(M1530-10-58) Gut Microbial Metabolites Regulate Bidirectional Transport and Intracellular Accumulation of Alzheimer’s disease Amyloid Beta Peptides at the Blood-Brain Barrier Endothelium

Purpose: Alzheimer's Disease (AD) is a common type of dementia observed in elderly population and amyloid beta (Aꞵ) deposition in the brain is one of its pathological hallmarks. Several published reports have identified significant changes in gut microbiota composition in AD patients and gut-brain axis (GBA) has attracted considerable attention in the scientific community ¹. The GBA is formed by the integration of gut and brain interactions mediated by gut microbial metabolites, hormones, immune transmitters and vagal nerve functions. One of the major gut microbial metabolites, short chain fatty acids (SCFAs) including acetate, propionate, butyrate and valerate, modulate such interactions between gut and the brain. Beneficial effects of the SCFAs on brain function have been widely reported. Specifically, high butyrate levels in blood were reported to improve cognitive functions and even reduce amyloid beta deposition in the brain of AD transgenic mice ². However, the biological mechanisms by which these metabolites impact Aꞵ deposition are yet to be characterized. Blood-brain barrier (BBB) is widely believed to maintain the dynamic equilibrium between blood and brain Aꞵ pools. Further, accumulation of toxic Aꞵ peptides in the BBB endothelium is believed to induce BBB dysfunction which aggravate AD progression. Therefore, it is critical to investigate the effects of various SCFAs on Aꞵ transport at the BBB and its accumulation within the endothelial cells

Methods: Flow cytometry and confocal microscopy studies were conducted to investigate the effects of sodium acetate (NaAc), propionate (NaPr) and butyrate (NaBu) on Aꞵ42 accumulation in the BBB endothelial cells. Briefly, human cerebral microvascular endothelial cells (hCMEC/D3) and primary porcine brain endothelial cells (pBEC) cells were pretreated with 5mM NaAc, NaPr or NaBu for 2 hours followed by co-incubation with fluorescein-labeled Aꞵ42 (F-Aꞵ42, 12.5µg/ml) for another 1 hour. Intracellular fluorescence was measured using flow cytometry (Figure 1A). For confocal microscopy, hCMEC/D3 cells were grown on 35-mm coverslip bottom dishes and treated as described in the flow cytometry study. After washing and fixation, cells were nucleic stained and then subjected to confocal microscopy imaging (Figure 1B). Impact of NaBu on the abluminal-to-luminal (A-L) versus luminal-to-abluminal (L-A) flux of Aꞵ42 across BBB cell monolayers was assessed using primary pBEC cultured on 12-mm Transwell inserts with 0.4-µm pores. Polarized cell monolayer was incubated with F-Aꞵ42 in tracer amount with or without 5mM NaBu on the luminal or abluminal side. Culture media was sampled from the contralateral side at multiple time points, and fluorescence was measured by a microplate reader. The permeabilities were calculated using the Fick’s first law (Figure 1C). Western blot studies were performed to investigate the effect of NaBu on expression of receptors/transporters that participate in Aꞵ flux. hCMEC/D3 cells were treated with NaBu for 24 hours followed by co-incubation with Aꞵ42  for 1 hour. Whole cell lysates were prepared and used for western blots. (Figure 1D).

Results: Treatment with 5mM NaPr and NaBu significantly decreased the cellular uptake of F-Aꞵ42 in hCMEC/D3 cells by 2-fold (p < 0.05, one-way ANOVA followed by Bonferroni multiple comparison test) (Figure 2A-B). Further, the reduction in F-Aꞵ42 uptake by pBEC cells was found to be dependent on NaBu dose (Figure 2C-D). In contrast, no significant differences in F-Aꞵ42 uptake were observed in the cells treated with NaAc. Confocal micrographs demonstrated lower intracellular accumulation of F-Aꞵ42 in cells treated with NaBu. Moreover, L-A influx of F-Aꞵ42 across the pBEC monolayer was decreased when the cells were treated with NaBu on the luminal side. To identify the underlying mechanisms by which NaBu decreased Aꞵ42 influx transport and intracellular accumulation at the BBB, protein expression of critical receptors that regulate Aꞵ42 trafficking, including the receptor for advanced glycation end products (RAGE) and P-glycoprotein (P-gp) was assessed by western blots in hCMEC/D3 cells. Incubation with NaBu for 24 hours significantly upregulated P-gp expression by 1.5 fold (Figure 3B), which is evident from the immuno-blots (Figure 3A). In contrast, the expression of RAGE receptor, which mediates Aꞵ42 uptake on the luminal side and subsequent transcytosis, was found to be reduced by NaBu treatment (Figure 3).

Conclusion: This study demonstrated for the first time that gut microbial metabolite butyrate could decrease the blood-to-brain influx of Aꞵ42 at the BBB and its accumulation within the endothelium. Further, the observed kinetic changes could be due to the upregulation of efflux transporter P-gp and downregulation of the RAGE receptor. These findings suggest potential mechanisms driving reduction in brain Aꞵ levels in AD animals upon treatment with butyrate, and substantiate the evidence that gut microbial metabolites could modulate brain pathophysiology.

References: 1. Dalile B, Van Oudenhove L, Vervliet B, Verbeke K. The role of short-chain fatty acids in microbiota-gut-brain communication. Nat Rev Gastroenterol Hepatol. 2019 Aug;16(8):461-478.
2. Fernando WMADB, Martins IJ, Morici M, Bharadwaj P, Rainey-Smith SR, Lim WLF, Martins RN. Sodium Butyrate Reduces Brain Amyloid-β Levels and Improves Cognitive Memory Performance in an Alzheimer's Disease Transgenic Mouse Model at an Early Disease Stage. J Alzheimers Dis. 2020;74(1):91-99.

Acknowledgments: This work was supported by National Institutes of Health/National Institute of Neurological Disorders and Stroke R01NS125437

Figure 1. Experimental design



Figure 2. NaBu and NaPr inhibits the accumulation of F-Aꞵ42 in BBB cell monolayers.



Figure 3. NaBu treatment upregulates P-gp expression but downregulates RAGE expression.