| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 From the Department of Ophthalmology and Visual Sciences, College of Medicine, and the 2 Department of Biological Sciences, College of Liberal Arts and Sciences, University of Illinois at Chicago; the 3 Research Service, Hines VA Hospital, Illinois 4 Departments of Neurology and 5 Ophthalmology, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois; the 6 Department of Ophthalmology, New England Eye Center, Boston, Massachusetts; and 7 Saint Louis University Eye Institute and the Cell and Molecular Biology Graduate Program, Saint Louis University School of Medicine, Missouri.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
METHODS. Three transgenic mouse lines were generated that express different levels of an opsin with three amino acid modifications at the C terminus. These modifications created an epitopic site that can be readily distinguished from the endogenous protein using a bovine opsin-specific antibody. Evidence of degeneration associated with opsin overexpression was provided by anatomic studies and electroretinogram (ERG) recordings. Western blot analysis was used to confirm the production of the transgenic opsin, and an enzyme-linked immunosorbent assay (ELISA) was used to determine the amounts of opsin overexpressed in each line. Immunocytochemistry was used to determine the cellular localization of transgenic opsin. Amounts of 11-cis retinal were determined by extraction and high-performance liquid chromatography (HPLC).
RESULTS. Opsin expression levels in the three lines were found to be 123%, 169%, and 222% of the level measured in nontransgenic animals, providing direct correlation between the level of transgene expression and the severity of the degenerative phenotype. In the lower expressing lines, ERG a-wave amplitudes were reduced to less than approximately 30% and 15% of normal values, whereas responses of the highest expressing line were indistinguishable from noise. In the lowest expressor, a 26% elevation in 11-cis retinal was observed, whereas in the medium and the high expressors, 11-cis retinal levels were increased by only 30% to 33%, well below the 69% and 122% increases in opsin levels.
CONCLUSIONS. The overexpression of normal opsin induces photoreceptor degeneration that is similar to that seen in many mouse models of retinitis pigmentosa. This degeneration can be induced by opsin levels that exceed by only approximately 23% that of the normal mouse retina. Opsin overexpression has potential implications in retinitis pigmentosa.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although photoreceptor degeneration resulting from opsin overexpression has been described, no characterization of the pattern and rate of the associated degeneration or the possible mechanism leading to the degeneration has been provided. In the present study, we investigated the correlation between the amount of opsin expressed and the rate of retinal degeneration. Furthermore, we investigated the mechanism through which overexpression of opsin can lead to photoreceptor cell death. To accomplish these goals, we used a transgene construct in which three amino acid substitutions were made at the C terminus to give the transgene bovine opsin-specific immunoreactivity. We refer to this as the Bouse (bovine and mouse) opsin transgenic construct. These substitutions, which have not been associated with any retinal disease, allowed the transgenic protein to be distinguished within the environment of the endogenous protein using the bovine opsin-specific monoclonal antibody (mAb) 3A6.15 16
Three transgenic mouse lines designated Bouse A, Bouse B,and Bouse C, were established from the injection of the Bouse opsin transgenic construct. Retinas of Bouse transgenic mice were examined at specific ages with electroretinography to define the functional competence of the retina, with immunocytochemistry to determine the cellular localization of transgenic opsin, and enzyme-linked immunosorbent assay (ELISA) to determine the opsin expression levels. The results confirm that the severity of degeneration depends on the level of opsin expression and demonstrates that an approximate 23% overexpression of opsin is sufficient to trigger photoreceptor degeneration.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As shown in Figure 1 , there are only three amino acid differences (D332E, A335T, and A337V) between mouse and bovine rhodopsin at the epitopic site recognized by mAb 3A6. Although the C terminus has been demonstrated to be crucial for the proper functioning of the molecule,18 these specific amino acids are not involved in any known function or associated with photoreceptor degeneration.
|
Southern blot analysis was used to determine the pattern of transgene integration and copy number in the three transgenic lines. Genomic DNA was isolated from tail clippings by use of a kit (QIAamp Tissue Kit; Qiagen, Valencia, CA). A 30-µg DNA aliquot extracted from each animal was digested with BamHI at 37°C overnight, electrophoresed in a 0.8% Tris-acetate agarose gel, and transferred onto a membrane (Nytran Plus; Schleicher & Schuell). A 4-kb P32-labeled EcoRI-SalI fragment of the 3' flanking sequences of the mouse opsin gene was used as a probe.17 This probe labels the endogenous opsin gene as well as the integrated transgene. By labeling the junction between the transgene and the surrounding DNA, this probe allowed the differentiation between the endogenous gene and the junctional fragment of the transgene. Hybridization and wash conditions were as described previously.23 Comparative densitometric measurements of the transgene and endogenous specific bands were used to determine the transgene copy numbers.
To express the Bouse transgene in the absence of endogenous opsin, Bouse B transgenic mice were mated to mice with targeted disruption of the opsin gene (opsin-/-), and the offspring were screened for the presence of the transgene (as described earlier) and the knockout construct.24 25 Animals that were heterozygous for both Bouse and the knockout mutation (i.e., opsin+/-) were mated to opsin-/- mice to produce animals that are heterozygous for Bouse and opsin-/-.
All experiments were approved by the local Institutional Animal Care and Use Committees and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Electroretinography
ERGs were recorded from the corneal surface of mice, as
described previously.22
In brief, after overnight dark
adaptation, mice were anesthetized with ketamine (80 mg/kg) and
xylazine (16 mg/kg) and placed on a heating pad after the pupils were
dilated (1% tropicamide; 2.5% phenylephrine HCl). Responses were
amplified (1–1000 Hz), averaged, and stored using a signal-averaging
system (Compact 4; Nicolet, Madison, WI). Strobe flash stimuli were
presented in a Ganzfeld (Nicolet), first in the dark and then
superimposed on a steady rod-desensitizing adapting field (1.3 log
candelas [cd]/m2), after a 7-minute interval
was allowed for light adaptation.26
Flash intensities were
controlled with neutral density filters (Wratten 96; Kodak, Rochester,
NY) and ranged from -3.0 to 1.0 log cd sec/m2,
calibrated with a photometer (model 550; EG&G, Gaithersburg, MD).
Histology and Morphometry
The morphologic appearance of the transgenic and normal retinas
was examined after fixation in mixed aldehyde fixative and tissue
processing, as described previously.27
Sections were
examined by microscope (Axioskope; Zeiss, Oberkochen, Germany).
For morphometric analysis, digital images of retinal cross sections were captured with a microscope (Olympus, Lake Success, NY) fitted with a digital camera (Sensus; Photometrics, Tucson, AZ). Photoreceptor nuclei were counted in a microscopic field that spanned 100 µm and was centered 300 µm from the edge of the optic nerve head. The 100-µm field was determined by an imaging system (MetaMorph; Universal Imaging, West Chester, PA). This measurement was performed on both sides of the optic nerve head for each section. No differences were found in the number of photoreceptor nuclei between these regions. Three sections from each of three retinas were examined for each time point.
Light Microscopic Immunocytochemistry
Immunocytochemical analysis was performed after a 4- to 16-hour
fixation of enucleated eyes in Davidson fixative28
29
and
subsequent removal of the anterior segment. Tissues were cryoprotected
with 30% sucrose in phosphate-buffered saline (PBS; 140 mM NaCl, 2.7
mM KCl, 10 mM Na2HPO4, and
1.76 mM KH2PO4), embedded
in optimal cutting temperature compound (OCT; Miles Diagnostics,
Elkhart, IN), and snap frozen in an isopentane-dry ice bath.
Six-micrometer sections were cut, picked up on glass slides, and
allowed to dry at room temperature. Sections were incubated with PBS
containing 5% normal goat serum and 0.1% bovine serum albumin (BSA)
for 30 minutes at room temperature. After three washes with PBS,
sections were incubated with the primary antibody in PBS containing 5%
goat serum, 0.1% BSA, and 140 mM NaCl (mAb 1D4 at 1:100 dilution at
4°C) or 500 mM NaCl (mAb 3A6 at 1:20 dilution at room temperature)
overnight. After primary antibody incubation, sections were rinsed in
PBS three times. The secondary antibody, fluorescein isothiocyanate
(FITC)–conjugated goat anti-mouse (Jackson ImmunoResearch, West Grove,
PA), was applied at 1:1000 dilution for 30 minutes at room temperature.
Sections were then washed three times with PBS, mounted in medium
(H-1000; Vector, Burlingame, CA), and viewed with an epifluorescence
microscope (Axioskope; Ziess).
Alternatively, hemisected eyes (anterior segment removed) were fixed in mixed aldehyde fixative (0.1 M sodium phosphate buffer [pH 7.4], containing 2.5% glutaraldehyde, 2.0% paraformaldehyde, and 0.025% CaCl2) and processed for embedding in resin (LR White; Electron Microscopy Sciences, Fort Washington, PA), essentially according to the method of Erickson et al.30 Thick sections (0.75 µm) were cut with a microtome and placed onto glass slides, followed by treatment for 30 minutes at room temperature with blocking buffer (PBS containing 1% [wt/vol]) radioimmunoassay grade BSA, 10% [vol/vol] normal goat serum, and 0.05% [vol/vol] Triton X-100). Tissue sections were exposed for 2 hours at room temperature, either to rabbit anti-bovine opsin IgG or to nonimmune rabbit serum (each diluted 1:500 with blocking buffer), rinsed briefly with PBS, and treated for 2 hours at room temperature with 1 nm colloidal gold–conjugated goat anti-rabbit IgG secondary antibody (AuroProbe One GAR; Amersham, Arlington Heights, IL; diluted 1:50 [vol/vol] with blocking buffer). Sections were rinsed three times (15 minutes each) with PBS, followed by fixation for 10 minutes at room temperature with 2% (vol/vol) glutaraldehyde in PBS and then rinsed with distilled water (twice, 5 minutes each). Silver intensification was performed with a kit (IntenSE M Silver Enhancement; Amersham), according to the directions of the manufacturer. Sections were then rinsed with distilled water, counterstained with 1% (wt/vol) toluidine blue in 1% (wt/vol) sodium borate, rinsed again with distilled water, air dried, and coverslipped (Permount; Fisher Scientific, Fairlawn, NJ). Sections were viewed and photographed with a photomicroscope (BH-2 with a x20 DPlanApo objective; Olympus).
Western Blot Analysis
Retinas were isolated, immediately frozen in liquid nitrogen,
and stored at -70°C until used. Retinal protein extracts were
prepared by homogenization of frozen tissue in PBS containing 1 mM
EDTA, 1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride (PMSF),
and a protease inhibitor cocktail (Mini Complete; Boehringer–Mannheim,
Indianapolis, IN). Protein concentrations of the homogenates were
determined with a BCA protein assay kit (Pierce, Rockford, IL), using
the BSA provided as a calibration standard.
Retinal extracts were combined with Laemmli sample buffer31 containing 0.2 M Tris (pH 6.8), 1 mM EDTA, 4% (wt/vol) sodium dodecyl sulfate (SDS), 20% (vol/vol) glycerol, 0.005% bromophenol blue, and 5% ß-mercaptoethanol. Twenty to 40 µg total protein aliquots of each sample (300 ng from bovine rod outer segment [ROS] extract) were resolved on a 12% SDS–polyacrylamide minigel31 and transferred32 to polyvinylidene (PVDF) membrane (Immunoblot; Bio-Rad, Hercules, CA) in a minigel apparatus (Transblot; Bio-Rad) at 200 V for 2 hours in 25 mM Tris, 192 mM glycine, 10% methanol, and 0.1% SDS. Membranes were blocked in 5% nonfat dry milk (Carnation, Glendale, CA) in TTBS (10 mM Tris [pH 7.5], 100 mM NaCl, 0.1% Tween-20) for 1 hour at room temperature with agitation. Primary antibody incubations (mAb 1D4 at 1:3000, mAb 3A6 at 1:1) were performed in 5% milk/TTBS (containing 0.01% Tween-20) for 16 hours, at 4°C for mAb 1D4 and at room temperature for mAb 3A6. Membranes were then washed five times for 5 minutes each at room temperature in TTBS, and incubated in a horseradish peroxidase–linked goat anti-mouse IgG (Pierce) for 1 hour at room temperature, at a dilution of 1:20,000 in 5% milk/TTBS. Membranes were then washed as described earlier. Blots were incubated in an enhanced chemiluminescence detection system (SuperSignal; Pierce) for 5 minutes, and then exposed to film (XAR; Kodak).
Enzyme-Linked Immunosorbent Assay
Protein extracts used for ELISA were prepared from individual
animals, as described for Western blot analysis. Control experiments
established the linear ranges for total protein loaded, primary and
secondary antibody dilutions, and color substrate concentration.
Aliquots (5–320 ng total protein from retinal homogenates from mice at
postnatal day [P]15) were diluted in PBS for a total sample volume of
50 µl, loaded in duplicates onto a 96-well cell culture dish (Costar,
Cambridge, MA), and allowed to bind overnight. Samples were rinsed
three times with water, and incubated for 2 hours with blocking buffer
(PBS, 0.05% Tween-20, 1 mM EDTA, 0.25% gelatin). Samples were rinsed
and allowed to incubate in primary antibody (partially purified mAb 1D4
at 1:1000 and anti-peripherin/rds33
polyclonal antibody at
1:500, diluted in blocking buffer) for 2 hours. Wells were rinsed as
described earlier, blocking buffer was added, incubation was performed
for 10 minutes, and the wells were rinsed again. Horseradish
peroxidase–labeled secondary antibodies were applied (goat anti-mouse
IgG and goat anti-rabbit IgG [Pierce] at a dilution of
1:10,000) in blocking buffer for 2 hours. Samples were rinsed,
blocked, and rinsed again as described. Then a 100-µl aliquot of
o-phenylenediamine substrate (1 mg/ml) in stable peroxide
buffer (Pierce) was added to each well and allowed to develop for 30
minutes. The reaction was stopped by adding 50 µl of 2.5 M sulfuric
acid to each well, and plates were read at 490 nm (Microplate
Autoreader, model EL309; Bio-Tek, Winooski, VT). All steps were
performed at room temperature.
The mean optical density readings from blank wells (no protein loaded) were subtracted from readings obtained for the remaining wells. These values were then averaged for duplicate wells. Final values were plotted versus total protein loaded for opsin as well as for peripherin/rds. The slope of the opsin plot (defined by optical density/total protein) was then divided by the slope of the peripherin/rds plot to obtain an opsin/peripherin/rds ratio. Opsin/peripherin/rds ratios from three trials were averaged; values for animals of the same line (i.e., normal and Bouse A, B, and C) were then averaged and divided by the mean value obtained for nontransgenic and breeder normal samples to arrive at values for opsin expression levels. The peripherin/rds ratio was used to correct for any degeneration.
Localization of Bouse opsin Anti-Opsin Antibody
ROS membranes were prepared from bovine retinas by discontinuous
sucrose density ultracentrifugation, per the method of
Papermaster.34
Purity was assessed by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE; broad band at molecular mass 38 kDa,
representing 90% of the Coomassie blue–stainable material) and
spectral ratio (A280/A498 =
1.8–2.0). Rhodopsin was purified from ROS membranes by lectin-affinity
column chromatography according to the method of Litman,35
using concanavalin (ConA)-Sepharose (Sigma, St. Louis, MO), and purity
again was assessed by SDS-PAGE and spectral ratio
(A280/A498 = 1.6).
Polyclonal antiserum was raised against purified bovine rhodopsin in
New Zealand White rabbits, using a standard protocol with adjuvant
(Hunter’s TitreMax; Sigma). Preimmune serum was obtained before
immunization with antigen. A purified IgG fraction of antiserum was
obtained by diethylaminoethyl (DEAE) chromatography (Affi-Gel Blue;
Bio-Rad), according to the manufacturer’s protocol. Titer was assessed
to be at least 1:5000 by standard dot blot assay.
Electron Microscopic Immunogold Cytochemistry
Electron microscopic immunogold cytochemistry was performed
essentially as described by Erickson et al.30
Briefly,
ultrathin sections (silver-gold) obtained from each of the tissue
blocks (embedded in LR White; Electron Microscopy Sciences) were placed
onto nickel grids and treated for 15 minutes at room temperature with
50 mM ammonium chloride, followed by blocking for 30 minutes at room
temperature with blocking buffer (composition described earlier). Grids
were exposed overnight at 4°C, either to rabbit anti-bovine opsin IgG
or to nonimmune rabbit serum (each diluted 1:250 with blocking buffer),
then rinsed briefly with PBS and treated for 2 hours at room
temperature with goat anti-rabbit IgG conjugated to 10 nm colloidal
gold (AuroProbe EM GAR G10; Amersham; diluted 1:50 with blocking
buffer). After a brief rinsing with PBS, sections were treated with 1%
glutaraldehyde (5 minutes at room temperature), rinsed serially with
PBS and distilled water, stained with uranyl acetate and lead citrate,
rinsed again with distilled water, exposed to
OsO4 vapors, and air dried. Sections were viewed
with an electron microscope (JEM-1200EX; JEOL, Tokyo, Japan) at an
accelerating voltage of 80 keV.
Retinoid Analysis
Analyses were performed on dark-reared P15 mice. Experiments
were performed under dim red light. Each animal was first anesthetized
with ketamine (0.15–0.18 mg/g) and xylazine (0.004–0.006 mg/g) and
then killed by cervical dislocation. Retinas were removed and then
homogenized in 500 µl of PBS supplemented with protease inhibitor
cocktail (one tablet protease inhibitor cocktail per 10 ml), using a
1-ml manual tissue grinder (Wheaton, Millville, NJ).
An aliquot (100 µl) of homogenate was added to 200 µl of formaldehyde (37% wt/vol aqueous solution), supplemented with isopropanol and water, and subsequently extracted with n-hexane, as previously described.36 This formaldehyde-based extraction procedure recovers both chromophoric and nonchromophoric 11-cis retinal.37 38 39 Another 100-µl aliquot of the homogenate was added to 400 µl of isopropanol, supplemented with water, and extracted with n-hexane.36 Levels of 11-cis retinal and other retinaldehydes were determined from results obtained with the formaldehyde-based extraction procedure. Data obtained from extractions in the absence of formaldehyde were used to determine levels of retinal.36 Each sample was then evaporated under nitrogen and redissolved in 200 µl n-hexane. Levels of retinoids in each sample were determined by normal-phase high-performance liquid chromatography (HPLC).36 The last 200-µl aliquot of homogenate was used for protein concentration determination and ELISA.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Electroretinography
To assess retinal function, ERGs were recorded from transgenic
mice and nontransgenic littermates. Figure 2A
presents dark-adapted ERGs obtained from representative 1-month-old
nontransgenic, Bouse A, and Bouse B mice to flash
stimuli that spanned a 4-log-unit range of intensity. At the lowest
flash intensity, the response of nontransgenic mice was dominated by
the positive-polarity b-wave, reflecting the activity of rod bipolar
cells.40
At the higher flash intensities, the b-wave was
preceded by the negative-polarity a-wave, which represents the mass
response of the rod photoreceptors.41
In the transgenic
mice, both a- and b-waves were reduced in amplitude at all stimulus
intensities. The magnitude of this reduction was greater in
Bouse B than Bouse A mice and was greatest in
Bouse C mice, in which ERG responses were not
distinguishable from the preflash baseline (data not shown). The bottom
panels present average intensity–response functions for the major ERG
components, a-wave (Fig. 2B)
and b-wave (Fig. 2C)
. In each transgenic
line, a- and b-wave amplitudes were well outside the 99% confidence
interval defined in nontransgenic mice (dashed lines). The magnitude of
this reduction was greatest in Bouse C mice and least in
Bouse A animals. Bouse B mice exhibited an
intermediate level of amplitude reduction. The amplitude of the cone
ERG b-wave was also reduced but to a lesser degree than that seen for
the rod ERG (data not shown). This reduction was greatest in
Bouse C mice and least in Bouse A and
Bouse B animals, implicating a secondary effect on cones of
the rod degeneration, as has been seen before in animal models of
retinitis pigmentosa8
10
13
42
43
and in patients with
retinitis pigmentosa who have rod-specific gene
defects.44
45
46
47
48
|
|
|
|
|
|
Relationship between Severity of Degeneration and Opsin Expression
To determine the level of opsin expression, retinas were examined
at P15 by ELISA. When mAb 1D4 was used, the values obtained for total
opsin reflect both Bouse and endogenous forms. These values
were normalized to those obtained for peripherin/rds, a
photoreceptor-specific structural protein localized in the OS, using a
polyclonal antibody (anti-mRDS-C).33
Because
peripherin/rds is an essential component of the OS,49
50
this normalization procedure accounted for the loss of OSs due to the
degenerative process. As shown in Table 1
, opsin expression levels for Bouse A,
Bouse B, and Bouse C mice were found to be,
respectively, 123%, 169%, and 222% of the level measured in
nontransgenic animals, indicating a correlation between the level of
transgene expression and the severity of the degenerative phenotype.
Although the Bouse A line exhibited the slowest rate of
degeneration, these results indicate that overexpression of opsin by
approximately 23% is sufficient to trigger photoreceptor cell death, a
finding in agreement with that previously reported by Sung et
al.11
This value may not set a lower limit for
opsin-induced photoreceptor degeneration, but it clearly demonstrates
the sensitivity of rod photoreceptors to opsin overexpression.
|
Effect of Light on the Rate of Retinal Degeneration
Because the overall amount of rhodopsin in Bouse
transgenic retinas was increased, the effects of light were
investigated. Several litters were reared from birth in constant
darkness, and ERG recordings were made at P30, followed by histologic
examination. As shown in Figure 8A
, the ERGs obtained from Bouse A mice were significantly
larger when these mice were reared in darkness than when treated under
cyclic light (P < 0.005, n = 9). In
comparison, dark rearing had a modest but insignificant benefit in
Bouse B mice (P > 0.10, n = 8)
and no effect in Bouse C animals (Fig. 8A)
.
|
Determining the Effects of the C-Terminal Modifications of
Bouse
To demonstrate that the C-terminal amino acid alterations made to
generate the Bouse epitope do not by themselves induce
photoreceptor degeneration, Bouse mice were bred with
opsin-/- mice.24
Because a minimum
of 50% of the wild-type amount of opsin is required to support the
morphogenesis of OSs,24
only Bouse B mice were
used. After extensive matings, mice that were heterozygous for the
Bouse B transgene and opsin-/- were
obtained. Examination of these animals indicated that their retinal
histology and ERG recordings were indistinguishable from those of
wild-type mice. As shown in Figure 9
, Bouse opsin alone supported the OS morphogenesis and
maintenance. Dark-adapted ERG a-waves were comparable with those of
wild-type mice (data not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A number of prior studies have shown that expression of mutant forms of opsins induce photoreceptor degeneration in transgenic mice.7 8 9 10 11 12 14 In one study, mice expressing as little as 10% more opsin transcript exhibited a notable reduction in the number of photoreceptors by 8 weeks of age,11 whereas in another study, expression levels as high as 100% over endogenous levels did not cause retinal degenerative changes.10 This discrepancy may result from the use of a semiquantitative method such as PCR to determine the steady state levels of opsin transcripts.10 11 Although we determined the amount of the protein rather than the transcript, we find our data more in agreement with the first study, because only approximately 20% overexpression of opsin was sufficient to cause functional deficits and structural abnormalities in ROSs. In this regard, it is worth noting that homozygous Bouse A mice, presumably expressing twice as much opsin (>40%), exhibited a more severe retinal degeneration than did their heterozygous counterparts (data not shown). Finally, it is of interest to note that no studies of opsin overexpression have reported a notable increase in the length or width of the ROS, despite the increase in opsin synthesis and the fact that overexpressed opsin is localized to the ROS.10 11 Nevertheless, careful morphometric analysis will be required to demonstrate conclusively whether or not ROS of mice overexpressing rhodopsin have abnormal dimensions.
The present results indicate that it is difficult to conclude that the degeneration is uniquely related to the particular gene defect that was introduced, without dissection of the deleterious effect induced by overexpression of opsin itself. Moreover, it is possible that a similar situation applies to other models in which a mutant opsin transgene is expressed either in pigs13 43 51 or rats.52 With mice, the development of opsin-/- lines24 25 provides the opportunity to distinguish between these two factors. The properties of a mutant opsin can be evaluated by expression in the opsin-/- background. In certain cases it may be more appropriate to express a transgene in opsin+/- heterozygotes, which express only 50% of the endogenous (wild-type) protein. This is determined by the levels of expression of the mutant transgenic protein. Although other techniques, including knock-in, have the potential to introduce a mutant rhodopsin gene in a manner that allows experimental results to be clearly interpreted, the present study emphasizes the need for the continued development of accurate animal models for human photoreceptor degeneration associated with opsin mutations.
The Degenerative Mechanism
There are several possible mechanisms by which the overexpression
of opsin could lead to death of rod photoreceptors. For example, the
overproduction of opsin could simply overwhelm the transport machinery,
and cell death would be initiated by mislocalized opsin molecules.
Although opsin mislocalization has been observed in transgenic mice
expressing pig51
or human10
11
opsins,
electron microscopic immunoanalysis of retinas of Bouse A
transgenic mice indicate that both endogenous and transgenic opsins
were properly localized to the OSs. These results argue against opsin
mislocalization as a major cause of cell death in Bouse
mice.
The relationship between opsin levels and the amount of 11-cis retinal in the retina was examined at P15, an age when OS has been elaborated and degeneration is minimal. In Bouse A mice, for which the overexpression of opsin was on average 23%, the amount of 11-cis retinal per unit density of ONL nuclei exceeded the average for nontransgenic mice by 26% (Table 1 , right column). Relative amounts of opsin were normalized to the amount of peripherin/rds in the analyzed sample because its levels at P10 were comparable in both Bouse transgenic and nontransgenic retinas as determined by western analyses (data not shown). Because at P15 retinal degeneration in Bouse C is already under way (data not shown), peripherin/rds was used to account for the loss of OS assuming that opsin and peripherin/rds disappear at the same rate with the reduction in the OS. The values for relative opsin level are effectively scaled to the number of photoreceptors present, and the level of opsin overexpression may be compared with the observed increase in 11-cis retinal in Bouse A mice. The results suggest that in Bouse A opsin overexpression leads to increase of 11-cis retinal sufficient to match the chromophore requirement of the 23% opsin overexpression. There would appear, however, to be a limit to such an induced increase in 11-cis retinal level. In the higher expressing Bouse B and C, a substantial percentage of opsin molecules have no 11-cis retinal chromophore. It has been suggested that free opsin arising in vitamin A deprivation, as well as some mutant forms of opsin that cannot bind 11-cis retinal, can excite the phototransduction cascade.53 It would be of interest in future studies to examine the effect in Bouse B and Bouse C mice of systemic vitamin A administration, a treatment that has been found in other studies of transgenic mice54 and human trials55 to promote recovery of visual sensitivity.
There are several other means through which overexpression of opsin could lead to cell death. For example, because some fraction of the overproduced opsin is coupled to the chromophore, the number of photobleachable rhodopsin molecules is increased. As a result, a given amount of light falling on the retina would be expected to activate relatively more rhodopsin molecules in Bouse retinas than it does in the normal retina. If there is a limited supply of proteins that are involved in deactivating the phototransduction cascade, the photoactivated rhodopsin may have a longer life—that is, the effect can be likened to prolonged light exposure.
Because dark rearing of Bouse A mice did not restore their ERGs to those of their normal counterparts, there must be another mechanism besides the increased amount of total rhodopsin that contributes to the cell death. Opsin constitutes 80% to 90% of the OS proteins,56 57 and because 10% of the OS is regenerated each day58 the cell must resynthesize 10% of total opsin daily, as well as all the other proteins that are required for the genesis of the OS. These other proteins include peripherin/rds, rom-1, the phototransduction cascade members as well as the proteins involved in packaging, transport and OS assembly. Because it appears that the vast majority of the available opsin is shipped to the OSs of Bouse mice, the increased synthesis of opsin presumably is also associated with an increased synthesis of other needed proteins. As a result, in Bouse mice, it is feasible that the photoreceptor cell is functioning at the threshold of its maximum translational capacity. The net result is a situation that is likely to be unsustainable and may ultimately lead to cell death.
It is interesting to note that the same situation does not appear to apply to cone photoreceptors. Shaaban et al.59 studied transgenic mice in which the human long-wavelength cone opsin gene was expressed in cones of transgenic mice using the human red-green cone opsin promoter. These mice exhibited no evidence of cone photoreceptor degeneration, even when the transgenic cone opsin was expressed at rod opsin levels that are known to compromise rod photoreceptors.10 11 This may be due to the considerably faster regeneration of the photobleached cone pigment when compared with rhodopsin.60 61 62 The explanation for this fundamental difference between rod and cone photoreceptors may provide important insights into the photoreceptor degeneration induced by opsin overexpression.
Clinical Implications
The results described here have implications for the understanding
of photoreceptor degeneration, particularly in retinitis pigmentosa.
Although much effort has been devoted to the identification of point
mutations in the coding regions of the opsin gene, it is possible that
some forms of retinitis pigmentosa may be due to an abnormally high
level of rhodopsin (or opsin) expression. To evaluate this possibility,
it is important to understand the regulatory mechanism that controls
the rate of opsin gene expression. In this regard it is interesting to
note that mutations in Crx, a regulatory element for opsin
expression, are linked to retinitis pigmentosa.63
64
65
It
is possible that a dominant positive mutation in crx could
lead to rhodopsin overexpression, thus causing a retinal degenerative
disorder. If this is the case, the Bouse mouse could provide
a useful model in which to conduct treatment trials, through
pharmacologic intervention, gene therapy, or other means.
In conclusion, the present results confirm that rod photoreceptor degeneration can be induced by overexpression of a normal opsin gene, a factor that should be considered when evaluating studies of mice expressing a mutant opsin transgene. These results also indicate that precise control of opsin expression is of critical importance in maintaining viable photoreceptors. As a consequence, these findings underscore the importance of completely identifying genes that regulate rhodopsin expression. They further indicate that identification of these genes is a potentially fruitful avenue for the identification of gene defects that may underlie retinitis pigmentosa and other retinal disorders.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Submitted for publication August 7, 2000; revised October 30, 2000; accepted November 15, 2000.
Commercial relationships policy: N.
Corresponding author: Muayyad R. Al-Ubaidi, Department of Cell Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, BMSB 781, Oklahoma City, OK 73104. muayyad-al-ubaidi{at}ouhsc.edu
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. Chakraborty, X.-Q. Ding, S. M. Conley, S. J. Fliesler, and M. I. Naash Differential requirements for retinal degeneration slow intermolecular disulfide-linked oligomerization in rods versus cones Hum. Mol. Genet., March 1, 2009; 18(5): 797 - 808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kondo, T. Sakai, K. Komeima, Y. Kurimoto, S. Ueno, Y. Nishizawa, J. Usukura, T. Fujikado, Y. Tano, and H. Terasaki Generation of a Transgenic Rabbit Model of Retinal Degeneration Invest. Ophthalmol. Vis. Sci., March 1, 2009; 50(3): 1371 - 1377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Mortimer, D. Xu, D. McGrew, N. Hamaguchi, H. C. Lim, S. J. Bowne, S. P. Daiger, and L. Hedstrom IMP Dehydrogenase Type 1 Associates with Polyribosomes Translating Rhodopsin mRNA J. Biol. Chem., December 26, 2008; 283(52): 36354 - 36360. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C.S. Tam, A.-S. Kiang, A. Kennan, P. F. Kenna, N. Chadderton, M. Ader, A. Palfi, A. Aherne, C. Ayuso, M. Campbell, et al. Therapeutic benefit derived from RNAi-mediated ablation of IMPDH1 transcripts in a murine model of autosomal dominant retinitis pigmentosa (RP10) Hum. Mol. Genet., July 15, 2008; 17(14): 2084 - 2100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Conley, M. Nour, S. J. Fliesler, and M. I. Naash Late-Onset Cone Photoreceptor Degeneration Induced by R172W Mutation in Rds and Partial Rescue by Gene Supplementation Invest. Ophthalmol. Vis. Sci., December 1, 2007; 48(12): 5397 - 5407. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Manavathi, S. Peng, S. K. Rayala, A. H. Talukder, M. H. Wang, R.-A. Wang, S. Balasenthil, N. Agarwal, L. J. Frishman, and R. Kumar Repression of Six3 by a corepressor regulates rhodopsin expression PNAS, August 7, 2007; 104(32): 13128 - 13133. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. S. Lee and J. G. Flannery Transport of Truncated Rhodopsin and Its Effects on Rod Function and Degeneration Invest. Ophthalmol. Vis. Sci., June 1, 2007; 48(6): 2868 - 2876. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Khanna, M. Akimoto, S. Siffroi-Fernandez, J. S. Friedman, D. Hicks, and A. Swaroop Retinoic Acid Regulates the Expression of Photoreceptor Transcription Factor NRL J. Biol. Chem., September 15, 2006; 281(37): 27327 - 27334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Wang, X.-H. Wen, Z. Ablonczy, R. K. Crouch, C. L. Makino, and J. Lem Enhanced Shutoff of Phototransduction in Transgenic Mice Expressing Palmitoylation-deficient Rhodopsin J. Biol. Chem., July 1, 2005; 280(26): 24293 - 24300. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Friedman, H. Khanna, P. K. Swain, R. DeNicola, H. Cheng, K. P. Mitton, C. H. Weber, D. Hicks, and A. Swaroop The Minimal Transactivation Domain of the Basic Motif-Leucine Zipper Transcription Factor NRL Interacts with TATA-binding Protein J. Biol. Chem., November 5, 2004; 279(45): 47233 - 47241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-Q. Ding, M. Nour, L. M. Ritter, A. F.X. Goldberg, S. J. Fliesler, and M. I. Naash The R172W mutation in peripherin/rds causes a cone-rod dystrophy in transgenic mice Hum. Mol. Genet., September 15, 2004; 13(18): 2075 - 2087. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nour, X.-Q. Ding, H. Stricker, S. J. Fliesler, and M. I. Naash Modulating Expression of Peripherin/rds in Transgenic Mice: Critical Levels and the Effect of Overexpression Invest. Ophthalmol. Vis. Sci., August 1, 2004; 45(8): 2514 - 2521. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. Otteson, Y. Liu, H. Lai, C. Wang, S. Gray, M. K. Jain, and D. J. Zack Kruppel-like Factor 15, a Zinc-Finger Transcriptional Regulator, Represses the Rhodopsin and Interphotoreceptor Retinoid-Binding Protein Promoters Invest. Ophthalmol. Vis. Sci., August 1, 2004; 45(8): 2522 - 2530. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yoshida, A. J. Mears, J. S. Friedman, T. Carter, S. He, E. Oh, Y. Jing, R. Farjo, G. Fleury, C. Barlow, et al. Expression profiling of the developing and mature Nrl-/- mouse retina: identification of retinal disease candidates and transcriptional regulatory targets of Nrl Hum. Mol. Genet., July 15, 2004; 13(14): 1487 - 1503. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Pittler, Y. Zhang, S. Chen, A. J. Mears, D. J. Zack, Z. Ren, P. K. Swain, S. Yao, A. Swaroop, and J. B. White Functional Analysis of the Rod Photoreceptor cGMP Phosphodiesterase {alpha}-Subunit Gene Promoter: Nrl AND Crx ARE REQUIRED FOR FULL TRANSCRIPTIONAL ACTIVITY J. Biol. Chem., May 7, 2004; 279(19): 19800 - 19807. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Tan, X.-Q. Ding, A. Saadi, N. Agarwal, M. I. Naash, and M. R. Al-Ubaidi Expression of Cone-Photoreceptor-Specific Antigens in a Cell Line Derived from Retinal Tumors in Transgenic Mice Invest. Ophthalmol. Vis. Sci., March 1, 2004; 45(3): 764 - 768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nour, A. B. Quiambao, M. R. Al-Ubaidi, and M. I. Naash Absence of Functional and Structural Abnormalities Associated with Expression of EGFP in the Retina Invest. Ophthalmol. Vis. Sci., January 1, 2004; 45(1): 15 - 22. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. F. Maison, A. E. Luebke, M. C. Liberman, and J. Zuo Efferent Protection from Acoustic Injury Is Mediated via alpha 9 Nicotinic Acetylcholine Receptors on Outer Hair Cells J. Neurosci., December 15, 2002; 22(24): 10838 - 10846. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
)
Nonlabeled photoreceptor presumed to be a cone. Abbreviations are as in
