CATHEPSIN B PLAY A CRITICAL ROLE IN AMYLOID β INFLUX VIA CEREBRAL ENDOTHELIAL CELLS DURING PORPHYROMONAS GINGIVALIS INFECTION

Abstract Background Cerebral amyloid angiopathy (CAA) is observed in more than 80% of Alzheimer’ s disease (AD) patients and receptor for advanced glycation end products (RAGE) mediates amyloid β (Aβ) influx into brain (Deane et al. 2003). We have found that cathepsin B (CatB) involves peripheral Aβ production during Porphyromonas gingivalis (P.g ) infection (Nie et al., 2019). Aims & Objectives In the present study, we explored the roles of CatB in Aβ transportation from periphery into the brain, known as “Aβ influx” during P.g infection. Method Fifteen-month-old female mice C57BL/6J were intraperitoneally injected with P.g (1×108 CFU/mouse) every 3 days and last for 3 consecutive weeks. The step-through passive avoidance test was used to test learning and memory function. Culture hCMEC/D3 cell line was infected by P. gingivalis (ATCC33277, MOI: multiplicity of infection=5). Real-time quantitative PCR, immunofluorescent staining and immunoblotting were used for gene and protein analysis Results In comparison to non-infection mice, systemic P. gingivalis infection for 3 consecutive weeks significantly increased the RAGE expression in the CD31-positive endothelial cells (2-fold increase) and the Aβ loads around the CD31-positive cells in the parenchyma of mice’ s brains (11-fold, increase). The RAGE expression in the CD31-positive cells was positively correlated with the Aβ loads. In addition, learning and memory function was significantly decreased in the P.g-infected mice than that of in PBS-injected mice. In compassion to uninfected hCMEC/D3 cells, RAGE expression was significantly increased in the P.g-infected hCMEC/D3 cells which was inhibited by pretreating with NFκ B inhibitor. On the other hand, CatB expression was increased in the P.g-infected hCMEC/D3 cells, and CatB specific inhibitor suppressed both Iκ Bα degradation and RAGE expression in the P.g-infected hCMEC/D3 cells. Using fluorescently labeled Aβ1-42, the amount of Aβ1-42 in basolateral medium of the P.g-infected hCMEC/D3 cells was 16-fold higher than that in uninfected cells, the P.g-increased Aβ1-42 in basolateral medium of culture hCMEC/D3 cells was inhibited by pretreating with RAGE specific inhibitor. Discussion & Conclusion These observations demonstrate that NF-κ B activation-dependent RAGE upregulation in cerebral endothelial cells mediates Aβ influx during P. gingivalis infection. CatB plays a critical role in Aβ influx via up-regulating RAGE expression and NF-κ B activation. Taking together with our previous findings of the involvements of CatB in Aβ generation in peripheral inflammatory macrophages during P.g infection (Nie et al. 2019), CatB would be a therapeutic target for preventing the Porphyromonas gingivalis infection-associated amyloidogenesis in AD. References 1.Deane, R., Du Yan, S., Submamaryan, R. K. et al. (2003) RAGE mediates amyloid β peptide transport across the blood-brain barrier and accumulation in brain. Nature edicine 9, 907-913.2. 2.Nie R, Wu Z, Ni J, Zeng F, Yu W, Zhang Y, Kadowaki T, Kashiwazaki H, Teeling JL,Yanmin Zhou Y. (2019) Porphyromonas gingivalis Infection Induces Amyloid β Accumulation in Monocytes/Macrophages. J. Alzheimer's Dis. 72 (2):479-494.