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Duplication 15q syndrome (Dup15q) is a genetic disorder caused by duplications of the 15q11.2-q13.1 region and is characterized by developmental delay, autism spectrum disorder, and treatment resistant epilepsy. Extra copies of the E3 ubiquitin ligase UBE3A and elevated levels of UBE3A expression in neurons are thought to be the primary cause of Dup15q phenotypes. However, animal models overexpressing UBE3A in neurons have not successfully recapitulated all aspects of Dup15q syndrome, especially epilepsy. Here, we used Drosophila melanogaster (fruit flies) to investigate Dup15q syndrome. In Chapter 2 we explored whether Dube3a, the Drosophila homolog of UBE3A, is imprinted in the fly brain. In mammals, UBE3A undergoes complex imprinting and is expressed only from the maternal allele in mature neurons. Prior to this work the imprinting status of Dube3a in flies was unclear. Here, we present evidence that Dube3a is not imprinted and is biallelically expressed in the fly. Next, in Chapter 3 we examined the interaction between Dube3a and HERC2. HERC2 is also an E3 ubiquitin ligase located in the 15q11.2-q13.1 critical region and is duplicated in all Dup15q individuals. HERC2 physically interacts with and stimulates the ubiquitin ligase activity of UBE3A in vitro. We found that Drosophila HERC2 appears to stimulate the ubiquitin ligase function of Dube3a, and Dube3a and HERC2 interact synergistically to impact phenotypes associated with Dup15q syndrome in vivo. Data presented in Chapter 3 suggests that genes other than UBE3A are important in generating the Dup15q phenotypes and should not be ignored when modeling Dup15q syndrome. In Chapter 4 we investigated how non-neuronal cells, specifically glial cells, contribute to the seizure phenotype of Dup15q syndrome. We found that elevated levels of Dube3a or UBE3A in glia causes seizures, while overexpression in neurons does not. These data are consistent with mammalian models in which UBE3A elevation in neurons does not generate seizures. Furthermore, overexpression of Dube3a in glia reduced protein levels of the Na+/K+ ATPase, ATPα. ATPα down regulation in glia is both necessary and sufficient to generate seizures. In Chapter 5 we further characterized our Dup15q epilepsy model and investigated cell type specific effects of Dube3a overexpression in glia compared to neurons using whole transcriptome and whole proteome analyses. We found that elevation of Dube3a in glia caused a cell non-autonomous down regulation of synaptic proteins in neurons while simultaneously causing a cell autonomous upregulation of glutathione Stransferases (GSTs) in glial cells. Finally, we showed that the upregulation of GSTs is common to multiple different Drosophila gliopathic seizure lines, not just our Dup15q epilepsy model. GSTs play a role in drug metabolism, and elevation of these enzymes may underlie the treatment resistant nature of some epilepsies including Dup15q syndrome. The results from these studies highlight the role that glial cell dysfunction may play in generating seizures in Dup15q syndrome and could ultimately provide novel avenues for epilepsy treatments. Document Type Dissertation Degree Name Doctor of Philosophy (PhD) Program Biomedical Sciences Research Advisor Lawrence T. Reiter, Ph.D.

Investigation of Cell-Type-Specific Effects and Synergistic Interactions Between Genes in Duplication 15q Syndrome