Alcohol use disorders (AUDs) imposing enormous economic and social burdens on society today. AUDs are now understood as a chronic nervous system disorder characterized by a physiological dependence on alcohol beyond voluntary control. While environmental factors contribute to alcohol use, recent genetic studies reveal that the heritability of alcoholism is around 50%, illustrating that common genetic -variation contributes significantly to the etiology of alcoholism. However, the major target mediating alcohol reward signaling and the synaptic mechanisms of alcohol reward are poorly understood. Recent genetic studies have identified alcoholism associated gene variants in patients, one being the single nucleotide polymorphism (SNP) of an adenine to a guanine at position 118 of human OPRM1, a gene encoding the mu opioid receptor (MOR). This results in a nonsynonymous substitution of asparagine (N) at residue 40 to an aspartate (D). Although heterologous expression systems and animal studies suggest altered receptor trafficking and ligand binding affinities, there have been no conclusive studies determining the underlying cellular and molecular mechanisms of the N40D SNP on alcohol reinforcement, especially in a human neuronal context. To better understand the functional consequences of this SNP, the objective of this study is to investigate the interaction of alcohol and opioid signaling in human neurons carrying allelic variants for MOR N40 or D40 at the cellular and synaptic level. Using the novel induced pluripotent stem (iPS) cell and induced neuron (iN) cell techniques, it is possible to elucidate how the N40D gene variant alters alcohol reward signaling in human neurons. Using iPS cell lines from multiple individuals carrying homozygous MOR D40 or N40 alleles, human neurons of excitatory, inhibitory, and dopaminergic subtypes will be derived using the iN cell technology. To characterize the functional impact of this SNP, biochemical and morphological analyses examining changes in MOR expression, localization/trafficking, and degrees of glycosylation will be conducted using western blotting and immunohistochemistry. To give insight into interactions between alcohol exposure and MOR activation, presynaptic and dendritic markers will be analyzed to quantify synapse numbers and dendritic branching in alcohol-nave, 1-day and 7-day alcohol and/or DAMGO treated iNs. I will also use electrophysiology to elucidate how the MOR N40 or D40 genotype differentially affects the more detailed functional parameters of synaptic transmission in iNs with or without alcohol and/or MOR activation. Our system using iPS cells carrying human gene variants provides the unique opportunity to dissect the interactions of alcohol exposure and MOR activation on synapse functionality in human neurons. As the long-term goal of this project is to understand the effect of human gene variants on alcohol sensitivity and synaptic function, results of this research will likely provide novel information about how to devise therapies for a heterogeneous group of AUDs.
While environmental factors contribute to the economic and social burdens of alcohol use disorders (AUDs) today, recent genetic studies have identified alcoholism-associated gene variants in patients, particularly in the gene encoding the human mu opioid receptor (MOR). The objective of this study is to use induced pluripotent stem cells and induced neurons from subjects carrying allelic variants in the MOR to investigate the interaction between alcohol and opioid signaling in human neurons. A detailed understanding of how this gene variant affects alcohol sensitivity in humans will likely provide novel information about the involvement of opioid signaling in AUDs.
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