Investigating AMD mechanisms using primary RPE cultures and donor tissue

Stimuli that activate inflammasomes include mitochondrial (mt) reactive oxygen species, release of damaged mtDNA into the cytosol, or disruption of cellular ionic balance. These stimuli can arise from mt dysfunction, which may be a key pathogenic event in age-related macular degeneration (AMD). This hypothesis is consistent with increased mtDNA damage reported in macular retinal pigment epithelium (RPE) of human donors with AMD. To investigate inflammasome activation, we developed primary cultures of RPE from donors with or without AMD. Cells express the RPE-specific proteins bestrophin, CRALBP, and RPE65, are pigmented, express ZO-1 at cell margins and Na-K ATPase apically. These results show our primary cultures phenocopy the RPE in vivo. To examine inflammasome activation, cells were stimulated with LPS as a priming event, then treated with either the proteasome inhibitor MG132 to initiate formation of protein aggregates or rotenone to generate reactive oxygen species. We observed differences between cells from control and AMD donors in IL-18 and IL-1beta production and also caspase-1 cleavage, the prototypic marker of inflammasome activation. These results suggest AMD affects inflammasome activation in the RPE. Commercial interest