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

基本信息

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
项目类别：
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&apos s Disease

项目摘要

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) 中铁的积累通过成像、组织病理学和生化方法证实了 SNc 中的铁沉积。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 PD 病理学的后果尚不清楚 LRRK2 中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。 LRRK2的生理和病理功能尚未完全阐明。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-蛋白质相互作用方面可能存在很大差异。我们小组的最新研究结果表明 G2019S 结果与激酶活性有关。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 注射进行纯合敲入小鼠诱发这些变化。铁稳态是否是 LRRK2 突变的保守特征。此外，尚不清楚 G2019S 之外的情况是否为内源性杂合。 LRRK2 突变足以驱动铁的基础增加。最后，PD 患者脑内铁沉积量增加。在黑质内观察到 DA 神经元中各种 LRRK2 突变的影响完全是因此，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。使用选择性成像探针和高内涵成像将 LRRK2 突变 iPSC 转化为皮质和 DA 神经元。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。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，这些实验将严格且明确地进行。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 LRRK2 的所有致病性突变都集中在人类皮质神经元和 DA 神经元之间。十多种 Rab GTP 酶的磷酸化增加，包括 Rab8a 和 Rab10。 PD 病因学的磷酸化尚不清楚，LRRK2 依赖性磷酸化被认为是捕获 Rabs 的诱因。处于 GDP 约束状态，有效抑制了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。如果成功的话，这种探索性的 R21 足以挽救 LRRK2 突变皮质和 DA 神经元中的铁变化。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。