Mutant rhodopsin transgene expression on a null background.
OBJECTIVE
To study mechanisms leading to photoreceptor degeneration in mouse models for autosomal dominant retinitis pigmentosa (adRP) based on the rhodopsin P23H mutation.


METHODS
Mice of a transgenic line expressing a rhodopsin triple mutant, V20G, P23H, and P27L (GHL), were mated with rhodopsin (rho) knockout mice. Littermates of various ages and genotypes (GHL+rho+/+, GHL+rho+/-, and GHL+rho-/-) were examined for outer nuclear layer thickness and outer segment formation (histology), fate of mutant rhodopsin (immunocytochemistry), and photoreceptor function (electroretinogram; ERG).


RESULTS
Mice expressing GHL-rhodopsin in the absence of wild-type rhodopsin had severe retinopathy, which was nearly complete by postnatal day (P)30. GHL-rhodopsin formed homodimers nearly exclusively on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, whereas wild-type rhodopsin predominantly formed monomers. Expression level of mutant rhodopsin in predegenerate (P10) GHL+rho-/- retinas was low, approximately 10% to 25% of normal levels. No elaboration of disc membrane or outer segment formation was observed at any time point examined. The mutant rhodopsin was found mostly in perinuclear locales (endoplasmic reticulum; ER) as evidenced by colocalization using the antibodies Rho1D4 and calnexin-NT.


CONCLUSIONS
GHL-rhodopsin dimerizes, localizes to the ER, and fails to transport and support outer segment formation. Additionally, the mutant protein does not support a scotopic ERG a-wave and accelerates photoreceptor degeneration over that occurring with the rhodopsin knockout alone. These findings indicate a cytotoxic effect of the mutant protein, probably elicited by an unfolded protein response.
Frederick
J M
JM
John A. Moran Eye Center, University of Utah Health Science Center, 50 North Medical Drive, Salt Lake City, UT 04132, USA. jfrederi@hsc.utah.edu
Krasnoperova
N V
NV
Hoffmann
K
K
Church-Kopish
J
J
Rüther
K
K
Howes
K
K
Lem
J
J
Baehr
W
W
D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004596:Electroretinography; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005838:Genotype; D015151:Immunoblotting; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D016253:Microscopy, Immunoelectron; D009154:Mutation; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism / Q000648:ultrastructure*; D012162:Retinal Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012243:Rhodopsin / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes / Q000235:genetics*
D017931:DNA Primers; D012243:Rhodopsin