Exploration of the role of TBK1 in autophagy and Amyotrophic Lateral Sclerosis

Tumor necrosis factor receptor-associated factor NF-KB (TANK) binding kinase 1 (TBK1) was recently identified as a novel Amyotrophic Lateral Sclerosis (ALS) gene in numerous independent human genetic studies. TBK1 is a non-canonical IkB kinase, playing a role in autophagy and the innate immune response. Over 150 TBK1 mutations along the entire protein length, predicted to cause TBK1 haploinsufficiency, have been found in patients with ALS and ALS-frontotemporal dementia (FTD). TBK1 mutations are found in approximately 4% of all ALS, ALS-FTD and FTD cases, but exactly how these mutations are contributing towards disease is unclear. It is hypothesised that TBK1 mutations contribute to ALS disease pathology due to impaired autophagy, in particular mitophagy. These two processes are crucial for homeostasis and are involved in numerous neurodegenerative diseases; basal autophagy levels are also high in neurons. Given that aggregate formation is a pathological feature of ALS, removal of these aggregates by autophagy is of particular relevance in this disease. The main aim of this work is to investigate pathways regulated by TBK1 in a ‘neuronal-like’ setting, and to elucidate how TBK1 mutations contribute to the pathogenesis of ALS. This work shows that TBK1 phosphorylation is elevated upon autophagy induction in NSC-34 cells, a cell-line commonly used in ALS research. Inhibition or depletion of TBK1 results in two distinct autophagy phenotypes – autophagy blockage and autophagy reduction. Using proteomic approaches, this work has explored the TBK1 signalling pathways further and identified a novel upstream kinase of TBK1, Nek1, another recently discovered ALS-gene. This not only links two ALS genes in the same pathway regulating autophagy, but also identifies a novel role for Nek1 in autophagy. This work has implications in the ALS field as expansion of both the potential roles of TBK1 and Nek1 mutations in ALS has been conducted, giving further insight into how they may be contributing to ALS pathology.