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.

总结 随着外显子组测序的出现，越来越多的儿童被鉴定为从头缺失。 线粒体分裂所必需的大GT3-发动蛋白相关蛋白1（DRP 1）中的功能突变; 这些突变导致严重的神经发育表型如发育迟缓，视神经萎缩， 和癫痫性脑病。虽然已经确定线粒体分裂是一个重要的前体， 发育中的皮质快速变化的代谢需求，目前还不清楚如何识别突变， DRP 1的不同结构域独特地破坏该过程。F-肌动蛋白和内质网（ER）形成一个 复合物通过在形成前预收缩线粒体膜来引发线粒体分裂 DRP 1寡聚体。DRP 1突变对蛋白质与F-肌动蛋白和ER相互作用的影响从未被证实。 在发育中的皮层细胞类型中进行了研究。本提案侧重于测试DRP 1的机制 在分裂位点的蛋白质相互作用以及对皮质神经元的下游效应上的功能障碍 分化和成熟。我们的目标是通过利用诱导多能性的力量来接近这些差距。 在GT3或茎结构域中携带DRP 1突变的干细胞（iPSC），以模拟细胞命运变化 与早期皮质发育有关我们将在功能上评估这些具有突变体的iPSC的能力。 DRP 1采用神经祖细胞命运并发展为活性皮质神经元的定量分析， 神经突生长和分支、钙瞬变记录和同步突触放电。了解 突变形式的DRP 1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配对，以捕获组装中的变化， 这个裂变机器的分解。成功地完成这些目标将提高我们对 线粒体分裂在皮质发育过程中的作用，以及在这一过程的哪个阶段扰动 成为高致病性。此外，这些结果可以帮助阐明不同的患者症状， 基于特定DRP 1突变的结果，可能导致线粒体疾病的个体化治疗 疾病