study the role of TDP-43 in pathogenesis of TDP-43 proteinopathy. Using cell lines and primary neuron culture, we plan to systematically examine effects of wild-type and disease-mutant TDP-43 on morphology and function of mitochondria, as well as identify mitochondrial proteins interacting with TDP-43. With our established TDP-43 transgenic fly model, we then confirm the protein candidate(s) mediating TDP-43 mitochondrial translocation and dissect molecular and cellular mechanisms of TDP-43 induced mitochondrial damage. Finally, we are going to screen small molecular compounds using our modified microfluidic chamber to identify drugs rescuing TDP-43 induced mitochondrial damage. This study will provide new insights into common pathogenic mechanisms underlying a range of neurodegenerative diseases associated with TDP-43 and information useful for future development of diagnostic and therapeutic tools for this group of devastating diseases.

RNA结合蛋白TDP-43参与RNA代谢与加工的多个过程，它的几十种突变与一系列常见并致命的神经退行性疾病密切相关，且病理组织中存在TDP-43阳性的异常蛋白聚集，这些疾病统称为TDP-43蛋白病，严重影响神经系统的正常功能，危害我国中壮及老年人群的健康。我们的前期研究发现，使用免疫电镜检测TDP-43蛋白病患者脑样本，含有TDP-43纤维沉淀的神经细胞出现严重的线粒体损伤。本项目利用细胞系和原代神经元转染，在细胞水平分析表达TDP-43突变体对线粒体形态、功能及能量代谢的影响，筛选与TDP-43相互作用的线粒体蛋白质；利用已建立的TDP-43转基因果蝇模型，在动物水平上确定介导TDP-43线粒体定位的蛋白质，研究其功能及它们相互作用引起线粒体损伤的机制；利用改进的微流控芯片高通量筛选挽救神经元轴突线粒体损伤的小分子药物，为预防和缓解TDP-43异常表达导致多种神经退行性病变提供理论依据。

TDP-43是一个多功能的DNA和RNA结合蛋白，由TARDBP基因编码，在细胞内的RNA转录、选择性剪接及mRNA稳定性调节等过程中发挥功能。在ALS (amyotrophic lateral sclerosis)和FTLD (frontotemporal lobar degeneration)患者大脑或脊髓受损区域的神经元和胶质细胞中，能检测到泛素化的蛋白质包涵体，TDP-43是其中的主要成分。而且20-50%的阿尔茨海默病（Alzheimer’s disease， AD）中有TDP-43蛋白的异常变化。在家族性的ALS病例中，已鉴定出40多个TARDBP基因的突变，它们多集中于该蛋白C端的甘氨酸富集区。关于TDP-43蛋白如何造成神经元细胞死亡及其引起神经退行性疾病的细胞和分子机制尚不明确。.本项目通过合作获得FTLD-TDP患者脑样本，结合前期建立的TDP-43蛋白病的细胞模型和转基因果蝇模型，对TDP-43进入线粒体引起线粒体损伤进行了系统性研究。研究结果显示TDP-43表达引起线粒体结构和功能缺陷，包括线粒体嵴结构严重损坏，膜电位下降，ROS产生增加，ATP合成降低。在细胞模型和转基因果蝇模型中，TDP-43进入线粒体并激活线粒体去折叠蛋白反应通路，而且下调此通路相关的线粒体蛋白酶LonP1使得线粒体内的TDP-43水平增加、并加重TDP-43诱导的线粒体损伤和退行性表型。这些结果证明了TDP-43诱导的线粒体损伤是TDP-43蛋白病的关键环节，不仅揭示了LonP1参与调节线粒体内TDP-43水平的重要作用，而且有助于我们深入理解线粒体损伤可能是多种神经退行性疾病的共有机制，阻断线粒体损伤可能有助于治疗这些毁灭性疾病。这些研究结果为核定位的RNA结合蛋白靶向线粒体提供了重要证据，为未来研发治疗衰老相关神经退行性疾病的诊断工具和治疗方法提供重要研究思路。