Mechanisms of Neural Stem Cells Regulation by Autophagy

 描述(申请人提供)：哺乳动物的大脑在组织动态平衡和损伤后保持产生新神经元的能力，这是由神经干细胞/祖细胞(NSCs)驱动的。这些研究的长期目标是了解自噬途径和基因在神经干细胞调控中的分子和细胞机制。自噬是维持细胞内稳态的一个高度保守的细胞过程，自噬功能障碍与许多疾病有关，包括神经退行性疾病。尽管近年来对自噬的研究越来越多，但对自噬在神经干细胞等组织干细胞调控中的作用和机制知之甚少。FIP200(FAK家族相互作用蛋白，200 kDa)最初是我们实验室发现的，后来发现它是ULK1/Atg13/FIP200复合体的一个组成部分，在自噬诱导过程中起重要作用。最近，我们发现FIP200的条件性敲除(CKO)导致了NSCs的耗尽和分化异常，为自噬在出生后NSCs中的作用提供了第一个证据。在初步研究中，我们发现ATG5或Atg16L1(自噬成熟所需的自噬基因)的缺失对NSCs没有影响，进一步的比较分析表明，FIP200，ATG5和Atg16L1 CKO小鼠的缺失导致了NSCs的p62聚集，而ATG5和Atg16L1的缺失没有。FIP200和P62双重CKO小鼠的产生和分析表明，P62聚集体的优先形成在触发超氧化物歧化增加导致神经干细胞缺陷的过程中发挥了关键作用，主要是通过损害SOD功能。此外，我们还发现，敲除Atg13(自噬诱导复合体中的FIP200伴侣)在体外抑制了NSC的自我更新，并确定了FIP200中与Atg13结合所需的582-585残基。然后，我们创建了一个新的FIP200敲入突变小鼠，其FIP200x等位基因与Atg13缺乏结合，并表明Ki/KI MEF在基础自噬和饥饿诱导的自噬中都存在缺陷。最后，对不同小鼠神经球的微阵列分析显示，当FIP200缺失时，多种与炎症相关的基因表达增加，而这些基因并不能通过P53失活来挽救。此外，在FIP200 CKO小鼠以及FIP200和P53双重CKO小鼠的SVZ中发现了更多的小胶质细胞渗透，这表明渗透的小胶质细胞可能是FIP200缺失后NSCs非P53依赖的异常分化的原因。基于这些初步研究和以前的研究，我们建议1)。研究p62聚集体在自噬调节NSC中的作用机制，2)。分析FIP200介导的自噬在神经干细胞调控中的作用。探讨小胶质细胞浸润增加在FIP200诱导的神经干细胞异常分化中的作用和机制。总之，这些研究将极大地促进我们对NSC自噬调节机制的理解，这可能有助于未来设计更有效的治疗神经退行性疾病和其他相关疾病的方法。

 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.