Abstract
Purpose: :
Central to the retinal pigment epithelial cell’s (RPE) role as a professional phagocyte is the regulated biogenesis and maturation of lysosomes which serve as the major degradative compartment, containing over 50 acid-dependent hydrolytic enzymes. Clearance of shed photoreceptor outer segments (POS) by the lysosomal system of the RPE is essential for function and viability of photoreceptors; impairment of POS clearance contributes to retinal diseases, including age related retinopathies. The purpose of these studies is to determine if lysosomogeneis and lysosomal hydrolase expression are correlated with light dependent phagocytic uptake of outer segments
Methods: :
Lysosome formation and lysosomal hydrolase maturation were followed in C57Bl6/J mice in response to light stimulated disk shedding. Mice were sacrificed at various times relative to light onset: -60 min, 0 min, 30 min, 1 h, 2 h and 6 h. Lysosome formation was evaluated by qPCR, immunoblotting and immunocytochemistry using endo-lysosomal markers. Lysosomal hydrolases were identified by qPCR and maturity and localization profiles by Western blot and immunocytochemistry, respectively.
Results: :
Lysosomogenesis appears to follow a biphasic pattern with early (15-30 min) and later peaks (6hrs). The primary degradative hydrolase, cathepsin D, followed a similar distribution profile while Cathepsin S, a protease required to process Cathepsin D, does not appear to show a biphasic expression profile. Levels of autophagy markers LC3, Atg5 and Atg8 were similarly compared and their intracellular distribution profile correlated with degradation of RDS and Rom-1.
Conclusions: :
Lysosomogenesis, a critical step in the degradation of ingested outer segment debris is likely a biphasic event, triggered in part by phagocytosis of outer segments. This observation is consistent with two pools of lysosome like organelles proposed by Bosch, et al., 1993 over a decade ago.
Keywords: phagocytosis and killing • retinal degenerations: cell biology • pH regulation/protons