title: Correlation between inactive cathepsin D expression and retinal changes in mcd2/mcd2 transgenic mice.
abstract: OBJECTIVE
  To investigate the correlation between the presence of the inactive cathepsin D (CatD) and retinal changes in mcd2/mcd2 transgenic mice.
  
  
  METHODS
  Computational modeling was used to examine whether CatD mutants maintain competitive substrate binding. D407 cells were transfected with pcDNACatDM1 or pcDNACatDM2, containing procathepsin D (pro-CatD) with 6-bp (CatDM1) or 12-bp (CatDM2) deletions, respectively, flanking the pro-CatD cleavage site, and the aspartic protease activity of the transfected cells was measured. Subsequently, transgenic mice (mcd2/mcd2) containing CatDM2 were generated. Relative transgene copy number and transcript levels in the previously produced mcd/mcd (carrying CatDM1) and mcd2/mcd2 mice were measured by quantitative real-time PCR. Western blot analysis and aspartic protease activity were used to characterize the mutated proteins. Retinal changes were described by using color fundus photography and fluorescein angiography, histology, immunohistochemistry, and electron microscopy.
  
  
  RESULTS
  Computational modeling of the CatDM1 and CatDM2 structures indicated that the substrate binding site was not altered. There was limited or no aspartic protease activity associated with CatDM1 and CatDM2 proteins, respectively. Mcd2/mcd2 animals contained a higher amount of inactive CatD than mcd/mcd or wild-type mice. Retinal abnormalities in mcd2/mcd2 mice developed at 3 months of age, earlier than in mcd/mcd mice. These changes included hypopigmentation, hyperfluorescence, retinal pigment epithelial (RPE) cell depigmentation or clumping, cell proliferation, and pleomorphism. Proliferating cells were identified as being of RPE origin.
  
  
  CONCLUSIONS
  This study demonstrated a correlation between the presence of the inactive CatD in RPE cells and the development of ophthalmoscopic, cellular, and histologic changes in the retina.
authors.affiliation: Department of Molecular Ophthalmology, Lions Eye Institute, Perth, Australia.
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.forename: Meliha
authors.initials: M
authors.lastname: Brankov
authors.forename: Mahindra T
authors.initials: MT
authors.lastname: Makhija
authors.forename: Terry
authors.initials: T
authors.lastname: Robertson
authors.forename: Erik
authors.initials: E
authors.lastname: Helmerhorst
authors.forename: John M
authors.initials: JM
authors.lastname: Papadimitriou
authors.forename: Piroska E
authors.initials: PE
authors.lastname: Rakoczy
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D015153:Blotting, Western; D002402:Cathepsin D / Q000737:chemistry / Q000502:physiology*; D003198:Computer Simulation; D004792:Enzyme Precursors / Q000502:physiology; D005451:Fluorescein Angiography; D017353:Gene Deletion; D006801:Humans; D007150:Immunohistochemistry; D008268:Macular Degeneration / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D015394:Molecular Structure; D010857:Pigment Epithelium of Eye / Q000201:enzymology* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D004792:Enzyme Precursors; D016282:Aspartic Acid Endopeptidases; C058345:procathepsin D; D002402:Cathepsin D