(M1130-04-23) Gut Microbial Metabolites Effect 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 in elderly with amyloid beta (Aꞵ) deposition in the brain as one of its pathological hallmarks. Cerebrovascular dysfunction is prevalent in 90% of AD patients and believed to be a significant contributor to AD progression. In addition, brain insulin resistance and gut microbiome alterations­­ have been reported to be major AD risk factors ^1,2. However, the interrelationship between cerebrovascular dysfunction, insulin resistance and gut dysbiosis in AD pathogenesis remains uncharacterized. One of the interactions between gut and the brain is mediated by gut microbial metabolites (GMM) including short chain fatty acid (acetate, propionate, butyrate) and Trimethylamine N-oxide (TMAO). High butyrate level in blood was reported to reduce Aꞵ deposition in AD brain but the underlying mechanisms are yet to be characterized ³. Our previous publications have demonstrated that Aꞵ trafficking at the blood-brain barrier (BBB), which lines the cerebrovascular lumen and clears Aꞵ from the brain, is regulated by insulin signaling. However, the effect of GMM on Aꞵ trafficking/accumulation and insulin signaling in the BBB remains unknown. Therefore, we hypothesize that gut microbiome metabolites could impact Aꞵ trafficking and accumulation at the BBB. We further hypothesize that the effect is mediated via insulin signaling pathway.

Methods: Flow cytometry studies were conducted to investigate the effect of gut microbial metabolites like sodium butyrate (NaB) and TMAO on the amyloid beta accumulation in the BBB endothelial cells. Human cerebral microvascular endothelial cells (hCMEC/D3) were pretreated with physiological concentration of NaB and TMAO (10µM) for 2h, 6h and 24h followed by co-incubation with FITC-labeled Aꞵ (F-Aꞵ₄₀, 25µg/ml, F-Aꞵ₄₂, 12.5µg/ml) for another 1 hour. To investigate the effect of insulin signaling on Aꞵ accumulation, the cells were pretreated with insulin signaling inhibitors (Tramatinib, PD98059) followed by incubation with F-Aꞵ_40/42 for another 1 hour. Intracellular fluorescence was measured using flow cytometry and data was analyzed using flow Jo. Western blots (WB) were performed to examine the effect of NaB on protein expression of receptors/transporters involved in Aꞵ transport at the BBB endothelium. HCMEC/D3 cell monolayers were pre-treated with NaB for 6h, 24h and lysates were probed for WB and the blots were probed for receptor for advanced glycation end products (RAGE), P-glycoprotein (P-gp), caveolin 1, calnexin, and vascular endothelial cadherin. Later, the effect of NaB and TMAO on insulin signaling pathway is investigated on BBB endothelial cells. HCMEC/D3 cells were pre-treated with NaB and TMAO for 2h, 6h followed by co-incubation with 50nM insulin for another 5min. Whole cell lysates were prepared and subjected to western blot to evaluate the protein phosphorylation of insulin receptor, ERK, AKT, RAGE, P-gp, Caveolin1, GAPDH.

Results: Treatment with NaB has decreased ((p < 0.05, one-way ANOVA, Bonferroni multiple comparison test) F-Aꞵ₄₀ uptake with 6h pretreatment only. It also reduced F-Aꞵ₄₂ accumulation at 2h and 6h. TMAO has prominently increased F-Aꞵ₄₀ uptake at 6h with no change at 2h and 24h. In contrast, it decreased F-Aꞵ₄₂ accumulation at 2h, 6h and 24h time points as shown in Fig 1. Western blot was performed to investigate the effect of NaB on BBB protein/transporters and RAGE was significantly decreased at 24h time point as shown in Fig 2A, B. Further, cells were pretreated with ERK inhibitors (Tramatinib, PD98059) followed by incubation with F-Aꞵ_40/42 for another 1 hour. Intracellular fluorescence was measured using flow cytometry and data was analyzed using flowJo software. Tramatinib increased F-Aꞵ₄₂ uptake (Fig 2D) and PD98059 decreased F-Aꞵ₄₀ uptake (Fig 2E). Also, effects of NaB and TMAO with insulin co-incubation were investigated. Both metabolites upregulated the phosphorylation of ERK and AKT in HCMEC/D3 cells as shown in Fig 2C and Fig 3. The results suggest that TMAO has differential effects on the accumulation of F-Aꞵ₄₀ and F-Aꞵ₄₂ in the brain. While TMAO treatment increased F-Aꞵ₄₀ levels, it decreased F-Aꞵ₄₂ accumulation like NaB. Interestingly, treatment with an ERK inhibitor had opposing effects on F-Aꞵ₄₀ and F-Aꞵ₄₂ uptake. In particular, inhibition of ERK decreased F-Aꞵ₄₀ levels but increased F-Aꞵ₄₂ accumulation. These findings suggest that NaB and TMAO might be activating insulin signaling pathway (AKT, ERK) to modulate Aꞵ peptide accumulation in the brain.

Conclusion: In current study, we have identified the possible interaction between gut microbial metabolites and insulin signaling pathway in regulating amyloid beta accumulation. NaB decreased F-Aꞵ_40/42 accumulation in BBB endothelial cells and down-regulated RAGE expression. TMAO down-regulated F-Aꞵ₄₂ uptake but upregulated F-Aꞵ₄₀ uptake in HCMEC/D3 cells. NaB and TMAO when co-incubated with insulin increased phosphorylation of AKT, ERK. F-Aꞵ₄₀ uptake is decreased and F-Aꞵ₄₂ is increased when cells were treated with ERK inhibitor. All these results suggest that gut microbial metabolites might be communicating via insulin signaling pathway to regulate Aꞵ deposition in BBB endothelial cells. In future, more studies can be performed to identify molecular mechanism involved in the transportation of amyloid beta peptides.

References: 1. Christian Hölscher, Insulin Signaling Impairment in the Brain as a Risk Factor in Alzheimer’s Disease Front Aging Neurosci. 2019; 11: 88.
2. 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.
3. 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.

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

Fig 1: Fold change of intracellular median fluorescence intensity (MFI) in hCMEC/D3 cells treated with Butyrate/TMAO for different duration.

Fig 2: A, B. NaB pre-treatment for 24h down-regulated RAGE expression in whole membrane HCMEC/D3 lysates with blots in A and quantification in B. C. NaB pre-treatment for 6h increased AKT phosphorylation in whole cell lysates in HCMEC/D3 cells. D. Tramatinib treatment, (ERK) inhibition increased AB42 uptake. E. PD98059 treatment, (ERK) inhibition decreased AB40 uptake.

Fig 3: A, B. TMAO pre-treatment for 2h and 6h up-regulated phosphorylation of ERK in whole membrane HCMEC/D3 lysates with blots in A and quantification in B. C. TMAO pre-treatment for 2h increased AKT phosphorylation in whole cell lysates in HCMEC/D3 cells.