ABSTRACT Frontotemporal dementia (FTD) is the second most common form of dementia in patients under the age of 65 years. FTD is characterized by atrophy of the frontal and temporal lobes of the brain, personality changes and language impairment. FTD also shares some of the same genetic features as amyotrophic lateral sclerosis (ALS). Genetic advances have identified mutations in more than a dozen genes which can cause pure FTD, pure ALS or both. However, how these genetic mutations contribute to the pathogenesis of FTD remain to be characterized. Significant to this proposal, one gene found mutated in both disorders is the TANK binding protein 1 (Tbk1). Previous studies suggest that haploinsufficiency of TBK1 (reduced TBK1protein levels) is causative for FTD and ALS. TBK1 is a multifunctional kinase which can phosphorylate a variety of substrates in order to exert its functions in a few cellular processes including innate immune system. However, our understanding of the physiological functions of TBK1 in neurons and how the loss of TBK1 contributes to neurodegeneration remains limited. To generate preliminary data, we used Tbk1 shRNA to knock down Tbk1 gene expression in rat primary neurons and explore neuronal functions of TBK1. We also used the CRISPR technique to generate human TBK1 knockout induced pluripotent stem cells (iPSC), and differentiated them into cortical-like neurons, one cell type affected in FTD. In both types of neurons, we found that TBK1 reduction impairs mitochondrial dynamics and causes mitochondrial fragmentation. By analyzing proteomic data, we found a cluster of TBK1 interacting proteins related to mitochondria. One of these is the mitochondria fusion protien OPA1, related to mitochondrial dynamics. Our further studies demonstrated that: 1) TBK1 is localized on and co-purifies with pure mitochondria; 2) Permeabilization and digestion assays suggested that a portion of TBK1 can locate inside of mitochondria as TBK1 is more resistant to digestion than the mitochondrial outer membrane protein TOM20; 3) TBK1 proteomic data demonstrated that TBK1 physically interacts with mitochondria import proteins such as TOM70, TIM50 and TIM44; 4) OPA1 immunoprecipitation also pulls down TBK1, and OPA1 has predicted phosphorylation sites by TBK1; 6) TBK1 reduction also impairs OPA1 processing in both type of neurons; 7) TBK1 reduction decreases the mitochondrial membrane potential and causes an abnormal dendritic spine phenotype. Based on these exciting preliminary studies, we hypothesize that TBK1 plays an important role in controlling mitochondrial dynamics and function through the interaction with OPA1, and the loss of TBK1-OPA1 interaction causes mitochondrial and neuronal dysfunction. Our proposed study will be the first mechanistic study investigating the effect of TBK1 reduction on mitochondrial dynamics/function and neuronal function, and will likely reveal a novel role of TBK1 in the regulation of mitochondrial dynamics/function....