Introduction: Heterozygous loss-of-function mutations in TANK-binding kinase 1 (TBK1) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (1,2), two devastating and interlinked neurodegenerative disorders that form a continuous ALS-FTD disease spectrum. Whilst TBK1 haploinsufficiency has been shown to contribute to cell autonomous neuronal dysfunction, the impact of TBK1 loss-of-function in microglia is less well understood. We aimed to investigate the function of TBK1 in human microglia and understand how loss of TBK1 activity may intrinsically impair microglial function to contribute to ALS-FTD pathophysiology.

Methods: Using a combination of in-house gene editing, the JAX/iNDI collection (3,4) and well-established differentiation protocols (5), we generated isogenic wild-type (TBK1^WT/WT) and homozygous TBK1 knockout (TBK1^-/-) human pluripotent stem cell-derived microglia in two independent genetic backgrounds (HUES9 human embryonic stem cells and KOLF2.1J induced pluripotent stem cells). Using TMT LC-MS/MS, we performed unbiased proteomic and phosphoproteomic characterisation of TBK1^WT/WT and TBK1^-/- microglia, both at baseline and following immune stimulation (LPS/IFNγ, 100ng/ml, 24 hours) (HUES9: N=4, 1 clone x 4 replicates). Subsequently, we performed targeted functional assays to investigate the cellular consequences of microglial TBK1 loss (HUES9: N=8, 2 clones x 4 replicates; KOLF2.1J: N=4-12, 1-3 clones x 4 replicates). Statistical significance was determined using unpaired t-tests or two-way ANOVA, with post-hoc testing and correction for multiple comparisons as appropriate.

Results: We found that the proteome and phosphoproteome of TBK1^-/- microglia was significantly dysregulated, both at baseline and to a greater extent following immune stimulation, with numerous differentially expressed proteins and phosphopeptides being identified (padj<0.01, log2(FC)<-0.6 or >0.6). Functional enrichment analysis highlighted vesicle trafficking as a key dysregulated pathway. Using live cell timelapse microscopy, we visualised the internalisation and processing of pHrodo-tagged cargo, including myelin, dead SH-SY5Ys and dextran. We found that TBK1^-/- microglia displayed deficient internalisation and/or processing of several cargo specifically in immune stimulated conditions. Additionally, using sandwich ELISAs on cell culture supernatants, we observed an increase in IL-1α and IL-1β secretion in TBK1^-/- microglia specifically upon immune stimulation.

Conclusions: We found that TBK1 loss-of-function resulted in widespread cell autonomous microglial dysfunction, including global dysregulation of the microglial proteome and phosphoproteome, immune dysregulation and endocytic dysfunction, which were exacerbated or apparent only upon immune stimulation. Our data suggests that TBK1 loss-of-function mutations may contribute to ALS-FTD pathogenesis via cell autonomous microglial dysfunction. More broadly, our data adds to emerging evidence of a failure of microglia to mount an appropriate inflammatory response in ALS-FTD.