Mechanisms of Neural Stem Cells Regulation by Autophagy

自噬调节神经干细胞的机制

• 批准号: 9001627
• 负责人: JUN-LIN GUAN
• 金额: $ 34.56万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9001627
• 关键词: Ablation; Adult; Affect; Alleles; Autoimmune Diseases; Autophagocytosis; Autophagosome; Binding; Biological; Brain; Brain Injuries; Brain region; Cell Maintenance; Cell physiology; Cells; Code; Complex; Data; Defect; Disease; Embryo; Family; Functional disorder; Future; Generations; Genes; Goals; Hippocampus (Brain); Homeostasis; In Situ; In Vitro; Infiltration; Inflammation; Injury; Knock-in Mouse; Knock-out; Knockout Mice; Laboratories; Maintenance; Malignant Neoplasms; Mediating; Microarray Analysis; Microglia; Modeling; Molecular; Mus; Mutant Strains Mice; Mutate; Nerve Degeneration; Neurodegenerative Disorders; Neurons; PTK2 gene; Pathway interactions; Phenotype; Play; Process; Proteins; Regulation; Research; Role; SOD2 gene; Starvation; Stem cells; Superoxides; Tissues; base; comparative; dentate gyrus; design; effective therapy; in vivo; inhibition of autophagy; insight; interest; lateral ventricle; mimetics; mouse model; mutant; nerve stem cell; neurogenesis; neuromechanism; neuroregulation; novel; postnatal; public health relevance; regenerative; relating to nervous system; response; self-renewal; stem; subventricular zone

 DESCRIPTION (provided by applicant): The mammalian brain maintains the capacity to generate new neurons for tissue homeostasis and after injuries, which is driven by neural stem/progenitor cells (NSCs). The long term goal of the proposed studies is to understand the molecular and cellular mechanisms of autophagy pathway and genes in the regulation of NSCs. Autophagy is a highly conserved cellular process for maintenance of cellular homeostasis, and dysfunctions in autophagy have been implicated in many disorders, including neurodegenerative diseases. Despite increasing research on autophagy in recent years, little is known about the role and mechanisms of autophagy in the regulation of tissue stem cells such as NSCs. FIP200 (FAK-family Interacting Protein of 200 kDa) was initially identified in our laboratory and later shown as one component of the ULK1/Atg13/FIP200 complex essential for autophagy induction. Very recently, we showed that conditional knockout (cKO) of FIP200 resulted in the depletion of NSCs and their aberrant differentiation, providing the first evidence for a role of autophagy in postnatal NSCs. In prelim studies, we found that autophagy inhibition by deletion of Atg5 or Atg16L1 (autophagy genes required for autophagosome maturation) did not affect NSCs, and further comparative analyses of FIP200, Atg5 and Atg16L1 cKO mice suggested that FIP200, but not Atg5 and Atg16L1, deletion caused p62 aggregation in NSCs. Generation and analysis of a FIP200 and p62 double cKO mice suggested that the preferential p62 aggregates formation plays a crucial role in triggering aberrant superoxide increase leading to defective NSCs primarily by impairing SOD functions. In addition, we showed that knockdown of Atg13 (FIP200 partner in the autophagy induction complex) inhibited NSC self-renewal in vitro, and identified residues 582-585 in FIP200 required for its binding to Atg13. We then created a novel FIP200 knock-in mutant mice containing FIP200x allele lacking binding to Atg13 and showed that KI/KI MEFs were defective in both basal and starvation-induced autophagy. Lastly, microarray analysis of neurospheres from various mice revealed elevated expression of multiple genes associated with inflammation upon FIP200 deletion, which were not rescued by p53 inactivation. Moreover, an increased infiltration of microglia was found in the SVZ of FIP200 cKO mice, as well as FIP200 and p53 double cKO mice, suggesting an interesting possibility that the infiltrating microglias may be responsible for the p53-independent, aberrant differentiation of NSCs upon deletion of FIP200. Based on these preliminary and previous studies, we propose to 1). investigate the mechanisms of p62 aggregates in mediating NSC regulation by autophagy, 2). analyze FIP200-mediated autophagy function in the regulation of NSCs, and 3). explore the role and mechanisms of elevated microglia infiltration in the aberrant differentiation of NSCs upon FIP200 ablation. Together, these studies will significantly advance our understanding of the mechanisms of NSC regulation by autophagy that may contribute to future design of more effective therapies for neurodegenerative and other related diseases.

 描述（由申请人提供）：哺乳动物大脑在神经干/祖细胞（NSC）的驱动下保持产生新神经元以实现组织稳态和损伤后的能力。拟议研究的长期目标是了解分子机制。自噬途径和基因在神经干细胞调控中的细胞机制 自噬是维持细胞稳态的高度保守的细胞过程，自噬功能障碍与许多疾病有关，包括尽管近年来对自噬的研究不断增加，但我们实验室最初发现了自噬在调节 NSC 等组织干细胞中的作用和机制。后来被证明是自噬诱导所必需的 ULK1/Atg13/FIP200 复合物的一个组成部分。最近，我们发现了条件敲除 (cKO)。 FIP200 导致 NSC 耗竭及其异常分化，为自噬在出生后 NSC 中的作用提供了第一个证据。在初步研究中，我们发现通过删除 Atg5 或 Atg16L1（自噬体成熟所需的自噬基因）不会抑制自噬。对 NSCs 的影响，对 FIP200、Atg5 和 Atg16L1 cKO 小鼠的进一步比较分析表明FIP200（而非 Atg5 和 Atg16L1）缺失导致 NSC 中 p62 聚集 FIP200 和 p62 双 cKO 小鼠的生成和分析表明，优先的 p62 聚集体形成在触发异常超氧化物增加中起着至关重要的作用，主要通过损害 SOD 导致缺陷 NSC。此外，我们还发现 Atg13（FIP200 参与自噬诱导）的敲除。复合物）在体外抑制 NSC 自我更新，并鉴定了 FIP200 中与 Atg13 结合所需的残基 582-585，然后我们创建了一种新型 FIP200 敲入突变小鼠，其含有缺乏与 Atg13 结合的 FIP200x 等位基因，并显示 KI/KI MEF。最后，对不同小鼠神经球的微阵列分析显示多个相关基因的表达升高。 FIP200 缺失后出现炎症，p53 失活无法挽救这种炎症。此外，在 FIP200 cKO 小鼠以及 FIP200 和 p53 双 cKO 小鼠的 SVZ 中发现小胶质细胞浸润增加，这表明浸润的小胶质细胞可能会增加。根据这些初步和之前的研究，FIP200 缺失导致 NSC 发生不依赖于 p53 的异常分化。我们建议：1) 研究 p62 聚集体通过自噬介导 NSC 调节的机制，2) 分析 FIP200 介导的自噬功能在 NSC 调节中的作用和机制。总之，这些研究将显着促进我们对自噬调节 NSC 机制的理解，这可能有助于未来更有效的设计。神经退行性疾病和其他相关疾病的治疗。