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）：癫痫是神经元激发和抑制之间失衡的障碍。但是，已经确定了导致人癫痫的几种基因，但是，由于基本的复杂遗传学，大多数基因仍然未知。成人“适合”的小鼠在编码Dynamin-1的基因中携带一个新型突变的副本，这是一种与神经元内内吞作用和突触膜回收的大型GTPase，经历了复发性特发性缘和富幅性癫痫发作。除了共济失调和神经感觉缺陷外，具有两种副本的合适青少年都具有更严重的癫痫发作。有趣的是，dynamin-1 null突变体虽然神经系统受损，但并未显示出这些特殊的异常。因此，对新合适的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，该突变驻留在Dynamin-1的替代外显子中，编码两种同工型之一，并且对体外的蛋白质组装和内吞作用具有显着的负面影响。我们的第一个目的是通过更详细地确定正常和突变同生对原代神经元细胞内吞作用的后果，通过将野生型和突变的dynatin-1 cDNA引入培养物中的dynamin-1 null原发性神经元以及确定的神经元细胞系中。内吞作用和突触囊泡回收将在转染的神经元培养物以及适当的突触蛋白定位中进行检查。第二个目的是询问合适的突变是否对突触形成和成熟有效。通过定量和突触后标志物的定位和定位，将在转染的原发性神经元培养物和细胞系中评估突触发生。适合的小鼠是一种新颖而有趣的特发性癫痫的模型，这是源于一个基因，它已经有很多理解，但是没有直接与癫痫直接相关的基因。适当的人将使我们对至少一种可能涉及特发性癫痫病的非常复杂的遗传学涉及的途径的见解。公共卫生相关性：作为大脑“兴奋性”的疾病，癫痫是神经元激发和抑制之间复杂平衡的毁灭性故障。适当的Dynamin-1介导的神经元内吞作用的破坏会导致神经元传播缺陷。研究Dynamin-1的功能和有助于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发作产生，并可能为未来的疗法提供新的方法。