Genetic and molecular network of the calcium/calmodulin-dependent serine protein kinase CASK

钙/钙调蛋白依赖性丝氨酸蛋白激酶 CASK 的遗传和分子网络

基本信息

批准号：
280629181
负责人：
Professor Dr. Hans-Jürgen Kreienkamp
金额：
--
依托单位：
Institut für Humangenetik
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/280629181?language=en
关键词：
Genetic molecular network calcium calmodulin

项目摘要

Next-generation sequencing is a powerful approach for unravelling the molecular basis of Mendelian disorders. Recently, we and others identified mutations in the X-linked CASK (calcium/calmodulin-dependent serine protein kinase) gene in females and males with intellectual disability with or without additional clinical features. In synapses of the central nervous system, CASK contributes to the formation of the presynaptic specialization, and to postsynaptic delivery of NMDA receptors. CASK is also redistributed to the neuronal nucleus where it binds the transcription factor Tbr1 and activates the target genes Reelin and NR2B (encoding a regulatory subunit of NMDA receptors). As NMDA receptors function as switches for memory formation by gating synaptic plasticity, the contribution of CASK to expression and transport of NMDA receptors strongly suggests a critical role of CASK in synaptic plasticity, learning and memory. Our current understanding indicates that a complex genetic and molecular synaptic network determines the phenotypic outcome of individuals with a CASK mutation. Phenotypic variability suggests that there are a number of modifier genes which affect CASK-related pathways. On the protein and cellular level, CASK acts at three different locations in neurons in different protein complexes, and it is currently unclear which of these complexes is relevant for disease. Our goals are to1) establish a genotype-phenotype correlation in males with a CASK mutation by analyzing CASK transcripts and protein in patient-derived cells.2) identify modifier genes responsible for differences in phenotypic expression of females with a heterozygous deleterious CASK mutation. We will use an extreme phenotype study design by defining individuals who are at both ends of a phenotype distribution. Whole exome sequencing on individuals of both groups will help to identify rare sequence variants enriched in one group.3) characterize the effect of CASK missense mutations (p.Y268H, p.L354P, p.P396S, p.Y723C and p.W914R) on transcriptional regulation of specific target genes of the CASK-Tbr1 transcriptional activator complex. We will carry out dual luciferase reporter assays in primary cortical neurons derived from Cask knockout mice expressing wild-type or mutant CASK. 4) analyze the effect of CASK missense mutations on specific protein-protein interactions and on intracellular trafficking of neurotransmitter receptors. CASK wildtype and mutants will be expressed in neurons derived from Cask-deficient mice, and the effect of CASK mutations on synaptogenesis and spinogenesis will be analyzed by immunocytochemistry and confocal microscopy. We will also determine the cell surface level of NMDA receptors by using live-cell surface immunostaining and biotinylation assays.5) study the in vivo effects of a selected CASK mutation in a Cask knock-in mouse, which will be analyzed with respect to synapse formation, neuronal morphology, and behavior.

下一代测序是揭示孟德尔疾病分子基础的有力方法。最近，我们和其他人在有或没有其他临床特征的智力障碍女性和男性中发现了 X 连锁 CASK（钙/钙调蛋白依赖性丝氨酸蛋白激酶）基因的突变。在中枢神经系统的突触中，CASK 有助于突触前特化的形成以及 NMDA 受体的突触后传递。 CASK 还重新分布到神经元核，在那里它与转录因子 Tbr1 结合并激活靶基因 Reelin 和 NR2B（编码 NMDA 受体的调节亚基）。由于 NMDA 受体通过门控突触可塑性充当记忆形成的开关，因此 CASK 对 NMDA 受体表达和运输的贡献强烈表明 CASK 在突触可塑性、学习和记忆中的关键作用。我们目前的理解表明，复杂的遗传和分子突触网络决定了具有 CASK 突变的个体的表型结果。表型变异表明存在许多影响 CASK 相关途径的修饰基因。在蛋白质和细胞水平上，CASK 在不同蛋白质复合物中的神经元的三个不同位置发挥作用，目前尚不清楚这些复合物中的哪一个与疾病相关。我们的目标是：1) 通过分析患者来源的细胞中的 CASK 转录本和蛋白质，建立具有 CASK 突变的男性的基因型-表型相关性。2) 识别导致具有杂合有害 CASK 突变的女性表型表达差异的修饰基因。我们将通过定义位于表型分布两端的个体来使用极端表型研究设计。对两组个体进行全外显子组测序将有助于鉴定一组中富集的罕见序列变异。3) 表征 CASK 错义突变的影响（p.Y268H、p.L354P、p.P396S、p.Y723C 和 p.W914R） CASK-Tbr1 转录激活复合物特定靶基因的转录调控。我们将在源自表达野生型或突变型 CASK 的 Cask 敲除小鼠的原代皮质神经元中进行双荧光素酶报告基因测定。 4) 分析CASK错义突变对特定蛋白质-蛋白质相互作用和神经递质受体细胞内运输的影响。 CASK野生型和突变体将在来自Cask缺陷小鼠的神经元中表达，并且将通过免疫细胞化学和共聚焦显微镜分析CASK突变对突触发生和棘发生的影响。我们还将通过使用活细胞表面免疫染色和生物素化测定来确定 NMDA 受体的细胞表面水平。5) 研究 Cask 敲入小鼠中选定的 CASK 突变的体内影响，并将针对突触进行分析形成、神经元形态和行为。