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ALS and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy

View ORCID ProfileOlivia Harding, View ORCID ProfileChantell S. Evans, View ORCID ProfileJunqiang Ye, View ORCID ProfileJonah Cheung, View ORCID ProfileTom Maniatis, View ORCID ProfileErika L.F. Holzbaur
doi: https://doi.org/10.1101/2021.04.12.439050
Olivia Harding
1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD
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Chantell S. Evans
1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
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Junqiang Ye
3Department of Biochemistry and Molecular Biophysics, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032
4Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, NY 10027
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Jonah Cheung
5Special Projects Group, New York Structural Biology Center, New York, NY 10027
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Tom Maniatis
3Department of Biochemistry and Molecular Biophysics, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032
4Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, NY 10027
6New York Genome Center, New York, NY 10013
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Erika L.F. Holzbaur
1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD
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  • For correspondence: holzbaur@pennmedicine.upenn.edu
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ABSTRACT

TANK-binding kinase 1 (TBK1) is a multi-functional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD), including missense mutations that disrupt the ability of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), auto-activate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presense of either WT- or kinase inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals.

SIGNIFICANCE STATEMENT Missense mutations in TANK-binding kinase 1 (TBK1) have various biophysical and biochemical effects on the molecule, and are associated with the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). TBK1 plays an essential role in clearing damaged mitochondria. Here, we investigate the impact of 10 ALS-linked TBK1 mutations on the critical early stage of mitophagy. We find that both TBK1 recruitment and kinase activity contribute to the clearance of the damaged mitochondria. Furthermore, in neurons, expression of TBK1 mutants alone affects mitochondrial network health. Our investigation utilizes disease-linked mutations to further refine the current model of mitophagy, identifying crosstalk between the regulatory kinases TBK1 and ULK1, and providing new insights into the roles of TBK1 in neurodegenerative pathogenesis.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • http://doi.org/10.5281/zenodo.4670341

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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ALS and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy
Olivia Harding, Chantell S. Evans, Junqiang Ye, Jonah Cheung, Tom Maniatis, Erika L.F. Holzbaur
bioRxiv 2021.04.12.439050; doi: https://doi.org/10.1101/2021.04.12.439050
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ALS and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy
Olivia Harding, Chantell S. Evans, Junqiang Ye, Jonah Cheung, Tom Maniatis, Erika L.F. Holzbaur
bioRxiv 2021.04.12.439050; doi: https://doi.org/10.1101/2021.04.12.439050

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