Dynamin-1 Mutation in a Genetic Epilepsy Model: Isoform-Specific Roles

遗传性癫痫模型中的 Dynamin-1 突变：异构体特异性作用

基本信息

批准号：
7787947
负责人：
REBECCA M BOUMIL
金额：
$ 8.7万
依托单位：
JACKSON LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7787947
关键词：
Address Adolescent Adult Age Age-Months Alternative Splicing Amino Acid Substitution Ataxia Biological Assay Brain Cell Line Cells Cellular Morphology Cellular biology Complementary DNA Complex Data Defect Development Disease Dominant-Negative Mutation Dynamin I Endocytosis Epilepsy Equilibrium Exons Fluorescent Dyes Future Generations Genes Genetic Guanosine Triphosphate Phosphohydrolases Homo Human In Vitro Inhibitory Synapse Knock-out Knockout Mice Learning Lesion Mammalian Cell Mediating Microscopy Missense Mutation Modeling Molecular Biology Mus Mutant Strains Mice Mutate Mutation Neurons Pathway interactions Phenotype Population Process Protein Isoforms Proteins Public Health RNA Splicing Recurrence Recycling Reporting Role Seizures Synapses Synaptic Membranes Synaptic Receptors Synaptic Transmission Synaptic Vesicles Tonic - clonic seizures Variant experience insight mutant nervous system disorder neuronal excitability neurotransmission novel novel strategies public health relevance stem synaptic function synaptogenesis uptake

项目摘要

DESCRIPTION (provided by applicant): Epilepsy is a disorder of imbalance between neuronal excitation and inhibition. Several genes that are responsible for human epilepsy have been identified, however, most remain unknown due to the underlying complex genetics. Adult "fitful" mice carrying one copy of a novel mutation in the gene encoding Dynamin-1, a large GTPase involved in endocytosis and synaptic membrane recycling in neurons, experience recurrent idiopathic limbic and tonic-clonic seizures. Fitful adolescents with both copies mutated have more severe seizures, in addition to ataxia and neurosensory deficits. Interestingly, Dynamin-1 null mutants, while neurologically impaired, do not show these particular abnormalities. Study of the new fitful mutation therefore provides a unique opportunity to better understand the relationship between normal synaptic function and neurological disease. Preliminary data show that the mutation resides in an alternative exon of Dynamin-1, encoding one of two isoforms, and that it has a dominant negative effect on protein assembly and endocytosis in vitro. Our first aim is to determine in more detail the consequences of normal and mutant isoforms on endocytosis in primary neuronal cells, by introducing wild-type and mutated Dynamin-1 cDNA into Dynamin-1 null primary neurons in culture as well as established neuronal cell lines. Endocytosis and synaptic vesicle recycling will be examined in transfected neuronal cultures as well as proper synaptic protein localization. The second aim is to ask if the fitful mutation has an effect on synapse formation and maturation. Synaptogenesis will be evaluated in transfected primary neuronal cultures and cell lines by quantification and localization of pre- and post-synaptic markers. The fitful mouse is a novel and intriguing model for generalized idiopathic epilepsy, stemming from a gene about which there is already a great deal of understanding but none so far tied directly to epilepsy. Fitful will give us insight to at least one pathway that is likely to be involved in the very complex genetics that underlie idiopathic epilepsies. PUBLIC HEALTH RELEVANCE: As a disorder of "excitability" of the brain, epilepsy is a devastating malfunction of the intricate balance between neuronal excitation and inhibition. Disruption of proper Dynamin-1 mediated endocytosis in neurons results in defective neuronal transmission. Studying how Dynamin-1 functions and contributes to the stability of synapse dynamics will help us to understand how perturbation of endocytosis leads to seizure generation and may provide for novel approaches to future therapies.

描述（由申请人提供）：癫痫是一种神经元兴奋和抑制之间不平衡的疾病。一些与人类癫痫有关的基因已被确定，但由于潜在的复杂遗传学，大多数基因仍然未知。成年“间歇性”小鼠携带编码 Dynamin-1 基因的一个新突变副本，Dynamin-1 是一种参与神经元内吞作用和突触膜回收的大型 GTP 酶，它们会经历反复的特发性边缘和强直阵挛癫痫发作。两个拷贝都发生突变的间歇性青少年除了共济失调和神经感觉缺陷外，还会出现更严重的癫痫发作。有趣的是，Dynamin-1 无效突变体虽然神经受损，但并未表现出这些特殊的异常。因此，对新的间歇性突变的研究为更好地了解正常突触功能与神经系统疾病之间的关系提供了独特的机会。初步数据表明，该突变存在于 Dynamin-1 的另一个外显子中，编码两种异构体之一，并且它对体外蛋白质组装和内吞作用具有显着的负面影响。我们的第一个目标是通过将野生型和突变的 Dynamin-1 cDNA 引入培养的 Dynamin-1 无效原代神经元以及已建立的神经元细胞系中，更详细地确定正常和突变亚型对原代神经元细胞内吞作用的影响。将在转染的神经元培养物中检查内吞作用和突触小泡回收以及适当的突触蛋白定位。第二个目的是询问断断续续的突变是否对突触形成和成熟有影响。将通过突触前和突触后标记的量化和定位来评估转染的原代神经元培养物和细胞系中的突触发生。断断续续的小鼠是一种新颖而有趣的全身性特发性癫痫模型，它源于一种基因，人们对此已经有了很多了解，但迄今为止还没有与癫痫直接相关的基因。 Fitful 将使我们了解至少一种可能与特发性癫痫背后的非常复杂的遗传学有关的途径。公共健康相关性：作为一种大脑“兴奋性”障碍，癫痫是神经元兴奋和抑制之间复杂平衡的破坏性功能障碍。神经元中 Dynamin-1 介导的内吞作用的破坏会导致神经元传递缺陷。研究 Dynamin-1 如何发挥作用并促进突触动力学的稳定性将有助于我们了解内吞作用的扰动如何导致癫痫发作，并可能为未来的治疗提供新方法。