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Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.

Caspases cleave over 1500 substrates in the human proteome in both lethal and non‐lethal scenarios. However, reports of the physiological consequences of substrate cleavage are limited. Additionally, the manner in which caspase cleaves only a subset of substrates in the non‐lethal scenario remains to be elucidated. BubR1, a spindle assembly checkpoint component, is a caspase substrate in humans, the physiological function of which remains unclear. Here, we found that caspases, especially Drice, cleave Drosophila BubR1 between the N‐terminal KEN box motif and C‐terminal kinase domain. By using proximity labelling, we found that Drice, but not Dcp‐1, is in proximity to BubR1, suggesting that protein proximity facilitates substrate preference. The cleaved fragments displayed altered subcellular localization and protein–protein interactions. Flies that harboured cleavage‐resistant BubR1 showed longer duration of BubR1 localization to the kinetochore upon colchicine treatment. Furthermore, these flies showed extended lifespan. Thus, we propose that the caspase‐mediated cleavage of BubR1 limits spindle assembly checkpoint and organismal lifespan. Our results highlight the importance of the individual analysis of substrates in vivo to determine the biological significance of caspase‐dependent non‐lethal cellular processes. [ABSTRACT FROM AUTHOR]

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage. Author summary: The innate immune system is conserved among multicellular organisms and has developed the mechanisms to recognize various dangers including infection and non-infectious damage. In Drosophila, the Toll pathway, an innate immune signaling pathway, is activated in response to tissue damage. Toll activation requires the use of serine protease (SP) cascades to activate the Toll ligand Spätzle. In this study, we explored SPs and damage-associated molecules involved in the sterile activation of the Toll pathway using a fly model, where apoptosis deficiency of wing epidermal cells causes damage. Genetic analyses demonstrated that paralogous SPs Hayan and Persephone contribute to Toll activation in apoptosis-deficient flies. Furthermore, we found that hydrogen peroxide, a representative damage-associated molecule, activates Persephone in cells and that apoptosis-deficient wings produce reactive oxygen species (ROS). Our findings highlight the importance of ROS in initiating the SP cascade for Toll activation in response to tissue damage. [ABSTRACT FROM AUTHOR]