Dual roles of Nell-1 in craniofacial bones and brain through interaction with Cntnap4

项目总结 Nell-1因其在颅面部骨骼发育中的成骨作用而被广泛研究。然而，尽管 它在大脑中的高表达，一直以来对Nell-1的S在神经系统中的作用缺乏了解 直到最近发现Nell-1和Cntnap4之间存在类似配体-受体的相互作用。Cntnap4是一个PRE- 突触膜蛋白--全球缺失Cntnap4差异性抑制GABA能输出和增强 多巴胺能传递，并可在小鼠中诱导自闭症谱系障碍(ASD)样行为。值得注意的是， Enu诱导的Nell-1单倍体缺陷小鼠表现出类似于Cntnap4突变体的ASD样行为 老鼠。同时，Cntnap4或Nell-1在脑神经脊细胞(CNCC)中的失活导致 非常相似的头盖骨缺损症。此外，Nell-1和Cntnap4在中国的新的共定位研究 小鼠和人脑显示出广泛的神经组织分布和高水平的共定位表达。 因此，这一建议假设Nell-1在大脑和头面部骨骼(CB)中具有双重作用，通过 Nell-1和Cntnap4之间的一种新的相互作用以及Nell-1/Cntnap4功能轴的破坏将不会 不仅会导致CB缺陷，还会干扰大脑中的神经传递。这项提案推进了三个阶段 目的：利用两种新的牙线系小鼠研究Nell-1/Cntnap4在CB和脑中的功能轴 行、Nell-1fl/fl和Cntnap4fl/fl。目标1将首先确定Nell-1和Cntnap4的组织分布和表达 在野生型(WT)小鼠的正常发育和生长过程中，CB和大脑中都有。然后目标1将具体说明 利用组织特异性基因敲除(KO)研究Nell-1和Cntnap4对CB和脑的功能贡献 1Wnt1KO和Cntnap4Wnt1KO小鼠通过Wnt1-Cre靶向CNCCs来源的CB和三叉神经节(TG)，以及 Nell-1PV-2AKO和Cntnap4PV-2AKO小鼠靶向小白蛋白阳性(PV)GABA能神经元 用PV-2A-Cre测定Tg。这些KO小鼠的CB生长和行为变化将与 受检者的骨骼、脑和颅面神经骨骼界面的形态变化 感觉纤维支配丘脑。目标2将在Cntnap4的背景下破译Nell-1‘S的成骨特性 Nell-1蛋白修复Cntnap4Wnt1KO小鼠CB缺陷的实验研究使用NELL-1作为 作为WNT/β-连环蛋白信号的激动剂，AIM2将决定整合素β1、NELL-1结合伙伴和糖原 合成酶β-3β是β-连环蛋白磷酸化的重要调节因子，对Nell-1/Cntnap4有调节作用 相互作用介导的Wnt/β-catenin信号在CNCC成骨过程中的激活。Aim 3将调查Nell- 1/Cntnap4相互作用对Nell-1PV-2AKO和Cntnap4PV-1PV-2AKO和Cntnap4PV-1PV-2AKO的GABA能传递和ASD样行为的影响 2AKO小鼠。AIM 3将在Nell-1/β-nap4相互作用的背景下进一步评估Wnt/Cntnap4-连环蛋白信号- 介导的GABA释放，以及GSK3β抑制剂与NELL-1可能的相加或协同作用 小鼠神经细胞。影响：最终目标是揭示新发现的Nell-1/Cntnap4相互作用 可能转化为开发Nell-1作为一种治疗人类骨骼和神经病理的新疗法。

PROJECT SUMMARY Nell-1 has been studied extensively for its osteogenic role in craniofacial skeletal development. However, despite its high expression in the brain, there has been a lack of understanding of Nell-1's function in the nervous system until the recent identification of a ligand-receptor like interaction between Nell-1 and Cntnap4. Cntnap4 is a pre- synaptic membrane protein whereby global Cntnap4 loss differentially inhibits GABAergic output and augments dopaminergic transmission, and can induce autism spectrum disorder (ASD)-like behaviors in mice. Remarkably, ENU-induced Nell-1 haploinsufficient mice exhibit ASD-like behaviors similar to those seen in Cntnap4 mutant mice. Meanwhile, inactivation of either Cntnap4 or Nell-1 in cranial neural crest cells (CNCCs) gives rise to remarkably similar calvarial bone defects. Moreover, novel co-localization studies of Nell-1 and Cntnap4 in mouse and human brains revealed broad neural tissue distribution and high levels of co-localized expression. Consequently, this proposal hypothesizes that Nell-1 has dual roles in the brain and craniofacial bones (CB) via a novel interaction between Nell-1 and Cntnap4, and that disruption of the Nell-1/Cntnap4 functional axis will not only induce deficits in CB, but also interfere with neural transmission in the brain. This proposal advances three specific aims to investigate the Nell-1/Cntnap4 functional axis in the CB and brain using two new floxed mouse lines, Nell-1fl/fl and Cntnap4fl/fl. AIM 1 will first define the tissue distribution and expression of Nell-1 and Cntnap4 in both the CB and brain during normal development and growth in wild type (WT) mice. AIM 1 will then specify the functional contributions of Nell-1 and Cntnap4 to the CB and brain using tissue-specific knockout (KO) Nell- 1Wnt1KO and Cntnap4Wnt1KO mice targeting CNCCs-derived CB and trigeminal ganglions (TG) by Wnt1-Cre, and Nell-1PV-2AKO and Cntnap4PV-2AKO mice targeting parvalbumin positive (PV+) GABAergic neurons in the brain and TG by PV-2A-Cre. The CB growth and behavioral changes in these KO mice will be correlated with morphological changes in the examined bones, brain, and the craniofacial neuroskeletal interface where TG sensory fibers innervate the CB. AIM 2 will decipher Nell-1's osteogenic property in the context of Cntnap4 during the repair and regeneration of CB defects in Cntnap4Wnt1KO mice using Nell-1 protein. With Nell-1 serving as an agonist of Wnt/β-catenin signaling, AIM 2 will determine how integrin β1, a Nell-1 binding partner, and glycogen synthase kinase-3β (GSK3β), a crucial regulator of β-catenin phosphorylation, modulate Nell-1/Cntnap4 interaction-mediated Wnt/β-catenin signaling activation during CNCC osteogenesis. AIM 3 will investigate Nell- 1/Cntnap4 interaction on brain GABAergic transmission and ASD-like behaviors in Nell-1PV-2AKO and Cntnap4PV- 2AKO mice. AIM 3 will further assess Wnt/β-catenin signaling in the context of Nell-1/Cntnap4 interaction- mediated GABA release, along with the possible additive or synergistic effects of GSK3β inhibitors with Nell-1 in mouse neuronal cells. IMPACT: The ultimate goal is to reveal how the newly identified Nell-1/Cntnap4 interaction may translate into developing Nell-1 as a novel therapeutic to treat human skeletal and neurological pathologies.