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

项目摘要/摘要 线粒体是不断经历分裂(碎裂)和融合的动态信号细胞器 (伸长)使其结构适应细胞的要求。DRP1(Dynamin-Related Protein 1)是一种GTP酶 在线粒体分裂中起着至关重要的作用。基因新生杂合性错义突变患者 它编码DRp1，DNM1L，出现神经发育症状。去审问分子 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 DRp1不同结构域突变患者的成纤维细胞和IPSC衍生模型 临床上完全不同的情况。G32A突变位于Drp1的GTP酶结构域，与 小头畸形。R403C突变位于DRp1的茎结构域，导致进行性严重癫痫。 具体目标1介绍了迄今揭示Drp1突变对线粒体结构影响的进展 以及使用患者来源的成纤维细胞的代谢功能。患者细胞显示拉长的线粒体结构 并且削弱了电子传输链(ETC)的耦合效率。具体目标2(本计划的F99阶段 提案)将探索这些发现在神经发育中的后果，使用患者衍生的诱导 多能干细胞(IPSC)模型：三维脑器官和二维神经 祖先文化。将使用共聚焦成像和质量细胞术(CyTOF)来确定患者是否发生突变 导致皮质生成早期细胞命运的改变。此外，多电极阵列(MEA)技术将 用来研究患者衍生的脑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的机制将使我们深入了解 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 申请者成为一名独立的学术研究人员，研究代谢之间的相互作用 信号和神经发育。K00阶段将为申请者提供代谢分析方面的培训 利用碳示踪和体外生化分析以及专业发展和网络 运营一个独立的研究实验室所必需的。总体而言，本提案概述的工作将使 具备在新陈代谢领域取得长足进步的技术专长和专业技能的候选人 在神经发育方面。

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.