Angiogenin对progranulin基因A9D突变所致神经元损伤中的作用机制研究

Progranulin (PGRN) mutations have been reported to be associated with neurodegenerative diseases. Among those mutations, one of the mutations in PGRN gene exon 1 introduces a charged amino acid in the hydrophobic core of the signal peptide at residue 9 (PGRN A9D) do not affect its mRNA level, but causes the cytoplasm retardation of PGRN, cell apoptosis and redistribution of cellular angiogenin (ANG, a neuroprotective protein) from nuclear to stress granules. It is revealed that ANG modulates the production of tiRNA, inhibits protein synthesis, and promotes cytoplasmic stress granules assembly, thus activating a cytoprotective stress response program to preventing cell apoptosis under stress conditions. To better understand the protective functions and mechanisms of angiogenin in progranulin A9D mutation induced neuron injury, this project aims to introduce PGRN A9D mutation on neuron cells by CRISPR/CAS9 method, and illustrates the role of ANG in PGRN A9D induced neuron injury by altering the ANG level. Furthermore, we will explore underlying mechanisms related to this process, such as the ANG mediated tiRNA formation and PI3K/AKT/mTOR signaling pathway. We believe our project could elucidate how PGRN and ANG act in concert to regulate the progress of neurodegeneration, and promote a novel therapeutic strategy of neurodegenerative diseases.

颗粒蛋白前体（progranulin，PGRN）基因突变已在多种神经退行性疾病中被报道。其中PGRN信号肽错义突变A9D不影响其mRNA水平，但会导致该蛋白滞留于细胞质中，引起细胞凋亡；同时，该突变还会改变神经元保护因子血管生成素（angiogenin，ANG）在细胞内的定位，从细胞核转移至细胞质的应激小体中。已有研究显示，细胞质中的ANG能降解tRNA形成tiRNA抑制细胞内整体蛋白质的翻译水平，促进应激小体形成，激活细胞内应激保护机制。本项目拟利用CRISPR/CAS9定点突变技术结构PGRN基因A9D突变的神经元细胞，通过改变ANG的水平来明确其对神经元损伤的保护作用；进一步，利用生化和分子技术探索ANG介导的tiRNA形成和PI3K/AKT/mTOR信号通路在其中的作用。本研究将有助于明确ANG和PGRN如何共同在神经退行性疾病发生发展中发挥作用，进而为其成为临床治疗靶点提供依据。

颗粒蛋白前体（PGRN）基因突变已经在多种神经退行性疾病中被陆续报道。其中，PGRN信号肽错义突变A9D只影响该蛋白质的成熟与分泌，其具体的病理生理学后果和机制有待阐明。本项目在神经母细胞瘤细胞株SH-SY5Y中高表达了PGRN基因A9D，发现该突变确实使得PGRN蛋白不能分泌，且主要滞留在细胞质的应激小体上，导致细胞凋亡。进一步，该突变体会改变了神经元保护因子血管生成素（ANG）在细胞内的定位，从细胞核转移至细胞质的应激小体中，从而降解tRNA形成tiRNA保护细胞免受凋亡。本项目不仅揭示了PGRN基因A9D突变引起神经退行性疾病的一种可能机制，而且阐释了tiRNA的调控作用，提示ANG介导的tiRNA可能成为临床治疗神经退行性疾病的潜在靶点。通过本项目研究，已发表SCI收录论文1篇，会议摘要3篇，申请国家发明专利1项，培养博士后1名、博士研究生3名、硕士研究生2名，圆满完成原计划的研究目标和预期指标。