Modulating expression of peripherin/rds in transgenic mice: critical levels and the effect of overexpression.
OBJECTIVE
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.
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, 73104, USA.
May
M
Nour
Xi-Qin
XQ
Ding
Heidi
H
Stricker
Steven J
SJ
Fliesler
Muna I
MI
Naash
D000818:Animals; D015153:Blotting, Western; D004596:Electroretinography; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D015151:Immunoblotting; D007381:Intermediate Filament Proteins / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D064531:Peripherins; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism* / Q000648:ultrastructure; D011233:Precipitin Tests; D012162:Retinal Degeneration / Q000378:metabolism* / Q000473:pathology
D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D064531:Peripherins; C578996:Prph2 protein, mouse