SUMMARY Non-alcoholic fatty liver disease (NAFLD) is a spectrum of disorders initiated by steatosis that can progress to nonalcoholic steatohepatitis (NASH) and cirrhosis. Although it is the leading cause of liver disease in the US, there are no targeted therapeutic interventions, highlighting the critical need for identifying molecular processes underlying its pathology. Transmembrane protein 55B (TMEM55B) is a PI(4,5)P2 phosphatase located on the lysosome that has been shown to impact lysosome levels and localization. In preliminary studies, we found that TMEM55B ASO treatment caused hepatic steatosis in C57BL/6J mice after 6 weeks on western diet, while TMEM55B knockout mice developed NASH after 12 weeks on a high-fat, cholesterol and fructose supplemented (GAN) diet. We have identified several effects of TMEM55B knockdown that can promote NAFLD: 1) accumulation of lipids within lysosomes, indicative of impaired fatty acid (FA) mobilization, 2) impaired mitochondrial FA oxidation together with reduced mitochondrial volume, mitochondrial fragmentation, and increased oxidative stress; and 3) in vivo inhibition of hepatic triglyceride secretion. The lysosome plays a critical role in sorting and processing lipids, where proper lysosome motility is essential for their ability to mobilize FA from lipid droplets and maintain mitochondrial homeostasis. TMEM55B plays a key role in regulating lysosomal positioning, thus our central hypothesis is that loss of TMEM55B promotes NAFLD onset and progression through lysosomal effects on hepatic FA mobilization and trafficking, mitochondrial FA oxidation, and triglyceride secretion. In Aim 1, we will determine whether TMEM55B impacts FA mobilization and mitochondrial function through its ability to impact lysosomal localization. We will also examine the impact of TMEM55B on mitochondrial morphology and dysfunction. Hepatic triglyceride (TG) is primary secreted within VLDL, which is generated through a multi-step process of lipidating nascent APOB with FA mobilized from lipid droplets. In Aim 2A we will test whether TMEM55B knockdown prevents lipidation of nascent VLDL and test the dependency of these effects on the lysosome. Men are well-known to have higher NAFLD risk than pre-menopausal women, however the basis for this sex difference is poorly understood. We found that TMEM55B knockdown caused steatosis in male, but not female, mice, and that loss of hepatic TMEM55B inhibited TG secretion to a much greater degree in male versus female mice. The sex-dependent differences in TG secretory defects were attributed to differences in sex hormones. In Aim 2B we will evaluate the interaction between TMEM55B and sex hormones on TG secretion in male and female mice, and in estrogen receptor knockout mice. Finally, in Aim 3 we will evaluate the effect pharmacologic and genetic inhibition of TMEM55B on the development and progression of NAFLD. Understanding the mechanism(s) by which loss of TMEM55B impacts multiple crit...