The impact of mitochondrial and peroxisomal fission dynamics on metabolic signaling during corticogenesis.

线粒体和过氧化物酶体裂变动力学对皮质生成过程中代谢信号的影响。

• 批准号: 10393920
• 负责人: Gabriella Lou Puig Robertson
• 金额: $ 3.23万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10393920
• 关键词: Address; Affect; Biochemical; Biological; Biological Assay; Biology; Brain; Carbon; Cells; Cerebrum; Clinical; Coupling; Crista ampullaris; Cytometry; Defect; Development; Developmental Delay Disorders; Disease; Dominant-Negative Mutation; Dynamin; Dynamin I; Electron Transport; Embryo; Encephalopathies; Epilepsy; Fibroblasts; Generations; Genes; Glycolysis; Guanosine Triphosphate Phosphohydrolases; Human; Image; Immunohistochemistry; Impairment; In Vitro; Knock-out; Knowledge; Laboratory Research; Lead; Maintenance; Measures; Metabolic; Metabolism; Microcephaly; Microscopy; Missense Mutation; Mitochondria; Molecular; Morphology; Muscle; Mutation; Neurodevelopmental Impairment; Neurons; Organelles; Organoids; Patients; Phase; Phenotype; Play; Postdoctoral Fellow; Production; Prosencephalon; Proteins; Protons; Regulation; Research; Research Personnel; Research Project Grants; Resolution; Role; Running; Seizures; Signal Transduction; Structure; Symptoms; System; Technical Expertise; Technology; Therapeutic; Training; Transmission Electron Microscopy; Work; confocal imaging; effective therapy; experience; induced pluripotent stem cell; insight; metabolic profile; mouse model; multi-electrode arrays; mutant; nerve stem cell; neurodevelopment; neurogenesis; neurological pathology; neuron development; neuronal excitability; novel; patch clamp; peroxisome; post-doctoral training; self-renewal; skills; stem cell model; therapeutic target; three-dimensional modeling; tool; two-dimensional

PROJECT SUMMARY/ABSTRACT Mitochondria are dynamic signaling organelles that constantly undergoes fission (fragmentation) and fusion (elongation) to adapt its structure to the demands of the cell. DRP1 (dynamin-related protein 1) is a GTPase that plays a crucial role in mitochondrial fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with neurodevelopmental symptoms. To interrogate the molecular mechanisms by which DRP1 mutations cause neurodevelopmental defects, we are utilizing patient-derived fibroblasts and iPSC-derived models from patients with mutations in different domains of DRP1 who present with clinically disparate conditions. The G32A mutation lies in the GTPase domain of DRP1 and is associated with microcephaly. The R403C mutation lies in the stalk domain of DRP1 and causes progressively severe epilepsy. Specific Aim 1 presents the progress thus far to uncover the impact of DRP1 mutations on mitochondrial structure and metabolic function using patient-derived fibroblasts. Patient cells display elongated mitochondrial structure and impaired coupling efficiency of the electron transport chain (ETC). Specific Aim 2 (the F99 phase of this proposal) will explore the consequences of these findings in neurodevelopment using patient-derived induced pluripotent stem cell (iPSC) models: three-dimensional cerebral organoids and two-dimensional neural progenitor cultures. Confocal imaging and mass cytometry (CyTOF) will be used to determine if patient mutations lead to change of cell fate in early corticogenesis. Further, multi-electrode array (MEA) technology will be leveraged to examine the development of neuronal network activity in patient-derived brain organoids. Understanding the mechanism by which these mutations cause neurological pathology will give insight into the role of mitochondrial dynamics in neurodevelopment. Specific Aim 3 (the K00 phase of this proposal) will develop the applicant into an independent academic researcher investigating the interactions between metabolic signaling and neurodevelopment. The K00 phase will provide the applicant with training in metabolic analysis using carbon tracing and in vitro biochemical assays as well as the professional development and networking necessary to run an independent research laboratory. Overall, the work outlined in this proposal will equip the candidate with both the technical expertise and professional skills to make great strides in the field of metabolism in neurodevelopment.

项目总结/摘要 线粒体是一种动态的信号细胞器，不断经历分裂（碎裂）和融合 （伸长）以使其结构适应细胞的需求。DRP 1（动力蛋白相关蛋白1）是一种GT3， 在线粒体分裂中起着至关重要的作用基因新发杂合错义突变患者 编码DRP 1，DNM 1 L，呈现神经发育症状。去审问那些 DRP 1突变导致神经发育缺陷的机制，我们正在利用患者来源的 成纤维细胞和iPSC衍生的模型，这些模型来自具有DRP 1不同结构域突变的患者， 临床上完全不同的病症G32 A突变位于DRP 1的GTdR结构域，与 小头畸形R403 C突变位于DRP 1的茎域，并导致进行性严重癫痫。 具体目标1介绍了迄今为止发现DRP 1突变对线粒体结构的影响的进展 和代谢功能。患者细胞显示延长的线粒体结构 并且损害电子传递链（ETC）的偶联效率。具体目标2（F99阶段） 建议）将探讨这些结果在神经发育的后果，使用患者衍生的诱导 多能干细胞（iPSC）模型：三维脑类器官和二维神经 祖先文化将使用共聚焦成像和质谱细胞术（CyTOF）来确定患者突变 导致皮质生成早期细胞命运改变。此外，多电极阵列（MEA）技术将被广泛应用。 用于检查患者来源的脑类器官中神经元网络活动的发展。 了解这些突变导致神经病理学的机制将有助于深入了解 线粒体动力学在神经发育中的作用。将制定具体目标3（本提案的K 00阶段） 申请人成为一个独立的学术研究人员，调查代谢之间的相互作用， 信号传导和神经发育。K 00阶段将为申请人提供代谢分析培训 使用碳示踪和体外生化分析以及专业发展和网络 有必要运行一个独立的研究实验室。总的来说，本提案中概述的工作将使 具有技术专长和专业技能的候选人，在代谢领域取得长足进步 在神经发育中。