Deciphering high function autism using mice with human de novo ANK2 mutations

使用携带人类 ANK2 新突变的小鼠解读高功能自闭症

基本信息

批准号：
9429362
负责人：
G Vann Bennett
金额：
$ 23.88万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-18 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9429362
关键词：
ANK2 gene Ankyrins Arrhythmia Autistic Disorder Axon Behavior Behavioral Binding Brain Brain region Cell Adhesion Molecules Cellular Assay Cellular biology Cherry - dietary Cognition Collaborations Computer Assisted Core Facility Data Development Developmental Delay Disorders Diffusion Magnetic Resonance Imaging Electrophysiology (science)Exhibits Exocytosis Exons Frameshift Mutation Genes Goals Growth Cones Human Image Image Analysis Immunofluorescence Immunologic Injection of therapeutic agent Intellectual functioning disability Label Length Metabolic syndrome Missense Mutation Modeling Molecular Motor Mouse Strains Mus Mutant Strains Mice Mutation Nervous system structure Neural Cell Adhesion Molecule L1 Neurons Neurosciences Neurosecretory Systems Nonsense Mutation Phenotype Prosencephalon Psychology Reproducibility Research Research Personnel Resources Role Seizures Sensory Signal Transduction Social Behavior Symptoms Synapses Three-dimensional analysis Variant Yin anxiety-like behavior autism spectrum disorder axon growth base brain morphology cell type conditional mutant human data in vivo insight knock-down mouse model mutant myelination polypeptide postnatal premature relating to nervous system synaptogenesis

项目摘要

The long-term goal of this research is to resolve molecular and cellular substrates for high-functioning autism. We will develop mouse models for human de novo mutations in ANK2, one of the few genes associated with autism in the absence of intellectual disability, developmental delay, or seizures. ANK2 encodes 220 kDa ankyrin-B (ankB) which is widely expressed, and 440 kDa ankB which is found only in neurons and is localized to unmyelinated axons and growth cones. The distribution of human de novo mutations suggests that mutation of 440 kDa ankB is sufficient to cause high function autism. We have generated mice modeling a human de novo ANK2 frameshift mutation that selectively depletes 440 kDa ankB. We will explore the hypothesis that mutation/deficiency of 440 kDa ankB selectively perturbs both local and long-range neural connectivity through altered axonal function as follows. 1. Resolve the molecular basis for increased axon growth/branching observed in 440 kDa ankB mutant neurons, and establish a cellular assay to evaluate human 440 kDa ankB variants. Neurons cultured from mice lacking 440 kDa ankB exhibit increased axon length and branching. We will explore the roles in this phenomenon of cytoskeletal dynamics and exocytosis. We also will develop a rescue strategy to evaluate functional consequences of human variants of 440 kDa ankB on axon growth/branching. 2. Determine consequences of 440 kDa ankB mutation for establishment of local and long-range neural connectivity during postnatal brain development. 440 kDa ankB-deficient mice exhibit increased cortical synaptogenesis as well as loss of interaction with the cell adhesion molecule L1CAM. Since L1CAM requires ankyrin-binding activity for long distance axon pathfinding, we hypothesize that 440 kDa ankB-deficient neurons will exhibit abnormal axon targeting. We will evaluate local and long-range targeting of axons of during development in 440 kDa ankB-deficient mice using a) diffusion tensor imaging (DTI) b) imaging of axons of cortical neurons sparsely labeled by stereotactic injection of AAV GFP/Cherry constructs; c) analysis of excitatory and inhibitory forebrain synapses during development using immunofluorescence and electrophysiology. 3. Determine a reproducible behavioral phenotype for 440 kDa ankB mutant mice. In collaboration with Drs. William Wetsel (Director of the Duke Mouse Behavioral and Neuroendocrine Analysis Core Facility) and Henry Yin (Duke Department of Psychology and Neuroscience) and co-investigators on this proposal, we will evaluate social behavior, cognition, sensory-motor gating, and repetitive/anxiety-like behavior. In addition, in collaboration with Henry Yin, we will use computer-assisted analysis of 3D imaging to develop an unbiased reproducible behavior profile. These studies of cellular and in vivo consequences of de novo ANK2 mutation promise to provide the first mechanistic insights into high functioning forms of autism.

这项研究的长期目标是解决高功能自闭症的分子和细胞基质。我们将开发人类 ANK2 新生突变的小鼠模型，ANK2 是与 ANK2 相关的少数基因之一。不存在智力障碍、发育迟缓或癫痫发作的自闭症。 ANK2 编码 220 kDa 锚蛋白-B (ankB) 广泛表达，440 kDa ankB 仅在神经元中发现且定位无髓鞘轴突和生长锥。人类新生突变的分布表明，突变 440 kDa ankB 足以引起高功能自闭症。我们已经生成了模拟人类 de 的小鼠 novo ANK2 移码突变选择性耗尽 440 kDa ankB。我们将探讨以下假设： 440 kDa ankB 的突变/缺陷通过以下方式选择性地扰乱局部和远程神经连接轴突功能改变如下。 1. 解决轴突生长/分支增加的分子基础在 440 kDa ankB 突变神经元中观察到，并建立细胞测定法来评估人类 440 kDa ankB 变体。从缺乏 440 kDa ankB 的小鼠中培养的神经元表现出轴突长度增加和分枝。我们将探讨细胞骨架动力学和胞吐作用在这种现象中的作用。我们也会制定救援策略来评估 440 kDa ankB 人类变体对轴突的功能影响生长/分枝。 2. 确定 440 kDa ankB 突变对建立局部和出生后大脑发育过程中的远程神经连接。 440 kDa ankB 缺陷小鼠表现出皮质突触发生增加以及与细胞粘附分子 L1CAM 相互作用的丧失。自从 L1CAM 需要锚蛋白结合活性来进行长距离轴突寻路，我们假设 440 kDa ankB 缺陷的神经元将表现出异常的轴突靶向。我们将评估本地和远程目标使用 a) 扩散张量成像 (DTI) b) 成像观察 440 kDa ankB 缺陷小鼠发育过程中的轴突通过立体定向注射 AAV GFP/Cherry 构建体稀疏标记的皮质神经元轴突； c) 使用免疫荧光分析发育过程中的兴奋性和抑制性前脑突触电生理学。 3. 确定 440 kDa ankB 突变小鼠的可重复行为表型。在与博士的合作。 William Wetsel（杜克大学小鼠行为和神经内分泌分析主任核心设施）和亨利·尹（杜克大学心理学和神经科学系）以及共同研究人员建议中，我们将评估社会行为、认知、感觉运动门控和重复/焦虑样行为。此外，我们将与 Henry Yin 合作，利用 3D 成像的计算机辅助分析来开发无偏见的可重复行为概况。这些关于从头 ANK2 的细胞和体内后果的研究突变有望为高功能自闭症形式提供第一个机制见解。