SUMMARY At least 3.4 million people in the USA live with epilepsy. Despite a rapid increase in newly approved anti-seizure drugs in the last decades, epilepsy is still intractable in about thirty percent of all cases. Alternative and conceptually novel therapeutic strategies are therefore urgently needed. One group of promising novel treatment targets are microRNAs, small noncoding RNAs that suppress the translation and induce the degradation of their target mRNAs. MicroRNAs often target several components of the same pathway, making them powerful regulators of biological processes. While this is an advantage when trying to treat complex diseases like epilepsy, a disadvantage is the increased potential for side effects. This is a major obstacle reducing enthusiasm for the use of microRNA as treatment targets in brain disorders. We will address this issue by using cell type-specific strategies to test the hypothesis that microRNAs achieve specificity in controlling multiple aspects of brain function by mediating their diverse roles through different cell types, brain circuits, and cell type-specific targets, which could be leveraged to optimize treatment strategies. Most preclinical studies targeting microRNAs in epilepsy have used cell type-unspecific antagomir (antisense oligonucleotide) approaches. These studies revealed that several microRNAs shown to be crucial for seizure control in epilepsy also affect neuronal morphology under healthy conditions, potentially reducing their value as treatment targets. The proposed research will follow conceptually novel approaches to overcome this problem by using microRNA sponges. MicroRNA sponges sequester and functionally inhibit microRNAs and can, in contrast to antagomirs, be expressed under the control of cell type-specific promoters, thus inhibiting microRNAs only in select target cell populations in the brain. Taking two epilepsy-relevant microRNAs as examples, we will first use microRNA sponges under the control of neuron subtype- and astrocyte-specific promoters combined with adeno-associated viral gene transfer to test if the effects of the microRNAs on seizure susceptibility and neuronal morphology can be separated by cell types in the brain (aim 1). To provide insight into the underlying mechanisms and the cell type-specific mRNA targets of the microRNAs, we will combine the microRNA sponges with transgenic mice that Cre-dependently express epitope-tagged RNA-induced silencing complex (RISC) allowing for the specific isolation of RISC-associated mRNA from only those cells that express the viral sponge (aim 2). Comparing the RISC-associated transcriptome from cells transduced with scrambled versus microRNA-specific sponges we will experimentally identify cell type-specific microRNA targets. Our strategy will take advantage of the multiplex function of microRNAs regulating hundreds of mRNAs while maximizing specificity by manipulating microRNAs in the cell types that are the most relevant for the...