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.

项目概要/摘要 线粒体是动态信号细胞器，不断经历裂变（断裂）和融合 （伸长）以使其结构适应细胞的需求。 DRP1（动力相关蛋白 1）是一种 GTP 酶， 在线粒体裂变中起着至关重要的作用。基因中存在新杂合错义突变的患者 编码 DRP1、DNM1L，出现神经发育症状。询问分子 DRP1 突变导致神经发育缺陷的机制，我们正在利用患者来源的 来自 DRP1 不同域突变患者的成纤维细胞和 iPSC 衍生模型 临床上不同的情况。 G32A 突变位于 DRP1 的 GTPase 结构域中，并与 小头畸形。 R403C 突变位于 DRP1 的茎结构域，可导致进行性严重的癫痫。 具体目标 1 展示了迄今为止揭示 DRP1 突变对线粒体结构影响的进展 和使用患者来源的成纤维细胞的代谢功能。患者细胞显示出细长的线粒体结构 电子传输链（ETC）的耦合效率受损。具体目标 2（本次的 F99 阶段） 提案）将利用患者衍生的诱导神经发育来探索这些发现的后果 多能干细胞（iPSC）模型：三维大脑类器官和二维神经细胞 祖文化。共聚焦成像和质谱流式细胞仪 (CyTOF) 将用于确定患者是否发生突变 导致早期皮质发生中细胞命运的改变。此外，多电极阵列（MEA）技术将 用于检查患者来源的大脑类器官中神经元网络活动的发育。 了解这些突变引起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。具体目标 3（本提案的 K00 阶段）将制定 申请人成为一名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。 K00阶段将为申请人提供代谢分析方面的培训 使用碳追踪和体外生化测定以及专业发展和网络 运行一个独立的研究实验室是必要的。总体而言，本提案中概述的工作将使 兼具技术专长和专业技能的候选人，在新陈代谢领域取得长足进步 在神经发育方面。