Identifying cortical circuit changes in Akap11 mutant mice

项目摘要/摘要 开发有效治疗精神分裂症(SCZ)等精神疾病的主要障碍 双相情感障碍(BD)是指缺乏明确的潜在神经生物学机制。多条证据 来自人类研究和尸检组织的研究表明，包括细胞类型在内的皮质回路发生了变化 构成以及局部和远程突触连接。然而，这一假设很难检验。 直接由于缺乏对大脑皮层回路进行系统和公正分析的工具，缺乏强有力的 可以在老鼠身上模拟的遗传风险因素。在这项提案中，我们将开发标准化方法来 使用空间转录学和病毒突触追踪相结合的方法测量大脑皮质回路。我们将应用这一点 通过最近的大规模外显子组测序研究确定的基因的方法，这些研究已经确定了罕见的丢失- 功能突变与疾病密切相关。在与BD和SCZ相关的基因中， Akap11，其基因产物调节PKA，一种控制神经元转录的中央信号分子 和可塑性，以及GSK3β，建议在BD中进行锂治疗。我们将使用多路传输的错误稳健 用荧光原位杂交技术对皮质细胞及其受体的转录特性进行分类 Akap11突变小鼠的板层分布及其改变方式(目标1)。我们还将优化条件 用于使用复制缺陷狂犬病病毒的远程和局部突触跟踪，以探索 Akap11突变小鼠的突触连接(目标2)。狂犬病突触输入的局部标记将是 结合MerFish-Based来识别细胞类型的特定连接性变化。这些实验将 确定Akap11缺失导致的细胞组成和连接性的变化，并提出假设 疾病背后的电路机制。重要的是，这项提议还将建立一条分析管道 用于表征可应用于许多疾病相关变种的大脑皮层回路。未来 使用这一管道的研究将确定跨高置信度风险因素的汇聚电路机制， 最有可能导致疾病，并为新疗法提供有希望的靶点。

Project Summary/Abstract A major barrier towards developing effective therapeutics for psychiatric diseases like schizophrenia (SCZ) and bipolar disorder (BD) is the lack of a clear underlying neurobiological mechanism. Multiple lines of evidence from human studies and post-mortem tissue implicate changes to the cortical circuit, including cell-type composition and local and long-range synaptic connectivity. However, this hypothesis has been difficult to test directly due to a lack of tools to carry out systematic and unbiased analyses of cortical circuitry a lack of strong genetic risk factors that can be modeled in mice and. In this proposal, we will develop standardized methods to measure the cortical circuit using spatial transcriptomics combined with viral synaptic tracing. We will apply this method to genes identified by recent, large-scale exome sequencing studies that have identified rare loss-of- function mutations strongly associated with disease. Among those genes implicated in both BD and SCZ is Akap11, whose gene product regulates PKA, a central signaling molecule that controls neuronal transcription and plasticity, and GSK3β, the proposed target of lithium treatment in BD. We will use multiplexed error-robust fluorescent in situ hybridization (merFISH) to classify the transcriptional identity of cortical cells and their laminar distribution and how they are altered in Akap11 mutant mice (Aim 1). We will also optimize conditions for both long-range and local synaptic tracing using replication-defective rabies viruses to explore alterations to synaptic connectivity in Akap11 mutant mice (Aim 2). Local labeling of synaptic inputs with rabies will be combined with merFISH-based to identify cell-type specific changes in connectivity. These experiments will identify changes in cellular composition and connectivity caused by loss of Akap11 and generate hypotheses on the circuit mechanisms underlying disease. Importantly, this proposal will also establish an analysis pipeline for characterizing the cortical circuit that can be applied across many disease-associated variants. Future studies using this pipeline will identify convergent circuit mechanisms across high-confidence risk factors that are most likely to cause disease and provide promising targets for new therapies.