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

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ANK 2从头突变的小鼠模型，ANK 2是少数与ANK 2相关的基因之一。 没有智力障碍、发育迟缓或癫痫发作的自闭症。ANK 2编码220 kDa 广泛表达的ankB-B（ankB）和仅在神经元中发现并定位于神经元的440 kDa ankB 到无髓鞘轴突和生长锥。人类新生突变的分布表明， 440 kDa的ankB足以导致高功能自闭症。我们制造了模拟人类死亡的老鼠， novo ANK 2移码突变，选择性地消耗440 kDa ankB。我们将探讨一个假设， 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.解决增加轴突生长/分支的分子基础 在440 kDa ankB突变体神经元中观察到，并建立细胞测定法以评估人440 kDa ankB突变体神经元中的 ankB变体。从缺乏440 kDa ankB的小鼠培养的神经元表现出轴突长度增加， 分支我们将探讨细胞骨架动力学和胞吐作用在这一现象中的作用。我们也将 开发拯救策略，以评估轴突上440 kDa ankB的人类变体的功能后果 生长/分支。2.确定440 kDa ankB突变对建立局部和 长距离神经连接在出生后的大脑发育。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L1 CAM的相互作用丧失。以来 L1 CAM需要锚定结合活性来进行长距离轴突寻路，我们假设440 kDa的L1 CAM是一种具有锚定结合活性的蛋白。 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，在440 kDa ankB缺陷小鼠中发育期间的轴突 c）通过立体定向注射AAV GFP/Cherry构建体稀疏标记的皮质神经元的轴突; 使用免疫荧光和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突变小鼠的可重现行为表型。在 与William Wetsel博士（杜克小鼠行为和神经内分泌分析主任）合作 核心设施）和亨利尹（杜克大学心理学和神经科学系）和共同研究者对此 建议，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，在与亨利尹合作，我们将使用计算机辅助分析的三维成像，以开发一个 无偏见的可重复的行为特征。这些研究的细胞和体内后果从头ANK 2 突变有望为自闭症的高功能形式提供第一个机械的见解。

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.