Caspase-3 Cleaves Extracellular Vesicle Proteins During Auditory Brainstem Development.

Forrest Weghorst,Karina S Cramer,Lisa L Cunningham,Mehron Dhillon,Melanie Barzik,Paul D Gershon,Yeva Mirzakhanyan,Kian Samimi

doi:10.3389/fncel.2020.573345

Abstract

Sound localization requires extremely precise development of auditory brainstem circuits, the molecular mechanisms of which are largely unknown. We previously demonstrated a novel requirement for non-apoptotic activity of the protease caspase-3 in chick auditory brainstem development. Here, we used mass spectrometry to identify proteolytic substrates of caspase-3 during chick auditory brainstem development. These auditory brainstem caspase-3 substrates were enriched for proteins previously shown to be cleaved by caspase-3, especially in non-apoptotic contexts. Functional annotation analysis revealed that our caspase-3 substrates were also enriched for proteins associated with several protein categories, including proteins found in extracellular vesicles (EVs), membrane-bound nanoparticles that function in intercellular communication. The proteome of EVs isolated from the auditory brainstem was highly enriched for our caspase-3 substrates. Additionally, we identified two caspase-3 substrates with known functions in axon guidance, namely Neural Cell Adhesion Molecule (NCAM) and Neuronal-glial Cell Adhesion Molecule (Ng-CAM), that were found in auditory brainstem EVs and expressed in the auditory pathway alongside cleaved caspase-3. Taken together, these data suggest a novel developmental mechanism whereby caspase-3 influences auditory brainstem circuit formation through the proteolytic cleavage of extracellular vesicle (EV) proteins.

Highlights

The auditory system depends on specialized neural circuits that faithfully preserve sound information from the ears to higher-order auditory structures, processing various aspects of the sound environment along the way
We performed intraventricular injections of the caspase-3/7 inhibitor z-DEVD-fmk or vehicle solution at developmental stages when cleaved caspase-3 is expressed in nucleus magnocellularis (NM) axons and nowhere else in the auditory brainstem, namely embryonic days (E) 9 and 10, corresponding to Hamburger-Hamilton stages 35 and 36 (Hamburger and Hamilton, 1951; Rotschafer et al, 2016)
Despite this evidence that auditory brainstem EV (ABEV) proteins are Auditory Brainstem Caspase-3 (ABC3) substrates, we found that both procaspase-3 and active, cleaved caspase-3 were depleted in ABEVs compared to crude brainstem lysates (Figures 6A–C; Dunn’s multiple comparisons tests, p = 0.056 and p = 0.033, respectively), suggesting that proteolysis of ABEV proteins does not occur in ABEVs, but rather that caspase-3 cleaves ABEV proteins either before their loading into extracellular vesicles (EVs) or after EV uptake by recipient cells

Summary

Introduction

The auditory system depends on specialized neural circuits that faithfully preserve sound information from the ears to higher-order auditory structures, processing various aspects of the sound environment along the way. Other features, such as the spatial location of sound sources, are instead rapidly calculated by circuits in the auditory hindbrain and midbrain (Nelken, 2008) One such circuit in the auditory brainstem exploits interaural time differences (ITDs), discrepancies in the arrival time of sounds between the ears, to localize sound sources in horizontal space (Carr and Konishi, 1990; Overholt et al, 1992). Many species use this circuit to distinguish sounds arising from sources as close as one spatial degree, a feat that requires accurate detection of ITDs of less than 10 microseconds. Such extreme functional precision leaves little room for error in auditory

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