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，海马齿状回(DG)是大脑中一种独特的结构。持续的成人神经发生，即神经干细胞(NSCs)产生和整合新神经元，保证了个人学习和适应不断变化的环境的能力。虽然神经发生是DG高水平可塑性的主要原因，但最近的数据表明，小生境星形胶质细胞的积极参与。成年神经干细胞也会产生星形胶质细胞，尽管数量比神经元少。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，成年海马体中神经和星形细胞发生之间的平衡也保持不变。这些发现暗示着存在一种严格调控成年神经干细胞命运决定的机制，并引发了这样一个问题：哪些因素对控制神经元与星形胶质细胞的比例起作用？转录因子Sox9在神经干细胞发育过程中作为神经元/神经胶质细胞开关的主要调节者出现。为了揭示成体神经发生和星形胶质细胞之间的平衡机制，我将利用遗传小鼠模型，分别在成年海马神经干细胞中缺失和过度表达Sox9，并根据形态和免疫组织学标准分析新生细胞的特性。为了揭示Sox9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Sox9缺失和过表达的动物，通过比较RNA测序方法确定Sox9的下游靶点。随后的体外实验旨在通过转导下游靶向shRNA或过表达的NSCs来确认和挽救Sox9操纵所介导的效应。除了NSC的内在调节外，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星形胶质细胞？在这里，我的目标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。我预计Sox9的过度表达会增加星形发生的发生。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，而不是以新生神经元为代价。随后，将通过应用多西环素将Sox9的表达恢复到野生型水平。利用这个系统，我将研究星形胶质细胞对DG的过度填充是否会影响海马神经干细胞的细胞命运决定。我的研究将极大地促进我们对控制成年NSC行为从而控制海马可塑性的内在和外在调控机制的理解。这为更好地了解神经胶质和神经病理表型及其对脑疾病的贡献提供了重要的前提条件。

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.