Abstract
Purpose:
Recent studies have suggested that a proportion of mutant P23H protein is retained in the rod photoreceptor ER where it is folded, but is incorrectly glycosylated and unstable. It has been proposed that this leads to ER stress and photoreceptor cell death. The mechanistic framework underlying photoreceptor cell death is still not fully understood. We have previously shown that there is an inflammatory component to retinal degeneration in P23H transgenic rat model. We hypothesize that inflammasome activation is a major contributor to P23H retinal cell death.
Methods:
Inflammasome activation was analyzed by histology, TUNEL staining, ELISA, immunohistochemistry and western blotting in P23H rat retinal samples at P21, P45 and P120. Small molecule drugs were used to target specific parts of the inflammasome pathway in vivo and functional assessment of the therapeutic benefit was measured by electroretinography. Immunohistochemical images were obtained by confocal scanning microscopy.
Results:
Up-regulation of the RIP1/RIP3 complex resulted in the translocation of phosphorylated DRP1 to the mitochondria of rod photoreceptors, resulting in the production of reactive oxygen species (ROS). Inflammasome components NLRP3 and activated caspase-1 were up-regulated in genetically normal cones, whereas their expression was absent from wildtype controls. The mature form of two downstream pro-inflammatory cytokines, Il-1ß and Il-18, were present in mutant retinal tissue extracts. NLRP3 was also up-regulated in activated microglia that had infiltrated the mutant retina. Treatment with either necrostatin-1s or N-acetylcysteine decreased NLRP3 expression, reduced reactive oxygen species, inhibited IL-1β expression, inhibited cone cell death, reduced the infiltration of activated microglia and prevented attenuation of the microvasculature in the retina. In contrast 3-aminobenzamide (an inhibitor of caspase-independent cell death) had no effect on inhibiting retinal degeneration.
Conclusions:
Collectively, this novel data provides compelling evidence that inflammasome activation in the neurosensory retina is a critical component in the pathogenesis of rod and cone photoreceptor degeneration in the P23H model of retinitis pigmentosa. Limiting the inflammasome-driven response will be crucial in preserving retinal integrity when designing future therapeutic options.