Project Summary/Abstract Frontotemporal dementia (FTD) is characterized by the unrelenting loss of cortical neurons that manifests clinically as devastating changes in the behavior, language, and personality of affected individuals. Amyotrophic lateral sclerosis (ALS) is a related neurodegenerative disease that results in rapidly progressive motor deficits and eventual paralysis. Disease-modifying treatments for FTD and ALS remain elusive. Loss of function variants in TBK1, which encodes a multifunctional protein kinase, represent one of the most common genetic causes of FTD, ALS, and combined ALS/FTD. TBK1 has been implicated in innate immunity, apoptosis, neuroinflammation, and autophagy. One of its key substrates is optineurin (OPTN), which functions in selective autophagy, and haploinsufficiency of OPTN has been strongly linked to familial ALS/FTD. This suggests that disruption of selective autophagy, which acts to maintain protein homeostasis and organelle quality control, is sufficient to cause neurodegeneration. However, selective autophagy has not been well- characterized in neurons and how reduced TBK1 activity leads to the loss of excitatory neurons remains unclear. Our preliminary efforts to systematically evaluate the effects of TBK1 loss-of-function, including unbiased phospho-proteomics, indicate that TBK1 regulates the phosphorylation of numerous proteins involved in autophagy and lysosomal pathways. Additionally, we have identified interactions between OPTN and specific organelles. Our central hypothesis is that TBK1 controls OPTN and additional selective autophagy cargo receptors to target proteins and organelles for degradation and maintain neural proteostasis. The overall objective of this proposal is to integrate new stem cell-based models of ALS/FTD with advanced proteomics for a comprehensive understanding of TBK1-associated autophagy pathways in human neurons. In this proposal we aim to: 1) Identify novel TBK1 protein substrates in human stem-cell derived neurons and characterize the effects of TBK1 loss on neural regeneration; 2) Define the consequences of OPTN loss and disease-associated variants in autophagy; 3) Construct selective autophagy cargo receptor protein-protein interaction networks in neurons and assess their contributions to a form of secretory autophagy. Our long- term goal is to better understand selective autophagy to support the development of novel targeted therapeutic approaches to modulate this pathway in age-related neurodegeneration.