PROJECT SUMMARY Dopamine (DA) plays key roles in the central nervous system by regulating a variety of physiological states, including cognition, motivation, and attention. While DA dysfunction is known to be involved in several neuropsychiatric disorders, its role in the pathogenesis of Autism Spectrum Disorders (ASD) as well as comorbidities associated with ASD (e.g. Attention Deficit Hyperactivity Disorder) are largely unknown. The DA transporter (DAT) is a key constituent of DA signaling in the CNS and the principal regulator of DA homeostasis, mediating the active re-uptake of DA from the synapse following its release upon action potential depolarization. The research proposed focuses on understanding how rare variants (RVs) in the human DAT (hDAT) gene (SLC6A3) identified in several ASD subjects cause aberrant hDAT function and disrupted DA- associated behaviors. Importantly, this work will determine how RVs in hDAT support DA dysfunction, a complication associated with ASD. Our laboratory recently defined the first ASD identified de novo mutation in hDAT. This mutation located in the seventh transmembrane domain, a region critical to DA uptake and efflux, dramatically alters hDAT function both in vitro and in vivo. Notably, this de novo DAT mutation alters DA neurotransmission and DA-associated behaviors as observed in Drosophila melanogaster. The work presented in this proposal aims to create a novel set of discoveries exploiting the structural, functional and behavioral findings from the analysis of new hDAT variants identified in multiple individuals with ASD. We will (S.A. #1) engineer the hDAT ASD variants to determine their putative structural and functional deficits in heterologous expression systems. We will generate transgenic Drosophila lines expressing hDAT wildtype or hDAT variants specifically in DAergic neurons to determine the functional impairments of these hDAT variants in whole, intact Drosophila brains. We will use (S.A. #2) these transgenic Drosophila lines to evaluate the behavioral significance of hDAT dysfunction supported by these hDAT variants.