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

Nell-1 通过与 Cntnap4 相互作用在颅面骨和大脑中发挥双重作用

• 批准号: 10674475
• 负责人: Chia Soo
• 金额: $ 47.01万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10674475
• 关键词: Adult; Agonist; Behavior; Behavior assessment; Behavioral; Binding; Bone Growth; Bone Regeneration; Brain; Breeding; Calvaria; Cell surface; Cells; Cephalic; Craniosynostosis; Data; Defect; Development; Drug or chemical Tissue Distribution; Embryo; Ethylnitrosourea; Event; Exhibits; Fiber; Frontal bone structure; GABA Agonists; Goals; Growth and Development function; Histology; Human; Integrins; Interneurons; Intervention; Knock-out; Knockout Mice; Knowledge; Ligands; Link; LoxP-flanked allele; Measurement; Mediating; Membrane; Membrane Proteins; Molecular; Morphology; Mus; Mutant Strains Mice; Natural regeneration; Neonatal; Nervous System; Neural Crest Cell; Neurons; Osteoblasts; Osteogenesis; Outcome; Output; Parietal bone structure; Parvalbumins; Pathogenesis; Patients; Phosphorylation; Process; Property; Proteins; Role; Sensory Ganglia; Signal Transduction; Skeletal Development; Skeletal system; Specific qualifier value; Structure of trigeminal ganglion; Synaptic Transmission; Testing; Therapeutic; Tissues; Transgenic Organisms; Translating; WNT Signaling Pathway; Wild Type Mouse; autism spectrum disorder; beta catenin; bone; bone healing; bone repair; brain tissue; comparative; craniofacial; craniofacial bone; gamma-Aminobutyric Acid; glycogen synthase kinase 3 beta; healing; improved; inhibitor; member; microCT; neural; neurological pathology; novel; novel therapeutics; osteogenic; osteogenic protein; overexpression; postnatal; premature; presynaptic; receptor; repaired; skeletal; skeletal tissue; spatiotemporal; suture fusion; transmission process; Adult; Agonist; Behavior; Behavior assessment; Behavioral; Binding; Bone Growth; Bone Regeneration; Brain; Breeding; Calvaria; Cell surface; Cells; Cephalic; Craniosynostosis; Data; Defect; Development; Drug or chemical Tissue Distribution; Embryo; Ethylnitrosourea; Event; Exhibits; Fiber; Frontal bone structure; GABA Agonists; Goals; Growth and Development function; Histology; Human; Integrins; Interneurons; Intervention; Knock-out; Knockout Mice; Knowledge; Ligands; Link; LoxP-flanked allele; Measurement; Mediating; Membrane; Membrane Proteins; Molecular; Morphology; Mus; Mutant Strains Mice; Natural regeneration; Neonatal; Nervous System; Neural Crest Cell; Neurons; Osteoblasts; Osteogenesis; Outcome; Output; Parietal bone structure; Parvalbumins; Pathogenesis; Patients; Phosphorylation; Process; Property; Proteins; Role; Sensory Ganglia; Signal Transduction; Skeletal Development; Skeletal system; Specific qualifier value; Structure of trigeminal ganglion; Synaptic Transmission; Testing; Therapeutic; Tissues; Transgenic Organisms; Translating; WNT Signaling Pathway; Wild Type Mouse; autism spectrum disorder; beta catenin; bone; bone healing; bone repair; brain tissue; comparative; craniofacial; craniofacial bone; gamma-Aminobutyric Acid; glycogen synthase kinase 3 beta; healing; improved; inhibitor; member; microCT; neural; neurological pathology; novel; novel therapeutics; osteogenic; osteogenic protein; overexpression; postnatal; premature; presynaptic; receptor; repaired; skeletal; skeletal tissue; spatiotemporal; suture fusion; transmission process

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.

项目摘要 NELL-1已广泛研究其在颅面骨骼发育中的成骨作用。但是，需求 它在大脑中的高表达，缺乏对Nell-1在神经系统中的功能的了解 直到最近对配体受体的鉴定，例如NELL-1和CNTNAP4之间的相互作用。 CNTNAP4是 突触膜蛋白，全局CNTNAP4损失不同地抑制了GABA能量和增强 多巴胺能传播，可以诱导小鼠的自闭症谱系障碍（ASD）类似行为。值得注意的是 ENU诱导的NELL-1单倍弹性小鼠暴露于与CNTNAP4突变体类似的ASD类似行为 老鼠。同时，颅神经rest细胞中的CNTNAP4或NELL-1灭活会导致 非常相似的颅骨缺损。此外，NELL-1和CNTNAP4的新型共定位研究 小鼠和人的大脑显示出广泛的神经元组织分布和高水平的共定位表达。 因此，该提议假设Nell-1通过大脑和颅骨（CB）具有双重作用 NELL-1和CNTNAP4之间的一种新型相互作用，以及NELL-1/CNTNAP4功能轴的破坏将不会 仅诱导CB中的定义，但也干扰了大脑的神经传播。该提议提出了三个 具体目的是使用两只新的Floxed小鼠研究CB和大脑中NELL-1/CNTNAP4功能轴 线，NELL-1FL/FL和CNTNAP4FL/FL。 AIM 1将首先定义NELL-1和CNTNAP4的组织分布和表达 在正常发育和野生型（WT）小鼠的正常发育和生长过程中，在CB和大脑中。 AIM 1然后指定 NELL-1和CNTNAP4使用组织特异性敲除（KO）Nell-的功能贡献对CB和脑的功能贡献 1WNT1KO和CNTNAP4WNT1KO小鼠针对CNCCS衍生的CB和三叉神经节（TG），Wnt1-Cre和 NELL-1PV-2AKO和CNTNAP4PV-2AKO小鼠靶向脑中的白蛋白阳性（PV+）GABA能神经元 和PV-2A-CRE的TG。这些KO小鼠的CB生长和行为变化将与 所检查的骨骼，大脑和颅面神经骨骼界面的形态变化，其中TG 感觉纤维支配CB。 AIM 2将在CNTNAP4的背景下破译Nell-1的成骨特性 使用NELL-1蛋白的CNTNAP4WNT1KO小鼠中CB缺陷的修复和再生。 Nell-1充当 Wnt/β-catenin信号传导的激动剂，AIM 2将确定整合素β1，NELL-1结合伴侣和糖原如何如何 合酶激酶-3β（GSK3β）是β-catenin磷酸化的关键调节剂，调节NELL-1/CNTNAP4 CNCC成骨发生过程中相互作用介导的Wnt/β-catenin信号传导激活。 AIM 3将调查Nell- NELL-1PV-2AKO和CNTNAP4PV-中的1/CNTNAP4相互作用在脑GABA能传播和类似ASD的行为上 2ako小鼠。 AIM 3将在NELL-1/CNTNAP4相互作用的背景下进一步评估Wnt/β-catenin信号传导 介导的GABA释放，以及具有NELL-1 IN的GSK3β抑制剂的添加剂或协同作用 小鼠神经元细胞。影响：最终目标是揭示新鉴定的NELL-1/CNTNAP4相互作用如何 可以转化为开发NELL-1作为治疗人骨骼和神经病理学的新型治疗方法。