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

项目摘要 Nell-1在颅面骨骼发育中的成骨作用已被广泛研究。但尽管 由于Nell-1在大脑中的高表达，人们对Nell-1在神经系统中的功能缺乏了解 直到最近鉴定出Nell-1和Cntnap 4之间的配体-受体样相互作用。cntnap 4是一个前- 一种突触膜蛋白，Cntnap 4的整体缺失可不同程度地抑制GABA能输出并增强GABA能的表达 多巴胺能传递，并且可以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单倍缺陷小鼠表现出与Cntnap 4突变体相似的ASD样行为 小鼠同时，在颅神经嵴细胞（CNCC）中Cntnap 4或Nell-1的失活引起 非常相似的颅骨缺损此外，Nell-1和Cntnap 4在细胞中的新的共定位研究， 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，该提议假设Nell-1在脑和颅面骨（CB）中具有双重作用， Nell-1和Cntnap 4之间的一种新的相互作用，并且Nell-1/Cntnap 4功能轴的破坏不会 不仅诱导CB的缺陷，而且还干扰大脑中的神经传递。该提案提出了三个 本研究的目的是利用两种新的floxed小鼠研究Nell-1/Cntnap 4在CB和脑中的功能轴 细胞系Nell-1 fl/fl和Cntnap 4fl/fl。AIM 1将首先定义Nell-1和Cntnap 4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和脑中均存在这种情况。AIM 1将指定 使用组织特异性敲除（KO）Nell-1和Cntnap 4对CB和脑的功能贡献， 通过Wnt 1-Cre靶向CNCC衍生的CB和三叉神经节（TG）的1 Wnt 1 KO和Cntnap 4 Wnt 1 KO小鼠，以及 靶向脑中小清蛋白阳性（PV+）GABA能神经元的Nell-1 PV-2AKO和Cntnap 4PV-2AKO小鼠 和TG通过PV-2A-Cre。这些KO小鼠中的CB生长和行为变化将与 形态学变化的检查骨，脑，颅面神经骨骼界面，其中TG 感觉纤维支配CB。AIM 2将解释Nell-1在Cntnap 4背景下的成骨特性， 使用Nell-1蛋白修复和再生Cntnap 4 Wnt 1 KO小鼠中的CB缺陷。Nell-1作为 作为Wnt/β-catenin信号传导的激动剂，AIM 2将决定整合素β1（Nell-1结合伴侣）和糖原 合成酶激酶3β（GSK 3 β）是β-catenin磷酸化的重要调节因子，调节Nell-1/Cntnap 4 CNCC成骨过程中相互作用介导的Wnt/β-连环蛋白信号传导激活。AIM 3将调查内尔- 1/Cntnap 4相互作用对Nell-1 PV-2AKO和Cntnap 4PV-2AKO中脑GABA能传递和ASD样行为的影响 2只AKO小鼠。AIM 3将在Nell-1/Cntnap 4相互作用的背景下进一步评估Wnt/β-连环蛋白信号传导。 沿着GSK 3 β抑制剂与Nell-1的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/Cntnap 4相互作用是如何发生的。 可能转化为开发Nell-1作为治疗人类骨骼和神经病理学的新疗法。

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.