Abstract
The role of mitophagy, a process that allows the removal of damaged mitochondria from cells, remains unknown in multiple sclerosis (MS), a disease that is found associated with dysfunctional mitochondria. Here we have qualitatively and quantitatively studied the main players in PINK1-mediated mitophagy in peripheral blood mononuclear cells (PBMCs) of patients with relapsing–remitting MS. We found the variant c.491G>A (rs550510, p.G140E) of NDP52, one of the major mitophagy receptor genes, associated with a MS cohort. Through the characterization of this variant, we discovered that the residue 140 of human NDP52 is a crucial modulator of NDP52/LC3C binding, promoting the formation of autophagosomes in order to drive efficient mitophagy. In addition, we found that in the PBMC population, NDP52 is mainly expressed in B cells and by ensuring efficient mitophagy, it is able to limit the production of the proinflammatory cytokine TNF-α following cell stimulation. In sum, our results contribute to a better understanding of the role of NDP52 in mitophagy and underline, for the first time, a possible role of NDP52 in MS.
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Data availability
All the software used is freely available. The scripts, input, and outputs files generated during the study will be freely available in a GitHub repository associated with our publication (https://github.com/ELELAB/LC3C_NDP52_GE_mutant). The MD trajectories will be available in Open Science Framework (OSF): https://osf.io/48wzq/.
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Acknowledgements
We wish to thank Dr Christian Behrends for kindly providing us the construct encoding HA-LC3C; Prof. Honglin Luo for the gift of 3x-Flag-NDP52 plasmid; Dr Anna Kabanova for granting the execution of some revision experiments in her laboratory; Krenare Bruqi for her technical assistance and D.Hodder for his proofreading. We are grateful to Meike Crecelius, Natalia Rogova, and Viktoria Morasch for their help with cloning of NDP52 fragments for ITC and NMR experiments, protein/peptide sample preparation, and ITC data collection.
Funding
This work was supported by grants: ROCHE (Roche per la ricerca 2017) and 5XMILLE Italian Ministry of Health (2017) to FS. The study was partially supported by the Italian Ministry of Health (Progetto di ricerca Finalizzata RF-2018-12366111) and by the Italian Foundation for Multiple Sclerosis (Fism Progetto Speciale 2018/S/5) to LB. The work of NW, VD, and VVR was supported by the DFG-funded Collaborative Research Centre on Selective Autophagy (SFB 1177 “Molecular and Functional Characterization of Selective Autophagy”), Germany. VD and VVR also received funding from Structural Genomic Consortium (SGC). The SGC is a registered charity (no: 1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Diamond, Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and USA), Merck & Co (aka MSD outside Canada and USA), Pfizer, São Paulo Research Foundation-FAPESP, Takeda and Wellcome [106169/ZZ14/Z]. The work of VC was supported by Italian Foundation of Multiple Sclerosis (FISM grant 2017/R/08) and by Ministry of Health, Progetto Giovani Ricercatori (GR-2016-02362380). The work of MK, MT, ML, FL, VD, and EP is supported by Danmarks Grundforskningsfond (DNRF125) and a Carlsberg Foundation Distinguished Fellowship (CF18-0314) to EP’s group. Moreover, the project has been supported by a Netaji Subhash ICAR international fellowship, Government of India to MK to work in EP group. The calculations described in this paper were performed using the DeiC National Life Science Supercomputer Computerome at DTU (Denmark), and a DeiC-Pilot Grant on Abacus (Denmark).
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FS designed the project. ADR and FS wrote the paper. ADR performed analysis in MS patients (Fig. 1), on B cells (Fig. 5a–d) and performed the cloning of NDP52GE. TM investigated the role of NDP52 variants in the TLR signaling (Supplementary Fig. 1) and helped FS in performing Immunofluorescences of Figs. 2, 3, and 5e–g. FS characterized the binding between LC3C and NDP52 variants, performed the mitophagy analysis and Fig. 5e–g. MC helped FS in the characterization of LC3C-NDP52 interaction. PDA participated in the amplification of NDP52-LIR motif from MS patients and HD donors. DFA and LB provided PBMCs of MS patients and the LCL cell line. VC provided PBMCs of HD and MS patients and helped in preparing some cDNAs (Fig. 1). DFA performed FACS analysis (TMRM) and sorted monocytes, B and T cells from healthy patients (Fig. 5a). GC, RC, and VC performed the genetical study. MK, MT, ML, FL, VD, and EP performed Fig. 3e, f. The ITC and NMR experiments were designed by VD and VVR, and performed by NW and FL (Fig. 3 and Supplementary Fig. 2). ADR, EP, VVR, EG, and FS analyzed data. All authors commented on the final draft of the paper.
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Di Rita, A., Angelini, D.F., Maiorino, T. et al. Characterization of a natural variant of human NDP52 and its functional consequences on mitophagy. Cell Death Differ 28, 2499–2516 (2021). https://doi.org/10.1038/s41418-021-00766-3
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DOI: https://doi.org/10.1038/s41418-021-00766-3
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