Cannabinoid Signaling Interactions During Axon Development in situ

摘要 大麻植物大麻和大麻是妇女最常使用的滥用物质。 生育年龄。但是，令人担忧的是，孕妇频繁使用大麻可能会导致持久的 在宫内暴露的儿童的认知和神经行为问题。它们的作用机制 大麻植物中的大麻素对发育中的大脑结构和功能的影响不是很好 明白了。先前在动物模型中的研究表明，主要的大麻素受体CB1R影响 通过各种分子因子，如Netrin受体DCC， PKA和RhoA。然而，我们对CB1R的基本机制缺乏全面的了解 在大脑发育过程中影响神经元回路的形成。多年来，我们的实验室确定 Wnt和粘附素因子-catenin在视觉投射发育中的信号机制 非洲爪蛙模型脊椎动物的蝌蚪。这是研究神经元回路的理想系统。 形成，因为它与人类有很强的遗传相似性，易于分子和基因操作 单个视网膜神经节细胞和分子信号改变的单个视网膜神经节细胞的成像 直接在它们的原生环境中。我们现在建议确定CB1R是否抑制Wnt/Caherin/- 连环蛋白信号调节生长锥、丝状足突和轴突的原位寻路功能。这项建议 来源于其他研究表明CB1R抑制WNT并破坏癌细胞中-连环蛋白的稳定，以及 Wnt介导的-连环蛋白失稳下调粘附素细胞与细胞间的黏附，以及我们最近的研究 已发表的论文显示CB1R和-连环蛋白相反地调节视网膜中的生长锥丝状足孔 神经节细胞。我们将测试两个目标：1)量化轴突寻路和生长锥体缺陷 CB1R的原位操作。我们之前公布的数据表明，药物操纵 CB1R干扰生长锥丝状足突和轴突的原位丛生。我们现在将评估细胞是否- 单个视网膜神经节细胞CB1R自主(基于吗啡)功能丧失改变多发性 生长锥体和轴突寻径参数。2)建立CB1R和CB1R之间的功能相互作用 视网膜神经节细胞中的WNT/钙粘连蛋白信号。我们将确定CB1R缺失的表型效应- 在生长锥丝状足突和轴突寻路特征上的功能缺失是通过在 典型的Wnt信号通路(Axin，APC)及其细胞-细胞黏附关键分子突变体 复合体(-连环蛋白、-连环蛋白)。这些数据将决定一种新的和必要的信号机制 大麻素在脊椎动物模型神经元回路发育中的作用。考虑到这些信号的保守性 这些结果也可能为内源性大麻素的形成建立一个基本的机制。 人类胎儿的神经元回路，以及增加的产前大麻暴露如何扰乱 神经回路的建立，并导致儿童持续的认知和神经行为缺陷。

Abstract The cannabis plants marijuana and hashish are the most commonly used substances of abuse by women of reproductive age. But, concerningly, frequent use of cannabis by pregnant women may result in lasting cognitive and neuro-behavioral issues in children that were exposed in utero. The mechanisms by which cannabinoids in cannabis plants influence the structure and function of the developing brain are not well understood. Previous studies in animal models indicate that the main cannabinoid receptor, CB1R, affects formation of neuronal circuits by signaling though various molecular factors such as the Netrin receptor DCC, PKA and RhoA. However, we lack comprehensive knowledge of essential mechanisms by which CB1R influences neuronal circuit formation during brain development. For many years, our laboratory determined signaling mechanisms for Wnt and Cadherin factor, -catenin, in development of the visual projection in tadpoles of the vertebrate frog model Xenopus laevis. This is an ideal system for studying neuronal circuit formation because of its strong genetic similarity to humans, amenability to molecular and genetic manipulation in single retinal ganglion cells and imaging of individual retinal ganglion cells with altered molecular signaling directly in their native environment. We now propose to determine whether CB1R inhibits Wnt/Cadherin/- catenin signaling to regulate growth cone filopodia and axon pathfinding features in situ. This proposal stems from work from others showing that CB1R inhibits Wnt and destabilizes -catenin in cancer cells, and Wnt mediated destabilization of -catenin downregulates Cadherin cell-cell adhesion, and our recently published paper showing that CB1R and -catenin oppositely modulate growth cone filopodia in retinal ganglion cells. We will test two aims: 1) Quantify axon pathfinding and growth cone defects following manipulation of CB1R in situ. Our previously published data showed that pharmacological manipulation of CB1R perturbs growth cone filopodia and axon fasciculation in situ. We will now assess whether cell- autonomous (morpholino based) loss-of-function of CB1R in individual retinal ganglion cells alters multiple growth cone and axon pathfinding parameters. 2) Establish functional interactions between CB1R and Wnt/Cadherin signaling in retinal ganglion cells. We will determine whether phenotypic effects of CB1R loss- of-function on growth cone filopodia and axon pathfinding features are rescued by expression of factors in the canonical Wnt signaling pathway (Axin, APC) and mutants of key players in Cadherin cell-cell adhesive complex (-catenin, -catenin). This data will determine a novel and essential signaling mechanism for cannabinoids in neuronal circuit development in a vertebrate model. Given the conservation of these signaling pathways, these results may also establish a fundamental mechanism for endocannabinoids in formation of neuronal circuits in human fetuses, and for how increased prenatal cannabis exposure could disrupt establishment of neuronal circuits, and lead to persistent cognitive and neurobehavioral deficits in children.