Differential Effects of Alfaxalone on Oxidative Stress Enzyme Activities in APPNL-G-F Knock-In Mice

Abstract

Oxidative stress plays important role in the development and progression of Alzheimer’s disease (AD), promoting mitochondrial dysfunction, impaired antioxidant response capacity, and metabolic disturbances in both central and peripheral tissues (Chen and Zhong, 2014). Neuroactive steroid Alfaxalone (ALX), a synthetic analog of Allopregnanolone, exhibits antioxidant and anti-inflammatory properties (Serrao et al., 2022), yet its systemic redox effects, particularly across peripheral organs, remain poorly understood. Given the pronounced sex-related differences in AD vulnerability and redox biology, the present study examined how chronic ALX administration modulates antioxidant enzyme activities in APPNL-G-F knock-in (APP KI) mice and their non-KI littermates (non-KI). At the age of 9 months, male and female APP KI and non-KI mice (9 months) received subcutaneous subanesthetic (10 mg/kg) or anesthetic (40 mg/kg) doses of ALX for four weeks (once per week). Activities of CuZnSOD, MnSOD, catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were measured in liver, kidneys, heart, and erythrocytes. Non-parametric statistics (Kruskal-Wallis, Mann-Whitney U) were used to assess sex-, dose- and genotype-specific effects. The goal was to identify whether ALX exerts organ-specific and sex-dependent effects on redox homeostasis and whether these effects differ in the presence of AD-like pathology. ALX induced pronounced and highly selective redox alterations, with the strongest effects in the liver and kidneys, while the heart and erythrocytes remained largely resistant to the treatment. In the liver, cytosolic CuZnSOD remained stable, however, mitochondrial MnSOD increased exclusively in APP KI females treated with the anesthetic ALX dose, suggesting mitochondrial sensitivity in AD-like amyloidosis. CAT activity decreased in females of both genotypes and with both ALX doses, whereas in males no alterations were observed. Pathology increased hepatic GPx in females, and ALX (both doses) lowered these levels toward control levels, suggesting partial functional restoration. GR activity showed minimal modulation across groups. Overall, hepatic redox responses to ALX were predominantly female-specific, with notable mitochondrial engagement. Kidneys demonstrated a distinct response pattern. While CuZnSOD remained unchanged, MnSOD activity exhibited opposite shifts in non-KI vs APP KI females, reflected by the decrease in non-KI and an increase in APP KI females following an anesthetic ALX dose. CAT levels were elevated in APP KI females but normalized following 40 mg/kg ALX, further supporting its restorative potential. GR activity showed robust modulation with both doses increasing GR in healthy males, whereas only the higher dose increased GR in APP KI males. In females, high-dose ALX elevated GR regardless of genotype. However, GPx activity remained unchanged. These findings highlight complex renal redox remodeling shaped by sex, disease state, and ALX dose. In summary, chronic ALX treatment did not produce uniform systemic antioxidant effects but instead drives distinct, organ-selective and sex-dependent redox modulation, with the strongest impact observed in females and APP KI mice. The lack of ALX-induced effects on cardiac and erythrocyte antioxidant profiles also indicates that systemic oxidative stress was not generally altered but restricted to specific organs with well-known metabolic burden. ALX particularly influences mitochondrial antioxidant pathways (MnSOD) and glutathione cycling (GR), suggesting potential therapeutic relevance for targeting peripheral oxidative stress in AD. These findings underscore the importance of incorporating sex and organ specificity when evaluating redox-active therapeutics in neurodegenerative disease models.Gotti S, editor. Abstracts of invited lectures and free contributions : 13th International Meeting - Steroids and Nervous System; 2026 Febr 21-25; Torino, Italy. Torino: Fondazione Cavalieri Ottolenghi; 2026. p. 96-7