March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Rab38 Deficiency Leads To Loss Of Cathepsin D And a Defect In Rhodopsin Degradation In Mouse Retinal Pigment Epithelium
Author Affiliations & Notes
  • Tanya Tolmachova
    Molecular Medicine, NHLI, Imperial College London, London, United Kingdom
  • Miguel C. Seabra
    Molecular Medicine, NHLI, Imperial College London, London, United Kingdom
  • Footnotes
    Commercial Relationships  Tanya Tolmachova, None; Miguel C. Seabra, None
  • Footnotes
    Support  WT093445MA
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1132. doi:
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      Tanya Tolmachova, Miguel C. Seabra; Rab38 Deficiency Leads To Loss Of Cathepsin D And a Defect In Rhodopsin Degradation In Mouse Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1132.

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Abstract

Purpose: : Rabs are small GTP-binding proteins that regulate intracellular vesicular traffic. There are 60 known mammalian Rabs, some of which regulate general steps and are expressed ubiquitously, while others perform a specialised function and are expressed in a more restricted pattern. Rab38 was shown in the past to be expressed in pigmented cells such as melanocytes and retinal pigment epithelium (RPE) and be important for the delivery of tyrosinase to melanosomes. Deficiency in Rab38 leads to a significant reduction in a number of mature melanosomes in the RPE of the Rab38 mutant mice (chocolate). The aim of this study was to investigate the role of Rab38 in the phagocytosis of rod outer segments (ROS) in mouse RPE cells.

Methods: : RPE cells were isolated from the eyes of Rab38 cht/cht, Rab38 cht/+ and C57Bl6 mice and maintained in culture. Phagocytosis was studied in primary mouse RPE cultures using FITC-labelled mouse ROS and data was collected by FACS. Degradation of rhodopsin was followed using antibody specific to the C-terminus (1D4) in FITC-positive cells. Secretion of cytokines was measured in primary RPE cultures by ELISA. The protein level of rhodopsin and cathepsin D was analysed by FACS and Western blotting in RPE cells collected from the mouse eyes 6 hours after the light onset.

Results: : In primary Rab38cht/cht RPE cultures fed with FITC-labelled ROS we detected a significant reduction in the rate of rhodopsin degradation in comparison to Rab38 cht/+ and C57Bl6 RPE cultures. This result was confirmed by FACS in non-cultured RPE cells where we detected a significant increase in rhodopsin staining in Rab38cht/cht RPE in comparison to Rab38 cht/+ and C57Bl6 RPE. Cathepsin D level was significantly reduced in Rab38cht/cht, in comparison to Rab38 cht/+ and C57Bl6. Secretion of IL-6 (but not LIF) was specifically upregulated in Rab38cht/cht in comparison to Rab38 cht/+ and C57Bl6 mice.

Conclusions: : Rab38 is important for rhodopsin degradation. Cathepsin D is severely reduced in the absence of Rab38 which suggests that Rab38 might be involved in cathepsin D trafficking.

Keywords: retinal pigment epithelium • phagocytosis and killing • cytokines/chemokines 
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