Modulating expression of peripherin/rds in transgenic mice: critical levels and the effect of overexpression

Invest Ophthalmol Vis Sci. 2004 Aug;45(8):2514-21. doi: 10.1167/iovs.04-0065.

Abstract

Purpose: Mutations in the photoreceptor-specific protein peripherin/rds are associated with multiple retinal diseases. To date, attempts to achieve complete structural and functional rescue in animal models of peripherin/rds-induced retinal degeneration have not been successful. Gene therapy-directed approaches have been hindered by the haploinsufficiency phenotype, which dictates well-regulated expression of peripherin/rds protein levels.

Methods: Using a transgenic mouse line expressing wild-type peripherin/rds (NMP), the authors evaluated the critical in vivo level of peripherin/rds needed to maintain photoreceptor structure and ERG function and assessed the consequences of peripherin/rds overexpression in both rods and cones by Western blot and immunoprecipitation analyses, immunohistochemistry, electron microscopy, and electroretinography. The NMP transgene included a C-terminal modification (P341Q) to facilitate detection of the transgenic protein in the presence of wild-type peripherin/rds, using the monoclonal antibody 3B6.

Results: Peripherin/rds protein levels in NMP homozygotes were approximately 60% of wild-type levels. Western blot and immunoprecipitation analyses confirmed normal biochemical properties of the NMP protein when compared with wild-type peripherin/rds. Immunohistochemistry demonstrated appropriate localization of transgenic peripherin/rds protein to the disc rim region of photoreceptor outer segments. Total peripherin/rds levels in the retina were modulated by crossing NMP transgenic mice into different rds genetic backgrounds. A positive correlation was observed between peripherin/rds expression levels and the structural and functional integrity of photoreceptor outer segments. Overexpression of peripherin/rds caused no detectable adverse effects on rod or cone structure and function.

Conclusions: These findings may have significant implications regarding therapeutic intervention in peripherin/rds-associated retinal diseases.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Electroretinography
  • Female
  • Fluorescent Antibody Technique, Indirect
  • Gene Expression Regulation / physiology*
  • Immunoblotting
  • Intermediate Filament Proteins / genetics*
  • Intermediate Filament Proteins / metabolism
  • Male
  • Membrane Glycoproteins / genetics*
  • Membrane Glycoproteins / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Nerve Tissue Proteins / genetics*
  • Nerve Tissue Proteins / metabolism
  • Peripherins
  • Photoreceptor Cells, Vertebrate / metabolism*
  • Photoreceptor Cells, Vertebrate / ultrastructure
  • Precipitin Tests
  • Retinal Degeneration / metabolism*
  • Retinal Degeneration / pathology

Substances

  • Intermediate Filament Proteins
  • Membrane Glycoproteins
  • Nerve Tissue Proteins
  • Peripherins
  • Prph2 protein, mouse