Harry Perkins Institute of Medical Research, Centre for Medical Research, University of Western Australia, Nedlands, Western Australia, Australia; Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia; Perron Institute for Neurological and Translational Science, Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Nedlands, Western Australia, Australia; Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Health Research Building, Discovery Way, Murdoch, WA, Australia.
School of Health Sciences, Notre Dame University, Fremantle, Western Australia, Australia.
Mol Cell Neurosci. 2020 Oct;108:103539. doi: 10.1016/j.mcn.2020.103539. Epub 2020 Aug 21.
Mutations affecting SQSTM1 coding for p62 and TANK-Binding Kinase 1 (TBK1) have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TBK1 is a serine-threonine kinase that regulates p62's activity as an autophagy receptor via phosphorylation and also has roles in neuroinflammatory signalling pathways. The mechanisms underlying ALS and FTLD pathogenesis as a result of TBK1 mutations are incompletely understood, however, loss of TBK1 function can lead to dysregulated autophagy and mitophagy. Here, we report that an ALS-associated TBK1 variant affecting the kinase domain, p.G175S, is defective in phosphorylation of p62 at Ser-403, a modification critical for regulating its ubiquitin-binding function, as well as downstream phosphorylation at Ser-349. Consistent with these findings, expression of p.G175S TBK1 was associated with decreased induction of autophagy compared to wild type and reduced degradation of the ALS-linked protein TDP-43. Expression of wild type TBK1 increased NF-κB signalling ~300 fold in comparison to empty vector cells, whereas p.G175S TBK1 was unable to promote NF-κB signalling above levels observed in empty vector transfected cells. We also noted a hitherto unknown role for TBK1 as a suppressor of oxidative stress (Nrf2) signalling and show that p.G175S TBK1 expressing cells lose this inhibitory function. Our data suggest that TBK1 ALS mutations may broadly impair p62-mediated cell signalling, which ultimately may reduce neuronal survival, in addition TDP-43 was not efficiently degraded, together these effects may contribute to TBK1 mutation associated ALS and FTLD pathogenesis.
影响 SQSTM1 编码的 p62 和 TANK 结合激酶 1（TBK1）的突变与肌萎缩侧索硬化症（ALS）和额颞叶变性（FTLD）有关。TBK1 是一种丝氨酸-苏氨酸激酶，可通过磷酸化调节 p62 作为自噬受体的活性，并且在神经炎症信号通路中也具有作用。然而，TBK1 突变导致 ALS 和 FTLD 发病机制的机制尚未完全了解，但是 TBK1 功能的丧失会导致自噬和线粒体自噬失调。在这里，我们报告了一种与 ALS 相关的 TBK1 变体，该变体影响激酶结构域，p.G175S，在 p62 的 Ser-403 处的磷酸化作用受损，该修饰对于调节其泛素结合功能以及下游的 Ser-349 磷酸化作用至关重要。与这些发现一致，与野生型相比，表达 p.G175S TBK1 与自噬的诱导减少以及 ALS 相关蛋白 TDP-43 的降解减少有关。与空载体细胞相比，表达野生型 TBK1 可使 NF-κB 信号增加约 300 倍，而 p.G175S TBK1 无法使 NF-κB 信号增加到空载体转染细胞中观察到的水平以上。我们还注意到 TBK1 作为氧化应激（Nrf2）信号的抑制剂的先前未知作用，并表明表达 p.G175S TBK1 的细胞丧失了这种抑制功能。我们的数据表明，TBK1 ALS 突变可能广泛损害 p62 介导的细胞信号转导，最终可能降低神经元的存活，此外，TDP-43 不能有效降解，这些影响共同导致 TBK1 突变相关的 ALS 和 FTLD 发病机制。
