Butyrate Regulates the Blood-Brain Barrier Transport and Intraendothelial Accumulation of Alzheimer's Disease Amyloid-Beta Peptides.

Alzheimer's disease (AD) is characterized by the pathological deposition of amyloid β (Aβ) peptides as amyloid plaques and by cerebrovascular dysfunction, both of which drive AD progression. Butyrate, a gut microbial metabolite, is reduced in AD patients, and its supplementation has been shown to improve cognition and reduce the amyloid burden in animal models. However, the underlying mechanisms are unclear. Our previous studies demonstrated that insulin signaling regulates Aβ transport kinetics at the blood-brain barrier (BBB). In this study, we investigated whether butyrate reduces intraendothelial Aβ accumulation and improves BBB integrity through modulation of insulin signaling pathways. The effect of butyrate on Aβ42 accumulation was assessed by flow cytometry in polarized BBB endothelial cell culture models. Activation of insulin signaling and expression of key BBB constituents involved in Aβ transport and accumulation [p-glycoprotein (P-gp), the receptor for advanced glycation end products (RAGE)], as well as BBB integrity (tight junction protein, claudin-5), were evaluated by using Western blotting and confocal microscopy. The roles of insulin signaling nodes, including AKT, ERK, mTOR, and p38 phosphorylation, were investigated by using specific inhibitors MK2206, Trametinib, Rapamycin, and VX-745, respectively. In addition, the effect of butyrate on BBB receptors and transporters involved in Aβ trafficking was examined in vivo in mouse brains colonized with butyrate-producing bacteria via immunohistochemistry. Butyrate significantly decreased endothelial Aβ42 accumulation, an effect associated with the activation of insulin signaling, particularly AKT and ERK phosphorylation. Inhibitor studies established the critical role of these pathways, as co-incubation with MK2206 (AKT inhibitor) or Trametinib (ERK inhibitor) reversed the protective effect of butyrate and increased Aβ42 accumulation, whereas inhibition of mTOR or p38 had no significant effect. Moreover, butyrate restored Aβ-induced reductions in the P-gp efflux transporter expression and claudin-5 tight junction protein levels. These findings were supported by in vivo studies demonstrating upregulation of the tissue inhibitor of metalloproteinases-2 (TIMP-2), a protein associated with AKT activation and extracellular matrix stabilization in mice colonized with butyrate-producing bacteria. In conclusion, our data demonstrate that butyrate reduces Aβ42 uptake at the BBB endothelium by activating the AKT and ERK arms of the insulin signaling pathway. These changes may also enhance BBB integrity by increasing claudin-5 expression, stabilizing the extracellular matrix, and upregulating TIMP-2. Collectively, this study highlights butyrate as a potential therapeutic modulator of AD-associated BBB dysfunction.