Epigenetic Regulation of Adult Hippocampal Neurogenesis

了解成人大脑具有在一生中产生新神经元的先天能力，将成人神经发生的研究推向了科学研究的前沿。虽然许多因素，如衰老和运动，已被确定为调控成年海马神经发生，但目前我们对细胞自主调节海马神经前体发育从而促进海马神经发生的分子机制尚缺乏了解。由于表观遗传学已经成为整合外部信号以确定内在基因表达的分子界面，我们的长期目标是了解表观遗传学机制是如何细胞自主地调控成年海马神经发生的。基于我们最近对组蛋白乙酰转移酶CBP(CREB结合蛋白)在神经干细胞生物学调控中的作用的研究成果，我们假设CBP以细胞自主和CREB结合依赖的方式在体内调节成人神经前体的发育，最终促进成年海马神经发生和海马依赖记忆。为了验证这一假设，我们将追求以下三个目标。目的1：确定CBP在体内调节成体神经前体发育的细胞自主作用。我们将使用可诱导的条件性CBP基因敲除小鼠来确定CBP调控的海马神经前体发育的各个阶段，以及CBP对海马神经发生和海马依赖记忆的贡献。目的：确定CREB结合在CBP调控的成体神经前体发育中的重要性。我们将使用CBPKIX小鼠(缺乏结合CREB的能力)来评估CREB结合对CBP介导的海马神经前体在体内发育的重要性及其对海马神经发生和海马依赖记忆的贡献。目的3：确定CBP对成年海马神经前体细胞Ser436磷酸化的下游靶点。我们将使用CBPS436A小鼠(在Ser436缺乏非典型的蛋白激酶C介导的磷酸化)来鉴定成年海马神经前体中aPKC-CBP途径的直接下游靶点，因为我们最近的研究表明，aPKC-CBP途径对于整个成年期维持动态平衡的神经发生是必不可少的。为此，我们将进行激光帽显微切割，分离含有丰富神经前体的海马体颗粒下带(SGZ)组织，进行RNA测序和染色质免疫沉淀分析(ChIP)。两种检测方法的结合将使我们能够确定CBP S436磷酸化直接调控的靶基因。阐明这种表观遗传机制将促进我们对海马可塑性的分子基础的理解，包括海马神经发生和学习记忆。

Knowing the adult human brain has the innate ability to produce new neurons throughout life has propelled the study of adult neurogenesis to the forefront of scientific research. While numerous factors, such as aging and exercise, have been identified to regulate adult hippocampal neurogenesis, there is currently a gap in our knowledge with respect to the molecular mechanisms that cell-autonomously modulate hippocampal neural precursor development to contribute to hippocampal neurogenesis. Since epigenetics has emerged as a molecular interface that integrates extrinsic signals to determine intrinsic gene expressions, our long term goal is to understand how epigenetic mechanisms cell-autonomously regulate adult hippocampal neurogenesis. On the basis of our recent fruitful research regarding the role of a histone acetyltransferase, CBP (CREB binding protein) in regulating neural stem cell biology, we hypothesize that CBP regulates adult neural precursor development in vivo in a cell-autonomous and CREB binding-dependent manner, ultimately contributing to adult hippocampal neurogenesis and hippocampal-dependent memory. To test the hypothesis, we will pursue the following three aims. Aim 1: Determine the cell-autonomous effect of CBP in regulating adult neural precursor development in vivo. We will use inducible conditional CBP knock-out mouse lines to identify the stages of hippocampal neural precursor development that are regulated by CBP and its contribution to hippocampal neurogenesis and hippocampal-dependent memory. Aim 2: Determine the importance of CREB binding in CBP-regulated adult neural precursor development in vivo. We will use CBPKIX mouse (lacking the ability to bind CREB) to assess the importance of CREB binding for CBP-mediated hippocampal neural precursor development in vivo and its contribution to hippocampal neurogenesis and hippocampal-dependent memory. Aim 3: Identify downstream targets of CBP upon Ser436 phosphorylation in adult hippocampal neural precursors. We will use CBPS436A mouse (lacking the atypical protein kinase C-mediated phosphorylation at Ser436) to identify the direct downstream targets of the aPKC-CBP pathway in adult hippocampal neural precursors, since our recent study shows that the aPKC-CBP pathway is essential to maintain homeostatic neurogenesis throughout adulthood. To do that, we will perform Laser Cap Microdissection to isolate hippocampal subgranular zone (SGZ) tissues, which contain enriched neural precursors, to conduct RNA-sequencing analysis and Chromatin immunoprecipitation assay (ChIP). The combination of both assays will allow us to identify the target genes that are directly regulated by CBP S436 phosphorylation. Elucidation of such epigenetic mechanisms will advance our understanding of the molecular basis of hippocampal plasticity including hippocampal neurogenesis and learning and memory.