Genetics of Amphetamine Preference

––– PROJECT SUMMARY/ABSTRACT ––––––––––––––––– R21/R33: Genetics of Amphetamine Preference. –––– Substance used disorder (SUD) is still highly prevalent in the US, and the recent pandemic has made its prevalence increase even more. Genetic factors account for a substantial driving force for the development of SUD, and in recent years ~50 human genes have been associated with psychostimulant use in numerous genetic studies. Many of these genes have not previously been studied for their role in SUD, neither in human samples, nor in model organisms. Thus, their causative role in driving the development of SUD has yet to be elucidated. Furthermore, some associations have highlighted single nucleotide polymorphisms between two human genes, rendering the question of causality even harder to determine. This gap has also been highlighted by funding agencies, and it has led NIDA to issue a Program Announcement for the “Functional Validation and/or Characterization of Genes or Variants Implicated in Substance Use Disorders” (NIDA PAR- 23-041). The vinegar fly, Drosophila melanogaster, has been a genetic model organism for more than a hundred years. Major strides have been made in the last 15 years studying the behavioral responses to alcohol and other drugs of abuse in flies. This applies regarding the characterization of novel genes and pathways that are also conserved in humans, and it applies for the development of new assays that resemble endophenotypes of addiction more closely as well. Based on our recent development of a robust experience- dependent amphetamine preference (EDAP) assay with high throughput, we here propose to test 20 human candidate SUD genes for their in vivo role in the development of amphetamine preference (Aim1/R21 phase). In order to progress to the R33 phase of the proposal, we will validate ≥1 human candidate SUD gene for an amphetamine preference phenotype in concordant, independent replication experiments that use two distinct tools of genetic manipulation, for example, one short-guide RNA for CRISPR/Cas9-mediated knock out generation and one RNA-interference line to cause gene knock down. In the following R33 phase we propose to functionally characterize 1-3 candidate SUD genes. We will test predicted interaction partners for their role in amphetamine preference, identify the conserved neurotransmitter systems that require these genes for wild- type amphetamine preference, and will determine effects on RNA levels and on genome accessibility. Together, these data will both validate genes implicated in SUD and lead to the “identification of new targets for future addiction therapeutics” (NIDA PAR-23-041).