Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss accompanied by cytoplasmic localization of TDP-43 proteins and their insoluble accumulations. Haploinsufficiency of TBK1 has been found to associate with or cause ALS. However, the cell-autonomous mechanisms by which reduced TBK1 activity contributes to human motor neuron pathology remain elusive. Here, we generated a human cellular model harboring loss-of-function mutations of TBK1 by gene editing and found that TBK1 deficiency was sufficient to cause TDP-43 pathology in human motor neurons. In addition to its functions in autophagy, we found that TBK1 interacted with endosomes and was required for normal endosomal maturation and subsequent lysosomal acidification. Surprisingly, TDP-43 pathology resulted more from the dysfunctional endo-lysosomal pathway than the previously recognized autophagy inhibition mechanism. Restoring TBK1 levels ameliorated lysosomal dysfunction and TDP-43 pathology and maintained normal motor neuron homeostasis. Notably, using patient-derived motor neurons, we found that haploinsufficiency of TBK1 sensitized neurons to lysosomal stress, and chemical regulators of endosomal maturation rescued the neurodegenerative process. Together, our results revealed the mechanism of TBK1 in maintaining TDP-43 and motor neuron homeostasis and suggested that modulating endosomal maturation was able to rescue neurodegenerative disease phenotypes caused by TBK1 deficiency.

TDP-43 subcellular mislocalisation is correlated with loss of optineurin binding for frontotemporal dementia and amyotrophic lateral sclerosis associated TBK1 missense variants

“…Mislocalisation of neuronal TDP-43 from the nucleus to the cytoplasm is a key pathogenic process observed in the majority of ALS and ∼50% of FTD patients [3], including those with TBK1 mutations [4,36,37]. TBK1 knockdown human cellular and mouse models have also demonstrated TDP-43 cytoplasmic mislocalisation [38,39]. Here we assess for the first time the effect of TBK1 missense variants on this process.…”

The role of autophagic kinases in regulation of axonal function

“…Another link between TBK1 and endolysomal trafficking is that TBK1 directly phosphorylates Rab7a, a critical regulator of late endosomes. In fact, TBK1 loss of function in human iPS MNs and TBK1 patient-derived human MNs led to a reduction of Rab7a and deficient lysosomal activity ( Hao et al, 2021 ). In axons, loss of TBK1 in human iPS MNs led to overactive spontaneous firing and impaired axonal regeneration, suggesting a link between impaired TBK1 regulation of endolysosomal trafficking and axonal dysfunction in ALS ( Hao et al, 2021 ).…”

“…In fact, TBK1 loss of function in human iPS MNs and TBK1 patient-derived human MNs led to a reduction of Rab7a and deficient lysosomal activity ( Hao et al, 2021 ). In axons, loss of TBK1 in human iPS MNs led to overactive spontaneous firing and impaired axonal regeneration, suggesting a link between impaired TBK1 regulation of endolysosomal trafficking and axonal dysfunction in ALS ( Hao et al, 2021 ). These data indicate that ALS-causing TBK1 mutations may cause dysregulation of axonal function through multiple pathways, including autophagosome formation, mitophagy, and endolysosomal trafficking.…”

Two FTD-ALS genes converge on the endosomal pathway to induce TDP-43 pathology and degeneration

“…Loss of TBK1 impairs endosome maturation in human motor neurons ( 22 ). We asked whether TBK1 sequestration also impaired this pathway.…”

“…TBK1-mediated endosomal defects induce TDP-43 pathology in human motor neurons ( 22 ). Rare pTDP-43 inclusions are also observed in mice expressing poly(GA) ( 15 ).…”