Non-Apoptotic Functions of Caspase-3 in Brainstem Development

ABSTRACT 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, with incomplete lamination. 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. Given the extent of anatomical circuit disruption that occurs when caspase-3 is inhibited, we will investigate the effects of caspase-3 inhibition on maturation of auditory brainstem synapses. We will test auditory brainstem responses in hatchlings

after caspase-3 inhibition to determine effects on hearing. 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. 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 how caspase-3 regulates the protein content of EVs. We will

test whether pharmacological disruption of EV formation impairs auditory development. 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.