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

海马体是大脑中不断产生新的齿状颗粒细胞(GC)的区域。 在一生中。成年出生的GC的发育和它们与先前存在的回路的整合受 环境和局部电路的电活动，并在老化的大脑中改变。要了解如何 GC整合的发生以及它是如何受到活动和衰老的调制的，剖析精确的 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，但经常在哈佛的阿洛塔实验室应用。正在加入 解决这一具体问题的努力是自然的下一步。这项提议的首要目标是利用 脑发育中转录图谱和生物信息学分析的策略得到了验证 Arlotta实验室和Schinder实验室在成人出生的GC功能表征方面的专业知识，以建立 发现控制成人和衰老电路可塑性的新分子的实验流水线 大脑，这包括测试单个蛋白质的作用达到前所未有的分子水平 和功能细节。在目标1中，我们建议生成一个管道来揭示转录因子，表观遗传 调节基因，或控制GC成熟和整合过程中发育过渡的效应基因。 拟议的实验流水线对于所有三个目标都是相似的：(I)FACS-净化出生日期成年出生的GC 不同的阶段；(2)使用两种互补形式的RNA转录描述每个种群 测序；(Iii)生物信息识别可能控制阶段的转录因子或表观遗传调控因子 通过体内敲除或过表达对候选分子进行功能测试。 使用同样的方法，然后我们将研究特定阶段的转录组动力学是如何在 从老化的海马体中提取GCS，以识别并从功能上测试可能是 对他们的长期发展负责(目标2)。最后，我们将寻求确定分子介体 活动介导的GC在成人和衰老大脑中发育的加速。(目标3)。这笔赠款将 进一步说明Fogarty国际中心扩大Schinder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，以及 在Leloir和哈佛研究界成员之间建立合作网络。这项工作将 显著扩展Leloir的能力，以应用最先进的转录简档(目前有限， 整个阿根廷)，以解决与大脑功能和疾病有关的新问题，在当地得到一个 生物信息学家，该项目的联合调查员。因此，拟议的工作流程不仅将使 通过应用最先进的分子工具在迅达实验室进行新一代分子研究 而且还通过人员培训来转让专业知识和诀窍，从而使这些 技术和方法将在Leloir研究所广泛用于日常使用。

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.