Understanding the effects of mitochondrial fission disruption during early cortical development

摘要 随着外显子组测序的出现，越来越多的儿童被确认为从头开始丢失 线粒体分裂动力蛋白相关蛋白1(Drp1)所必需的大GTP酶的功能突变； 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、 以及癫痫脑病。尽管已经确定线粒体分裂是一种重要的先兆 快速变化的新陈代谢需求，目前还不清楚如何识别突变 DRP1的不同结构域独特地破坏了这一过程。F-肌动蛋白和内质网形成 通过在形成前预先收缩线粒体膜来为线粒体的分裂做准备的复合体 DRp1寡聚体。DRp1突变对F-肌动蛋白和内质网相互作用的影响 已经在发育中的皮质的细胞类型中进行了研究。这项提案的重点是测试Drp1的机制 分裂部位蛋白质相互作用的功能障碍及其对皮质神经元的下游影响 分化和成熟。我们的目标是通过利用诱导多能性的力量来填补这些差距。 在GTPase或茎结构域中携带Drp1突变的干细胞(IPSCs)模拟细胞命运的变化 与早期皮质发育有关。我们将从功能上评估这些突变的ipscs的能力。 DRP1采用神经前体细胞的命运和进展来定量分析活跃的皮质神经元 轴突生长和分支、钙瞬变记录和同步突触放电。要理解 在分裂过程中，突变形式的Drp1如何与F-肌动蛋白和内质网相互作用，我们将使用实时超分辨率 AiryScan显微镜与细胞内免疫沉淀相结合，以捕捉组装和 拆解这台核裂变机器。成功完成这些目标将提高我们对 线粒体分裂在皮质发育中的作用以及这一过程的哪个阶段受到干扰 变得高度致病。此外，这些结果可以帮助了解不同的患者症状和 基于特定Drp1突变的结果，可能导致对线粒体的个体化治疗 疾病。

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.