Axonal Cleaved Caspase-3 Regulates Axon Targeting and Morphogenesis in the Developing Auditory Brainstem

Abstract

Caspase-3 is a cysteine protease that is most commonly associated with cell death. Recent studies have shown additional roles in mediating cell differentiation, cell proliferation and development of cell morphology. We investigated the role of caspase-3 in the development of chick auditory brainstem nuclei during embryogenesis. Immunofluorescence from embryonic days E6–13 revealed that the temporal expression of cleaved caspase-3 follows the ascending anatomical pathway. The expression is first seen in the auditory portion of VIIIth nerve including central axonal regions projecting to nucleus magnocellularis (NM), then later in NM axons projecting to nucleus laminaris (NL), and subsequently in NL dendrites. To examine the function of cleaved caspase-3 in chick auditory brainstem development, we blocked caspase-3 cleavage in developing chick embryos with the caspase-3 inhibitor Z-DEVD-FMK from E6 to E9, then examined NM and NL morphology and NM axonal targeting on E10. NL lamination in treated embryos was disorganized and the neuropil around NL contained a significant number of glial cells normally excluded from this region. Additionally, NM axons projected into inappropriate portions of NL in Z-DEVD-FMK treated embyros. We found that the presence of misrouted axons was associated with more severe NL disorganization. The effects of axonal caspase-3 inhibition on both NL morphogenesis and NM axon targeting suggest that these developmental processes are coordinated, likely through communication between axons and their targets.

Caspase-3 Cleaves Extracellular Vesicle Proteins During Auditory Brainstem Development

“…Because the ABC3 dataset was obtained from embryonic brainstems not undergoing significant cell death (Rotschafer et al, 2016), we hypothesized that databases representing samples that are primarily apoptotic in nature (MEROPS, CASBAH, and the apoptotic DegraBase substrates) would contain fewer ABC3 substrates than sets more comparable to the present study insofar as caspase activity is localized to synapses (Victor et al, 2018) or is not associated with apoptosis (untreated DegraBase substrates). Our hypothesis was supported in that the proportion of ABC3 substrates in apoptosis-only datasets (67 / 837 = 8.0%) was less than that of non-apoptotic substrates, regardless of whether the proteins in non-apoptotic datasets were also found in apoptotic sets (24 / 116 = 20.7%; Figure 3, Fisher's Exact Test, Odds ratio = 2.998, p < 0.0001).…”

“…To identify the targets of non-apoptotic caspase-3 activity in the embryonic chick auditory brainstem, we searched for likely caspase product peptides that were abolished by caspase-3 inhibition using the strategy shown in Figure 1. Specifically, 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 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). We then used tandem mass spectrometry to characterize auditory brainstem proteins and peptides.…”

“…We previously reported a requirement for the activity of the apoptotic protease caspase-3 for proper chick ITD circuit assembly (Rotschafer et al, 2016). This role was first suggested by the sequential, ascending expression pattern of active caspase-3 in neuronal processes of the chick auditory brainstem throughout embryonic development: First in axons of the auditory nerve (AN) on embryonic days (E) 6-7; then in axons of the AN synaptic target, nucleus magnocellularis (NM) on E8-10; and finally in dendrites of the NM synaptic target, nucleus laminaris (NL) on E11-13.…”

The Role of Apoptotic Signaling in Axon Guidance

“…Slit-N is neurotrophic [97], and it will be interesting to see whether Slit-FL can directly activate caspases, perhaps via p38 MAPK signaling as suggesting by zebrafish studies [66]. Additionally, activated caspase activity has been observed in the developing auditory brainstem within several segments of navigating axons as well as their terminal branches within targets where it is proposed to limit unnecessary axonal arborization, because inhibition of caspase activity causes axon branches to spread into inappropriate target tissue [18]. Although much less is known about axon branching within targets, caspase activity appears to be an important regulator of not only primary axons but also their terminal branches.…”

Characterization of the caspase family in zebrafish

“…The role of caspase activity in axon guidance was first suggested by the observation that growth cone responses to netrin or lysophosphatidic acid were blocked in the presence of caspase inhibitors in vitro [ 34 ]. Since then, caspases have been shown to regulate NCAM-dependent axon outgrowth [ 35 ], axon targeting in the auditory brainstem and the olfactory bulb [ 36 , 37 ], and retinal axon arborization [ 38 ]. Caspases have also emerged as playing a crucial role in the refinement of neuronal connectivity by regulating axonal and dendritic pruning [ 39 – 44 ].…”