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：
7688612
负责人：
RICHARD E MAINS
金额：
$ 33.3万
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/7688612
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状刺的形成和功能中起关键作用。智力低下与单个Rho蛋白的缺陷，鸟嘌呤核苷酸交换因子（GEFS）激活Rho蛋白和活化Rho蛋白的下游靶标有关。大型，双Rho GEF蛋白的表达缺陷与阿尔茨海默氏病大脑中的iNOS水平升高以及精神分裂症患者验尸前额叶皮层的脊柱密度降低有关。此外，卡利林与Disc1（候选精神分裂症基因）和HAP1（亨廷顿相互作用者）相互作用。单核苷酸多态性预测卡利林功能的杂合子数量很大，只有一个表达的拷贝或正常拷贝和一个突变的拷贝。这些观察结果是分析小鼠的令人信服的案例，在发育过程中和成年人中都可以操纵卡利林的表达。哺乳动物还表达三重奏，这是一种高度同源但非冗余的基因。果蝇和秀丽隐杆线虫中的单个kalirin/三人基因在神经系统内部和外部起着至关重要的作用。基于我们在培养神经元的研究，卡利林在轴突启动和产物和树突生长中起着核心作用。 Kalirin，Kalirin-7的主要成人剪接变体的过表达增加了在金字塔神经元和正常aspiny中间神经元中的树突状棘的形成。 Kalirin-7和Kalirin-7（由不同启动子产生的N末端截断变体）的表达减少导致脊柱形成和维持的缺陷。我们生成了小鼠模型，其中可以改变Kalirin-7和Kalirin-7的表达。缺乏Kalirin-7/ Kalirin-7（KAL7KO）独有的单一外显子的小鼠出生于预期频率的一半，但幸存到成年和繁殖。该外显子（KAL7+/KO）的杂合小鼠的杂合子降低了Kalirin-7和Kalirin-7水平，并在突触传播中显示出缺陷。在超微结构水平上，KAL7KO小鼠的正常兴奋性突触数量减少，以及在正常小鼠中未见的许多异常突触特征。当在较高的零迷宫中进行测试时，KAL7+/KO和KAL7KO小鼠显示出焦虑样行为的分级降低。 KAL7外显子被LOX-P位点（KAL7CKO）包围的小鼠允许组织特异性，发育调节的消除Kalirin-7/ Kalirin-7。这些小鼠将使用行为测试，突触前和后突触后元素的形态评估，切片的电生理记录以及亚细胞分数的生化分析。可以通过表达外源性kalirin-7来挽救由KAL7KO小鼠制备的海马神经元中的脊柱形成，从而可以详细分析Kalirin-7和孤立的SEC14P，Spectrin样，DH和pH结构域的作用。 KAL7在脊柱形成中的作用将评估Shank3，Glur2和Neuroligin-1等蛋白质。将使用KAL7KO小鼠评估六个已知的人kalirin-7突变体挽救脊柱形成和突触功能的能力。公共卫生相关性：在树突状刺上的兴奋性突触解释了神经元之间发生的许多沟通。树突状棘的数量和形状对发育线索，环境刺激和荷尔蒙变化有反应。脊柱形态的变化在学习和记忆中起关键作用，很明显，许多信号通路会影响脊柱的形成和功能。 Kalirin-7是Rho家族的小型GTP结合蛋白的激活剂，被定位于树突状棘，是已知能够增加树突状刺数的少数因素之一。我们计划使用我们生成的Kalirin-7基因敲除小鼠来阐明控制脊柱形成和结构的途径。