Mechanisms of Neural Stem Cells Regulation by Autophagy

 描述（由申请人提供）：哺乳动物大脑保持产生新神经元的能力，用于组织稳态和损伤后，这是由神经干细胞/祖细胞（NSC）驱动的。本研究的长期目标是了解神经干细胞自噬途径的分子和细胞机制以及调控神经干细胞自噬的基因。自噬是维持细胞稳态的高度保守的细胞过程，并且自噬功能障碍已经涉及许多疾病，包括神经退行性疾病。尽管近年来对自噬的研究越来越多，但对自噬在组织干细胞如神经干细胞的调控中的作用和机制知之甚少。FIP 200（FAK-family Interacting Protein of 200 kDa）最初是在我们的实验室中鉴定的，后来被证明是自噬诱导所必需的ULK 1/Atg 13/FIP 200复合物的一个组分。最近，我们发现FIP 200的条件性敲除（cKO）导致NSC的耗竭及其异常分化，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Atg 5或Atg 16 L1（自噬体成熟所需的自噬基因）的缺失来抑制自噬并不影响NSC，并且对FIP 200、Atg 5和Atg 16 L1 cKO小鼠的进一步比较分析表明，FIP 200而不是Atg 5和Atg 16 L1的缺失引起NSC中的p62聚集。FIP 200和p62双cKO小鼠的产生和分析表明，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功能导致有缺陷的NSC。此外，我们发现Atg 13（自噬诱导复合物中的FIP 200伴侣）的敲低抑制体外NSC自我更新，并鉴定了FIP 200中与Atg 13结合所需的残基582-585。然后，我们创建了一种新的FIP 200敲入突变小鼠，其含有缺乏与Atg 13结合的FIP 200 x等位基因，并表明KI/KI MEFs在基础和饥饿诱导的自噬中均存在缺陷。最后，来自各种小鼠的神经球的微阵列分析揭示了在FIP 200缺失后与炎症相关的多个基因的表达升高，这些基因不能通过p53失活来挽救。此外，在FIP 200 cKO小鼠以及FIP 200和p53双cKO小鼠的SVZ中发现小胶质细胞浸润增加，这表明一种有趣的可能性，即浸润的小胶质细胞可能是FIP 200缺失后NSC的p53非依赖性异常分化的原因。基于这些初步的和以前的研究，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FIP 200介导的自噬在神经干细胞调控中的作用; 3）.探讨FIP 200消融后小胶质细胞浸润增加在神经干细胞异常分化中的作用和机制。总之，这些研究将大大促进我们对自噬调节NSC机制的理解，这可能有助于未来设计更有效的神经退行性疾病和其他相关疾病的治疗方法。

 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.