Molecular mechanisms and functions underlying the role of Fto in mu opioid actions - Project Summary/Abstract Mu opioids, such morphine and fentanyl, remain in the mainstream for moderate and severe pain management. However, they also produce many side-effects such as tolerance and addiction, leading to the development of opioid use disorder, a main cause of the opioid epidemic and climbing opioid overdose death. Mechanisms underlying mu opioid actions, particularly their side-effects such as tolerance and reward, are extremely complex and involve multiple distinct systems or diverse signaling pathway. Fat mass and obesity-associated protein (FTO) was initially identified as a demethylase of N6-methyladenosine (m6A), a modified nucleotide in mRNA. Later, it was found that Fto exhibits significantly higher catalytic activity in demethylation of N6,2'-O- dimethyladenosine (m6Am) compared to m6A. Accumulating evidence suggests that FTO influences gene expression by modulating mRNA stability, alternative splicing, translation, and epigenetic modulation. Notably, FTO has emerged as an important molecular player in various neuronal functions, and neuropsychiatric disorders. Recently, we investigated the involvement of FTO in mu opioid actions, specifically tolerance and reward in mice. Our preliminary data demonstrated that Fto depletion or inhibition significantly reduced morphine and fentanyl tolerance and morphine reward measured by conditioned place preference (CPP). Furthermore, depleting Fto in the nucleus accumbens shell (NAcSh) by microinjecting AAV-Cre in Ftofl/fl mice or the dorsal root ganglion (DRG) by inducing Cre expression in the DRG by tamoxifen in Ftofl/fl:Advillin-CreER2 mice led to attenuation of morphine CPP and tolerance, respectively. Additionally, we performed a preliminary transcriptome study using the NAcSh of Fto-NAcSh-depleted mice, which revealed alterations in gene expression and alternative splicing. Collectively, these strongly support an overarching hypothesis that Fto contributes to mu opioid reward and tolerance in the NAcSh and DRG, respectively, through distinct molecular mechanisms. FTO inhibition or modulation of a key molecule or pathway downstream of FTO in a specific region may therefore represent a new approach for preserving mu opioid analgesia while mitigating specific unwanted side effects. To test this hypothesis, we propose the following three specific aims. Aim 1. Determine the role of Fto in the NAcSh and DRG on mu opioid tolerance and reward by CPP and intravenous self-administration. Aim 2. Determine if the role of Fto in the NAcSh and DRG on mu opioid reward and tolerance is mediated by increased m6Am or m6A by using double-floxed mouse models, Ftofl/flPcif1fl/fl and Ftofl/flMettl3fl/fl. Aim 3. Investigate molecular mechanisms underlying the role of Fto in the NAcSh and DRG on mu opioid reward and tolerance using several cutting-edge approaches, including m6Am/m6A mapping, miCLIP, SLAM-seq, GLORI, ribosome profiling and RNA-seq. The proposed studies promise to provide new insights into in vivo molecular mechanisms in mu opioid action regarding RNA methylation and identify new targets downstream of FTO, which may have therapeutic potentials for developing a novel strategy to reduce mu opioid tolerance and reward in pain management.
