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 ...