Dysregulation of iron homeostasis by mutant LRRK2 in human neurons

项目摘要 铁在帕金森病（PD）患者的黑质（SNc）中的蓄积， 通过影像学、组织病理学和生物化学方法证实。而铁在SNc中的沉积 与患者运动症状的严重程度相关，无论是疾病的早期促发事件还是 人类PD病理学的后果尚不清楚。LRRK 2的多个错义突变导致常染色体 显性PD和最近的数据链路野生型LRRK 2信号传导到更常见的散发性PD。的 LRRK 2的生理和病理功能尚未完全阐明。PD连锁突变可以是 发现于激酶结构域（G2019 S，I2020 T）和具有GT3功能的ROC/COR双链蛋白 （R144C/G/H，Y1699C）。这些突变在蛋白质-蛋白质相互作用方面可以有很大的不同， 激酶活性，但每一个都与PD相关。我们小组最近的研究结果表明，G2019 S的结果 在体内和体外的铁稳态异常中。然而，到目前为止，我们只考虑了这一个突变， 并依赖于异源过表达系统或颅内LPS注射在纯合敲入 老鼠来唤起这些变化。铁稳态异常是否是LRRK 2突变的保守特征 G2019 S之后的问题尚未解决。此外，还不知道内源性杂合是否 LRRK 2突变足以驱动铁的基础增加。最后，在PD患者脑铁沉积中， 在黑质内观察到，DA神经元中各种LRRK 2突变的影响完全是 未知因此，在目标1中，我们将区分WT、激酶结构域和非激酶结构域 LRRK 2突变型iPSC进入皮质和DA神经元。使用选择性成像探针和高含量成像， 我们将评估人WT和LRRK 2突变神经元中的细胞质和线粒体铁负荷。总 细胞铁将通过ICP-MS定量，LRRK 2激酶活性在这些作用中的作用将通过 通过选择性药理学抑制进行检查。其次，我们将探讨细胞铁的下游效应 通过测定铁相关因子和ROS的表达。这些实验将严格而明确地 确定LRRK 2中的多样性杂合PD突变是否驱动铁稳态异常，以及这是否 在人类皮质和DA神经元之间的作用不同。LRRK 2中的所有致病突变都集中在 超过12种Rab GTP酶的磷酸化增加，包括Rab 8a和Rab 10。这一贡献 磷酸化与PD病因的关系尚不清楚。LRRK 2依赖性磷酸化被认为是捕获Rabs的关键。 在GDP约束状态下，有效地抑制了它们的功能。我们小组最近发表的初步数据 Rab 8a与铁代谢的关系因此，在目标2中，我们将确定Rab 8a表达式是否唯一 足以挽救LRRK 2突变皮质和DA神经元中的铁变化。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LRRK 2依赖性表型，并提供令人信服的数据来支持 未来R 01探索神经元和神经胶质中LRRK 2驱动的铁稳态异常的病理后果。

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.