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底物。 我们将建立人类ANK2基因从头突变的小鼠模型，ANK2是为数不多的与 没有智力残疾、发育迟缓或癫痫发作的自闭症。ANK2编码220 kDa 广泛表达的ankyrin-B(AnkB)和仅在神经元中发现并定位的440 kDa ankB 无髓鞘轴突和生长锥体。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。我们已经产生了模拟人类死亡的小鼠 Novo ANK2移码突变，选择性耗尽440 kDa ankB。我们将探讨这样的假设 440 kDa ankB的突变/缺失通过选择性地干扰局部和远程神经连接 改变轴突功能如下。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，并建立一种细胞检测方法来评估人类440 kDa AnkB变种。缺乏440 kDa ankB的小鼠培养的神经元显示轴突长度增加和 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。我们也会 制定救援策略以评估人类440 kDa ankB变异对轴突的功能后果 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 增加皮质突触发生以及失去与细胞黏附分子L1CAM的相互作用。自.以来 L1CAM需要骨架蛋白结合活性来进行长距离轴突寻路，我们假设440 kDa AnkB缺陷神经元将表现出异常的轴突靶向。我们将评估局部和远程目标 用a)扩散张量成像(DTI)b)成像研究440 kDa ankB缺陷小鼠发育过程中轴突的分布 立体定向注射AAV GFP/Cherry结构稀疏标记的皮质神经元轴突；c) 用免疫荧光和免疫荧光技术分析发育中兴奋性和抑制性前脑突触 电生理学。3.确定440 kDa ankB突变小鼠的可复制行为表型。在……里面 与William Wetsel博士(杜克大学小鼠行为和神经内分泌分析主任)合作 核心设施)和Henry Yen(杜克大学心理和神经科学系)以及共同研究人员 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，我们将与尹鸿龄合作，利用计算机辅助分析3D成像来开发一种 不偏不倚的可重现的行为模式。对从头ANK2的细胞和体内影响的研究 突变有望提供对高功能自闭症形式的第一个机械性洞察。

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.