From Sox9 to astrocytes - Intrinsic and extrinsic influences that regulate the “neuron/glial switch” in adult neural stem cells of the hippocampus

由于其连通性和可塑性，海马的齿状回（DG）是大脑中独特的结构。持续的成人神经发生，即神经干细胞（NSC）产生和整合新神经元，保证了个体学习和适应不断变化的环境的能力。虽然神经发生主要是负责高水平的可塑性DG，最近的数据表明，一个积极参与的小生境星形胶质细胞。成体NSC也产生星形胶质细胞，尽管数量低于神经元。有趣的是，我们小组的初步工作显示，即使在自愿跑步等刺激下，成年海马体中神经发生和星形发生之间的平衡也保持不变。这些发现意味着存在一种机制，严格调节成人神经干细胞的命运决定，并导致问题，哪些因素是负责控制神经元星形胶质细胞的比例？转录因子Sox 9在发育过程中成为NSC中神经元/神经胶质开关的主调节因子。为了揭示成年神经发生和星形细胞发生之间的平衡是介导的机制，我将使用遗传小鼠模型，删除和过表达Sox 9在成年海马神经干细胞，分别和分析的身份，新产生的细胞的形态学和免疫组化标准。为了揭示Sox 9在神经干细胞中发挥促星形胶质细胞或抗神经元作用的机制，我将首先使用Sox 9缺失和过表达动物在比较RNA测序方法中鉴定Sox 9的下游靶点。随后的体外实验旨在通过用下游靶向shRNA或过表达构建体转导NSC来确认和拯救由Sox 9操纵介导的作用。除了神经干细胞的内在调节，新生神经元也影响干细胞的行为。这在多大程度上适用于新生的星形胶质细胞？在这里，我的目的是评估新生神经元和星形胶质细胞之间的比例失衡是否会影响海马神经干细胞的命运选择。我认为Sox 9的过度表达会增加星形细胞的形成。因此，我将使用这个系统来增加成年DG中新生星形胶质细胞的数量，以牺牲新生神经元为代价。随后，通过应用强力霉素将Sox 9表达恢复至野生型水平。使用这个系统，我将研究是否过度的DG的星形胶质细胞影响海马神经干细胞的细胞命运的决定。我的研究将显著促进我们对控制成年NSC行为的内在和外在调节机制的理解，从而促进海马可塑性。这为更好地理解神经胶质和神经病理表型及其对脑部疾病的贡献提供了重要的先决条件。

Due to its connectivity and plasticity, the dentate gyrus (DG) of the hippocampus is a unique structure in the brain. Persisting adult neurogenesis, the generation and integration of new neurons by neural stem cells (NSCs), warrants an individual´s ability to learn and adapt to a changing environment. While neurogenesis is predominantly responsible for the high level of plasticity in the DG, recent data indicate an active participation of niche astrocytes. Adult NSCs also generate astrocytes, although in lower numbers than neurons. Interestingly, preliminary work of our group revealed that the balance between neuro- and astrogenesis remains constant in the adult hippocampus even upon stimuli such as voluntary running. These findings imply the existence of a mechanism that tightly regulates the fate decision of adult NSCs and leads to the question, which factors are responsible in controlling the neuron-to-astrocyte ratio? The transcription factor Sox9 emerged as a master-regulator of the neuron/glial switch in NSCs during development. In order to reveal mechanisms by which the balance between adult neurogenesis and astrogenesis is mediated, I will use genetic mouse models to delete and overexpress Sox9 in adult hippocampal NSCs, respectively, and analyze the identity of newly generated cells by morphological and immunohistological criteria. In order to reveal mechanisms by which Sox9 exerts pro-astroglial or anti-neuronal effects in NSCs, I will first identify downstream targets of Sox9 in a comparative RNA-sequencing approach using Sox9 deleted and overexpressing animals. Subsequent in vitro experiments aim to confirm and rescue effects mediated by Sox9 manipulation by transducing NSCs with either downstream target-shRNA or -overexpressing constructs. Apart from NSC-intrinsic regulation, also newborn neurons affect stem cell behavior. To which extend does this apply to newborn astrocytes? Here, I aim to assess if an imbalance in the ratio between newborn neurons and astrocytes affects the fate choice of hippocampal NSCs. I expect an increase in astrogenesis upon overexpression of Sox9. Therefore, I will use this system to increase the number of newborn astrocytes in the adult DG at the expense of newborn neurons. Subsequently, will restore Sox9 expression to wildtype levels by application of Doxycyclin. Using this system, I will study whether overpopulation of the DG by astrocytes affects cell fate decisions of hippocampal NSCs. My research will significantly promote our understanding of intrinsic and extrinsic regulatory mechanism that control adult NSC behaviour and hence hippocampal plasticity. This presents an important prerequisite to better understand glio- and neuropathological phenotypes and their contribution to brain disease.