PROJECT SUMMARY In the United States, the prevalence of opioid use and opioid use disorder (OUD) more than doubled in recent years. There has been an increase in opioid-related overdose deaths with nearly 92,000 reported in 2020. This has led to an unprecedented current crisis of OUD and overdose deaths resulting from indiscriminate use of opiates. While opioids are potent analgesics and provide relief from pain, they are also prone to be addictive. This crisis is further worsened due to the availability of illicit and more potent synthetic opiates like Fentanyl. The current FDA-approved drugs for OUD are both inadequate and have adverse effects. Therefore novel mechanism-based drug discovery approaches are urgently required for OUD and also to prevent overdose deaths resulting from respiratory depression. The heritability of substance use disorder is estimated to be greater than 50% based on twin, family, and adoption studies, and yet few modulating genes have been evaluated. Mu-opioid receptor (MOR) signaling is the major pathway responsible for both pain relief and euphoric effects of opioids. It is important to note that morphine inhibits nuclear translocation of TFEB, a master regulator of the autophagy-lysosome pathway (ALP), thereby reducing autophagic activity. Interestingly, we also found that MOR is colocalized with TFEB in the subcellular neuronal membranes, and most importantly also physically interact with each other as shown by coimmunoprecipitations. Also, opioids induce significant damage to neurons with reduced synaptic plasticity, and TFEB is known to protect against neurodegeneration in vivo in the brain, especially the dopaminergic neurons. Even more important, the most frequent cause of overdose death due to opioids is opioid-induced respiratory depression (OIRD) as well as damage to the lung tissue. Interestingly, TFEB overexpression can decrease inflammation and mitochondrial damage in the lung tissue thereby protecting against acute lung injury. Based on this overwhelming evidence we hypothesize that “As a master regulator of ALP, TFEB plays a pivotal role in the mitigation of opioid tolerance and dependence by enhancing synaptic plasticity in the brain”. In specific aim 1, we will use SH-SY5Y cells and striatal primary neurons to verify whether TFEB overexpression or siRNA-mediated knockdown alters morphine-, fentanyl-, DAMGO, and Methadone-induced MOR desensitization, internalization, and stability. Specific aim 2 is an in vivo study designed to assess whether TFEB or its activator TPI-132 influences MOR agonist-induced analgesia, dependence, tolerance, respiratory depression, and withdrawal symptoms using flag-TFEB, TFEB-/- mice and wild-type mice after sub- chronic exposure to morphine and fentanyl. To increase rigor, we have included two cell types, multiple opioids, different time points, doses. If TFEB indeed mitigates opioid addiction and tolerance, TPI-132 that can activate TFEB and autophagy may be developed as novel and excellent therapy for OUD and overdose deaths

PROJECT NARRATIVE In the United States, the number of deaths resulting from opioid over dose is rapidly increasing and has reached epidemic proportions in recent years. The available FDA-approved treatment options for opioid over doses and withdrawal symptoms are limited and inadequate. Here we want to test whether TFEB, a master regulator of autophagy could reduce opioid-induced withdrawal symptoms as well as tolerance, dependence and respiratory depression by enhancing synaptic plasticity based on solid evidence that TFEB physically interacts with opioid receptors.