Purpose
Accumulation of amyloid beta peptides (A-beta) has been shown to be a potential contributing factor for the development of age-related macular degeneration (AMD). Recent work has shed light on the role of the pro-inflammatory macromolecular complex of the NLRP3-inflammasome in the pathogenesis of geographical atrophy (GA). Our previous studies have shown that A-beta increases the expression of the thioredoxin-interacting protein (TXNIP), a constituent of the NLRP3-inflammasome complex. In this study we wanted to determine whether A-beta stimulation of retinal pigmented epithelial cells (ARPE19) promoted the expression, activation and protein-protein interaction of constituents of the NLRP3-inflammasome.
Methods
Human retinal pigmented epithelial cells (ARPE19) were stimulated for 24 hours with 10μM of A-beta 1-42 or 10μM of the reverse peptide A-beta 42-1, which was used as control. Western blotting and immunoprecipitation analyses were performed to determine the expression and protein-protein interaction of the inflammasome constituent TLR4, NLRP3, ASC and TXNIP and to determine the expression of A-beta receptor (RAGE). NLRP3-activation was determined by measuring caspase1 activity. RAGE inhibition was achieved by transfecting ARPE19 with specific siRNA.
Results
A-beta treatment of ARPE19 promoted the expression of RAGE, TLR4, NLRP3 and TXNIP. Immunocytochemistry revealed that A-beta stimulated NLRP3 interaction with ASC and TXNIP and this effect was followed by increased caspase1 activity. Selective blockade of RAGE, by RNA silencing, blocked A-beta -induced activation of NLRP3-inflammasome in ARPE19, as assessed by monitoring caspase 1 activity.
Conclusions
The specific role of A-beta in the pathogenesis of wet AMD or GA is still controversial. Our data demonstrating that A-beta can promote RPE cells damage by inducing the NLRP3-inflammasome, further confirm a clear role of A-beta in the early pathogenesis of AMD.
Keywords: 412 age-related macular degeneration •
701 retinal pigment epithelium •
557 inflammation