Butyrate regulates the blood-brain barrier transport and intra-endothelial accumulation of Alzheimer’s disease Amyloid-beta peptides

Alzheimer’s disease (AD) is characterized by the pathological deposition of amyloid beta (Aꞵ) proteins as amyloid plaques, tau aggregates, and cerebrovascular dysfunction that drive disease progression. Butyrate, a gut microbial metabolite, has been found to be reduced in AD patients; butyrate supplementation improved cognition and decreased amyloid burden in animal models. However, the precise underlying mechanisms are unclear. Our previous studies have demonstrated that insulin signaling impacts Aꞵ transport kinetics at the blood-brain barrier (BBB). In this study, we investigated the effect of butyrate treatment on intra-endothelial Aꞵ accumulation and BBB integrity by modulating the insulin signaling pathway. The effect of butyrate on Aꞵ accumulation was assessed by flow cytometry in BBB cell culture models. Insulin signaling activation and the expression of various receptors and transporters at the BBB were evaluated by Western blots and confocal microscopy. The roles of various molecular mediators were confirmed using specific inhibitors (MK2206, Trametinib, Rapamycin, VX-745). The effect of butyrate on the expression of BBB receptors and transporters that play a critical role in Aꞵ trafficking was examined in mouse brains colonized with butyrate-producing bacteria via immunohistochemistry. Butyrate significantly decreased Aβ42 accumulation in endothelial cells. This effect was associated with insulin signaling pathway activation, particularly AKT and ERK phosphorylation. Inhibitor studies established the critical role of these specific arms, as co-incubation with MK2206 (AKT inhibitor) or Trametinib (ERK inhibitor) reversed the protective effect of butyrate and increased Aβ42 accumulation. However, mTOR and p38 inhibitors did not show a similar effect. In addition, butyrate restored P-glycoprotein efflux transporter expression and claudin-5 tight junction protein levels that were reduced with Aβ treatment. These effects were supported by in vivo work, which demonstrated the upregulation of Tissue Inhibitor of Metalloproteinases-2 (TIMP-2). This protein is associated with AKT activation and extracellular matrix stabilization in mice colonized with butyrate-producing bacteria. In conclusion, we have demonstrated that butyrate decreases Aβ42 uptake at the BBB endothelium by activating the AKT and ERK arms of the insulin signaling pathway. These changes may also improve the integrity of BBB tight junctions by increasing claudin-5 expression and extracellular matrix, and by upregulating TIMP-2 expression. This study highlights butyrate’s potential as a therapeutic modulator of AD-related BBB dysfunction.