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Published April 9, 2021 | Version v1
Journal article Open

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

  • 1. Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD.
  • 2. Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
  • 3. Department of Biochemistry and Molecular Biophysics, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032; Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, NY 10027
  • 4. Special Projects Group, New York Structural Biology Center, New York, NY 10027
  • 5. 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
  • 6. 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.

Description

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.

Notes

We would like to thank Dr. Mariko Tokito for re-engineering the TBK1 constructs with various tags, and the entire Holzbaur group for invaluable discussion. We gratefully acknowledge Project ALS for initiating this collaboration and Project ALS (Grant ID 2018-03) and NINDS (NS060698) for supporting this work. The study is also funded by the joint efforts of The Michael J. Fox Foundation for Parkinson's Research (MJFF) and the Aligning Science Across Parkinson's (ASAP) initiative. MJFF administers the grant ASAP-000350 on behalf of ASAP and itself. C.S.E. was supported by the Howard Hughes Medical Institute Hanna H. Gray Fellowship.

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1C_FR-halo-TBK1_dsRed2-mito_YFP-Parkin_cccp 2cell1.ome.tiff

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