Protocols for "LRRK2 mutations block NCOA4 trafficking upon iron overload leading to ferroptotic death"

Andres Goldman; Mai Nguyen; Heidi  McBride

Apr 13, 2026

Protocols for "LRRK2 mutations block NCOA4 trafficking upon iron overload leading to ferroptotic death"

DOI

https://dx.doi.org/10.17504/protocols.io.x54v9q4b4l3e/v1

Andres Goldman^1,2,
Mai Nguyen^1,2,
Heidi McBride^1,2

¹Montreal Neurological Institute, McGill University 3801 University Ave, Montreal, Quebec;
²Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA

Lilia Rodriguez

Université de Montréal

DOI: https://dx.doi.org/10.17504/protocols.io.x54v9q4b4l3e/v1

External link: https://www.biorxiv.org/content/10.1101/2025.08.25.672135v1

Collection Citation: Andres Goldman, Mai Nguyen, Heidi McBride 2026. Protocols for "LRRK2 mutations block NCOA4 trafficking upon iron overload leading to ferroptotic death". protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9q4b4l3e/v1

License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this collection and it's working

Created: April 13, 2026

Last Modified: April 13, 2026

Collection Integer ID: 314922

Keywords: ASAPCRN, iron homeostasis within the raw macrophage cell line, altered iron homeostasi, iron dysregulation in pd, iron homeostasi, dysregulation of iron, iron dysregulation, rich repeat protein kinase, elevated levels of ferritin, ferritin heavy chain, mutations in lrrk2, ferritinophagy, lrrk type ii kinase inhibitor, reduction of ferritin heavy chain, lrrk2g2019s mutant cell, ferritin, membrane trafficking pathway, iron chelation, homozygous mutant lrrk2g2019, iron overload, phosphorylated rab8, related protein, parkinson, rab8 at the plasma membrane, iron, mutation, proteomics analysis, activating mutation

Funders Acknowledgements:

Aligning Science Across Parkinson’s

Grant ID: ASAP-025171

Abstract

Altered iron homeostasis has long been implicated in Parkinson's Disease (PD), although the mechanisms have not been clear. Given the critical role of PD-related activating mutations in LRRK2 (leucine-rich repeat protein kinase 2) within membrane trafficking pathways we examined the impact of a homozygous mutant LRRK2G2019S on iron homeostasis within the RAW macrophage cell line with high iron capacity. Proteomics analysis revealed a dysregulation of iron-related proteins in steady state with highly elevated levels of ferritin light chain and a reduction of ferritin heavy chain. LRRK2G2019S mutant cells showed efficient ferritinophagy upon iron chelation, but upon iron overload there was a near complete block in the degradation of the ferritinophagy adaptor NCOA4. These conditions lead to an accumulation of phosphorylated Rab8 at the plasma membrane, which is selectively inhibited by LRRK type II kinase inhibitors. Iron overload then leads to increased oxidative stress and ferroptotic cell death. These data implicate LRRK2 as a key regulator of iron homeostasis and point to the need for an increased focus on the mechanisms of iron dysregulation in PD. 

Files

01_Cell_culture_and_reagents.docx

02_LRRK2G2019S_proteomics_analysis.docx

03_Cell_lysates_SDS-PAGE_and_immunoblotting.docx

04_Iron_quantification.docx

05_Aconitase_activity_assay.docx

06_Generation_of_CRISPR_KI_KO.docx

07_Immunofluorescence_and_live_imaging.docx

$Icon representing the file 08_Biochemical_fractionation_Triton_X100.docx$

08_Biochemical_fractionation_Triton_X100.docx

09_Quantification_condensate_number_and_size.docx

10_Quantification_FerroOrange_fluorescence.docx

11_Quantification_lysosomal_size_and_number.docx

12_Quantification_lysosomal_proteolytic_activity.docx

13_Quantification_pRAB8_NRF2_subcellular_distribution.docx

14_Growth_curve.docx