Dissecting the role of one neuronal RhoGEF amongst many: the Kalirin-7 null mouse

剖析一种神经元 RhoGEF 在众多神经元中的作用：Kalirin-7 null 小鼠

基本信息

批准号：
7892324
负责人：
RICHARD E MAINS
金额：
$ 32.97万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-30 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7892324
关键词：
3&apos Untranslated Regions APEX1 gene Accounting Actins Adult Affect Alzheimer&apos s Disease Anxiety Axon Behavior Binding Biochemical Brain Caenorhabditis elegans Cells Code Communication Coupled Cues Cytoskeleton Data Dendritic Spines Development Drosophila genus DsRed Elements Excitatory Synapse Exons Family Frequencies Genes Genetic Genetic Transcription GluR2 subunit AMPA receptor Growth Guanine Nucleotide Exchange Factors Hippocampus (Brain)Hormonal Change Human In Vitro Individual Interneurons Knockout Mice Learning Maintenance Mammals Membrane Membrane Protein Traffic Memory Mental Retardation Methods Morphology Mus Mutate Mutation Nervous system structure Neurons PH Domain Pathway interactions Patients Play Prefrontal Cortex Protein Isoforms Proteins RNA Splicing Recovery Role Sampling Schizophrenia Shapes Signal Pathway Single Nucleotide Polymorphism Site Slice Spectrin Stimulus Structure Subcellular Fractions Synapses Synaptic Transmission Testing Tissues Transcript Variant Vertebral column base behavior test density filamin hippocampal pyramidal neuron human APEX1 protein human EMS1 protein human Huntingtin protein in vivo mouse model mutant neuroligin 1 neuron development platelet protein P47 promoter public health relevance receptor recombinase response rho rho GTP-Binding Proteins spinophilin synaptic function

项目摘要

DESCRIPTION (provided by applicant): Rho GTPases play key roles in neuronal development and in the formation and function of dendritic spines. Mental retardation is associated with deficits in individual Rho proteins, in the guanine nucleotide exchange factors (GEFs) that activate Rho proteins and in downstream targets of activated Rho proteins. Deficits in expression of Kalirin, a large, dual Rho GEF protein, are associated with increased iNOS levels in Alzheimer's disease brain and with decreased spine density in post-mortem prefrontal cortex from schizophrenic patients. In addition, Kalirin interacts with DISC1 (a candidate schizophrenia gene) and HAP1 (a Huntingtin interactor). Single nucleotide polymorphisms predict a significant number of individuals heterozygous for Kalirin function, with only one expressed copy or a normal and a mutated copy. These observations make a compelling case for analyzing mice in which expression of Kalirin can be manipulated both during development and in the adult. Mammals also express Trio, a highly homologous, but non- redundant gene. The single Kalirin/Trio gene in Drosophila and C. elegans plays an essential role within and outside of the nervous system. Based on our studies in cultured neurons, Kalirin plays a central role in axon initiation and outgrowth and in dendritic growth. Over-expression of the major adult splice variant of Kalirin, Kalirin-7, increases the formation of dendritic spines in pyramidal neurons and in normally aspiny interneurons. Reductions in the expression of Kalirin-7 and Kalirin-7 (an N-terminally truncated variant generated from a different promoter) result in deficits in spine formation and maintenance. We generated mouse models in which expression of Kalirin-7 and Kalirin-7 can be varied. Mice lacking the single exon unique to Kalirin-7/ Kalirin-7 (Kal7KO) are born at half the expected frequency, but survive to adulthood and reproduce. Mice heterozygous for this exon (Kal7+/KO) have diminished levels of Kalirin-7 and Kalirin-7 and show deficits in synaptic transmission. At the ultrastructural level, Kal7KO mice have a reduced number of normal excitatory synapses, plus many aberrant synaptic profiles not seen in normal mice. When tested in the elevated zero maze, Kal7+/KO and Kal7KO mice show a graded decrease in anxiety-like behavior. Mice in which the Kal7 exon is surrounded by lox-p sites (Kal7CKO) allow tissue-specific, developmentally regulated elimination of Kalirin-7/ Kalirin-7. These mice will be assessed using behavioral tests, morphological assessment of pre- and post-synaptic elements, electrophysiological recordings of slices and biochemical analysis of subcellular fractions. Spine formation in hippocampal neurons prepared from Kal7KO mice can be rescued by expressing exogenous Kalirin-7, allowing detailed analysis of the role of Kalirin-7 and the isolated Sec14p, spectrin-like, DH and PH domains. The role of Kal7 in spine formation in response to proteins such as Shank3, GluR2 and Neuroligin-1 will be assessed. The ability of the six known human Kalirin-7 mutants to rescue spine formation and synaptic function will be assessed using the Kal7KO mice. PUBLIC HEALTH RELEVANCE: Excitatory synapses onto dendritic spines account for much of the communication that goes on between neurons. The number and shape of dendritic spines respond to developmental cues, environmental stimuli and hormonal changes. Changes in spine morphology play key roles in learning and memory and it is clear that many signaling pathways affect spine formation and function. Kalirin-7, an activator of small GTP binding proteins of the Rho family, is localized to dendritic spines and is one of a small number of factors known to be capable of increasing the number of dendritic spines. We plan to use the Kalirin-7 knockout mouse that we generated to elucidate the pathway(s) controlling spine formation and structure.

描述（由申请人提供）：Rho GTPases 在神经元发育以及树突棘的形成和功能中发挥关键作用。精神发育迟滞与个体 Rho 蛋白、激活 Rho 蛋白的鸟嘌呤核苷酸交换因子 (GEF) 以及激活 Rho 蛋白下游靶标的缺陷有关。 Kalirin（一种大型双 Rho GEF 蛋白）的表达缺陷与阿尔茨海默病大脑中 iNOS 水平的增加以及精神分裂症患者死后前额皮质中脊柱密度的降低有关。此外，Kalirin 与 DISC1（一种候选精神分裂症基因）和 HAP1（一种亨廷顿蛋白相互作用蛋白）相互作用。单核苷酸多态性预测大量 Kalirin 功能杂合个体，只有一个表达拷贝或一个正常拷贝和一个突变拷贝。这些观察结果为分析小鼠提供了令人信服的理由，在小鼠中，Kalirin 的表达在发育过程中和成年期都可以被操纵。哺乳动物也表达 Trio，一种高度同源但非冗余的基因。果蝇和线虫中的单一 Kalirin/Trio 基因在神经系统内外发挥着重要作用。根据我们对培养神经元的研究，Kalirin 在轴突起始和生长以及树突生长中发挥着核心作用。 Kalirin 的主要成体剪接变体 Kalirin-7 的过度表达会增加锥体神经元和正常无刺中间神经元中树突棘的形成。 Kalirin-7 和 Kalirin-7（由不同启动子产生的 N 末端截短变体）表达的减少会导致脊柱形成和维持的缺陷。我们生成了 Kalirin-7 和 Kalirin-7 表达可以变化的小鼠模型。缺乏 Kalirin-7/ Kalirin-7 (Kal7KO) 特有的单个外显子的小鼠出生频率只有预期的一半，但能存活到成年并繁殖。该外显子杂合子 (Kal7+/KO) 的小鼠 Kalirin-7 和 Kalirin-7 水平降低，并显示突触传递缺陷。在超微结构水平上，Kal7KO 小鼠的正常兴奋性突触数量减少，并且存在许多正常小鼠中未见的异常突触特征。在高架零迷宫中进行测试时，Kal7+/KO 和 Kal7KO 小鼠的焦虑样行为逐渐减少。 Kal7 外显子被 lox-p 位点 (Kal7CKO) 包围的小鼠允许组织特异性、发育调节的 Kalirin-7/ Kalirin-7 消除。将使用行为测试、突触前和突触后元素的形态学评估、切片的电生理记录和亚细胞部分的生化分析来评估这些小鼠。通过表达外源 Kalirin-7 可以挽救 Kal7KO 小鼠海马神经元中的脊柱形成，从而可以详细分析 Kalirin-7 和分离的 Sec14p、血影蛋白样、DH 和 PH 结构域的作用。将评估 Kal7 在响应 Shank3、GluR2 和 Neuroligin-1 等蛋白质的脊柱形成中的作用。将使用 Kal7KO 小鼠评估六种已知的人类 Kalirin-7 突变体拯救脊柱形成和突触功能的能力。公共健康相关性：树突棘上的兴奋性突触负责神经元之间进行的大部分通信。树突棘的数量和形状对发育线索、环境刺激和荷尔蒙变化做出反应。脊柱形态的变化在学习和记忆中发挥着关键作用，很明显，许多信号通路影响脊柱的形成和功能。 Kalirin-7 是 Rho 家族小 GTP 结合蛋白的激活剂，定位于树突棘，是已知能够增加树突棘数量的少数因子之一。我们计划使用我们生成的 Kalirin-7 敲除小鼠来阐明控制脊柱形成和结构的途径。