PROJECT SUMMARY We found that neurons secrete a soluble protein that protects neurons under cell stress conditions in a paracrine manner. Remarkably, this protein is derived from the C-terminus of the endoplasmic reticulum (ER) membrane bound transcription factor CREB3L2. Proteolytic cleavage liberates the N-terminal transcription factor and leads to the secretion of the ER luminal domain into the extracellular space. Our preliminary data indicate that the production and secretion of this C-terminal domain is triggered by cell stress and that it acts by increasing mitochondrial function via sonic hedgehog (Shh) signaling. In this project we are testing the general hypothesis that CREB3L2-C is a paracrine component of the adaptive neuronal stress response. We will culture primary rat cortical neurons and expose them to various cell stress triggers, including oxidative stress, ER stress, proteasome inhibition, and glucose-deprivation. We will determine whether the activation of the integrated stress response pathway is necessary and sufficient to upregulate the synthesis and cleavage of CREB3L2. We will investigate how the luminal C-terminal domain of CREB3L2 is secreted from cells and whether its secretory pathway is linked to cell stress. To investigate how the secreted C-terminal domain of CREB3L2 affects neurons, we will purify it and apply it to primary cortical neurons. We will focus on its proposed role in strengthening Shh signaling and determine whether it forms complexes with Shh and its receptor Patched-1 on neurons. We will determine whether application of the secreted, C-terminal domain of CREB3L2 is sufficient to increase Shh signaling and whether enhanced Shh signaling is necessary for its effects on cell survival and mitochondrial function. To assess the effect of the secreted C-terminal domain of CREB3L2 on mitochondria, we will employ a variety of tests to quantify changes to mitochondrial mass, abundance of mitochondrial proteins, and mitochondrial function in neurons under normal conditions and upon induction of oxidative stress, ER stress, proteasome inhibition, and glucose-deprivation. The cleavage of CREB3L2 activates necessarily two distinct signaling molecules, the intracellular transcription factor and the secreted C-terminus. We will use chromatin immunoprecipitation with sequencing in combination with RNA sequencing to determine the transcriptional targets of CREB3L2 in neurons under baseline and induced stress conditions, to determine whether both signaling molecule act cooperatively in the adaptive cell stress response. Lastly, we will use a previously generated conditional CREB3L2 knockout mouse to study the relative roles of the cell autonomous (i.e. the transcription factor) and the non-cell autonomous arm of CREB3L2 signaling in neuronal stress response in vitro and in vivo. Together, the results from this project will uncover a novel intercellular signaling pathway that is activated in response to cell stress in n...