Understanding the effects of mitochondrial fission disruption during early cortical development

了解早期皮质发育过程中线粒体裂变破坏的影响

基本信息

批准号：
10535949
负责人：
Tierney Baum
金额：
$ 3.2万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-05 至 2025-05-04
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10535949
关键词：
Actins Address Adopted Affect Apoptosis Binding Brain Calcium Cell Line Cell model Cells Child Complex Defect Development Developmental Delay Disorders Dynamin Dynamin I Endoplasmic Reticulum Epilepsy Event Exhibits F-Actin Feedback Fibroblasts Functional disorder Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Human Hydrolysis Immunoprecipitation Impairment Investigation Knowledge Lead Light Membrane Metabolic Microcephaly Microscopy Mitochondria Mitochondrial Diseases Modeling Molecular Morphology Mutation Nature Neurites Neurodevelopmental Deficit Neurodevelopmental Disorder Neuronal Differentiation Neurons Optic Atrophy Organelles Outcome Pathogenicity Pathologic Pathology Patients Phenotype Process Proteins Resolution Role Seizures Site Symptoms Synapses Testing Therapeutic Total Internal Reflection Fluorescent axon growth base cell motility cell type constriction de novo mutation early childhood epileptic encephalopathies exome sequencing improved induced pluripotent stem cell insight loss of function mutation mitochondrial fitness mitochondrial membrane mutant nerve stem cell neurodevelopment neurogenesis neuron development novel receptor stem cells synaptogenesis therapeutic target trafficking

项目摘要

Summary With the advent of exome sequencing, a growing number of children are being identified with de novo loss of function mutations in the large GTPase essential for mitochondrial fission - Dynamin Related Protein 1 (DRP1); these mutations result in severe neurodevelopmental phenotypes such as developmental delay, optic atrophy, and epileptic encephalopathies. Though it is established that mitochondrial fission is an essential precursor to the rapidly changing metabolic needs of the developing cortex, it is not understood how identified mutations in different domains of DRP1 uniquely disrupt this process. F-actin and the endoplasmic reticulum (ER) form a complex to prime the mitochondria for fission by pre-constricting the mitochondrial membrane prior to formation of DRP1 oligomers. The effect of DRP1 mutations on protein interactions with F-actin and the ER has never been studied in cell types of the developing cortex. This proposal focuses on testing the mechanism of DRP1 dysfunction both on protein interactions at sites of fission as well as downstream effects on cortical neuron differentiation and maturation. We aim to approach these gaps by leveraging the power of induced pluripotent stem cells (iPSCs) harboring DRP1 mutations in either the GTPase or stalk domains to model cell-fate changes associated with early cortical development. We will functionally assess the capacity for these iPSCs with mutant DRP1 to adopt a neural progenitor fate and progress to active cortical neurons using quantitative analysis of neurite outgrowth and branching, calcium transient recording, and synchronous synaptic firing. To understand how mutant forms of DRP1 interact with F-actin and the ER during fission, we will use live super-resolution Airyscan microscopy paired with in-cell immunoprecipitation to capture changes in the assembly and disassembly of this fission machinery. Successful completion of these aims will improve our understanding of the role of mitochondrial fission during cortical development and at which stages of this process perturbations become highly pathogenic. Furthermore, these results could help shed light on variable patient symptoms and outcomes based on specific DRP1 mutations, possibly leading to individualized therapeutics for mitochondrial disease.

概括随着外显子组测序的出现，越来越多的儿童被确定患有从头丧失的线粒体分裂所必需的大 GTP 酶的功能突变 - Dynamin 相关蛋白 1 (DRP1)；这些突变导致严重的神经发育表型，例如发育迟缓、视神经萎缩、和癫痫性脑病。尽管已确定线粒体裂变是线粒体分裂的重要前体发育中的皮质的代谢需求迅速变化，目前尚不清楚如何识别突变 DRP1 的不同域会独特地破坏这一过程。 F-肌动蛋白和内质网 (ER) 形成复合物通过在形成前预收缩线粒体膜来启动线粒体裂变 DRP1 寡聚体。 DRP1 突变对蛋白质与 F-肌动蛋白和 ER 相互作用的影响从未已在发育皮质的细胞类型中进行了研究。本提案重点测试DRP1的机制裂变位点的蛋白质相互作用以及对皮质神经元的下游影响的功能障碍分化和成熟。我们的目标是通过利用诱导多能的力量来弥补这些差距 GTPase 或茎结构域中含有 DRP1 突变的干细胞 (iPSC) 可模拟细胞命运的变化与早期皮质发育有关。我们将从功能上评估这些具有突变体的 iPSC 的能力 DRP1 采用神经祖细胞命运并通过定量分析发展为活跃的皮质神经元神经突生长和分支、钙瞬态记录和同步突触放电。要了解 DRP1 的突变形式如何在裂变过程中与 F-肌动蛋白和 ER 相互作用，我们将使用实时超分辨率 Airyscan 显微镜与细胞内免疫沉淀相结合，可捕获组装和组装过程中的变化拆卸这个裂变机器。成功完成这些目标将提高我们对线粒体裂变在皮质发育过程中的作用以及该过程的哪些阶段受到干扰变得高致病性。此外，这些结果可以帮助揭示不同的患者症状和基于特定 DRP1 突变的结果，可能导致线粒体的个体化治疗疾病。