The effects of methylphenidate and atomoxetine on Drosophila brain at single-cell resolution and potential drug repurposing for ADHD treatment

Abstract The stimulant methylphenidate (MPH) and the non-stimulant atomoxetine (ATX) are frequently used for the treatment of attention-deficit/hyperactivity disorder (ADHD); however, the function of these drugs in different types of brain cells and their effect on related genes remain largely unknown. To address these questions, we built a pipeline for the simultaneous examination of the activity behavior and transcriptional responses of Drosophila melanogaster at single-cell resolution following drug treatment. We captured the hyperactivity-like behavior using EasyFlyTracker following the administration of each drug. Subsequently, unsupervised clustering analysis of scRNASEQ for 82,917 cells yielded 28 primary cell clusters representing the major cell types in adult Drosophila brain. Indeed, both neuronal and glial cells responded to MPH and ATX treatment. Further analysis of differentially expressed genes revealed distinct transcriptional changes associated with these two drugs. For instance, two well-studied dopamine metabolism genes ( Dop2R and DopEcR ) were responsive to MPH but not to ATX at their optimal doses, in addition to genes involved in dopamine metabolism pathways such as Syt1 , Sytalpha , Syt7 , and Ih in different cell types. MPH also suppressed the expression of genes encoding other neurotransmitter receptors and synaptic signaling molecules in many cell types, while the responsive effects of ATX were much weaker. In addition to monoaminergic neuronal transmitters, other neurotransmitters have also shown a similar pattern with respect to a stronger effect associated with MPH than with ATX. Moreover, we identified four distinct glial cell subtypes responsive to the two drugs and detected a greater number of differentially expressed genes associated with ensheathing and astrocyte-like glia. Furthermore, our study provides a rich resource of candidate target genes for drug repurposing, which can be found at ADHDrug (http://adhdrug.cibr.ac.cn/). In conclusion, we propose a fast and cost-efficient pipeline to explore the underlying molecular mechanisms of ADHD drug treatment in Drosophila brain at single-cell resolution, which may further facilitate drug repurposing applications.