PROJECT SUMMARY/ABSTRACT The long-term objective of this proposal is to develop non-opioid novel molecular targets to reduce the pain suffering and mortality attributed to sickle cell disease (SCD), by identifying to upstream messengers of acute and chronic pain signaling. Mitochondrial retention in SCD red blood cells is recognized now to be a major contribution in SCD pathogenesis. However, the downstream molecular mechanisms related to pain and mitochondrial retention is not investigated. Our preliminary data suggested that dysregulated autophagy byproducts of Cathepsins, GABARAPs and synucleins released during lysis of RBCs of SCD red blood cells have potential to trigger pain episodes in SCD patients. Here, we hypothesized that specific autophagy proteins including CTSO, CTSW and SNCB along with other known DAMP signaling molecules generated from SCD RBCs contribute pain in SCD through interaction with pain signaling molecules in endothelium and neurons. This notion is supported by preliminary data indicating that SCD RBCs with increased levels of autophagy proteins not only in SCD RBCs but also in plasma compared to controls. To address this hypothesis, two aims are proposed. The first aim will determine whether the pain severity of SCD is associated with expression and activity levels of autophagy proteins in both pediatric and adults SCD patients. The second aim is to determine whether inhibiting autophagy proteins can reduce pain signaling and pain behaviors in an SCD mouse model and characterize the molecular interaction of autophagy proteins Synucleins (SNCA, SNCB), Cathepsins (CTSB, CTSO. CTSW) and GABARAPs (L1 and L2) with pain signaling molecules. It is anticipated that the result of these studies will make understand the unique mechanisms of pain in SCD and will ultimately lead to clinical trials to assess new pharmacological interventions to reduce the need for opiates to manage pain.