Extracellular Cu/Zn superoxide dismutases (SODs) are critical for balancing the level of reactive oxygen species in the extracellular matrix of eukaryotes. In the present study we have detected constitutive SOD activity in the haemolymph and defensive secretions of different leaf beetle species. Exemplarily, we have chosen the mustard leaf beetle, Phaedon cochleariae, as representative model organism to investigate the role of extracellular SODs in antimicrobial defence. Qualitative and quantitative proteome analyses resulted in the identification of two extracellular Cu/Zn SODs in the haemolymph and one in the defensive secretions of juvenile P. cochleariae. Furthermore, quantitative expression studies indicated fat body tissue and defensive glands as the main synthesis sites of these SODs. Silencing of the two SODs revealed one of them, PcSOD3.1, as the only relevant enzyme facilitating SOD activity in haemolymph and defensive secretions in vivo. Upon challenge with the entomopathogenic fungus, Metarhizium anisopliae, PcSOD3.1-deficient larvae exhibited a significantly higher mortality compared to other SOD-silenced groups. Hence, our results serve as a basis for further research on SOD regulated host-pathogen interactions. In defensive secretions PcSOD3.1-silencing affected neither deterrent production nor activity against fungal growth. Instead, we propose another antifungal mechanism based on MRJP/yellow proteins in the defensive exudates.Using oxygen in aerobic metabolism provides more energy per glucose than glycolysis does and hence constitutes an evolutionary advantage. Anyhow, molecular oxygen can be reduced to highly reactive by-products, termed reactive oxygen species (ROS), that can inflict damage on almost all molecules 1 . Oxygen is therefore a stringent selection criterion acting on organisms to sustain its reactive variants 2-4 . O 2 • − is considered the "primary" ROS, which can further interact with other molecules to generate "secondary" ROS including hydroxyl radicals (• OH), hydroperoxyl radicals (HOO• ) or hydrogen peroxide (H 2 O 2 ) 5,6 . While organisms evolved antioxidant activity to counteract the deleterious effects (termed oxidative stress) of these oxygen variants, they have also developed mechanisms to use ROS as vital components in cellular signalling pathways or immune response 5,7,8 .Superoxide dismutases (SOD, E.C. 1.15.1.1), common to all kingdoms of life, serve as first-line antioxidant enzymes 6,9 . They dismutate O 2 • − into O 2 and H 2 O 2 with diffusion-limited rate constants. SODs are classified according to their metal co-factors and localization 10 . In eukaryotes two intracellular forms are known. The dimeric Cu/Zn-binding SODs (SOD1; 32 kDa) are localized in the cytoplasm, outer mitochondrial space and nucleus 11 , whereas the tetrameric MnSODs (SOD2; 89 kDa) is exclusively present in the inner mitochondrial space 12 . The most recently discovered members of the SOD family are the tetrameric Cu/Zn SODs (SOD3; EC-SOD; 135 kDa) [13][14][15][16] functioning in the ext...

Extracellular vesicles (EVs), including small EVs (sEVs), are involved in neuroinflammation and neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Yet, increased neuroinflammation can also be detected in the aging brain, and it is associated with increased glial activation. Changes in EV concentration are reported in aging tissues and senescence cells, suggesting a role of EVs in the process of aging. Here, we investigated the effect of peripheral sEVs from aged animals on neuroinflammation, specifically on glial activation. sEVs were isolated from the peripheral blood of young (3 months) and aged (24 months) C57BL/6J wildtype mice and injected into the peripheral blood from young animals via vein tail injections. The localization of EVs and the expression of selected genes involved in glial cell activation, including Gfap, Tgf-β, Cd68, and Iba1, were assessed in brain tissue 30 min, 4 h, and 24 h after injection. We found that sEVs from peripheral blood of aged mice but not from young mice altered gene expression in the brains of young animals. In particular, the expression of the specific astrocyte marker, Gfap, was significantly increased, indicating a strong response of this glial cell type. Our study shows that sEVs from aged mice can pass the blood-brain barrier (BBB) and induce glial cell activation.

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