AMD-compatible lysosomal changes in Rab38-deficient mouse model

Abstract

Purpose: Accumulation of lipofuscin deposits, lysosome disregulation and inflammation are hallmarks of age-related macular degeneration (AMD). Retinal pigment epithelium (RPE) plays a major role in AMD pathology. Age-related changes in the normal RPE and AMD mechanisms are not fully understood. Rab38 is a small GTPase that is important for biogenesis of lysosome-related organelles. The purpose of the study was to assess lysosomal function in Rab38-deficient (chocolate) mouse, including expression of lysosomal proteins LAMP-1 and cathepsins, accumulation of autofluorescent deposits and inflammatory cytokine upregulation.

Methods: RPE cells were isolated from the eyes of young (3-4 months) and old (12-15 months) C57Bl6 and Rab38-deficient (chocolate) mice. LAMP-1 and cathepsin gene and protein expression were analysed in freshly isolated RPE by real-time PCR, FACS analysis and Western blotting. Cathepsin activity was measured in freshly isolated RPE cells and primary RPE cultures established from 3-week old mouse eyes. IL-6 and MCP-1 cytokine secretion was measured by ELISA. Transmission electron microscopy was used.

Results: We detected significant downregulation of LAMP-1 gene and protein expression in chocolate mice in comparison to C57Bl6 animals. While LAMP-1 expression significantly increased with age in C57Bl6 animals, in chocolate mice there was no significant difference between young and old age groups. Activity of cathepsin H was increased in the lysates of freshly isolated chocolate RPE cells as well as in chocolate RPE cultures. Activity of cathepsin D was also increased in chocolate RPE cell culture lysates. Furthermore, maturation of cathepsin D was delayed in chocolate RPE. We detected accumulation of autofluorescent material in RPE cells isolated from old chocolate mice in comparison to old C57Bl6 and young chocolate animals. Electron microscopy identified abnormalities in old chocolate mice such as cytoplasmic deposits, autophagosomes, elongated mitochondria. This was accompanied by significant upregulation of IL-6 in freshly isolated chocolate RPE cells and RPE cultures.

Conclusions: We identified lysosomal defects in Rab38-deficient (chocolate) mice, which correlated with accumulation of autofluorescent material with age and upregulation of proinflammatory cytokine IL-6. Our results suggest that Rab38 is important for lysosomal function in aged RPE and may play a role in AMD pathogenesis.

Keywords: 701 retinal pigment epithelium • 412 age-related macular degeneration • 695 retinal degenerations: cell biology