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From the Departments of 1 Molecular and Cell Biology, University of California, Berkeley; 2 Vision Science, School of Optometry, University of California, Berkeley; and 3 Ophthalmology and Anatomy, University of California School of Medicine, San Francisco.
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Abstract |
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METHODS. Retinas from transgenic rats expressing truncated rhodopsin (Ser334ter) were examined by light and electron microscopic immunocytochemistry at several time points. Retinal degeneration in transgenic rats raised in darkness was evaluated by quantification of outer nuclear layer thickness and by electroretinography.
RESULTS. Mutant rhodopsin was found at inappropriately high levels in the plasma membrane and cytoplasm of Ser334ter rat photoreceptors. When the cell death rate was high this mis-sorting was severe, but mis-sorting attenuated greatly at later stages of degeneration, as the cell death rate decreased. The distributions of two other outer segment proteins (the cGMP-gated channel and peripherin) were examined and found to be sorted normally within the photoreceptors of these rats. Raising Ser334ter transgenic rats in darkness resulted in minimal rescue from retinal degeneration.
CONCLUSIONS. Because dark rearing Ser334ter rats results in little rescue, it is concluded that constitutive activation of the phototransduction cascade does not contribute significantly to photoreceptor cell death in this rat model. The nature of the rhodopsin sorting defect and the correlation between the severity of mis-sorting and rate of cell death indicate that truncated rhodopsin may cause apoptosis by interfering with normal cellular machinery in the post-Golgi transport pathway or in the plasma membrane.
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Introduction |
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The C-terminal tail of rhodopsin has two established functions and, therefore, is a potential site of origin for two types of cellular defects. First, this region has been shown to be involved in the sorting of rhodopsin from the photoreceptor inner segment, where protein synthesis and other cellular maintenance occur, to the outer segment, which is the site of phototransduction. Evidence for this is provided by the abnormal protein distributions found in transgenic mice expressing Gln344ter truncated rhodopsin, which lacks the last five amino acids of the C-terminal tail.2 Mice expressing rhodopsin with the substitution mutation Pro347Ser also have sorting abnormalities, revealed by the presence of extracellular vesicles near the connecting cilium between the inner and outer segments.3 Second, the phosphorylation of serine and threonine residues within the last 15 amino acids of the C-terminal tail and the subsequent binding of arrestin to rhodopsin are centrally involved in the deactivation of the protein after light absorption (reviewed in Ref. 4) . The presence of rhodopsin that lacks these 15 amino acids in another strain of transgenic mice (Ser334ter mice) leads to responses to a dim light flash that last significantly longer than normal.5
Photoreceptor death in retinitis pigmentosa is believed to occur by apoptosis,6 7 8 9 and sorting failures and prolonged rhodopsin activation have been postulated to cause cell death in human patients. However, the precise mechanism for the induction of apoptosis is not known for any rhodopsin mutation. Mutations of the C-terminal domain occur in human retinitis pigmentosa patients, and these could interfere with either rhodopsin sorting or the quenching of the light response. Other mutations, like the substitutions Lys296Glu and Gly90Asp, are thought to result in forms of rhodopsin that can be constitutively active, but it is not clear that this defect leads to retinal degeneration.10 11 12
The goal of this study was to evaluate the contributions of improper rhodopsin sorting and constitutive activation to rod cell death in transgenic rats with the truncated Ser334ter form of rhodopsin. These rats, and transgenic rats with the substitution mutation Pro23His, used as nontruncation controls throughout this study, undergo severe retinal degeneration during the first several postnatal months. We first performed Western blot analysis to ensure that the introduction of the transgene did not cause exorbitant amounts of total opsin expression, which itself can cause degeneration.2 13 We then characterized the distributions of rhodopsin and other outer segment proteins to determine how abnormal sorting might lead to apoptosis in transgenic rats and monitored the extent of abnormal rhodopsin localization over the course of retinal degeneration to determine whether we could establish a correlation with cell death rates. Finally, we assayed for retinal rescue in Ser334ter rats raised in darkness to determine whether preventing prolonged photoresponses could slow photoreceptor cell death.
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Methods |
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Western Blot Analysis
Retinas from postnatal day 15 (P15) rats were dissected and
homogenized by sonication in 5 mM Tris–acetate buffer with 65 mM NaCl,
2 mM MgCl2, and protease inhibitors. Proteins
were solubilized by a 4-hour incubation with 2% digitonin at 4°C and
deglycosylated by an overnight incubation at room temperature with
N-glycosidase F.
Western blot analysis with several serial protein dilutions was performed to locate a concentration within the linear range of detection. Protein in solution with sample dye, with B-ME and sodium dodecyl sulfate for denaturation, was loaded and separated electrophoretically on a 12% polyacrylamide gel. Protein in the gel was then wet-transferred overnight to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA). The membrane was blocked, probed with the N-terminal rhodopsin antibody rho4D2, washed, and then probed with horseradish peroxidase–conjugated secondary antibody (Sigma, St. Louis, MO). Labeling was detected with a Renaissance enhanced chemiluminescence system (NEN Life Science Products, Boston, MA) and hyperfilm-ECL X-ray film (Amersham Life Science, Arlington Heights, IL). Densitometric analysis was performed on NIH image software (available at rsb.info.nih.gov/nih-image). Analysis of relative levels of protein expression was performed on samples with 5 µg of protein per lane. Results from two lanes from each of 4 rats were averaged before relative levels of expression were compared.
Immunocytochemistry
In all immunocytochemical experiments, rats were killed soon after
the beginning of the 12-hour light cycle. Tissue was processed using
the progressive lowering of temperatures method15
for
immunofluorescent labeling on Lowicryl plastic sections
(Polysciences, Inc., Warrington, PA). First, retinas were removed and
fixed at 0°C in a solution of 3.6% formaldehyde and 0.5%
glutaraldehyde in phosphate buffered saline (PBS). The tissue was then
dehydrated in a graded alcohol series over a period of 5 hours, and the
temperature simultaneously lowered to -50°C using a low temperature
embedding unit (Bal-tec LTE 020; Furstentum, Liechtenstein). The tissue
was embedded at -50°C in Lowicryl K11M resin and polymerized with
ultraviolet light. Semi-thin sections were cut and labeled overnight
with primary antibodies at a dilution of 1:300, and for 2 hours with
fluorescein-conjugated secondary antibodies (Sigma). Prolong Antifade
(Molecular Probes, Eugene, OR) was used as a mounting medium to reduce
fluorescein photobleaching. For each condition, labeling was performed
on sections from at least two rats. Propidium iodide staining at a
dilution of 1:20,000 was performed on some plastic sections after
antibody labeling.
Immunofluorescence on agarose sections was performed by first fixing dissected retinas in 4% formaldehyde for 30 minutes, washing in PBS, and embedding in 5% agarose in PBS. Sections were then cut at a thickness of 150 µm, and antibody labeling was performed as for plastic sections.
Tissue for immunoelectron microscopy was processed by freezing dissected retinas at high pressure using a Bal-Tec HMP 010 system (Furstentum, Liechtenstein), freeze-substituting for 3 days in acetone, and embedding in LR White resin.16 Ultra-thin sections were cut and labeled overnight in primary antibodies, and for 2 hours with gold-conjugated secondary antibodies (Sigma). Sections were stained with 3% uranyl acetate and photographed at 12,000x. Quantification of subcellular labeling densities was performed by counting gold particles in a minimum of four locations, each with an area of 18 µm2.
Antibodies rho4D2, rho1D4, pmc1D1, and per3B6 were generous gifts from Robert Molday (Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver). Antibody R215N was a generous gift from Paul Hargrave (Department of Ophthalmology, University of Florida, Gainesville).
Retinal Morphometry
The thickness of the outer nuclear layer (ONL) was quantified as
previously described17
to obtain degeneration curves for
the transgenic rat strains. In brief, rats were killed with an
inhalation overdose of carbon dioxide and perfused through the heart
with 2.0% formaldehyde and 2.5% glutaraldehyde in PBS. Heads were
removed and immersed in fixative for 12 hours. Eyes were then
enucleated and bisected along the vertical meridian through the optic
nerve head. Eye halves were osmicated, dehydrated, and embedded in
Epon–Araldite resin. One-micrometer-thick cross sections of the
retina, spanning from the ora serrata to the optic nerve head on both
the inferior and superior sides, were cut and stained with toluidine
blue. Fifty-four panretinal measurements of ONL thickness were made
using a camera lucida, digitizing pad, and quantification software
(Bioquant; R&M Biometrics, Nashville, TN).
Estimates of photoreceptor death rates in transgenic rats were made by first determining from the degeneration curves the number of micrometers of ONL thickness lost in the week centered around a given age and dividing by seven to obtain an approximation of micrometers lost per day. The number of photoreceptors each micrometer represents was estimated by dividing 17 million (the approximate number of photoreceptors in a wild-type rat retina)18 by the thickness of a wild-type ONL at the given age. The percent of the remaining photoreceptors that die in a day was calculated by dividing the number of micrometers lost in 1 day by the thickness on that day.
Electroretinography
Animals were dark-adapted for 12 hours, anesthetized with a
ketamine (87 mg/kg) and xylazine (13 mg/kg) mixture, and placed on a
heating pad. The pupils were dilated with atropine, and the corneas
were locally anesthetized with proparacaine. Electroretinograms (ERGs)
were recorded from both eyes with coiled chloride silver wire
electrodes,19
contacting the corneas through a layer of
clear gel (Gonak; Akorn, Abita Springs, LA). Brief (<1 msec) flashes
of light from a xenon flashtube (Novatron, Dallas, TX) were filtered
(W47B blue filter) and presented as diffuse full-field illumination
with an intensity of -0.15 log cd/sec · m2.
A-wave amplitudes were quantified by measurement on averaged ERGs from
the baseline amplitude to the peak of the a-wave. B-wave amplitudes
were measured starting from the peak of the a-wave.
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Results |
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To help determine the nature of the sorting defect of Ser334ter rhodopsin we performed quantitative immunoelectron microscopy, comparing the densities of rhodopsin labeling between wild-type and S334ter-4 rat photoreceptors at four different locations within the cell (Fig. 3A ). Labeling densities in wild-type and S334ter-4 rat photoreceptors are not significantly different in the outer segments, endoplasmic reticulum (ER), and Golgi membranes. Labeling densities are fivefold higher in S334ter-4 rats than in wild-type rats in areas of the cytosol not associated with any visible membranes, but which would be expected to include protein transport vesicles. In the plasma membrane of the photoreceptor inner segments, labeling is 20-fold higher in S334ter-4 rats than in wild-type rats. Figure 3B shows the distribution of rhodopsin found in wild-type rats, and Figure 3C shows an example of the abnormally high plasma membrane labeling seen in the inner segments of S334ter-4 photoreceptors. Similarly, there is sparse labeling in the ONL and outer plexiform layer of wild-type photoreceptors (Figs. 3D and 3E) but significant labeling in these regions in S334ter-4 photoreceptors (Figs. 3F and 3G) .
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Contribution of Constitutive Activation to Cell Death
To determine the extent to which constitutive activation of the
phototransduction cascade contributes to degeneration in S334ter-4
animals, we compared retinas of rats raised in cyclic light to those of
rats raised in darkness. The decreased activation of rhodopsin in
darkness should prevent any cell death induced by the activity of the
phototransduction cascade, so rescue seen in dark-reared S334ter-4 rats
would be an indicator of the degeneration caused by this mechanism.
P23H-3 rats were used as nontruncation controls because this rhodopsin
mutation would not be expected to exhibit constitutive activation after
light absorption.
We quantified the thickness of the ONL as a measure of photoreceptor number20 at six different ages in wild-type, cyclic light–reared S334ter-4, and dark-reared S334ter-4 rats, and at one age (P60) in cyclic light- and dark-reared P23H-3 rats (Fig. 6) . Each data point in the graph represents the average of 162 measurements made from the retinas of three different rats. At later ages, the average ONL thickness of dark-reared S334ter-4 rats was slightly greater than that of cyclic light–reared S334ter-4 rats, but this difference did not rise to the level of statistical significance at any age. Similarly, at P60 the average ONL thickness of dark-reared P23H-3 control retinas was slightly, but not significantly, thicker than that of cyclic light–reared P23H-3 retinas. (Most of the decrease of ONL thickness in wild-type rats, as well as some of the decrease in the transgenic rats, is due to the thinning of the retina as the eye grows in size.)
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Discussion |
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The finding that S334ter-4 transgenic rats exhibit a sorting abnormality corroborates several previous reports, which indicate a role for the C-terminal tail in the proper sorting of rhodopsin.2 3 21 22 Most recently, Deretic et al.21 showed in a cell-free system that the last five amino acids of the C-terminal tail appear to regulate the packaging of the protein into appropriate vesicles in the trans-Golgi network. The subcellular distribution of mutant rhodopsin that we see—a level in the ER and Golgi comparable to that of normal rhodopsin in wild-type animals, a level 5-fold higher than normal in the cytosol, and a level 20-fold higher than normal in the plasma membrane—fits particularly well with this model. It seems likely that after correct folding and processing in the ER and Golgi, rhodopsin lacking its signal sequence is packaged into vesicles with no explicit destination or with a default destination of the plasma membrane. The higher labeling in the cytosol may reflect the abnormal presence of rhodopsin in these vesicles.
We found no evidence that Pro23His mutant rhodopsin mis-sorts in the photoreceptors of control P23H-3 rats. Results from different animal models that both conflict with ours23 and agree with ours24 with respect to the subject of the sorting of rhodopsin containing the Pro23His mutation have been published previously. In this article, the P23H-3 control serves to demonstrate that the introduction of a transgene does not in itself lead to mis-sorted rhodopsin.
Our most convincing evidence that abnormal rhodopsin sorting leads to apoptosis in the S334ter-4 transgenic rats is that the severity of mis-sorting correlates well with the rate of cell death at a given point in the degeneration of the retina (Fig. 5) . At P15, mis-sorting is severe and widespread, and we estimate that 620,000 photoreceptors are dying per eye per day at that time. This represents 3.9% of the photoreceptors present at this age. At P90, mis-sorting is much less prevalent, and we estimate that only 190,000 photoreceptors (2.4% of those still present) are dying per eye per day. It is possible that this correlation exists because the rhodopsin mutation causes some other cellular defect that induces apoptosis and exacerbates mis-sorting at times when the cell death rate is greatest. However, the simplest explanation is that more severe mis-sorting in the photoreceptors of young rats, perhaps caused by protein synthesis rates higher than those in adults, leads directly to higher cell death rates. LaVail25 has shown that outer segment disc synthesis in young mice is up to 1.6 times the adult level, contributing to the process of outer segment elongation during development, and Treisman et al.26 have shown that opsin synthesis rates are higher in young rats than in adults.
There are at least three possible mechanisms for the induction of apoptosis that are consistent with a sorting defect at or beyond the trans-Golgi network, and that would be expected to produce higher rates of cell death at times of more severe mis-sorting. First, mutant rhodopsin may overwhelm the normal vesicular machinery of the plasma membrane–bound pathway in these cells, interfering with the routing of legitimate cargo. This hypothesis can be contrasted with the proposed mechanism of cell death in some Drosophila rhodopsin mutations, in which improperly folded rhodopsin is retained in large quantities in the ER and Golgi, potentially interfering with protein synthesis and maturation.27 28 Second, the physical presence of high levels of mutant opsin in the plasma membrane may interfere with normal cellular processes, such as neurotransmitter release in the synaptic zone. Third, the degradation of large amounts of mis-sorted protein may cause a damaging increase in the metabolic load placed on photoreceptors.2
Interestingly, we find in general that normal rhodopsin and other membrane-bound proteins of the outer segment do not mis-sort concurrently with Ser334ter rhodopsin. They have abnormal distributions only in occasional cells, which often show clear signs of being engaged in the process of apoptosis. For example, when sections are probed with both the protein antibody and propidium iodide (a chromosomal marker that stains apoptotic nuclei), cells that show abnormal protein localization also frequently label with the propidium iodide (data not shown). From this we can conclude that apoptosis in the photoreceptors of the transgenic rats is not a result of disruption of the sorting of these other proteins of the outer segment and, instead, that a breakdown in the sorting of many proteins is one of the secondary effects of the cell death process.
Expression of the Ser334ter rhodopsin gene in mice results in photoreceptor flash responses that can be more than 20 times longer than normal,5 and comparable results have been found in the S334ter rats (Shimpei Nishikawa, Roy H. Steinberg, Matthew M. LaVail, unpublished observations, February 1998). We therefore believe that the phototransduction machinery in cyclic light–reared S334ter-4 rats is grossly overactive. We find that dark rearing of S334ter-4 rats results in minimal rescue from retinal degeneration, and none beyond that seen in P23H-3 rats. Dark rearing produced some rescue of the ERG in S334ter-4 rats, but we found equal or greater rescue in the ERG of dark-reared P23H-3 rats. This rescue may therefore reflect an increased sensitivity of any or all of the retina’s signaling pathway components after development in darkness, rather than an effect specific to a given mutation. Indeed, when wild-type albino rats are dark-reared, the total rhodopsin level in the eyes is approximately 50% greater than that in cyclic light–reared littermates, and the rod outer segments are approximately 25% longer in the dark-reared rats.29 Because we find little or no change in the amount of photoreceptor apoptosis, and no change in the ERG beyond that seen in the nontruncation control rats, we conclude that in the S334ter-4 transgenic rat prolonged rhodopsin activation does not contribute significantly to photoreceptor death.
A strong case has been made for the idea that deletion of the gene encoding the cGMP-gated channel leads to retinal degeneration because the lack of this protein produces photoreceptor activity like that which occurs in a constant light environment.30 31 However, conclusive evidence that rhodopsin mutations can lead to retinal degeneration by constitutively activating the phototransduction cascade remains elusive. For example, substitution mutations at amino acid position 296 have been shown to be constitutively active in vitro,12 and can lead to photoreceptor degeneration,32 33 but ERGs of Lys296Glu transgenic mice did not reveal in vivo evidence of constitutive activation.32 Furthermore, the night-blindness reported by human patients with a Gly90Asp substitution would be expected if this rhodopsin mutant were constitutively active, but Gly90Asp patients do not undergo severe photoreceptor degeneration.11 It may be that constitutive activation causes some degeneration in these cases, as pointed out by Lisman and Fain,31 and that the mild rescue we observed reflects a small contribution of constitutive activation to cell death in S334ter-4 rats. However, our results are more aligned with studies suggesting that rhodopsin mutations induce degeneration primarily through other kinds of functional defects.
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Acknowledgements |
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Footnotes |
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Submitted for publication July 6, 1999; revised December 1, 1999; accepted December 9, 1999.
Commercial relationships policy: N.
Corresponding author: John Flannery, Department of Vision Science, 360 Minor Hall, University of California at Berkeley, Berkeley, CA 94720-2020. flannery{at}socrates.berkeley.edu
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