Non-apoptotic functions of caspase-3 in neural development

项目总结

PROJECT SUMMARY Auditory function relies on highly specialized and precise neuronal connectivity. A significant challenge for the field of auditory neuroscience is to understand how these neural circuits form during development. Our previous work suggests an important function for caspase-3, a protease best known for its role in apoptosis. Cleaved (active) caspase-3 is present in the developing auditory brainstem prior to the period of programmed cell death. During embryonic development, it is first seen in auditory nerve axons, then in the synaptic target of these axons in nucleus magnocellularis (NM), then in the synaptic target of NM, in nucleus laminaris (NL) dendrites. Caspase-3 inhibition during development results in substantial errors in NM axon targeting and in structural abnormalities in NL. We propose to investigate the regulation of caspase-3 activation during development. We will examine the basis for the progression of caspase-3 activation through the auditory pathway and test the hypothesis that cleaved caspase-3 is necessary in auditory axons for activation of caspase-3 in their synaptic targets. Caspase-3 activation during apoptosis is activated by cell death signals and mitochondrial permeabilization. We hypothesize that during auditory development, caspase-3 is activated through a non-canonical pathway that is protective for cells with cleaved caspase-3 in their axons. We will test the function of upstream molecules and determine whether they influence development of the NM-NL pathway. We have begun to investigate the molecules through which caspase-3 influences auditory development. Our proteomics study revealed hundreds of proteins that are cleaved by caspase-3 in the developing auditory brainstem. We have identified several substrates that mediate axon growth. We will use caspase-uncleavable forms of these proteins to test their caspase-dependent functions in development. Gene ontology analysis revealed that the most abundant cellular localization category for caspase-3 substrates was exosomes/extracellular vesicles (EVs). This finding suggests an overarching model in which caspase-3 influences the composition of EVs, which in turn provide an effective means of local communication between cells during development. We will examine enriched EV samples using tandem mass spectrometry to determine which caspase-3 substrates are present in EVs. We will use an EV grafting strategy to investigate whether EVs can rescue developmental deficits in caspase-3 inhibited host embryos. Together, these studies will advance our understanding of neural circuit assembly in the developing auditory brainstem.