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Elucidating functions of the gamma-protocadherins in CNS development

阐明γ-原钙粘蛋白在中枢神经系统发育中的功能

基本信息

批准号：
10216809
负责人：
JOSHUA A WEINER
金额：
$ 37.77万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-01 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10216809
关键词：
Address Alanine Alleles Amino Acids Anxiety Behavior Binding Biochemical Biological Assay C-terminal CRISPR/Cas technology Cadherins Cell Adhesion Molecules Cell Communication Cell Culture Techniques Cells Cerebral cortex Complex Cytoplasmic Tail Dendrites Development Disease Electroporation Ensure Exons Family Focal Adhesion Kinase 1 Fragile X Syndrome Future Gene Cluster Genes Goals Grant Growth Hippocampus (Brain)Human Human Development Hypermethylation Immunoglobulins In Vitro Intellectual functioning disability Knock-out Knockout Mice Laboratories Link Lysine Mediating Memory Mental Depression Mental disorders Mission Molecular Mus Mutate Mutation Neurodevelopmental Disorder Neurons Neurosciences Nonsense Codon Normal Range Phenotype Phospholipids Phosphorylation Process Protein Family Protein Isoforms Protein Kinase C Proteins Public Health Publishing Regulation Research Retina Role Serine Signal Pathway Signal Transduction Stress Techniques Testing Therapeutic Transmembrane Domain United States National Institutes of Health Variant WNT Signaling Pathway Work autism spectrum disorder behavioral outcome cell cortex conditional knockout extracellular genome editing human disease in utero in vivo in vivo evaluation innovation knock-down knowledge base member neural circuit neuron development neuropsychiatric disorder neuroregulation novel overexpression prevent time use

项目摘要

Growth of a complex dendrite arbor is a prerequisite for neural circuit formation, and reduced arbor complexity in the cortex is a consistent finding in neurodevelopmental/neuropsychiatric disorders. For future therapeutic approaches to be conceivable, there is a critical need to elucidate the molecular mechanisms that regulate dendrite arborization. The γ-Protocadherins (γ-Pcdhs), a family of 22 cell adhesion molecules encoded by the Pcdhg gene cluster, are required for normal cortical dendrite arborization; the mechanisms through which their diverse isoforms act are, however, poorly understood. The long-term goal is to identify molecular mechanisms that control neural circuit formation. The objective of this renewal application is to elucidate how both isoform diversity and common signaling mechanisms contribute to γ-Pcdh regulation of dendrite arborization. The central hypothesis is that γ-Pcdhs regulate arborization through: 1) diverse homophilic cell-cell interactions; 2) isoform-specific signaling via unique variable cytoplasmic domains (VCDs); and 3) shared signaling via a common C-terminal motif. Three Aims are proposed, each premised on published work from the current grant period, and each utilizing novel Pcdhg alleles generated through CRISPR/Cas9 genome editing. Aim 1: Establish the importance of a shared γ-Pcdh C-terminal motif in dendrite arborization in vivo. PKC phosphory- lation of a serine within a lysine-rich C-terminal motif abrogates its ability to bind phospholipids, and prevents γ- Pcdhs from inhibiting FAK and promoting dendrite arborization in vitro. To address the importance of this in vivo, 2 new mouse lines will be examined for arborization, γ-Pcdh protein stability, and FAK regulation: PcdhgS/A, in which the serine is mutated to an alanine, preventing phosphorylation; and PcdhgCTD, in which a premature stop codon results in proteins lacking the motif. Aim 2: Identify isoform-specific mechanisms through which the γ-Pcdh-C3 VCD regulates dendrite arborization. The VCD of the γ-Pcdh-C3 isoform uniquely inhibits Wnt signaling by binding to Axin1, a known regulator of dendrite arborization. Using in utero electroporation, Axin1 knockdown, and a novel PcdhgC3KO single-isoform knockout mouse, a distinct role for C3 in dendrite arborization will be ascertained. Aim 3: Ascertain the importance of γ-Pcdh isoform diversity for dendrite arborization and behavior. It's unknown whether isoform diversity generated by the endogenous Pcdhg gene cluster is important arborization. To test this in vivo, multiple novel mouse lines in which varying numbers of Pcdhg exons have been disrupted, reducing potential isoform diversity, will be analyzed. Additionally, anxiety- and memory-associated tests will be carried out on Pcdhg null, single-isoform overexpression, and reduced diversity mice to link cellular phenotypes to behavioral outcomes. The proposed research is innovative, because it utilizes CRISPR/Cas9 genome editing techniques to comprehensively assess the effects of altered γ-Pcdh repertoire and signaling in vivo. It is significant, because future therapeutic approaches will rely critically on identifying the molecules and signaling mechanisms that control dendrite arbor complexity.

复杂树突的生长是神经回路形成的先决条件，在神经发育/神经精神障碍中的一致发现。对于未来的治疗为了使这些方法成为可能，迫切需要阐明调节这些蛋白质的分子机制。枝晶树枝状化γ-原钙粘素（γ-Protocadherins，γ-Pcdhs）是由γ-葡聚糖编码的22种细胞粘附分子家族。 Pcdhg基因簇是正常皮质树突树枝化所必需的; 然而，对ACT的不同同种型知之甚少。长期目标是确定分子机制控制神经回路的形成本更新申请的目的是阐明两种亚型 γ-Pcdh调控树突树枝化的信号机制多样性和共同性。的中心假设是γ-Pcdhs通过以下方式调节树枝状化：1）不同的嗜同性细胞-细胞相互作用; 2）通过独特的可变胞质结构域（VCD）的同种型特异性信号传导;和3）通过可变胞质结构域（VCD）的共享信号传导。共同的C-末端基序。提出了三个目标，每个目标都基于当前资助的已发表作品期间，并且每一个都利用通过CRISPR/Cas9基因组编辑产生的新型Pcdhg等位基因。目标1：确定共享γ-Pcdh C-末端基序在体内树突树枝化中的重要性。PKC磷酸化富含赖氨酸的C-末端基序内的丝氨酸的取代消除了其结合磷脂的能力，并防止γ-氨基化。 Pcdhs在体外抑制FAK和促进树突分枝。为了解决这个问题的重要性，在体内，将检查2个新小鼠系的树枝状化、γ-Pcdh蛋白稳定性和FAK调节： PcdhgS/A，其中丝氨酸突变为丙氨酸，防止磷酸化;和PcdhgCTD，其中丝氨酸突变为丙氨酸，防止磷酸化。过早的终止密码子导致蛋白质缺乏该基序。目的2：通过以下途径确定异构体特异性机制： γ-Pcdh-C3 VCD调节枝晶树枝状化。γ-Pcdh-C3亚型的VCD独特地抑制了 Wnt信号通过与Axin 1结合，Axin 1是一种已知的树突树枝化调节因子。使用子宫内电穿孔， Axin 1敲除和一种新的PcdhgC 3 KO单同种型敲除小鼠，C3在树突中的独特作用将确定树枝化。目的3：确定γ-Pcdh异构体多样性对树突的重要性树枝化和行为。目前尚不清楚内源性Pcdhg基因产生的异构体多样性是否聚类是重要的树状化。为了在体内测试这一点，使用了多种新的小鼠品系，其中不同数量的 Pcdhg外显子已被破坏，减少潜在的异构体多样性，将进行分析。此外，焦虑- 和记忆相关的测试将在Pcdhg无效，单一亚型过表达，和减少将细胞表型与行为结果联系起来。这项研究具有创新性，因为它利用CRISPR/Cas9基因组编辑技术来全面评估基因组改变的影响， γ-Pcdh库和体内信号传导。这很重要，因为未来的治疗方法将严重依赖于识别控制树突复杂性的分子和信号机制。

项目成果

期刊论文数量（23）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Akirin2 is essential for the formation of the cerebral cortex.

DOI：
10.1186/s13064-016-0076-8
发表时间：
2016-11-21
期刊：
Neural development
影响因子：
3.6
作者：
Bosch PJ;Fuller LC;Sleeth CM;Weiner JA
通讯作者：
Weiner JA

γ-Protocadherins Interact with Neuroligin-1 and Negatively Regulate Dendritic Spine Morphogenesis.

DOI：
10.1016/j.celrep.2017.02.060
发表时间：
2017-03-14
期刊：
Cell reports
影响因子：
8.8
作者：
Molumby MJ;Anderson RM;Newbold DJ;Koblesky NK;Garrett AM;Schreiner D;Radley JJ;Weiner JA
通讯作者：
Weiner JA

The γ-Protocadherins Interact Physically and Functionally with Neuroligin-2 to Negatively Regulate Inhibitory Synapse Density and Are Required for Normal Social Interaction.