Dysregulation of iron homeostasis by mutant LRRK2 in human neurons

人类神经元中突变型 LRRK2 导致铁稳态失调

• 批准号: 10592478
• 负责人: Adamantios Mamais
• 金额: $ 22.88万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10592478
• 关键词: AGFG1 gene; Address; Affect; Binding; Biochemical; Biological Assay; Brain; Brain region; CRISPR/Cas technology; Cells; Cytoplasm; Data; Deposition; Disease; Dyes; Etiology; Event; Future; Genes; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heterozygote; Homeostasis; Human; Image; In Vitro; Inductively Coupled Plasma Mass Spectrometry; Injections; Iron; Knock-in Mouse; LRRK2 gene; Link; Messenger RNA; Methods; Microscopy; Missense Mutation; Mitochondria; Mutation; Nerve Degeneration; Neuroglia; Neurons; Oxidative Stress; Parkinson Disease; Pathogenicity; Pathologic; Pathology; Patients; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Process; Production; Protein Family; Proteins; Publishing; Reporting; Role; Severities; Signal Transduction; Substantia nigra structure; System; Threonine; Up-Regulation; Western Blotting; autosome; dopaminergic neuron; experimental study; imaging probe; in vivo; induced pluripotent stem cell; iron metabolism; live cell imaging; motor symptom; mutant; novel; overexpression; pars compacta; pharmacologic; protein protein interaction; rab GTP-Binding Proteins; sporadic Parkinson' s Disease

Project Summary The accumulation of iron in the substantia nigra pars compacta (SNc) of Parkinson’s disease (PD) patients has been confirmed via imaging, histopathological, and biochemical methods. While iron deposition in the SNc correlates with severity of motor symptoms in patients, whether it is an early precipitating event in disease or a consequence of human PD pathology is unknown. Multiple missense mutations in LRRK2 cause autosomal dominant PD and recent data link wild-type LRRK2 signaling to the far more common sporadic PD. The physiological and pathological functions of LRRK2 have not been fully elucidated. PD-linked mutations can be found in the kinase domain (G2019S, I2020T) and the ROC/COR bidomain that harbors its GTPase function (R144C/G/H, Y1699C). These mutations can differ substantially in terms of protein-protein interactions and kinase activity, yet each are associated with PD. Recent findings from our group indicate that G2019S results in iron dyshomeostasis, both in vitro and in vivo. Thus far, however, we have only considered this one mutation and have relied on heterologous overexpression systems or intracranial LPS injection in homozygous knockin mice to evoke these changes. Whether iron dyshomeostasis is a conserved feature of LRRK2 mutations beyond G2019S has not been addressed. Furthermore, it is not known whether endogenous heterozygous LRRK2 mutation is sufficient to drive basal increases in iron. Lastly, in PD patient brain iron deposition is observed within the substantia nigra and the impact of various LRRK2 mutations in DA neurons is entirely unknown. Therefore, in Aim 1 we will differentiate a panel of WT, kinase-domain, and non kinase-domain LRRK2-mutant iPSCs into cortical and DA neurons. Using selective imaging probes and high content imaging, we will assess cytoplasmic and mitochondrial iron load in human WT and LRRK2 mutant neurons. Total cellular iron will be quantified by ICP-MS and the role of LRRK2 kinase activity in these effects will be examined by selective pharmacological inhibition. Secondly, we will explore downstream effects of cellular iron by assaying expression of iron-related factors and ROS. These experiments will rigorously and unambiguously determine whether diverse, heterozygous PD mutations in LRRK2 drive iron dyshomeostasis and whether this effect differs between human cortical and DA neurons. All pathogenic mutations in LRRK2 converge on the increased phosphorylation of over a dozen Rab GTPases, including Rab8a and Rab10. The contribution of this phosphorylation to PD etiology remains unknown. LRRK2-dependent phosphorylation is thought to trap Rabs in a GDP-bound state effectively inhibiting their function. Recent published and preliminary data from our group link Rab8a to iron metabolism. Therefore, in Aim 2 we will determine whether Rab8a expression is uniquely sufficient to rescue iron changes in LRRK2 mutant cortical and DA neurons. If successful, this exploratory R21 will codify a novel LRRK2-dependent phenotype in human neurons and provide compelling data to support a future R01 to explore the pathologic consequences of LRRK2-driven iron dyshomeostasis in neurons and glia.

项目概要 帕金森病 (PD) 患者黑质致密部 (SNc) 中铁的积累 已通过影像学、组织病理学和生化方法证实。而铁沉积在 SNc 与患者运动症状的严重程度相关，无论是疾病的早期诱发事件还是疾病 人类帕金森病病理学的后果尚不清楚。 LRRK2 的多个错义突变导致常染色体 显性 PD 和最近的数据将野生型 LRRK2 信号传导与更常见的散发性 PD 联系起来。这 LRRK2的生理和病理功能尚未完全阐明。 PD 相关突变可以是 存在于激酶结构域（G2019S、I2020T）和具有 GTPase 功能的 ROC/COR 双结构域中 （R144C/G/H、Y1699C）。这些突变在蛋白质-蛋白质相互作用和 激酶活性，但均与 PD 相关。我们小组的最新研究结果表明，G2019S 结果 体内和体外的铁稳态失调。然而到目前为止，我们只考虑了这一突变 并依赖异源过表达系统或颅内 LPS 注射进行纯合敲入 老鼠来引发这些变化。铁稳态失调是否是 LRRK2 突变的保守特征 G2019S 之后的问题尚未得到解决。此外，尚不清楚内源性杂合子是否 LRRK2 突变足以驱动铁的基础增加。最后，PD患者脑内铁沉积是 在黑质内观察到 DA 神经元中各种 LRRK2 突变的影响完全是 未知。因此，在目标 1 中，我们将区分一组 WT、激酶结构域和非激酶结构域 LRRK2 突变 iPSC 转化为皮质和 DA 神经元。使用选择性成像探针和高内涵成像， 我们将评估人类 WT 和 LRRK2 突变神经元的细胞质和线粒体铁负荷。全部的 细胞铁将通过 ICP-MS 进行定量，LRRK2 激酶活性在这些效应中的作用将是 通过选择性药理抑制进行检查。其次，我们将探讨细胞铁的下游影响 通过检测铁相关因子和 ROS 的表达。这些实验将严格而明确地 确定 LRRK2 中的多样化杂合 PD 突变是否会导致铁稳态失调，以及这是否 人类皮质神经元和 DA 神经元的作用不同。 LRRK2 的所有致病性突变均集中在 增加十多种 Rab GTP 酶的磷酸化，包括 Rab8a 和 Rab10。本次活动的贡献 磷酸化对 PD 病因的影响仍不清楚。 LRRK2 依赖性磷酸化被认为可以捕获 Rabs 处于 GDP 约束状态，有效抑制了它们的功能。我们小组最近发布的初步数据 将 Rab8a 与铁代谢联系起来。因此，在目标 2 中，我们将确定 Rab8a 表达是否是唯一的 足以挽救 LRRK2 突变皮质和 DA 神经元中铁的变化。如果成功的话，这个探索性的 R21 将在人类神经元中编码一种新的 LRRK2 依赖性表型，并提供令人信服的数据来支持 未来的 R01 旨在探索 LRRK2 驱动的神经元和神经胶质细胞中铁稳态失调的病理后果。