Protocols for "Mitochondrial Anchored Protein Ligase MAPL is an inflammatory rheostat that regulates immune signalling and cell death"

mai.nguyen; Jack Collier; Olesia Ignatenko; Sidong Huang; Michel Desjardins; heidi.mcbride

May 05, 2025

Protocols for "Mitochondrial Anchored Protein Ligase MAPL is an inflammatory rheostat that regulates immune signalling and cell death"

DOI

dx.doi.org/10.17504/protocols.io.8epv5x266g1b/v1

mai.nguyen^1,2,
Jack Collier^1,2,
Olesia Ignatenko^1,2,
Sidong Huang³,
Michel Desjardins⁴,
heidi.mcbride^1,2

¹Department of Neurology and Neuroscience, Montréal Neurological Institute, McGill University, Montréal, Canada;
²Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
³Department of Biochemistry, McGill University, Montréal, Canada;
⁴Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, Canada

Lilia Rodriguez

Université de Montréal

DOI: dx.doi.org/10.17504/protocols.io.8epv5x266g1b/v1

Collection Citation: mai.nguyen, Jack Collier, Olesia Ignatenko, Sidong Huang, Michel Desjardins, heidi.mcbride 2025. Protocols for "Mitochondrial Anchored Protein Ligase MAPL is an inflammatory rheostat that regulates immune signalling and cell death". protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x266g1b/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: September 08, 2023

Last Modified: May 05, 2025

Collection Integer ID: 87577

Keywords: ASAPCRN, pyroptosis

Funders Acknowledgements:

Aligning Science Across Parkinson’s (ASAP)

Grant ID: ASAP 000525

Abstract

Cell death is inhibited in cancers but increased in neurodegeneration, highlighting that its regulation is essential for human health. MAPL is an outer mitochondrial membrane SUMO ligase with roles in cancer and neurodegeneration in vivo, yet how MAPL controls cell death remains unclear. Combining genome-wide screening and cell biological approaches, we found that MAPL induces pyroptosis through an inflammatory pathway involving mitochondria and lysosomes. MAPL overexpression promotes mitochondrial DNA (mtDNA) trafficking in mitochondrial-derived vesicles (MDVs) to lysosomes, which are permeabilised in a process requiring gasdermin pores. This triggers release of mtDNA into cytosol, activating the DNA sensor cGAS, which is required for cell death. Additionally, multiple Parkinson’s disease-related genes, including VPS35 and LRRK2, also regulate MAPL-induced pyroptosis. Consistent with our findings, genetic deletion of MAPL, LRRK2 or VPS35 inhibited inflammatory cell death in primary macrophages, placing MAPL and the mitochondria to lysosome pathway at the nexus of immune signalling and cell death.  

Files

Culturing of cell lines.docx

Generation of Bone Marrow derived macrophages.docx

Transfection and generation of stable cell lines.docx

siRNA transfection.docx

Blue native PAGE to evaluate NLRP3 oligomer.docx

Cells lysates SDS-PAGE and immunoblotting.docx

Pyroptotic cell death .docx

Detection of extracellular cytokine.docx

Amplfication of adenovirus.docx

Protocol

Name

CRISPR-Cas9 Screening Protocol for Gene Perturbation Analysis

Version 1

Created by

Lilia RodriguezUniversité de Montréal

Immunofluorescence.docx

Image processing and quantification.docx

Live cell microscopy.docx

RNA isolation from tissues with TRIzol.docx

TEM for monolayer cells.docx

Isolation of cytosolic DNA.docx

Immunoisolation of lysosomes.docx

Public workspaceProtocols for "Mitochondrial Anchored Protein Ligase MAPL is an inflammatory rheostat that regulates immune signalling and cell death"

Protocols for "Mitochondrial Anchored Protein Ligase MAPL is an inflammatory rheostat that regulates immune signalling and cell death"