Project Summary We have established the existence of an endoplasmic reticulum (ER) stress responsive and caspase 2 (Casp2) dependent pathway that leads to activation of sterol regulatory element binding proteins 1 and 2 (SREBP1/2), the master regulators of fatty acid and cholesterol metabolism. The Casp2-dependent pathway operates independently of SREBP cleavage activation protein (SCAP), the major activator of canonical SREBP signaling and the target for its feedback inhibition and negative regulation by sterols and other lipids. SCAP-dependent SREBP signaling is activated when sterol concentrations are low and during hepatic insulin resistance and may account for simple steatosis (NAFL), the benign form of non-alcoholic fatty liver disease (NAFLD). Conversely, the Casp2-dependent pathway is activated during hepatocyte ER stress and leads to non-alcoholic steatohepatitis (NASH), the aggressive form of NAFLD, characterized by liver damage and inflammation, and at advanced stages by loss of liver fat. Importantly, we discovered that the two pathways are engaged in negative crosstalk, such that SCAP-dependent SREBP activation attenuates ER stress, likely via ER membrane expansion, whereas activation of the Casp2-dependent pathway by the ER stress sensor and transducer IRE1 is associated with induction of INSIG2, an inhibitor of SCAP-dependent SREBP activation. Moreover, inhibition of SCAP-dependent SREBP signaling potentiates ER stress, which enhances IRE1 activation, resulting in elevated expression of INSIG2, Casp2 and PIDD1, a component of the PIDDosome complex, which includes Casp2 and the scaffold protein RAIDD. In addition to Casp2 activation, PIDD1 may target the PIDDosome complex to the ER membrane, thereby enabling cleavage of site 1 protease (S1P) by Casp2, the first step in the non-canonical SREBP activation pathway. We postulate that the mutually exclusive interactions between the two pathways contribute to the sharp transition from simple steatosis to NASH. We will test this hypothesis in three specific aims. 1. Test the hypothesis that elevated IRE1 activity, Casp2 activation and inhibition of SCAP- dependent SREBP activation control human NASH progression, using an extensive collection of NAFLD biopsies available at UCSD and a novel human liver spheroid model of NAFLD/NASH. 2. Test the hypothesis that INSIG2 induction inhibits SCAP-dependent SREBP activation and potentiates the Casp2- dependent pathway by enhancing ER stress and IRE1 activation. These studies will be conducted in mouse models and will deploy state-of-the-art high-content spatial transcriptomics. 3. Determine the role of IRE1 and its downstream effectors in NAFL to NASH progression in mouse models and human spheroids, and the suitability of these mediators to therapeutic targeting. Impact: these studies will shed new light on the mechanistic basis for Casp2-dependent SREBP activation and the role of ER stress, IRE1 and its effectors in NASH pathogenesis, laying the fo...