Ergothioneine, a dietary antioxidant improves amyloid beta clearance in the neuroretina of a mouse model of Alzheimer’s disease

**Background:** Alzheimer's disease (AD) is characterized by impaired clearance of amyloid beta (Aβ) protein, leading to plaque formation. Ergothioneine (Ergo) is a dietary antioxidant that accumulates in tissues via the OCTN1 transporter, which is highly expressed in cells prone to oxidative stress, including myeloid cells and ocular tissues. This study investigated whether Ergo enhances Aβ clearance in the neuroretina of 5XFAD mice, a transgenic AD model.

**Methods:** Age-matched groups of Ergo-treated 5XFAD (n=9), non-treated 5XFAD (n=9), and C57BL/6J wildtype (WT) controls (n=8) were used. Ergo (50 mg/kg) was administered orally 3 times per week for 8 weeks starting at 1.5 months of age. Wholemount neuroretinas (n=26) and eye cross-sections (n=18) were analyzed using immunofluorescence for OCTN1, Aβ (6E10), IBA1 (microglia/macrophages), GFAP (astrocytes), and AQP4 (water channel). Immunoreactivity was quantified by pixel counts and semi-quantitative manual counting of Aβ plaques and phagosome-like structures (PLS). Statistical analysis used Kruskal-Wallis H test with Dunn's post-hoc or one-way ANOVA with Games-Howell post-hoc.

**Key Results:** OCTN1 immunoreactivity in the whole eye was significantly lower in both non-treated and Ergo-treated 5XFAD compared to WT controls (p=0.002 for both). In wholemounts, 6E10 immunoreactivity was significantly higher in Ergo-treated 5XFAD and WT controls compared to non-treated 5XFAD (p<0.001 for both). However, the number of large Aβ deposits/plaques was significantly lower in Ergo-treated 5XFAD (mean 3.74 ± 0.37) than in non-treated 5XFAD (5.83 ± 0.75, p=0.036). IBA1-positive PLS (phagocytic macrophages) were significantly more numerous in Ergo-treated 5XFAD (1.29 ± 0.21) and WT controls (1.69 ± 0.31) compared to non-treated 5XFAD (0.65 ± 0.14, p=0.038 and p=0.009 respectively). In retinal cross-sections, 6E10 immunoreactivity was significantly lower in Ergo-treated 5XFAD compared to non-treated 5XFAD in both central (p<0.001) and peripheral (p=0.003) retina. AQP4 immunoreactivity was also significantly lower in Ergo-treated 5XFAD compared to non-treated 5XFAD (p<0.001). GFAP immunoreactivity in cross-sections was significantly higher in non-treated 5XFAD compared to Ergo-treated 5XFAD (p=0.001). Spearman correlation showed a stronger positive association between 6E10 and IBA1 in wholemounts of WT (ρ=0.603, p<0.001) and Ergo-treated 5XFAD (ρ=0.517, p<0.001) than in non-treated 5XFAD (ρ=0.359, p=0.018).

**Clinical Implications:** This study provides evidence that dietary ergothioneine can enhance Aβ clearance in the retina of an AD mouse model, potentially through recruitment of blood-derived phagocytic macrophages and perivascular drainage. The findings suggest that Ergo supplementation may be a nutritional strategy to reduce Aβ pathology in AD, with the retina serving as a accessible window to monitor therapeutic effects. The reduced OCTN1 expression in 5XFAD mice indicates that impaired Ergo uptake may contribute to AD pathology. Further research is needed to confirm these mechanisms in human AD and to explore Ergo as a preventive or adjunctive therapy.