Phospho-proteome profiling in human neurons reveals targets of TBK1 in ALS/FTD-Phospho-proteome profiling in human neurons reveals targets of TBK1 in ALS/FTD-associated autophagy networks [iNeurons]

Loss-of-function variants in TBK1, encoding a serine/threonine protein kinase, are strongly associated with sporadic and familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the targets of TBK1 in neurons are unknown, and how haploinsufficiency for TBK1 leads to age-related neurodegeneration remains unresolved. Here, we utilized sets of isogenic induced pluripotent stem cells (iiPSCs) with loss of TBK1 or loss of optineurin (OPTN) for quantitative proteomics and phospho-proteomics across tens of thousands of phospho-sites in proliferating stem cells and induced excitatory neurons. We found that TBK1 sustains the abundance and phosphorylation of its interacting adapter proteins, AZI2/NAP1, TBKBP1, and TANK1, in the cellular context of neurons. Moreover, TBK1 regulates the phosphorylation of selective autophagy proteins, particularly at novel phospho-serine residues within cargo receptors, and endo-lysosomal pathways, including the late-endosome GTPase RAB7A, in human neurons. Additionally, we observed that the disease-associated TBK1 G217R variant acts as a phospho-protein quantitative trait locus (ppQTL) for the phosphorylation status of familial ALS-associated proteins p62/SQSTM1 and OPTN, as well as GABARAPL2, an Atg8- family autophagy protein. Furthermore, we demonstrate that TBK1 is required for neurite outgrowth and lysosomal function. Finally, we provide a census of the phospho-proteome in nascent human neurons for further dissection of protein phosphorylation in neural-enriched proteins, such as tau, in neurological and psychiatric disorders. These studies provide a global view of TBK1 as a central point of convergence in ALS/FTD-linked endo-lysosomal networks that act in a cell-autonomous manner to maintain protein homeostasis in neurons.