PROJECT ABSTRACT Sound localization depends on the development of precise neural circuits in the auditory brainstem. Abnormal circuit assembly can contribute to auditory dysfunction in developmental disorders. However, the molecular mechanisms responsible for correct auditory brainstem circuit development remain largely unknown. Our lab has previously shown that caspase-3 activity is necessary for development of auditory brainstem circuits in the chick embryo. Throughout development, active caspase-3 is seen in axons and dendrites of auditory brainstem neurons in the ascending pathway of auditory information: first in auditory nerve axons; then in axons of their synaptic target, nucleus magnocellularis (NM); and finally in dendrites of NM’s synaptic target, nucleus laminaris (NL). Inhibition of caspase-3 activity when caspase-3 is present in NM axons results in NM axonal targeting errors, even though no apoptotic cell death occurs in the auditory brainstem until after this time period. These data suggest that caspase-3 is responsible for guiding NM axons in a non-apoptotic manner. To determine how caspase-3 influences NM axon guidance, I aimed to identify auditory brainstem caspase-3 substrates. I screened the peptidomes of caspase-3-inhibited and control brainstems for peptides that displayed a biochemical signature of caspase proteolysis (cleavage C-terminal of glutamate or aspartate residues) and that were observed only in control brainstems. The 421 peptides that fulfilled these two criteria hailed from 287 distinct proteins, which were enriched for several functional categories, including cytoskeletal regulatory proteins and RNA-binding proteins. Here I propose several experiments to test how caspase-3 cleavage of these substrate categories brings about correct auditory brainstem circuit development. In Aim 1, I propose to transfect NM with constructs expressing uncleavable forms of caspase-3 substrates involved in cytoskeletal regulation: myotrophin and fascin-1. Because I believe that caspase-3 inhibition causes NM axon targeting defects by preventing caspase-3 control of cytoskeletal regulation, I hypothesize that these uncleavable substrates will replicate axon targeting defects caused by global caspase-3 inhibition. In Aim 2, I will use UV cross-linking followed by orthogonal organic phase separation (OOPS) to purify RNA-bound proteins, protein-bound RNAs, and the remaining proteome and transcriptome from caspase-3-inhibited and control auditory brainstems. I will then use gene co-expression network analysis of these four datasets to probe the effect of caspase-3 proteolysis of RNA-binding proteins on gene expression in the auditory brainstem. These aims will thus clarify the role of caspase-3 with regard to the substrate categories revealed by my preliminary data, contributing to a fuller understanding of how the apoptotic pathway serves non- apoptotic functions during neurodevelopment and plasticity.