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

描述(由申请人提供)：Rho GTP酶在神经元发育和树突棘的形成和功能中发挥关键作用。智力低下与单个Rho蛋白、激活Rho蛋白的鸟核苷酸交换因子(GEF)和激活的Rho蛋白下游靶标的缺陷有关。Kalirin是一种大的、双Rho全球环境基金蛋白，其表达不足与阿尔茨海默病患者大脑中iNOS水平的升高以及精神分裂症患者死后前额叶皮质中脊柱密度的下降有关。此外，Kalirin还与DISC1(精神分裂症候选基因)和HAP1(亨廷顿相互作用因子)相互作用。单核苷酸多态预测有相当数量的个体具有Kalirin功能的杂合子，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一个令人信服的理由，在小鼠中，Kalirin的表达在发育过程中和成年鼠中都可以被操纵。哺乳动物也表达Trio，这是一种高度同源但非冗余的基因。在果蝇和线虫中，单一的Kalirin/Trio基因在神经系统内外发挥着重要的作用。根据我们对培养神经元的研究，Kalirin在轴突的起始和生长以及树突的生长中发挥着核心作用。Kalirin的主要成体剪接变体Kalirin-7的过度表达增加了锥体神经元和正常脊髓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端截断的变体)表达的减少导致脊柱形成和维持的缺陷。我们建立了可以改变Kalirin-7和Kalirin-7表达的小鼠模型。缺少Kalirin-7/Kalirin-7(Kal7KO)特有的单个外显子的小鼠出生的频率是预期的一半，但成年后存活并繁殖。该外显子杂合子(KAL7+/KO)的小鼠已经降低了Kalirin-7和Kalirin-7的水平，并显示出突触传递的缺陷。在超微结构水平上，Kal7KO小鼠的正常兴奋性突触数量减少，加上许多正常小鼠未见的异常突触轮廓。当在高架的零迷宫中测试时，KAL7+/KO和Kal7KO小鼠的焦虑样行为逐渐减少。KAL7外显子被LOX-p位点包围的小鼠(Kal7CKO)允许组织特异性的、发育调节的Kalirin-7/Kalirin-7的消除。这些小鼠将通过行为测试、突触前和突触后元素的形态评估、切片的电生理记录和亚细胞部分的生化分析进行评估。从Kal7KO小鼠制备的海马神经元中的脊柱形成可以通过表达外源Kalirin-7来挽救，从而可以详细分析Kalirin-7的作用以及分离的Sec14p、血影蛋白样结构域、DH和PH结构域。将评估KAL7在脊柱形成中对Shank3、GluR2和Neuroigin-1等蛋白质的反应中的作用。已知的6个人类Kalirin-7突变体挽救脊柱形成和突触功能的能力将通过Kal7KO小鼠进行评估。与公共健康相关：树突棘上的兴奋性突触占神经元之间交流的很大一部分。树突棘的数量和形状对发育线索、环境刺激和激素变化做出反应。脊柱形态的改变在学习和记忆中起着关键作用，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，是为数不多的能够增加树突棘数量的因子之一。我们计划使用我们培育的Kalirin-7基因敲除小鼠来阐明控制脊柱形成和结构的途径(S)。

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.