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.
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
J M
JM
Frederick
N V
NV
Krasnoperova
K
K
Hoffmann
J
J
Church-Kopish
K
K
Rüther
K
K
Howes
J
J
Lem
W
W
Baehr
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