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

• 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
• 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

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 随着外显节测序的出现，越来越多的儿童被从头丧失。 大型GTPase中的功能突变，对于线粒体裂变 - 动力学相关蛋白1（DRP1）所必需的功能突变； 这些突变导致严重的神经发育表型，例如发育延迟，视神经萎缩， 和癫痫性脑病。尽管确定线粒体裂变是 发育中的皮质的快速变化的代谢需求，尚不理解如何确定的突变 DRP1的不同域唯一破坏了此过程。 F-肌动蛋白和内质网（ER）形成A 复合物通过在形成之前预收缩线粒体膜来裂变线粒体裂变 DRP1低聚物。 DRP1突变对蛋白质与F-肌动蛋白相互作用和ER的影响从未 在发育中的皮质的细胞类型中进行了研究。该建议重点是测试DRP1的机制 裂变部位蛋白质相互作用的功能障碍以及对皮质神经元的影响 分化和成熟。我们的目标是利用诱导多能的力量来解决这些差距 干细胞（IPSC）在GTPase或Stalk结构域中含有DRP1突变，以建模细胞粘液变化 与早期皮质发育有关。我们将通过突变体功能评估这些IPSC的能力 DRP1采用神经祖细胞的命运并使用定量分析对活性皮质神经元进行进展 神经突生长和分支，钙瞬态记录以及同步突触射击。理解 DRP1突变形式如何与F-肌动蛋白和ER相互作用，我们将使用实时超分辨率 Airyscan显微镜与内部免疫沉淀配对以捕获组件的变化和 这种裂变机械的拆卸。这些目标的成功完成将提高我们对 线粒体裂变在皮质发育中的作用以及该过程扰动的阶段 变得高度致病。此外，这些结果可能有助于阐明可变的患者症状和 基于特定DRP1突变的结果，可能导致线粒体的个性化治疗剂 疾病。