Molecular principles of neuronal maturation and integration in the adult and aging brain

海马体是大脑中持续产生新的齿状颗粒细胞的区域 在生活中。成人出生的GC的发育及其与既存回路的整合受到以下因素的调节： 环境和局部电路的电活动，并在老化的大脑中改变。了解如何 GC整合的发生以及它是如何被活动和衰老调节的，关键是要分析GC整合的精确机制。 这些过程背后的分子机制。解决这些问题所需的技术 基本问题在阿根廷是不存在的，但在哈佛的阿洛塔实验室经常应用。加入 解决这一具体问题的努力是自然的下一步。该提案的总体目标是利用 转录谱分析和生物信息学分析在大脑发育中的策略， Arlotta实验室和Schinder实验室在成人出生GC功能表征方面的专业知识， 用于发现控制成人和衰老回路可塑性的新分子的实验管道 大脑，其中包括用于测试单个蛋白质的作用，以前所未有的分子水平的测定 功能细节。在目标1中，我们提出了一个管道，以揭示转录因子，表观遗传， 调控因子或效应基因控制沿着GC成熟和整合的发育转变。 所提出的实验管道对于所有三个目标是类似的：（i）FACS-纯化出生日期为成人出生的GC， 不同阶段;（ii）使用两种互补形式的RNA对每个群体进行转录分析 测序;（iii）生物信息学鉴定可控制阶段的转录因子或表观遗传调节因子 进展;和（iv）通过体内敲低或过表达功能性测试候选分子。 使用同样的方法，我们将研究阶段特异性转录组动力学是如何改变的， 从老化的海马体中提取GC，以识别和功能测试调节分子的变化， 负责其长期发展（目标2）。最后，我们将寻求确定分子介质的 活动介导的加速成人和衰老大脑中GC的发育。(Aim 3）。这笔赠款将 进一步实现了福格蒂国际中心扩大辛德博士实验室技术能力的目标， 包括提供设备，培训阿根廷研究生和博士后研究员， 在Leloir和哈佛研究团体成员之间建立学院网络。这项工作将 显著扩大Leloir应用最先进的转录组学分析的能力（目前有限， 在阿根廷各地），以解决与脑功能和疾病有关的新问题，由当地的 生物信息学家，他是该项目的共同研究者。因此，拟议的工作流程不仅可以 通过使用最先进的分子工具，在Schinder实验室进行新一代分子研究 和技术，而且还通过人员培训转让专门知识和专门技能， 技术和方法将在整个Leloir研究所日常使用。

The hippocampus is a region of the brain that continues to produce new dentate granule cells (GCs) throughout life. Development of adult-born GCs and their integration into preexisting circuits is modulated by environment and by electrical activity of the local circuits, and is altered in the aging brain. To understand how GC integration occurs and how it is modulated by activity and aging, it is crucial to dissect the precise molecular mechanisms underlying these processes. The technology required for addressing these fundamental problems is unavailable in Argentina, but routinely applied in the Arlotta lab at Harvard. Joining efforts to address this specific problem is a natural next step. The overarching goal of this proposal is to exploit the strategies for transcriptional profiling and bioinformatic analysis in brain development validated by the Arlotta lab and the Schinder lab's expertise in functional characterization of adult-born GCs to build an experimental pipeline for the discovery of new molecules controlling circuit plasticity in the adult and aging brain, that includes assays for testing the roles of individual proteins to an unprecedented level of molecular and functional detail. In Aim 1, we propose to generate a pipeline to reveal transcription factors, epigenetic regulators, or effector genes controlling the developmental transitions along GC maturation and integration. The proposed experimental pipeline is similar for all three Aims: (i) FACS-purify birth-dated adult-born GCs at different stages; (ii) transcriptionally profile each population using two complementary forms of RNA sequencing; (iii) bioinformatically identify transcription factors or epigenetic regulators that may control stage progression; and (iv) functionally test candidate molecules through in vivo knock-down or overexpression. Using this same approach, we will then investigate how stage-specific transcriptome dynamics are altered in GCs from the aging hippocampus, to identify and functionally test changes in regulatory molecules that may be responsible for their protracted development (Aim 2). Finally, we will seek to identify molecular mediators of activity-mediated acceleration in GC development in the adult and the aging brain. (Aim 3). This grant will further the aims of the Fogarty International Center in expanding the technical capacities of Dr Schinder's lab, including availability of equipment, training of Argentinian graduate students and postdoctoral fellows, and building collegial networks between members of the Leloir and Harvard research communities. This work will significantly expand the capacity of Leloir to apply state-of-the-art transcriptomic profiling (currently limited, throughout Argentina) to solve new problems related to brain function and disease, locally supported by a bioinformatician who is co-investigator in the project. The workflow proposed will therefore not only enable a new generation of molecular studies in the Schinder lab by implementing the most advanced molecular tools and technologies, but also transfer expertise and know-how through personnel training such that these technologies and approaches will become available for routine use at the Leloir Institute at large.