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1 From the University Eye Clinic, Zurich, Switzerland; the 2 Institute for Zoology, University of Mainz, Germany; and the 3 Department of Biological Science, Florida State University, Tallahassee.
Abstract
PURPOSE. To investigate whether regulation of rhodopsin levels as a response to changed lighting environment is performed by autophagic degradation of opsin in rod inner segments (RISs).
METHODS. Groups of albino rats were kept in 3 lux or 200 lux. At 10 weeks of age, one group was transferred from 3 lux to 200 lux, another group was switched from 200 lux to 3 lux, and two groups remained in their native lighting (baselines). Rats were killed at days 1, 2, and 3 after switching. Another group was switched from 3 lux to 200 lux, and rats were killed at short intervals after the switch. Numbers of autophagic vacuoles (AVs) in RISs were counted, and immunogold labeling was performed for opsin and ubiquitin in electron microscopic sections.
RESULTS. The number of AVs increased significantly after switching from 3 lux to 200 lux at days 1 and 2 and declined at day 3, whereas the reverse intensity change did not cause any increase. Early time points after change from 3 lux to 200 lux showed a significant increase of AVs 2 and 3 hours after switching. Distinct opsin label was observed in AVs of rats switched to 200 lux. Ubiquitin label was present in all investigated specimens and was also seen in AVs especially in 200-lux immigrants.
CONCLUSIONS. Earlier studies had shown that an adjustment to new lighting environment is performed by changes in rhodopsin levels in ROSs. Autophagic degradation of opsin or rhodopsin may subserve, at least in part, the adaptation to abruptly increased habitat illuminance by removing surplus visual pigment.
The light-sensitive rod outer segments (ROSs) are continually renewed by the shedding of their tips, degradation of the shed tips in the retinal pigment epithelium (RPE), and membrane synthesis at the ROS base.1 In rod inner segments (RISs), bulk degradation, termed autophagy, is observed.2 Autophagic vacuoles (AVs) that are ubiquitous in many cell types contain membrane-bound cytoplasmic components such as mitochondria, which are degraded by the lysosomal system in contrast to other cellular degradative routes.
Major parts of renewal follow a circadian rhythm3 and thus represent a highly regulated process. Inner segment autophagic degradation also shows rhythmicity, which persists in constant light but is rapidly abolished in constant darkness. In addition, both disc shedding and autophagy can be evoked by light pulses, further suggesting regulative functions of these processes.4
It has been proposed that those degradative processes may serve to adjust the light sensitivity of rods as a means to regulate the visual input stage for circadian rhythm regulation.5 Indeed, the adjustment to new lighting environment is in part performed by changes in disc-shedding patterns and is associated with the adaptation of rhodopsin levels in outer segment disks.6 7
In view of recent thinking that protein degradation constitutes an important mechanism of cellular regulation, including adaptation and control of timing of cellular programs,8 it is conceivable that opsin or rhodopsin or both are degraded by autophagy in photoreceptors under conditions in which adaptation is required. In the present study, we investigated whether an abruptly reversed lighting environment—a condition that would require adaptation—results in a modification of the number of AVs and whether these AVs contain rhodopsin. The existence of a ubiquitin-dependent pathway regulating phototransduction protein levels in mammalian ROSs has been shown previously.9 Therefore, we also tested for ubiquitin labeling in rods under our experimental conditions.
Materials and Methods
Animals
All experiments described herein conformed to the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research. Albino rats
(ZUR/SIV; Institute of Animal Breeding, University of Zurich,
Switzerland) were obtained as weanlings and were maintained in the
animal quarters of the University Hospital, Zurich, for 9 to 10 weeks.
Experimental Setup
Five groups were studied: one maintained at 3 lux (3-lux natives)
and one at 200 lux (200-lux natives) for the entire time, 1 group that
was switched to 3 lux from 200 lux (3-lux immigrants) for 3 days and
two groups that were switched to 200 lux from 3 lux (200-lux
immigrants) for 3 hours or for 3 days before death. All rats were
maintained on a 12-hour light–12-hour dark cycle with lights on at 6
AM and food and water provided ad libitum. Rats were moved into the
prospective new light regimen at the beginning of the preceding dark
period, and light onset in the new environment was at 6 AM.
The following parameters were studied in duplicate experiments: the number of AVs in RISs, immunostaining of opsin in AVs, and immunostaining of ubiquitin.
Native and immigrant rats were killed in dim red light between 10 and 11 AM, at a time when autophagic activity was expected to be high. One of the two 200-lux immigrant groups was killed at 15, 30, 45, 60, 120, and 180 minutes after the change in light regimen at day 1. Eyes were rapidly enucleated and placed in fixative for electron microscopy or immunogold staining.
For quantification of AVs the retinas from five groups of rats were analyzed (n = 2 rats per experimental parameter): 3- and 200-lux natives; 3- and 200-lux immigrants at days 1, 2, and 3 after switching; and 200-lux immigrants killed on the first day of switching at 0 (baseline) 15, 30, 45, 180, and 240 minutes after lights on.
Quantification of AVs by Electron Microscopy
After rats were killed and the eyes enucleated, the anterior half
of each eye was removed and the posterior half fixed in 2.5%
glutaraldehyde in 0.1 M cacodylate buffer and processed for electron
microscopic observation, as described.4
Particular care
was taken to prepare identical retinal areas (the central upper and the
central lower retinal regions) for the quantitative analysis.
Autophagic vacuoles are defined as membrane-bound, roundish bodies with
more or less degraded cytoplasmic contents.2
For each
retina, only one electron microscopic section mounted on a
carbon-coated, single-hole grid was quantified, to ensure that no
double counting of vacuoles occurred. In each section, 100
well-oriented RISs were counted. The area of counting comprised the
ellipsoid and myoid region up to the outer limiting membrane. The
perinuclear area and the synaptic body were excluded from counting.
Immunogold Labeling
Monoclonal antibodies against bovine rod opsin were kindly
provided by Paul A. Hargrave (University of Florida, Gainesville). The
clones used in the present study were B6-30a1, K16-155, and R2-15 and
were described by Adamus et al.10
The anti-opsin
monoclonal antibodies were applied separately, diluted (1:100 to 1:800)
in blocking solution (0.5% fish gelatin; Sigma, Deisenhofen, Germany)
or 0.1% ovalbumin (Sigma) in phosphate-buffered saline (PBS), or the
diluted antibodies were used in a cocktail of all three clones (1:1:1).
The monoclonal antibody against ubiquitin was purchased from
Boehringer, Ingelheim, Germany, and used in a dilution of 1:1000 in
blocking solution. This antibody recognizes both free and conjugated
ubiquitin.
After the animals were killed, retinas were removed through a slit in the cornea and fixed in 0.1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 hours at room temperature. Fixed tissue was dehydrated to 98% ethanol, embedded (LR White; Science Services, Munich, Germany), and polymerized at 4°C under UV light for 48 to 60 hours.
Ultrathin sections (60–70 nm) were collected on formvar-coated nickel
grids. Sections were first etched with saturated sodium periodate
(Sigma) at room temperature for 3 minutes. The grids were preincubated
with 0.1% Tween 20 in PBS and blocked with 50 mM NH4Cl in
PBS and in blocking solution. Sections were incubated with primary
antibodies (anti-opsin or anti-ubiquitin) diluted in blocking solution
at 4°C for 60 hours and washed once in PBS and twice in a mixture of
0.1% ovalbumin, 0.5% cold-water fish gelatin, 0.01% Tween 20, and
0.5 M NaCl in 10 mM phosphate buffer (pH 7.3). The sections were
incubated for 2 hours with goat anti-mouse IgG conjugated to nanogold
(Nanoprobes, Stony Brook, NY), diluted in 0.1% ovalbumin, 0.5% fish
gelatin, 0.01% Tween 20, and 0.5 M NaCl in 10 mM phosphate buffer (pH
7.3). Washed sections were postfixed in 2% glutaraldehyde for 10
minutes and air dried. The nanogold labeling was silver enhanced as
described by Danscher.11
The grids were then washed in
distilled water and stained with 2% ethanolic uranyl acetate for 10
minutes before observation by electron microscope (model 912
; Zeiss,
Oberkochen, Germany).
The following controls were applied: Primary or secondary antibodies were omitted, and secondary antibodies against antibodies differing from original primary antibodies and antibodies against different molecules such as anti-tubulins and anti-centrins were used. All those showed staining patterns different from those for ubiquitin.
Statistical Methods
Evaluation of differences in AV counts between natives and
immigrants during the first 3 days of switching the light regimens was
performed by one-way analysis of variance and Dunnett’s multiple
comparison tests. Evaluation of early time points in 200-lux immigrants
was performed by fitting the data points to a quadratic polynomial
A + Bx + Cx2.
Results
Quantification of AVs and Opsin Labeling
Figure 1
A represents a schematic drawing of a rod cell showing the main
localizations of AVs in RISs. Disc shedding and membrane renewal of
ROSs are also indicated. Figure 1B
depicts a schematic drawing of the
formation and degradation of AVs. Figure 2
A shows the effect on AV count of switching the habitat intensity from 3
lux to 200 lux. The 3-lux native animals had an average of 20 AVs/100
rods. On day 1 in 200-lux lighting, the number dramatically increased
(P < 0.01). The AV count peaked on day 2
(P < 0.01) and decreased to almost baseline on day 3
(P > 0.05). When 200-lux natives were switched to the
3-lux environment, the number of AVs did not decrease significantly,
remaining near baseline levels of approximately 20 AVs (Fig. 2B)
.
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In our experiments the number of AVs changed with sudden switches from lower to higher habitat light intensity. Whereas 200-lux immigrants showed a significant increase in AVs during the first 3 days, the 3-lux immigrants had numbers near and below baseline. The AVs were found to contain opsin. Furthermore, they contained ubiquitin, suggesting that their contents, including opsin, were destined for degradation by ubiquitin-regulated autophagy.12 In this and previous studies,2 membranes and vesicles of the Golgi apparatus were observed within AVs. Because newly synthesized opsin passes through the Golgi apparatus for glycosylation and is sequestered in transport vesicles13 it can be included in those AVs that contain transport vesicles and/or Golgi membranes.
Based on this and previous studies we conclude that the abrupt switching of habitat intensities requires rod adaptation to the new lighting regimens.14 15 The 200-lux immigrants adapted by reducing the optical density of rod cells, perhaps to avoid damage caused by absorption of "too many" photons. The 3-lux immigrants increased the optical density of their rods to achieve a "set number" of photons per day.14 This means that retinal rhodopsin levels must be upregulated for the 3-lux immigrants and downregulated for the 200-lux immigrants. Furthermore, this adaptation must be rapid to maintain retinal homeostasis and prevent damage.
Apart from rhodopsin levels, other photoreceptor components such as phospholipid composition, antioxidant levels, and phototransduction proteins have also been found to change as a function of an altered habitat light intensity.16 17 18 19 20 Such observations further emphasize the role of light as a modulator of photoreceptor function21 and the significance of processes regulating the optical density of ROSs.
That opsin is found in the AVs suggests that autophagic degradation of opsin is one possible way to regulate the rhodopsin content of ROSs. In particular, Penn and Williams,14 using retinal sections and Schremser and Williams7 using single rods, both showed that rhodopsin concentration in ROSs is inversely proportional to habitat light intensity. Thus, degradation of opsin in RIS may be one mechanism for controlling the rhodopsin content in ROSs. If so, the switch to higher light intensity, accompanied by a dramatic increase in AVs, is consistent with an initial downregulation of the opsin targeted to the ROSs.
Schremser and Williams6 did not observe an increase in disc shedding when rats were switched from 3 lux to 200 lux. Earlier studies demonstrated that bursts of disc shedding as well as autophagy could be evoked several times within 24 hours by light pulses followed by a dark period.22 Such an increase of phagosomes would rapidly reduce ROS length and rhodopsin levels. To date, it is unclear to what extent AV, disc shedding and/or the proteasome system contribute to the downregulation of rhodopsin levels. Whereas disc shedding removes the complete visual pigment from ROSs, AV and proteasome degradation would modify the amount of apoprotein to be inserted into the disc membrane after translation.
Schremser and Williams7 used a microspectrophotometer to study single rat rods and were able to show that the concentration in ROS tips was predictably different from that in the base if an animal was switched from 3 lux to 200 lux. Single ROSs were found with gradients of rhodopsin concentration in them that matched those of 200-lux immigrants at the base and those of 3-lux immigrants at the tip. Their findings demonstrated one of the end-stages of photostasis, and the current results provide the next level of explanation for those early observations.
In retrospect, the rapid dampening of the rhythm amplitude of AV in constant darkness found by Remé et al.4 can now be interpreted in terms of this proposed regulation: When switched into low photon fluxes, rod cells upregulate the rhodopsin content of their ROSs.6 7 In such an environment the demand on newly synthesized opsin would increase, and there would be little or no need to degrade opsins in the RIS by means of the AVs.
It can be inferred from our data that the prevention of damage is required for 200-lux immigrants. Their retinas reveal dilations and vesiculations of ROS tips as indicators of threshold light damage and opsin in the synaptic region and RIS plasma membrane as a possible sign of cellular injury. Similar mislocations of opsin were also found in photoreceptors of animal models of retinitis pigmentosa.23 In light of those and other observations, increased AV-removal of surplus opsin may also be present in light damage pathology when ROSs are injured or even absent. In our study, light damage was confined to the distal parts of ROSs and thus was considered as moderate threshold damage that is reversible within the regular ROS renewal cycle.24 This condition would still permit disc membrane synthesis at the base of ROSs but would require adaptation to avoid further damage. Therefore, conditions in our study may represent intermediate stages between pure adaptation and pure light damage. Furthermore, several studies did not detect any significant cell loss when animals were switched from 3 lux to 200 lux6 7 14
Ubiquitin label in AVs, in the cytoplasm and in ROSs was observed in all our specimens including baseline retinas. The AV label was distinct in 200-lux immigrants. There are two major ways of intracellular protein degradation, cytosolic degradation by the proteasome system, and lysosomal degradation by membrane endocytosis or by autophagy. Ubiquitin conjugation is known to target proteins to degradation by proteasomes. Ubiquitin appears to be present in all eukaryotic cells and degrades proteins including those with important regulatory functions such as transcription factors, the tumor suppressor p53 and cyclins. Numerous different conditions are known to use the ubiquitin pathway, those comprise cell differentiation and cell cycle, apoptosis, DNA repair, rapid stress responses, and signal transduction.25 26 27 Recent studies show that ubiquitination may target proteins also for lysosomal degradation, perhaps by a different type of ubiquitination than that used for proteasomes.12 28
Obin et al.9 suggested a role for ubiquitination of transducin and rhodopsin in the regulation of levels of phototransduction proteins. Notably, ubiquitination of transducin is modulated by light. They also suggested that removal of phototransduction proteins through ubiquitination is a protective measure against light damage.29 Naash et al.30 found that exposure to light higher than habitat intensity induces the ubiquitination of molecules in the inner retina. It was thus confirmed in their study that ubiquitin may act as a stress protein that helps to protect cells against damage. A similar condition may exist in our study for the 200-lux immigrants. The ubiquitination of opsin for proteasome and/or autophagic degradation may serve to regulate the level of rhodopsin and thus protect the retina against absorbing "too many" photons. Supporting the observations by Obin et al.,9 there was ubiquitin label in ROSs that may represent transducin destined for removal. This removal would reduce the capacity of ROSs to transduce the initial light signal.
The regulatory mechanism suggested by our results may have its counterpart in an invertebrate eye. Huber et al.31 studied the synthesis, maturation, and targeting of opsin to rhabdomeric membranes in the eye of the blow fly, Calliphora. They suggested that opsin without chromophore was degraded in photoreceptor cell bodies. Furthermore, AVs containing both rhodopsin and ubiquitin have been identified in the photoreceptor cells of opsin mutants of the fruit fly Drosophila before photoreceptor degeneration.32
In conclusion, our data show that autophagic degradation of opsin may contribute to the downregulation of rhodopsin levels in animals abruptly experiencing an increase of habitat light intensity. This adaptational regulation would enable the retina to reach the photostasis number of photons. Moreover, the downregulation of light absorption would help to prevent light-induced damage to photoreceptors. Further studies are needed to clarify in a quantitative manner the extent to which AV and other proteolytic pathways contribute to the regulation of opsin levels.
Acknowledgements
The authors thank Rainer Müller, University of Karlsruhe, for skillful technical assistance and Fabio Valeri, University Hospital Zürich, for valuable advice and performance of the statistical tests.
Footnotes
CER and UW contributed equally to this work.
Supported by an Alexander von Humboldt Stiftung Senior Scientist Award (TPW), Swiss National Science Foundation Grant 31-40791.94; the Suva Research Foundation, Luzern; the Ernst and Bertha Grimmke Foundation, Düsseldorf, Germany (CER); and Deutsche Forschungsgemeinschaft Grant Wo 548/3-1, FAUN-Stiftung, Nürnberg, Germany (UW).
Submitted for publication December 23, 1998; revised April 30, 1999; accepted May 27, 1999.
Proprietary interest category: N.
Corresponding author: Charlotte E. Remé, Department of Ophthalmology, University Hospital, 8091 Zurich, Switzerland. E-mail: chreme@opht.unizh.ch
References
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