Written by Tonya Mukherjee, Peer Reviewed by Dr. Karina S. Cramer and Edited by Kevin Liu
Hear? How? Thanks to our auditory system, we can hear and comprehend sounds. It consists of brain regions, auditory brain circuits, and external structures such as the outer, middle, and inner ear [1]. One part of the auditory system, the auditory brainstem, is important for identifying the locations of sound sources.
The chick auditory brainstem is an experimental model often used to examine sensory processing of the central nervous system (CNS). In all vertebrates, the central nervous system is made up of the spine and brain which process signals produced by the body to coordinate activity. Brain circuitry, including the auditory brainstem, are affected in neurodevelopmental disorders. Some of these disorders have been modeled in birds and rodents [2].
Caspase-3 is an important enzyme, a protein which causes a biochemical reaction to create products used by the body, involved in initiating apoptosis. Apoptosis is also known as cell death. Cell death seems unhealthy, but it is actually very important because it prevents damaged or mutated cells from replicating via the cell reproduction process of mitosis. It also prevents the overgrowth and excess of cells during a developmental process of programmed cell death. [3] Mutations in caspase-3 can lead to cancer and other diseases caused by cell overgrowth and mass production.
For a long time, caspase-3 was believed to only initiate and mediate apoptosis. However, recent research has revealed that caspase-3 performs non-apoptotic functions during the development of the auditory brainstem [2]. University of California, Irvine’s Dr. Karina S. Cramer and her lab have recently discovered that, during neurodevelopment of the auditory brainstem, caspase-3 targets and cleaves specific motifs, or recurring patterns in proteins, targeting cytoskeletal proteins to transition from apoptotic to non-apoptotic function [2] [4]. These proteins are responsible for the function of the cell cytoskeleton, a network of filaments and tubes which give the cell its shape and allow for cell movements [4].
“Caspase-3 localization was found ascending the auditory cell pathway. It transitions between apoptotic and non-apoptotic functions during early development of the synapses.” (Cramer, 2022). Overall, Cramer lab’s investigation led them to discover that caspase-3 switches between non-apoptotic and apoptotic function during the creation of synapses, or neural connections. In order to further test caspase-3’s function in the auditory brainstem, Cramer labs performed a caspase-3 inhibition experiment to discover that caspase-3 activity is necessary for neurodevelopment. “We treated embryos with caspase-3 inhibitors, and the inhibition resulted in severe axon targeting problems.” (Cramer, 2022) Axons are the cable-like projections of nerve cells (neurons) which carry electrical signals from the cell body, also called the core, of the neuron to the axon terminal, or the neuron’s other end [5].
Caspase-3 has also been shown to cleave extracellular vesicle (EV) proteins during auditory brainstem development. EVs are packages of material released from almost all cells. They deliver metabolic products from one cell to another [4]. Another database analysis study conducted by Dr. Cramer and others found that, in non-apoptotic contexts, auditory brainstem caspase-3 substrates–which are structures that fit into the caspase-3 enzyme active sites to create products–were enriched for proteins which are usually shown to be cleaved by caspase-3. When a substrate is enriched, it means the genes which are utilized by the substrate are more active or upregulated. Dr. Cramer and others identified several caspase-3 substrates in auditory brainstem EVs involved with axon guidance [6]. Caspase-3 substrates were also enriched for proteins associated with EVs. It is still unclear why this is the case [6]; however, it is suspected that caspase-3 may affect development of the auditory brainstem by modifying the cargo of EVs [7].
Overall, research suggests there is a neurodevelopmental mechanism in which caspase-3 and EV activity collaborate during axon guidance. This is thought to occur because caspase-3 substrates are involved in maintaining cytoskeleton proteins through assembly and disassembly of the cytoskeleton. This is done through the cleavage of certain auditory brainstem caspase-3 substrates, and this cleavage is initiated by alteration of cleavage site preference. This dual ability of caspase-3 is thought to create greater sensitivity and precision in neurogenesis guidance cues; neurogenesis guidance cues lead the direction of and manage the process of neuron growth [7].
Dr. Cramer and others believe there is much future research which can be done. Currently, the relationship between caspase-3 and EVs is not fully understood. It is unknown which EV proteins are important for caspase-3 function, and it is speculated that caspase-3 can cleave EV cargo to influence EV properties [7].
Furthermore, it is not fully understood why selective proteolysis–the process of breaking down proteins with enzymes–of caspase substrates occurs during non-apoptotic processes, but it is suspected that modifications of caspases and their substrates are necessary to alter cleavage site preferences. The Cramer Lab at UCI is currently in the process of investigating this. Dr. Cramer states: “We are actually conducting experiments using in-ovo electroporation to test the function of caspase-3 substrates. We made mutant forms of the caspase-3 target proteins without the cleavage site. They can’t be cleaved by caspase-3, so we hope to investigate and critically test if they (the cleavage sites) are important for mediating caspase-3 during auditory brainstem development.” (Cramer, 2022) In-ovo electroporation is a lab technique used to introduce new DNA in chick embryos [8]. Dr. Cramer hopes to utilize this technique to see if removing cleavage sites influences caspase-3 non-apoptotic function in auditory brainstem development.
Much investigation of caspase-3 is still needed to see how it is involved with non-apoptotic auditory brainstem development. These studies in the chick embryo add to a growing literature on non-apoptotic functions for caspases. Caspase-3 is a highly conserved protein with similar apoptotic functions across species. New studies will be needed to test the roles of caspase-3 in mammals and understand their functions in human development.
References:
- Peterson, D. C., Reddy, V., Hamel, R. N., (2021, August 11) Neuroanatomy, Auditory Pathway. StatPearls. https://www.ncbi.nlm.nih.gov/books/NBK532311/
- Weghorst, F., Mirzakhanyan, Y., Hernandez, K. L., Gershon, P. D., Cramer, K. S., (2022) Non-Apoptotic Caspase Activity Preferentially Targets a Novel Consensus Sequence Associated With Cytoskeletal Proteins in the Developing Auditory Brainstem. Front. Cell and Dev. Neurosci. 10:844844. Doi: 10.3389/fcell.2022.844844.
- Cell Death (2021, July 14) WEHI brighter together. https://www.wehi.edu.au/research/research-fields/cell-death#:~:text=Cell%20death%20is%20an%20important%20process%20in%20the%20body.,is%20made%20of%20dead%20cells.
- Cramer, Karina S., Interview, Conducted by Tonya Mukherjee, 11 April 2022.
- Britannica, T. Editors of Encyclopaedia (2011, September 26). axon. Encyclopedia Britannica. https://www.britannica.com/science/axon
- Weghorst, F., Mirzakhanyan, Y., Samimi, K., Dhilon, M., Barzik, M., Cunningham, L. L., Gershon, P. D., Cramer, K. S., (2020) Caspase-3 Cleaves Extracellular Vesicle Proteins During Auditory Brainstem Development. Front. Cell. Neurosci. 14:573345. Doi: 10.3389/fncel.2020.573345.
- Dehkordi, M. H., Munn, R. G. K., Fearnhead, H. O., (2022) Non-Canonical Roles of Apoptotic Caspases in the Nervous System. Frontiers in Cell and Dev. Bio. 10:840023[8] McLeannan, R., Kulesa, P. M., (2019) In Ovo Electroporation of Plasmid DNA and Morpholinos into Specific Tissues During Early Embryogenesis. Methods Mol Biol. 2019(1976): 71-82. Doi: 10.1007/978-1-4939-9412-0_6