Cannabinoid Signaling Interactions During Axon Development in situ

摘要 大麻植物、大麻和印度大麻脂是1999年妇女最常用的滥用物质， 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 认知和神经行为方面的问题。的机制 大麻植物中的大麻素对发育中的大脑结构和功能的影响并不好 明白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，我们缺乏对CB1R的基本机制的全面了解， 影响大脑发育过程中神经回路的形成。多年来，我们的实验室确定 Wnt和钙粘蛋白因子β-连环蛋白在视觉投射发育中的信号机制 脊椎动物蛙模型非洲爪蟾的蝌蚪。这是研究神经元回路的理想系统 由于其与人类的遗传相似性很强，易于进行分子和遗传操作， 在单个视网膜神经节细胞中和具有改变的分子信号传导的单个视网膜神经节细胞的成像中 直接在他们的原生环境中。我们现在建议确定CB1R是否抑制Wnt/钙粘蛋白/β-CD。 连环蛋白信号传导原位调节生长锥丝状伪足和轴突寻路特征。这项建议 源于其他人的工作，表明CB1R抑制Wnt并使癌细胞中的β-连环蛋白不稳定， Wnt介导的β-catenin不稳定下调钙粘蛋白细胞间粘附，我们最近发现， 已发表的论文表明，CB1R和β-连环蛋白相反地调节视网膜中生长锥丝状伪足， 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缺陷， CB1R的原位操作。我们以前发表的数据表明，药物操纵 CB1R干扰生长锥丝状伪足和轴突成束原位。我们现在要评估细胞是否- 在个体视网膜神经节细胞中CB1R的自主（基于吗啉代）功能丧失改变了多个 生长锥和轴突寻路参数。2)建立CB1R和 视网膜神经节细胞中的Wnt/钙粘蛋白信号传导。我们将确定CB1R缺失的表型效应- 在生长锥丝状伪足和轴突寻路特征上的of-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-functionsof-f 经典Wnt信号通路（Axin，APC）和钙粘蛋白细胞-细胞粘附中关键参与者的突变体 复合物（β-catenin，β-catenin）。这些数据将确定一种新的和必要的信号传导机制， 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 这些结果也可能建立内源性大麻素形成的基本机制， 人类胎儿的神经元回路，以及产前大麻暴露的增加如何破坏 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.