Alzheimer's disease (AD) is the most common form of dementia, and early alterations in the gut may contribute to disease progression. The current study focused on generating intestinal organoids from 3xTg-AD transgenic and Wild Type (WT) mice to investigate the role of the gut-brain axis in AD. Intestinal organoid cultures were produced through isolation of leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5+) crypt cells from 4-5-month-old or 1-year-old intestinal tissue. Transmission electron microscopy (TEM) analysis of aged 3xTg organoids demonstrated evidence of impaired gut epithelial viability compared to WT controls. In aged 3xTg organoids, enlarged intercellular spaces, disrupted cell adhesions, diminished tight junction complexes, cellular debris, and amyloid-like fibrils were prominent findings. Immunostaining also demonstrated decreased E-Cadherin and ZO1 expression, along with increased cellular and luminal Aβ in aged 3xTg organoids compared to young 3xTg and WT organoids and p-tau protein accumulation in aged 3xTg organoids compared to all other experimental groups. In the aged 3xTg cohort, increased immunoreactivity of inflammasome components, including IL-1β, ASC, and GSDMD, implicated pyroptosis as a potential mechanism of cell death. These results support the hypothesis that familial AD pathology includes pronounced effects on gut organoid viability, junctional integrity, and abnormal protein accumulation within the intestinal epithelium. This study provides early evidence of gut organoid abnormalities occurring independently of alterations in the AD brain. These findings suggest that the gut epithelium may warrant further investigation as a potential target for future therapeutic strategies in AD.
