Exp. Eycj Res. (1990) 51, 257-267
Opsin
Gene
Expression During Degeneration
IZHAK Department
NIR”,
of Pathology,
(Received
Early and Late in rds Mice
NEERAJ
AGARWAL
University
of Texas Health Science TX 78264, U.S.A.
7 1 August
1989
and accepted
AND
DAVID
in revised
Phases
of Retinal
S. PAPERMASTER
Center
form
at San Antonio,
23 January
San Antonio,
1990)
Opsin mRNA levels, opsin synthetic rates and localization of opsin were studied throughout the photoreceptor’slife spanin the rds mice.Mutant mice 11 daysto 11 monthsold were investigated.Opsin mRNA levels were studied by means of northern blot analysis. Opsin synthesiswas measuredby incorporationof [%]methionine into newly synthesizedopsinin vitro. Distributionof opsinin the retina was determinedby immunoelectronmicroscopy.OpsinmRNA was detectedin young as well as old retinas,and opsinsynthesiscould bedetectedat early phasesof degenerationbut not in late phases.The absenceof opsinsynthesisin older rdsmicemight be dueto translationaldown-regulationor someother defectin the capacity to synthesizeopsin.In young mice, opsinwas detectedin the subretinalspacein opsin-ladenvesicular membranes:such membraneswere absent from retinas of older mice. This disappearance parallelsthe cessationof opsinsynthesisand the consequentfailure to deliver opsinto the subretinal spacein retinas from older mice. Immunochemicalanalysisrevealedthe presenceof small amountsof opsinin all retinasup to 11 monthsof age.Immunoelectronmicroscopylocalizedthe residual opsin, mostly to the plasmamembranewhich envelopsthe nuclei and synaptic terminals.Theseopsin moleculesmight be a consequenceof very low levelsof opsinsynthesis,too low to be detectedby our assays,or may have beensynthesizedat an earlier ageand retainedin the plasmamembraneof the old mutant photoreceptors. Key words:rds mice; opsinmRNA ; opsin synthesis; immunocytochemistry; aging: inherited retinal degeneration. 1. Introduction In mice bearing the retinal degeneration slow (rds) mutation, rod and cone outer segmentsfail to develop (Sanyal and Jansen, 1981). The opsin content of the 3-4-week-old dystrophic retina is about 3 % of normal (Chalken et al., 1985). Our preliminary observations showed that in 11-day-old rds mice, opsin gene expression is not altered and declines slightly in the next month. At young ages, the photoreceptor cells produce opsin mRNA at a normal level and opsin is synthesized at a rate comparable to normal. The low level of opsin was attributed to the cell’s failure to incorporate opsin into a stable outer segment. Consequently the newly synthesized opsin-laden membranes that were released into the interphotoreceptor space were subjected to early and continuous phagocytosis by the adjacent pigment epithelium (Agarwal, Nir and Papermaster, 1988). The rds mutation is characterized by a slow photoreceptor cell death which is completed only by 1 yr of age (Sanyal and Hawkins, 1986). Although the rds gene was recently cloned, the function of the gene product is not known since the gene product does not resemble any known protein (Travis et al., 1989). (See ‘Note added at proof. ‘) Hence the cause of the * For correspondence at: Department of Pathology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive. San Antonio. TX 78284-7750. U.S.A.
00144835/90/090257+
11 503.00/O
photoreceptor cell death in the rds retina is not yet understood. It is apparent, however, that photoreceptor degeneration in the rds mouse is not due to critical cellular changes which precipitate rapid cell death in a relatively short time period such as the case in the rd mouse and RCS rat (la Vail, 198 1). Thus, unlike the other rodents with retinal dystrophy, a substantial number of photoreceptors in the rds mouse survive for a prolonged time in a diseasedretina. In the present study we explored whether these cells have significant alterations in opsin gene expression over the entire duration of the retinal degeneration as one measure of possibleeffects of long-term photoreceptor outer segment abnormalities.
2. Materials and Methods Animals BALB/c mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mutant 020/A rds/rds mice were obtained from Dr Janet Blanks, University of Southern California, Los Angeles, CA, from a colony that originated in the Netherlands and was distributed by Dr Sanyal. The animals were bred locally and maintained under a 12 hr/ 12 hr dark/light cycle with exposure to low (3-5 ft candles) light levels. The handling of the mice was in accordance with the ARVO resolution on the use of animals in research and NIH and AAALAC guidelines. Mice at various ages 0 1990 AcademicPressLimited
i NIR
were killed l-2 hr after the onset of light by exposure to co,.
Enucleated intact eyes were tixed overnight with 1 ‘%, glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer, pH 7.0 at 4°C. The eyes were bisected into two hemispheres through the optic nerve and post-fixed with 1 “/u 0~0, in 0.1 5 .LI phosphate buffer, pH 7.0. One hemisphere from each eye was oriented and embedded in epoxy resins as described by la Vail and Battelle (19 75). Sections 2 L,rn long, which include the full length of the retina from the optic nerve head to the far periphery, were stained with toluidine blue and viewed at 1000 x magnification. Photoreceptor nuclei were counted along 50 pm segments of the retina with the aid of an ocular micrometer. Measurements started at 100 !trn from the optic nerve head and continued at 100-l 50 /irn intervals to the far periphery. At each age, three to five eyes from different mice of separate litters were analyzed. cDNA
Probes
A 1.64 kb opsin cDNA probe, a gift of Dr Jeremy Nathans (Johns Hopkins University. Baltimore, MD), was purified from the flanking pBR322 plasmid sequences by EcoRI (BRL) digestion. Similarly, a 4-kb genomic probe for rat cytoplasmic p-actin (pAc 18.1, Nude1 et al., 1983) was purified from the pBR322 vector sequences by double digestion with restriction enzymes EcoRI and Hind111 (BRL). The opsin and ,$‘actin inserts were purified by running the digested DNA on 1% agarose gel and trapping the insert by DEAE paper (Schleicher and Schuell. Inc.) (Maniatis, Fritsch and Sambrook, 1982). Northern Blot Analysis Total RNA from BALB/c and rds retinas and BALB/c mouse liver was frozen in liquid N, stored at - 70°C and isolated by extraction in guanidinium isothiocyanate and centrifugation through a CsCl gradient, as described by Chirgwin et al. (1979). Purified total retinal RNA was separated by electrophoresis in 1.1% agarose formaldehyde denaturing gels. as described by Lehrach et al. ( 1977) and transferred to a nytran nylon membrane (Schleicher and Schuell, Inc.) for a period of 24 hr in 20 x SSC. Following the transfer, the nylon membrane was baked at 80°C for a period of 2 hr under vacuum. The blot was then subjected to hybridization with ““P-oligolabeled opsin cDNA or /Iactin genomic probes using the procedure described by Maniatis et al. (1982). After hybridization, the blot was subjected to a stringent wash procedure. For opsin transcripts, the blot was first washed with 2 x SSC+O.l % SDS at 20°C for a period of 30 min.
ET A:
The blot was further vzsashed in 0.1 x SSC’+ (), I “,, SDS at hS”C for a period of 2 Ilr with shaking. For @tin. dter washing at 20°C’ in 2 x SSC + 0.1 ‘,!;‘) SDS tier 30 min. the blot was washed in 0.2 Y SSC + 0.1 ‘I:, SDS at S(J”C for a period of 1 hr. Afterwards. the blots wet-r subjected to autoradiography using N-Omat S-ray tilm at -7 0 o C with an intensifying screen.
Six to eight retinas were incubated in methioninefree medium containing IO /rCi mll’ of [““S]methionine (NEN) for a period of 2 hr at 37°C with shaking, following the method of St Jules and O’Brien ( 1986). and as described in detail previously (Nir et al., 1989 I. After solubilizing the retinal homogenate and electrophoresis, a 10% SDS polyacrylamide gel (Laemmli. 1970) was stained with Coomassie blue, treated with 1 M sodium salicylate. dried under vacuum, and autoradiographed at -70°C. fffmmob1ottiffg
lmmunoblotting was conducted by a modification of the procedure of Towbin, Staehlin and Gordon ( 19 79 ). as described by Deretic and Hamm (1987), using the binding of mAb lD4 and the immunoperoxidase reaction to estimate the opsin content of retinas at various ages.
Post-embeddinglmmunocytochemicalProcedures Intact posterior eye-cups were fixed and the tissue strips were dehydrated and embedded in LK Gold (Polysciences)at 4°C. as describedby Nir et al. (1989). Thin sectionsof the retina were cut at different regions along the central to peripheral axis. For detection of opsin. two antibodies were used: (a) affinity-purified sheep anti-bovine opsin, a polyclonal antibody largely directed against the N-terminus of bovine opsin (Hargrave et al.. 1986), and (b) a monoclonal antibody (mAb) 1D4, directed against the C-terminus of bovine opsin (MacKenzie et al., 1986). kindly provided by Dr Robert Molday, [Jniversity of British Columbia, Vancouver. LR Gold-embeddedthin sectionswere immunolabeled, as described previously (Nir et al., 1989). The sections were stained with uranyl acetate and lead citrate before viewing in the electron microscope.
Scarming
Electron
Microscopy (SEM)
Intact eye-cups were fixed as describedabove. Tissue strips were treated with 1% 0~0, in 0.15 in phosphate buffer. pH 7.0, for 20 min at room temperature. Following dehydration in ethanol and freon 11 3. the tissueswere critical point-dried in CO,. The specimens were coated with golddpalladium and viewed in a Jeol 840 scanning electron microscope.
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FIG. 1. Quantitation of photoreceptor nuclear density. Retinas from BALB/c and rdsmice at various ages were analyzed. Nuclear density is expressed as the number of nuclei along a SO-pm length of retina. The data represent an average nuclear density in a region which extends from the optic nerve head to a distance of about 1500 pm. At each time period, several litters which differ slightly in age were analyzed. The range of ages (days) at each time point is specified,
3. Results Lossof PhotoreceptorCells The extent of photoreceptor cell losswas determined by measuring photoreceptor (PR) cell density. PR nuclei were counted in 50pm increments along the central-periphery axis of the retina. In normal mice the nuclear density of the outer nuclear layer dropped at the far periphery of the retina-at a distance of 200-300 km from the ora serrata. In the rds mice, the reduction in cell density was apparent at a distance of 300-500 pm from the ora serrata. As the mice aged, the reduction in ONL nuclear density began at sites closer to the center. In Fig. 1 the nuclear density is described in the region extending from the optic nerve to the mid-periphery, about 1500 pm from the optic nerve head. The peripheral region of the retina, in which nuclear density is reduced, was not included in the calculation of the mean nuclear density. The age points for which nuclear density was calculated are roughly the same ages for which opsin mRNA and synthesis were analyzed: 11 days, and 1, 3, 5. 6 and 7 months. The steepest drop in cell density in the rds retina (3 7%) occurred during the first month, to a cell density 63 % of that calculated in the 1 l-day-old retina (Fig. 1). In the next 2 months there was an additional lossof 28 % to a density of 3 5 % in 3-monthold retinas. In the next 3 months there was only a modest drop of 12% (as compared to a 63 % decrease
in the first 3 months). By 6 months the photoreceptor cell density was 23 % of that found in the 1 l-day-old rds retina. These remaining cells were gradually lost during the remaining 6 months of degeneration. In the control BALB/c retina there was only a small drop in photoreceptor density as the retina aged. By 6 months, the reduction was about 15 Y0of the maximal value at day 11 (Fig. 1). These results parallel the rates of cell loss determined by ONL thickness described by Hawkins, Jansen and Sanyal (1985). Opsin mRNA Levels Retinas from 11-day-old to 1-yr-old rds mice were analyzed for the presence of opsin mRNA and compared to normal BALB/c mice. The northern blot from one of the experiments is presented in Fig. 2. The opsin transcripts in the mouse consistsof five bands as previously described (Bowes and Farber, 198 7 : Baehr et al., 1988). The amount of opsin messagein the rds retina is reduced when compared to the BALB/c retinas. This reduction reflects the photoreceptor cell loss in the rds retina (Fig. 1). It should be noted, however, that all lanes illustrated in Fig. 1 were loaded with equal amounts of RNA (5 ,ug). thus a partial correction for cell losswas made. since aged rds retinas contain fewer photoreceptor cells. The opsin mRNA is observed to be at its highest levels in 1 I-90-day-old retinas. It is considerably reduced, but still can be clearly detected in 21~day-
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Bovine Retina
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M,* 10” 5.2 Kb 3.6 Kb 3.2 Kb -
97 -
2.6 Kb 2.0 Kb -
66 -
p-actin -
FIG. 2. Opsin mKNA content of I& retina. Total retinal RNA (5 j/g per lane) was electrophoresced. blotted, and probed with a “‘Poligo-labeled bovine opsin cDNA. Bovine opsin cDNA recognized five transcripts from both rrls and BALB/c retinas. Their size (kb) is indicated in the figure. Mouse liver and bovine retinas were used as controls. As expected. liver WA did not contain transcripts recognized by bovine opsin cDNA. whereas bovine retina had a single major band at 3.1 kb. The /r’-actin hybridization served as an internal control for the loading of total RNA in each lane. The opsin message is observed. although at reduced quantity. in all ages up to 2 10 days of age. There is no opsin mRNA detectable at 365 days which coincides with the total loss of photoreceptor cells at this age (see Fig. 1). Note that the five mRNA transcripts are affected differently during the degeneration.
old retinas. No opsin mRNA is detectable at 1 yr. Note that the individual mRNA transcripts are affected differently as the rds retina age. The 5.2 kb and 3.6 kb transcripts are reduced to a greater extent during late phases of the degeneration. The 5.2-kb band, in particular, which is at the samedensity as the 3.2-2.0 kb transcripts of 1l-day-old mice, is virtually lost from retinas of 2 lo-day-old mice. The total absenceof opsin mRNA by 365 days is in accord with the complete loss of all photoreceptors by that age (Sanyal and Hawkins, 1986). Opsin Synthesis Incorporation of [Ylmethionine into newly synthesized opsin was studied in 30-210 day-old rds retinas and compared to the incorporation in 30-day-old BALB/c retinas. A typical experiment is presented in Fig. 3. The different lanes in the gel were loaded with equal amounts of DNA, thus obtaining a partial correction for cell loss. In 30-day-old rds retinas, the labeled opsin band is of approximately the same density as the control BALB/c retina. By 90 days there is a substantial reduction in the amount of newly synthesized opsin, and in 1SO-day-old retinas opsin
42 -
Opsin +
[35S] - Autoradiograph (Coomassiegel) FIG. 3. In vitro opsin synthesis in rrls retina. SDS gels of I”“S]methionine-labeled proteins were subjected to autoradiography. The radiolabeled opsin band is indicated by the arrow. Each lane was loaded with the same amount of DNA. Note the considerable amount of newly synthesized opsin generated by young rds mice. By 150 days newly synthesized opsin is barely detectable. In the I SO- and 2 1 O-day-old retina opsin synthesis cannot be detected. although opsin mRNA is still present (cf. Fig. 2).
synthesis can be barely detected. By IX0 days. the opsin band density is no greater than background. Since the lanes of these ages were loaded with equal amounts of DNA (i.e. with increasing quantities of retinal tissue) in order to correct partially for photoreceptor cell loss, it appearsthat the absenceof newly synthesized opsin in rds mice older than 150 days represents actual cessation of opsin synthesis. The amount of opsin in the retinas of the various age groups was evaluated by immunoblot quantitation. The lanes were loaded with equal amounts of retinal tissue (0.13 retina per lane) sothat the changes in opsin content reflected the ongoing cell loss in the aging retinas (Fig. 4). Opsin was detectable. albeit at progressively lower levels. at all ages up to 1 yr. Localizntion of‘ Opsin Opsin in the interphotoreceptor space was detected in vesicular mebrane protiles and membrane whorls.
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Immunoblot FIG. 4. Immunochemical analysis of opsin content of rds retinas. The total amount of opsin on a per-retina basis is depicted in the immunoblot. Each lane was loaded with the equivalent of a 0.13 retina. The SDS gel was blotted on to nitrocellulose electrophoretically, and opsin was detected by mAb lD4 and peroxidase conjugates. The opsin band is shown by the arrow. Vesicular profiles were most abundant in young, llday-old to l-month-old retinas [Fig. 5(A)]. These structures appear to arise at the distal tip of the cilium. Budding of vesicular shaped membranes might reflect a failed attempt to form discs. A few photoreceptors in young retinas also projected elaborate membrane profiles from the distal ciliary tip. As the retinas aged the number of vesicular profiles dropped greatly. In these older retinas almost all of the opsin is localized in the elongated membranes which emanate from a few ciliary tips [Fig. S(B)]. Opsin is also detected throughout the various domains of the rod plasma membrane at all ages from 11 days to 11 months. The highest labeling density is observed along the plasma membrane which envelops nuclei and synaptic terminals (Fig. 6). Opsin is detected at a lower density also in the inner segment plasma membrane (data not shown). In normal BALB/c mice. labeling of the inner-segment plasma membrane and the perinuclear and synaptic terminal membranes was scanty, whereas outersegment plasma membranes were confluently labeled (data not shown). Similar observations in BALB/c mice were previously reported by Jansen et al. ( 19 8 7). Phngocytosis of Opsin-laden Membranes In young 1 l-day-old to l-month-old retinas, small opsin-laden inclusions were observed inside the PE cells (Fig. 7). These inclusions appear to be derived from the phagocytosis of opsin-laden vesicular structures which are abundant in the interphotoreceptor space at these ages. In older mice, opsin-containing vesicular inclusions in the PE are rarely seen (Fig. 8). This observation is in accordance with the major reduction in opsin-laden vesicular structures in interphotoreceptor space in older rds retinas.
Scanning electron microscopy (SEM) of Degenerating Photoreceptors SEM analysis of the rds retinas reveals the presence of cilia emerging from the apex of photoreceptor inner segments at all stages of degeneration. At early ages (Fig. 9), the cilium is decorated with a bulbous tip which probably reflects the process of vesicular budding which occurs in this domain [cf. Fig. S(A)]. In older mice the cilia elongate and are more slender, and the bulbous expansion of the ciliary tip is not as apparent as in younger retinas [Fig. 9(B)]. It should be noted, however, that during degeneration there was no preferential loss of cilia even in lo-month-old retinas. 4. Discussion In young (ll-day-old) rds mice, opsin gene expression is not reduced by the failure to form normal outer segments. Opsin mRNA levels and opsin synthesis rates of remaining photoreceptors are comparable to that observed in the normal mouse (Agarwal et al., 1988). Some of the newly synthesized opsin is sequestered in the photoreceptor plasma membrane (Nir and Papermaster, 1986 ; Jansen et al., 19 8 7 ; Usukura and Bok, 198 7). However. this domain is obviously limited in its capacity to store opsin. Most of the opsin is delivered to the distal ciliary tip, from which opsin-laden membranes appear to be shed and discarded into the interphotoreceptor space, where they are concomitantly phagocytosed by the adjoining pigment epithelium. While opsin mRNA is detectable in rds retinas up to 2 10 days, opsin synthesis can be clearly demonstrated only in mice up to 90 days of age. In 150-day-old mice the autoradiographs barely reveal incorporation of
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FIG. 5. Opsin localization in the interphotoreceptor space. A, Elcverl-day-old nls retina. Pclxt-embedding labeling with sheep anti-opsin and followed by rabbit anti-sheep IgG and goat anti-rabbit IgGlO nm gold. Opsin-laden vesicles (arrow) appear to bud from the tip of the connecting cilium (Cl. Those structures might represent an abortive attempt to form discmembranes (rod inner segment-RIX). r 15 600. B. One hundred and eighty-day-old retina. An elaborated membrane profile (arrow), extendsfrom the distal ciliary tip ICI. Note the completeabsenceof small opsin-containing \,csicles in the interphotoreceptor space (pigment epithelium-PE. rod inner segment-RIS). x 46 50( 1.
[35S]methionine into newly synthesized opsin. By 180 days opsin synthesis could no longer be observed even in overloaded gels. It should be noted that between 11 days and 90 days there was a h 3 % photoreceptor loss, whereas between 90 days and 150 days there was only an additional 8% cell loss (Fig. 1). Nevertheless. between 90 days and 150 days a major reduction in opsin synthetic capability was observed. Moreover, at 180 days. opsin synthesis could no longer be detected although a significant number of photoreceptors remain in the retina (21 cells per 50 /lrn of retinal length. as compared to 32 cells per 50 lrrn of retinal
length at 90 days. Fig. 1I. Thus the absence of opsin synthesis cannot be simply explained by the reduction in cell density to levels which were insufficient to produce newly synthesized opsin in quantities which could be detected by the experimental procedures. It is noteworthy that in the rLjs retina there is no preferential loss of rods (Sanyal, de Ruiter and Hawkins, 1980). Our immunocytochemical experiments confirm their conclusion. since almost all of the surviving photoreceptors were labeled with rodspecitic anti-opsin antibodies. Hence the reduced opsin synthesis does not reflect an increased proportion of
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FIG. 6. Localization of opsin in the rds mousesynaptic terminal and nuclear plasmamembranes.A, Twenty-one-day-old retina. Post-embedding labelingwith sheepanti-opsin.Opsinat a relatively high densityisdetectedalongthe plasmamembrane which envelopsthe axon (arrows) and synaptic terminal (S), x 40950. B, One hundred and thirty-five-day-old retina. Postembeddinglabelingwith mAb lD4. Denselabelingis seenon the plasmamembrane(arrow) which envelopsnucleus(N) and the synaptic terminal (R-synaptic ribbon), x 38000.
residual cones in the rds retina at late stages of the degeneration. Since the older rds mice contain opsin mRNA. the absence of newly synthesized opsin may indicate a reduction in the translation of the available opsin message.This might be due either to changes in the messageitself or to deficiencies in the translational machinery, e.g. partial loss of cytoplasmic organelles for membrane protein synthesis. It should be noted, however, that the structural components of the protein synthetic machinery, i.e. endoplasmic reticulum, Golgi apparatus and ribosomes, could be identified in the aging rds retinas. Thus the absenceof opsin synthesis cannot be simply a result of the absence of these cellular components. In young retinas immunoelectron microscopy localized opsin both to opsin-laden membranes in the
interphotoreceptor space and to the photoreceptor’s plasma membrane. In older retinas, the interphotoreceptor space was largely free of opsin-containing membranes, and the detectable opsin was localized almost exclusively in the photoreceptor plasma membrane. The highest density of opsin was observed in the region of plasma membrane which enveloped the nuclei and synaptic terminals. In mice older than 3 months, in which opsin synthesis was no longer detected, the remaining opsin which is observed in the plasma membrane might therefore represent ‘old’ molecules which were inserted in these domains at an early age and retained there. To evaluate this interpretation, turnover studies would be needed. They would be difficult to conduct, however, since the retinas are small, and only small numbers of truncated photoreceptors persist. Alternatively, levels of syn-
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FIG. 7. Phagocyrosis of opsin-laden membranes by pigment epithelium in a .!I-day-old with mAb lD4 followed by rabbit anti-mouse IgC; and goat anti-rabbit I&-I (1 nm gold. (arrows), apparently phagosomes. are seen in a pigment epithelium cell (IT). At this age opsin-containing \.esicles in the interphotoreceptor space (open arrows). some of which are
ti
pi
rtis retina. I’ost-embedding labeling Several opsin-containing inclusions there is still a relative abundance of trapped between PE microvilli (MV).
x 50600. thesis too low to be detected by our in vitro assay may be sufficient to sustain opsin content at detectable levels by immunoelectron microscopy. The detection of opsin in old rds retinas is in agreement with a previous report by Jansen et al. (1987). In searching for a comparable disassociation between opsin mRNA levels and opsin synthesis, it is interesting that in a DrosopMn mutant nina A. opsin levels are greatly reduced in the Rl-R6 photoreceptor cells, while opsin mRNA is maintained at normal levels (Zuker et al., 1988). In the Drosophiln rlina A mutant it was suggestedthat a defect exists in some aspectsof post-translational processingof opsin (Zuker et al., 1988). Despite these similarities, however, it is apparent that a defect exactly like nim A cannot account for the rds degeneration. The gene product encoded by r!ina A. a homolog of cyclosporin Abinding protein (Shieh et al., 1989). doesnot resemble the product encoded by the rds gene (Travis et al., 1989). Also. nitln A caused a reduction in opsin
content as soon as photoreceptors developed, as a consequence of reduced opsin synthesis, whereas rils mice losetheir capacity to translate opsin mRNA only after several months of degeneration. The genetic defect in the rrls retina is manifested initially by the inability to form outer segments. The defect results eventually in cell death. for reasons which are still not understood. Since the cells are lost progressively. it is apparent that ditierent cells are affected at different rates. Cellswhich persist in retinas of older mice appear to undergo functional changes which are absent at a younger age. The observation that opsin synthesis ceasesin older mice supports this interpretation. The function of the rds gene which was recently cloned is still unknown (Travis et al., 1989 ). Speculation as to the cause of cell death might be derived from the observation that about 65 x, of the photoreceptors die within the tirst 3 months and the remaining cells die gradually during the next Y months. It is obvious from the variation in onset ol
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FIG. 8. Phagocytosis of opsin in a 2 lo-day-old rds retina. Post-embedding labeling with mAb lD4. A large opsin-containing membrane whorl (*) is seen in close apposition to the pigment epithelium (PE). This structure might be attached to a cilium as seen in Fig. 5(B). Note the absence of opsin-laden vesicular structures in the interphotoreceptor space. A rare, small opsincontaining inclusion (arrow) is seen in the PE (rod inner segment-RIS), x 34 500.
photoreceptor cell death that the absence of outer segmentsby itself is not a sufficient cause of cell death. Moreover, in heterozygotic rds/ + mice which develop a shortened outer segment, cell death also occurs although at a slower rate (Hawkins et al., 1986). We propose for consideration therefore that the mutation may affect a key metabolic process, and that its manifestation as a defect of disc morphogenesisis only one of its consequences. The different rates of photoreceptor cell death may be explained if we consider, for example, that a key enzyme or receptor is affected by the mutation. If the enzyme or receptor determines the utilization of a ’ trophic factor ’ which is in limited
supply,
it will
result
initially
in rapid
cell
loss. Once the number of photoreceptors is reduced, the remaining cells can function for a longer period in an environment in which the competition for the limited ‘ trophic factor’ is reduced. 1'1
Although opsin synthesis fell to undetectable levels in S-month-old rds mice, opsin was detected in immunoblots of retina at all ages. The opsin level of l-month-old rds retinas is significantly lower in comparison to l-month-old BALB/c retina controls. This observation is compatible with the reported low steady-state level (3 %) of opsin in the mature rds retina (Chalken et al., 1985), which reflects the absence of outer segments and continuous phagocytosis of newly synthesized opsin-laden vesicles. As the animals aged, the immunoblots indicate further reduction in opsin content at rates which exceed the rates of photoreceptor cell loss. Thus, in addition to reduction in opsin due to cell loss,there is a reduction in opsin content per cell due to cessation of opsin synthesis in the remaining older photoreceptor cells. This interpretation is supported by immunocytochemical studies that demonstrate a reduced EEK 51
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FIG. 9. Scanning electron microscopy of rds retina. A, Thirty-day-old n(s retina. Hulbnus expansions at the ciliary tip (arrows) project from the inner segments (IS). Enlargement of the ciliary tip is probably due to the accumulation of membranes in this domain as seen in Fig. 5(A), x 10 200. B. Three-hundred-day-old rds retina. All of the inner segments (IS) in the field contain cilia. Expansion of the distal ciliary tip is not as apparent as in young retinas. * 9 %50.
amount of opsin in membranous structures in the interphotoreceptor space in older rds retinas. A large quantity of small opsin-laden vesicles in the interphotoreceptor space was observed in 1 l-day-old rds retinas, but declined as the mice aged. While vesicular profiles could still be seenin 3-month-old retinas, they were rare after 5 months. The decline in opsin in the interphotoreceptor spacecoincides with, and probably reflects the cessation of, opsin synthesis and of membrane budding at the distal ciliary tip in these older mice. Despite these changes, surviving inner segments continue to project apparently intact cilia into the interphotoreceptor spacethroughout their life span. Thus, the absence of delivery of opsin-laden membranes to the interphotoreceptor space in older mice is not a result of a preferential loss of cilia. In conclusion, during early and late phasesof the retinal degeneration, opsin gene expression at transcriptional and translational levels appears to be affected to a differing extent. While the opsin gene is still being transcribed in old rds mice. the absence of opsin synthesis in the older mutant mice may be due to some defect in translational or post-translational steps. The cessation of opsin synthesis in older mice probably accounts for the sharp reduction in opsincontaining membranes in the interphotoreceptor space and the scarcity of phagosomesin the pigment epithelium. Opsin persists, however, throughout the life of the photoreceptor cell in the plasma membrane
which envelops the inner segment, nucleus and synaptic terminal. Note Added at Proof Since submissionand acceptance of the manuscript, Connell. Boscom. McInnes and Molday (ARVO, 1990) reported that the rds gene encodes for peripherim. Acknowledgments Supported by NIH EY-6891 and EY-6892. Our thanks are extended to Nancy Ransom for excellent technical assistance, to Tammy Lowe for preparing the manuscript and to Z. D. Sharp for assistance in obtaining the /Cactin probe.
References Agarwal. N.. Nir. I. and Papermaster, D. S. ( 1988). Opsin gene expression in retinal dystrophy slow (r&i mice 1. Cell Biol. 107. 108a. (abstract). Baehr, W., Falk. J. D.. Bugra, K.. Triantafyllos, J. T. and McGinnis, J. F. (1988). Isolation and analysis of the mouse opsin gene. FEBS Lett. 238, 253-6. Bowes, C. and Farber, D. B. (1987). mRNAs coding for proteins of the cGMP cascade in the degenerative retina of the rd mouse. Exp. Eye Res. 45. 467-80. Chirgwin. J, M.. Przybyia. A. E.. MacDonald, R. J. and Rutter. W. J. ( 19 79 ). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Riochmistry 18. 5294-9.
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D. and Hamm, H. E. ( 198 7). Topographic analysis of antigenic determinants recognized by monoclonal antibodies to the photoreceptor guanyl nucleotide binding protein, transducin. J. Biol. Chem. 262, 1083947. Hawkins. R. K., Jansen. H. G. and Sanyal, S. (1985). Development and degeneration of retina in rds mutant mice : photoreceptor abnormalities in the heterozygotes. Exp. Eye Res. 41, 701-20. Hargrave, P. A., Adamus, G.. Arendt, A., McDowell, J. H., Wang, J., Szary, A., Curtis, D. and Jackson, R. W. (1986). Rhodopsin’s amino terminus is a principal antigenic site. Exp. Eye Res. 42. 363-73. Jansen. H. G.. Sanyal, S.. de Grip, W. J. and Schalken. J. J. ( 1987). Development and degeneration of retina in rds mutant mice: ultraimmunohistochemical localization of opsin. Exp. Eye Res. 44, 347-61. la Vail, M. M. ( 198 1). Analysis of neurological mutants with inherited retinal degeneration. invest. Ophthalmol. Vis. Sci. 21, 638-57. la Vail, M. M. and Battelle, B. A. (1975) Influence of eye pigmentation and light deprivation on inherited retina dystrophy in the rat. Exp. Eye Res. 21, 167-92. Laemmli, U. K. (19 70). Cleavage of structural protein during the assembly of the head of bacteriophage T,. Nuture 227, 680-S. Lehrach. H.. Diamond, D., Wozney. J. M. and Boedtker, H. (1977). RNA molecular weight determination by gel electrophoresis under denaturing condition, a critical reexamination. Biochemistry 16, 4743-51. MacKenzie, D.. Arendt. A., Hargrave. P.. McDowell, J. H. and Molday. R. S. (1984). Localization of binding sites for carboxyl terminal specific anti-rhodopsin monoclonal antibodies using synthetic peptides. Biochemistry 23, 6544-9. Maniatis. T.. Fritsch. E. F. and Sambrook, J. (1982). In Molecular Cloning: A Laboratory Manual. p. 202. Cold Spring Harbor Laboratory. Nir. I., Agarwal, N., Sagie, G. and Papermaster. D. S. (1989). Opsin distribution and synthesis in degenerating photoreceptors of rd mutant mice. Exp. Eye Res. 49, 403-22. Nir. I. and Papermaster, D. S. (1986). Immunocytochemical localization of opsin in the inner segment and ciliary plasma membrane of photoreceptor in retinas of rds mutant mice. Invest. Opkfkaimol. Vis. Sci. 27, 836-40. Deretic.
267 Nudel. Ll.. Zukut, R.. Shani, M., Neuman, S.. Levy, Z. and Yaffe. D. (1983). The nucleotide sequence of the rat cytoplasmic /&actin gene. Nucleic Acid Res. 11, 175971. Sanyal. S.. de Ruiter, A. and Hawkins. R. K. (1980). Development and degeneration of retina in rds mutant mice: light microscopy. J. Camp. Neural. 194. 193-207. Sanyal. S. and Hawkins, R. K. (1986). Development and degeneration of retina in rds mutant mice: effects of light on the rate of degeneration in albino and pigmented homozygous and heterozygous mutant and normal mice. Vision Res. 26, 1177-85. Sanyal. S. and Jansen, H. G. (1981). Absence of receptor outer segment in the retina of the rds mutant mouse. Neurosci. Letf. 21, 23-6. Schalken, J. J., Jaques, Janssen. J. M.. de Grip. W. J.. Hawkins, R. K. and Sanyal, S. (1985). Immunoassay of rod visual pigment (opsin) in the eyes of rds mutant mice lacking receptor outlet segments. Biockim. Biopkys. Acta 839, 122-6. Shieh. B.-H., Stamnes, M. A., Seavello. S.. Harris, G. L. and Zuker. C. S. (1989). The nina A gene required for visual transduction in Drosophila encodes for a homologue of cyclosporin A-binding protein. Nature 338. 67-70. St Jules, R. S. and O’Brien, P. J. (1986). The acylation of rat rhodopsin in vitro and in vivo. Exp. Eye Res. 43, 92940. Towbin. H.. Staehelin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4 3 504. Travis, G. H., Brennan, M. B., Danielson, P. E., Kozak, C. A. and Sutcliffe, J. G. (1989). Identification of a photoreceptor-specific mRNA encoded by the gene responsible for retinal degeneration slow (rds). Nature 338. 70-3. Usukura, J. and Bok. D. (1987). Changes in the localization and content of opsin during retinal development in the rds mutant mouse: immunocytochemistry and immunoassay. Exp. Eye Res. 45, 501-l 5. Zuker. C. S., Mismer, D., Hardy, R. and Rubin. G. M. ( 1988). Ectopic expression of a minor Drosophila opsin in the major photoreceptor class: distinguishing the role of primary photoreceptor and cellular context. Cell 55, 475-82.
Opsin
Gene
Expression During Degeneration
IZHAK Department
NIR”,
of Pathology,
(Received
Early and Late in rds Mice
NEERAJ
AGARWAL
University
of Texas Health Science TX 78264, U.S.A.
7 1 August
1989
and accepted
AND
DAVID
in revised
Phases
of Retinal
S. PAPERMASTER
Center
form
at San Antonio,
23 January
San Antonio,
1990)
Opsin mRNA levels, opsin synthetic rates and localization of opsin were studied throughout the photoreceptor’slife spanin the rds mice.Mutant mice 11 daysto 11 monthsold were investigated.Opsin mRNA levels were studied by means of northern blot analysis. Opsin synthesiswas measuredby incorporationof [%]methionine into newly synthesizedopsinin vitro. Distributionof opsinin the retina was determinedby immunoelectronmicroscopy.OpsinmRNA was detectedin young as well as old retinas,and opsinsynthesiscould bedetectedat early phasesof degenerationbut not in late phases.The absenceof opsinsynthesisin older rdsmicemight be dueto translationaldown-regulationor someother defectin the capacity to synthesizeopsin.In young mice, opsinwas detectedin the subretinalspacein opsin-ladenvesicular membranes:such membraneswere absent from retinas of older mice. This disappearance parallelsthe cessationof opsinsynthesisand the consequentfailure to deliver opsinto the subretinal spacein retinas from older mice. Immunochemicalanalysisrevealedthe presenceof small amountsof opsinin all retinasup to 11 monthsof age.Immunoelectronmicroscopylocalizedthe residual opsin, mostly to the plasmamembranewhich envelopsthe nuclei and synaptic terminals.Theseopsin moleculesmight be a consequenceof very low levelsof opsinsynthesis,too low to be detectedby our assays,or may have beensynthesizedat an earlier ageand retainedin the plasmamembraneof the old mutant photoreceptors. Key words:rds mice; opsinmRNA ; opsin synthesis; immunocytochemistry; aging: inherited retinal degeneration. 1. Introduction In mice bearing the retinal degeneration slow (rds) mutation, rod and cone outer segmentsfail to develop (Sanyal and Jansen, 1981). The opsin content of the 3-4-week-old dystrophic retina is about 3 % of normal (Chalken et al., 1985). Our preliminary observations showed that in 11-day-old rds mice, opsin gene expression is not altered and declines slightly in the next month. At young ages, the photoreceptor cells produce opsin mRNA at a normal level and opsin is synthesized at a rate comparable to normal. The low level of opsin was attributed to the cell’s failure to incorporate opsin into a stable outer segment. Consequently the newly synthesized opsin-laden membranes that were released into the interphotoreceptor space were subjected to early and continuous phagocytosis by the adjacent pigment epithelium (Agarwal, Nir and Papermaster, 1988). The rds mutation is characterized by a slow photoreceptor cell death which is completed only by 1 yr of age (Sanyal and Hawkins, 1986). Although the rds gene was recently cloned, the function of the gene product is not known since the gene product does not resemble any known protein (Travis et al., 1989). (See ‘Note added at proof. ‘) Hence the cause of the * For correspondence at: Department of Pathology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive. San Antonio. TX 78284-7750. U.S.A.
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photoreceptor cell death in the rds retina is not yet understood. It is apparent, however, that photoreceptor degeneration in the rds mouse is not due to critical cellular changes which precipitate rapid cell death in a relatively short time period such as the case in the rd mouse and RCS rat (la Vail, 198 1). Thus, unlike the other rodents with retinal dystrophy, a substantial number of photoreceptors in the rds mouse survive for a prolonged time in a diseasedretina. In the present study we explored whether these cells have significant alterations in opsin gene expression over the entire duration of the retinal degeneration as one measure of possibleeffects of long-term photoreceptor outer segment abnormalities.
2. Materials and Methods Animals BALB/c mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mutant 020/A rds/rds mice were obtained from Dr Janet Blanks, University of Southern California, Los Angeles, CA, from a colony that originated in the Netherlands and was distributed by Dr Sanyal. The animals were bred locally and maintained under a 12 hr/ 12 hr dark/light cycle with exposure to low (3-5 ft candles) light levels. The handling of the mice was in accordance with the ARVO resolution on the use of animals in research and NIH and AAALAC guidelines. Mice at various ages 0 1990 AcademicPressLimited
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were killed l-2 hr after the onset of light by exposure to co,.
Enucleated intact eyes were tixed overnight with 1 ‘%, glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer, pH 7.0 at 4°C. The eyes were bisected into two hemispheres through the optic nerve and post-fixed with 1 “/u 0~0, in 0.1 5 .LI phosphate buffer, pH 7.0. One hemisphere from each eye was oriented and embedded in epoxy resins as described by la Vail and Battelle (19 75). Sections 2 L,rn long, which include the full length of the retina from the optic nerve head to the far periphery, were stained with toluidine blue and viewed at 1000 x magnification. Photoreceptor nuclei were counted along 50 pm segments of the retina with the aid of an ocular micrometer. Measurements started at 100 !trn from the optic nerve head and continued at 100-l 50 /irn intervals to the far periphery. At each age, three to five eyes from different mice of separate litters were analyzed. cDNA
Probes
A 1.64 kb opsin cDNA probe, a gift of Dr Jeremy Nathans (Johns Hopkins University. Baltimore, MD), was purified from the flanking pBR322 plasmid sequences by EcoRI (BRL) digestion. Similarly, a 4-kb genomic probe for rat cytoplasmic p-actin (pAc 18.1, Nude1 et al., 1983) was purified from the pBR322 vector sequences by double digestion with restriction enzymes EcoRI and Hind111 (BRL). The opsin and ,$‘actin inserts were purified by running the digested DNA on 1% agarose gel and trapping the insert by DEAE paper (Schleicher and Schuell. Inc.) (Maniatis, Fritsch and Sambrook, 1982). Northern Blot Analysis Total RNA from BALB/c and rds retinas and BALB/c mouse liver was frozen in liquid N, stored at - 70°C and isolated by extraction in guanidinium isothiocyanate and centrifugation through a CsCl gradient, as described by Chirgwin et al. (1979). Purified total retinal RNA was separated by electrophoresis in 1.1% agarose formaldehyde denaturing gels. as described by Lehrach et al. ( 1977) and transferred to a nytran nylon membrane (Schleicher and Schuell, Inc.) for a period of 24 hr in 20 x SSC. Following the transfer, the nylon membrane was baked at 80°C for a period of 2 hr under vacuum. The blot was then subjected to hybridization with ““P-oligolabeled opsin cDNA or /Iactin genomic probes using the procedure described by Maniatis et al. (1982). After hybridization, the blot was subjected to a stringent wash procedure. For opsin transcripts, the blot was first washed with 2 x SSC+O.l % SDS at 20°C for a period of 30 min.
ET A:
The blot was further vzsashed in 0.1 x SSC’+ (), I “,, SDS at hS”C for a period of 2 Ilr with shaking. For @tin. dter washing at 20°C’ in 2 x SSC + 0.1 ‘,!;‘) SDS tier 30 min. the blot was washed in 0.2 Y SSC + 0.1 ‘I:, SDS at S(J”C for a period of 1 hr. Afterwards. the blots wet-r subjected to autoradiography using N-Omat S-ray tilm at -7 0 o C with an intensifying screen.
Six to eight retinas were incubated in methioninefree medium containing IO /rCi mll’ of [““S]methionine (NEN) for a period of 2 hr at 37°C with shaking, following the method of St Jules and O’Brien ( 1986). and as described in detail previously (Nir et al., 1989 I. After solubilizing the retinal homogenate and electrophoresis, a 10% SDS polyacrylamide gel (Laemmli. 1970) was stained with Coomassie blue, treated with 1 M sodium salicylate. dried under vacuum, and autoradiographed at -70°C. fffmmob1ottiffg
lmmunoblotting was conducted by a modification of the procedure of Towbin, Staehlin and Gordon ( 19 79 ). as described by Deretic and Hamm (1987), using the binding of mAb lD4 and the immunoperoxidase reaction to estimate the opsin content of retinas at various ages.
Post-embeddinglmmunocytochemicalProcedures Intact posterior eye-cups were fixed and the tissue strips were dehydrated and embedded in LK Gold (Polysciences)at 4°C. as describedby Nir et al. (1989). Thin sectionsof the retina were cut at different regions along the central to peripheral axis. For detection of opsin. two antibodies were used: (a) affinity-purified sheep anti-bovine opsin, a polyclonal antibody largely directed against the N-terminus of bovine opsin (Hargrave et al.. 1986), and (b) a monoclonal antibody (mAb) 1D4, directed against the C-terminus of bovine opsin (MacKenzie et al., 1986). kindly provided by Dr Robert Molday, [Jniversity of British Columbia, Vancouver. LR Gold-embeddedthin sectionswere immunolabeled, as described previously (Nir et al., 1989). The sections were stained with uranyl acetate and lead citrate before viewing in the electron microscope.
Scarming
Electron
Microscopy (SEM)
Intact eye-cups were fixed as describedabove. Tissue strips were treated with 1% 0~0, in 0.15 in phosphate buffer. pH 7.0, for 20 min at room temperature. Following dehydration in ethanol and freon 11 3. the tissueswere critical point-dried in CO,. The specimens were coated with golddpalladium and viewed in a Jeol 840 scanning electron microscope.
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FIG. 1. Quantitation of photoreceptor nuclear density. Retinas from BALB/c and rdsmice at various ages were analyzed. Nuclear density is expressed as the number of nuclei along a SO-pm length of retina. The data represent an average nuclear density in a region which extends from the optic nerve head to a distance of about 1500 pm. At each time period, several litters which differ slightly in age were analyzed. The range of ages (days) at each time point is specified,
3. Results Lossof PhotoreceptorCells The extent of photoreceptor cell losswas determined by measuring photoreceptor (PR) cell density. PR nuclei were counted in 50pm increments along the central-periphery axis of the retina. In normal mice the nuclear density of the outer nuclear layer dropped at the far periphery of the retina-at a distance of 200-300 km from the ora serrata. In the rds mice, the reduction in cell density was apparent at a distance of 300-500 pm from the ora serrata. As the mice aged, the reduction in ONL nuclear density began at sites closer to the center. In Fig. 1 the nuclear density is described in the region extending from the optic nerve to the mid-periphery, about 1500 pm from the optic nerve head. The peripheral region of the retina, in which nuclear density is reduced, was not included in the calculation of the mean nuclear density. The age points for which nuclear density was calculated are roughly the same ages for which opsin mRNA and synthesis were analyzed: 11 days, and 1, 3, 5. 6 and 7 months. The steepest drop in cell density in the rds retina (3 7%) occurred during the first month, to a cell density 63 % of that calculated in the 1 l-day-old retina (Fig. 1). In the next 2 months there was an additional lossof 28 % to a density of 3 5 % in 3-monthold retinas. In the next 3 months there was only a modest drop of 12% (as compared to a 63 % decrease
in the first 3 months). By 6 months the photoreceptor cell density was 23 % of that found in the 1 l-day-old rds retina. These remaining cells were gradually lost during the remaining 6 months of degeneration. In the control BALB/c retina there was only a small drop in photoreceptor density as the retina aged. By 6 months, the reduction was about 15 Y0of the maximal value at day 11 (Fig. 1). These results parallel the rates of cell loss determined by ONL thickness described by Hawkins, Jansen and Sanyal (1985). Opsin mRNA Levels Retinas from 11-day-old to 1-yr-old rds mice were analyzed for the presence of opsin mRNA and compared to normal BALB/c mice. The northern blot from one of the experiments is presented in Fig. 2. The opsin transcripts in the mouse consistsof five bands as previously described (Bowes and Farber, 198 7 : Baehr et al., 1988). The amount of opsin messagein the rds retina is reduced when compared to the BALB/c retinas. This reduction reflects the photoreceptor cell loss in the rds retina (Fig. 1). It should be noted, however, that all lanes illustrated in Fig. 1 were loaded with equal amounts of RNA (5 ,ug). thus a partial correction for cell losswas made. since aged rds retinas contain fewer photoreceptor cells. The opsin mRNA is observed to be at its highest levels in 1 I-90-day-old retinas. It is considerably reduced, but still can be clearly detected in 21~day-
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FIG. 2. Opsin mKNA content of I& retina. Total retinal RNA (5 j/g per lane) was electrophoresced. blotted, and probed with a “‘Poligo-labeled bovine opsin cDNA. Bovine opsin cDNA recognized five transcripts from both rrls and BALB/c retinas. Their size (kb) is indicated in the figure. Mouse liver and bovine retinas were used as controls. As expected. liver WA did not contain transcripts recognized by bovine opsin cDNA. whereas bovine retina had a single major band at 3.1 kb. The /r’-actin hybridization served as an internal control for the loading of total RNA in each lane. The opsin message is observed. although at reduced quantity. in all ages up to 2 10 days of age. There is no opsin mRNA detectable at 365 days which coincides with the total loss of photoreceptor cells at this age (see Fig. 1). Note that the five mRNA transcripts are affected differently during the degeneration.
old retinas. No opsin mRNA is detectable at 1 yr. Note that the individual mRNA transcripts are affected differently as the rds retina age. The 5.2 kb and 3.6 kb transcripts are reduced to a greater extent during late phases of the degeneration. The 5.2-kb band, in particular, which is at the samedensity as the 3.2-2.0 kb transcripts of 1l-day-old mice, is virtually lost from retinas of 2 lo-day-old mice. The total absenceof opsin mRNA by 365 days is in accord with the complete loss of all photoreceptors by that age (Sanyal and Hawkins, 1986). Opsin Synthesis Incorporation of [Ylmethionine into newly synthesized opsin was studied in 30-210 day-old rds retinas and compared to the incorporation in 30-day-old BALB/c retinas. A typical experiment is presented in Fig. 3. The different lanes in the gel were loaded with equal amounts of DNA, thus obtaining a partial correction for cell loss. In 30-day-old rds retinas, the labeled opsin band is of approximately the same density as the control BALB/c retina. By 90 days there is a substantial reduction in the amount of newly synthesized opsin, and in 1SO-day-old retinas opsin
42 -
Opsin +
[35S] - Autoradiograph (Coomassiegel) FIG. 3. In vitro opsin synthesis in rrls retina. SDS gels of I”“S]methionine-labeled proteins were subjected to autoradiography. The radiolabeled opsin band is indicated by the arrow. Each lane was loaded with the same amount of DNA. Note the considerable amount of newly synthesized opsin generated by young rds mice. By 150 days newly synthesized opsin is barely detectable. In the I SO- and 2 1 O-day-old retina opsin synthesis cannot be detected. although opsin mRNA is still present (cf. Fig. 2).
synthesis can be barely detected. By IX0 days. the opsin band density is no greater than background. Since the lanes of these ages were loaded with equal amounts of DNA (i.e. with increasing quantities of retinal tissue) in order to correct partially for photoreceptor cell loss, it appearsthat the absenceof newly synthesized opsin in rds mice older than 150 days represents actual cessation of opsin synthesis. The amount of opsin in the retinas of the various age groups was evaluated by immunoblot quantitation. The lanes were loaded with equal amounts of retinal tissue (0.13 retina per lane) sothat the changes in opsin content reflected the ongoing cell loss in the aging retinas (Fig. 4). Opsin was detectable. albeit at progressively lower levels. at all ages up to 1 yr. Localizntion of‘ Opsin Opsin in the interphotoreceptor space was detected in vesicular mebrane protiles and membrane whorls.
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Immunoblot FIG. 4. Immunochemical analysis of opsin content of rds retinas. The total amount of opsin on a per-retina basis is depicted in the immunoblot. Each lane was loaded with the equivalent of a 0.13 retina. The SDS gel was blotted on to nitrocellulose electrophoretically, and opsin was detected by mAb lD4 and peroxidase conjugates. The opsin band is shown by the arrow. Vesicular profiles were most abundant in young, llday-old to l-month-old retinas [Fig. 5(A)]. These structures appear to arise at the distal tip of the cilium. Budding of vesicular shaped membranes might reflect a failed attempt to form discs. A few photoreceptors in young retinas also projected elaborate membrane profiles from the distal ciliary tip. As the retinas aged the number of vesicular profiles dropped greatly. In these older retinas almost all of the opsin is localized in the elongated membranes which emanate from a few ciliary tips [Fig. S(B)]. Opsin is also detected throughout the various domains of the rod plasma membrane at all ages from 11 days to 11 months. The highest labeling density is observed along the plasma membrane which envelops nuclei and synaptic terminals (Fig. 6). Opsin is detected at a lower density also in the inner segment plasma membrane (data not shown). In normal BALB/c mice. labeling of the inner-segment plasma membrane and the perinuclear and synaptic terminal membranes was scanty, whereas outersegment plasma membranes were confluently labeled (data not shown). Similar observations in BALB/c mice were previously reported by Jansen et al. ( 19 8 7). Phngocytosis of Opsin-laden Membranes In young 1 l-day-old to l-month-old retinas, small opsin-laden inclusions were observed inside the PE cells (Fig. 7). These inclusions appear to be derived from the phagocytosis of opsin-laden vesicular structures which are abundant in the interphotoreceptor space at these ages. In older mice, opsin-containing vesicular inclusions in the PE are rarely seen (Fig. 8). This observation is in accordance with the major reduction in opsin-laden vesicular structures in interphotoreceptor space in older rds retinas.
Scanning electron microscopy (SEM) of Degenerating Photoreceptors SEM analysis of the rds retinas reveals the presence of cilia emerging from the apex of photoreceptor inner segments at all stages of degeneration. At early ages (Fig. 9), the cilium is decorated with a bulbous tip which probably reflects the process of vesicular budding which occurs in this domain [cf. Fig. S(A)]. In older mice the cilia elongate and are more slender, and the bulbous expansion of the ciliary tip is not as apparent as in younger retinas [Fig. 9(B)]. It should be noted, however, that during degeneration there was no preferential loss of cilia even in lo-month-old retinas. 4. Discussion In young (ll-day-old) rds mice, opsin gene expression is not reduced by the failure to form normal outer segments. Opsin mRNA levels and opsin synthesis rates of remaining photoreceptors are comparable to that observed in the normal mouse (Agarwal et al., 1988). Some of the newly synthesized opsin is sequestered in the photoreceptor plasma membrane (Nir and Papermaster, 1986 ; Jansen et al., 19 8 7 ; Usukura and Bok, 198 7). However. this domain is obviously limited in its capacity to store opsin. Most of the opsin is delivered to the distal ciliary tip, from which opsin-laden membranes appear to be shed and discarded into the interphotoreceptor space, where they are concomitantly phagocytosed by the adjoining pigment epithelium. While opsin mRNA is detectable in rds retinas up to 2 10 days, opsin synthesis can be clearly demonstrated only in mice up to 90 days of age. In 150-day-old mice the autoradiographs barely reveal incorporation of
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FIG. 5. Opsin localization in the interphotoreceptor space. A, Elcverl-day-old nls retina. Pclxt-embedding labeling with sheep anti-opsin and followed by rabbit anti-sheep IgG and goat anti-rabbit IgGlO nm gold. Opsin-laden vesicles (arrow) appear to bud from the tip of the connecting cilium (Cl. Those structures might represent an abortive attempt to form discmembranes (rod inner segment-RIX). r 15 600. B. One hundred and eighty-day-old retina. An elaborated membrane profile (arrow), extendsfrom the distal ciliary tip ICI. Note the completeabsenceof small opsin-containing \,csicles in the interphotoreceptor space (pigment epithelium-PE. rod inner segment-RIS). x 46 50( 1.
[35S]methionine into newly synthesized opsin. By 180 days opsin synthesis could no longer be observed even in overloaded gels. It should be noted that between 11 days and 90 days there was a h 3 % photoreceptor loss, whereas between 90 days and 150 days there was only an additional 8% cell loss (Fig. 1). Nevertheless. between 90 days and 150 days a major reduction in opsin synthetic capability was observed. Moreover, at 180 days. opsin synthesis could no longer be detected although a significant number of photoreceptors remain in the retina (21 cells per 50 /lrn of retinal length. as compared to 32 cells per 50 lrrn of retinal
length at 90 days. Fig. 1I. Thus the absence of opsin synthesis cannot be simply explained by the reduction in cell density to levels which were insufficient to produce newly synthesized opsin in quantities which could be detected by the experimental procedures. It is noteworthy that in the rLjs retina there is no preferential loss of rods (Sanyal, de Ruiter and Hawkins, 1980). Our immunocytochemical experiments confirm their conclusion. since almost all of the surviving photoreceptors were labeled with rodspecitic anti-opsin antibodies. Hence the reduced opsin synthesis does not reflect an increased proportion of
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FIG. 6. Localization of opsin in the rds mousesynaptic terminal and nuclear plasmamembranes.A, Twenty-one-day-old retina. Post-embedding labelingwith sheepanti-opsin.Opsinat a relatively high densityisdetectedalongthe plasmamembrane which envelopsthe axon (arrows) and synaptic terminal (S), x 40950. B, One hundred and thirty-five-day-old retina. Postembeddinglabelingwith mAb lD4. Denselabelingis seenon the plasmamembrane(arrow) which envelopsnucleus(N) and the synaptic terminal (R-synaptic ribbon), x 38000.
residual cones in the rds retina at late stages of the degeneration. Since the older rds mice contain opsin mRNA. the absence of newly synthesized opsin may indicate a reduction in the translation of the available opsin message.This might be due either to changes in the messageitself or to deficiencies in the translational machinery, e.g. partial loss of cytoplasmic organelles for membrane protein synthesis. It should be noted, however, that the structural components of the protein synthetic machinery, i.e. endoplasmic reticulum, Golgi apparatus and ribosomes, could be identified in the aging rds retinas. Thus the absenceof opsin synthesis cannot be simply a result of the absence of these cellular components. In young retinas immunoelectron microscopy localized opsin both to opsin-laden membranes in the
interphotoreceptor space and to the photoreceptor’s plasma membrane. In older retinas, the interphotoreceptor space was largely free of opsin-containing membranes, and the detectable opsin was localized almost exclusively in the photoreceptor plasma membrane. The highest density of opsin was observed in the region of plasma membrane which enveloped the nuclei and synaptic terminals. In mice older than 3 months, in which opsin synthesis was no longer detected, the remaining opsin which is observed in the plasma membrane might therefore represent ‘old’ molecules which were inserted in these domains at an early age and retained there. To evaluate this interpretation, turnover studies would be needed. They would be difficult to conduct, however, since the retinas are small, and only small numbers of truncated photoreceptors persist. Alternatively, levels of syn-
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FIG. 7. Phagocyrosis of opsin-laden membranes by pigment epithelium in a .!I-day-old with mAb lD4 followed by rabbit anti-mouse IgC; and goat anti-rabbit I&-I (1 nm gold. (arrows), apparently phagosomes. are seen in a pigment epithelium cell (IT). At this age opsin-containing \.esicles in the interphotoreceptor space (open arrows). some of which are
ti
pi
rtis retina. I’ost-embedding labeling Several opsin-containing inclusions there is still a relative abundance of trapped between PE microvilli (MV).
x 50600. thesis too low to be detected by our in vitro assay may be sufficient to sustain opsin content at detectable levels by immunoelectron microscopy. The detection of opsin in old rds retinas is in agreement with a previous report by Jansen et al. (1987). In searching for a comparable disassociation between opsin mRNA levels and opsin synthesis, it is interesting that in a DrosopMn mutant nina A. opsin levels are greatly reduced in the Rl-R6 photoreceptor cells, while opsin mRNA is maintained at normal levels (Zuker et al., 1988). In the Drosophiln rlina A mutant it was suggestedthat a defect exists in some aspectsof post-translational processingof opsin (Zuker et al., 1988). Despite these similarities, however, it is apparent that a defect exactly like nim A cannot account for the rds degeneration. The gene product encoded by r!ina A. a homolog of cyclosporin Abinding protein (Shieh et al., 1989). doesnot resemble the product encoded by the rds gene (Travis et al., 1989). Also. nitln A caused a reduction in opsin
content as soon as photoreceptors developed, as a consequence of reduced opsin synthesis, whereas rils mice losetheir capacity to translate opsin mRNA only after several months of degeneration. The genetic defect in the rrls retina is manifested initially by the inability to form outer segments. The defect results eventually in cell death. for reasons which are still not understood. Since the cells are lost progressively. it is apparent that ditierent cells are affected at different rates. Cellswhich persist in retinas of older mice appear to undergo functional changes which are absent at a younger age. The observation that opsin synthesis ceasesin older mice supports this interpretation. The function of the rds gene which was recently cloned is still unknown (Travis et al., 1989 ). Speculation as to the cause of cell death might be derived from the observation that about 65 x, of the photoreceptors die within the tirst 3 months and the remaining cells die gradually during the next Y months. It is obvious from the variation in onset ol
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FIG. 8. Phagocytosis of opsin in a 2 lo-day-old rds retina. Post-embedding labeling with mAb lD4. A large opsin-containing membrane whorl (*) is seen in close apposition to the pigment epithelium (PE). This structure might be attached to a cilium as seen in Fig. 5(B). Note the absence of opsin-laden vesicular structures in the interphotoreceptor space. A rare, small opsincontaining inclusion (arrow) is seen in the PE (rod inner segment-RIS), x 34 500.
photoreceptor cell death that the absence of outer segmentsby itself is not a sufficient cause of cell death. Moreover, in heterozygotic rds/ + mice which develop a shortened outer segment, cell death also occurs although at a slower rate (Hawkins et al., 1986). We propose for consideration therefore that the mutation may affect a key metabolic process, and that its manifestation as a defect of disc morphogenesisis only one of its consequences. The different rates of photoreceptor cell death may be explained if we consider, for example, that a key enzyme or receptor is affected by the mutation. If the enzyme or receptor determines the utilization of a ’ trophic factor ’ which is in limited
supply,
it will
result
initially
in rapid
cell
loss. Once the number of photoreceptors is reduced, the remaining cells can function for a longer period in an environment in which the competition for the limited ‘ trophic factor’ is reduced. 1'1
Although opsin synthesis fell to undetectable levels in S-month-old rds mice, opsin was detected in immunoblots of retina at all ages. The opsin level of l-month-old rds retinas is significantly lower in comparison to l-month-old BALB/c retina controls. This observation is compatible with the reported low steady-state level (3 %) of opsin in the mature rds retina (Chalken et al., 1985), which reflects the absence of outer segments and continuous phagocytosis of newly synthesized opsin-laden vesicles. As the animals aged, the immunoblots indicate further reduction in opsin content at rates which exceed the rates of photoreceptor cell loss. Thus, in addition to reduction in opsin due to cell loss,there is a reduction in opsin content per cell due to cessation of opsin synthesis in the remaining older photoreceptor cells. This interpretation is supported by immunocytochemical studies that demonstrate a reduced EEK 51
266
FIG. 9. Scanning electron microscopy of rds retina. A, Thirty-day-old n(s retina. Hulbnus expansions at the ciliary tip (arrows) project from the inner segments (IS). Enlargement of the ciliary tip is probably due to the accumulation of membranes in this domain as seen in Fig. 5(A), x 10 200. B. Three-hundred-day-old rds retina. All of the inner segments (IS) in the field contain cilia. Expansion of the distal ciliary tip is not as apparent as in young retinas. * 9 %50.
amount of opsin in membranous structures in the interphotoreceptor space in older rds retinas. A large quantity of small opsin-laden vesicles in the interphotoreceptor space was observed in 1 l-day-old rds retinas, but declined as the mice aged. While vesicular profiles could still be seenin 3-month-old retinas, they were rare after 5 months. The decline in opsin in the interphotoreceptor spacecoincides with, and probably reflects the cessation of, opsin synthesis and of membrane budding at the distal ciliary tip in these older mice. Despite these changes, surviving inner segments continue to project apparently intact cilia into the interphotoreceptor spacethroughout their life span. Thus, the absence of delivery of opsin-laden membranes to the interphotoreceptor space in older mice is not a result of a preferential loss of cilia. In conclusion, during early and late phasesof the retinal degeneration, opsin gene expression at transcriptional and translational levels appears to be affected to a differing extent. While the opsin gene is still being transcribed in old rds mice. the absence of opsin synthesis in the older mutant mice may be due to some defect in translational or post-translational steps. The cessation of opsin synthesis in older mice probably accounts for the sharp reduction in opsincontaining membranes in the interphotoreceptor space and the scarcity of phagosomesin the pigment epithelium. Opsin persists, however, throughout the life of the photoreceptor cell in the plasma membrane
which envelops the inner segment, nucleus and synaptic terminal. Note Added at Proof Since submissionand acceptance of the manuscript, Connell. Boscom. McInnes and Molday (ARVO, 1990) reported that the rds gene encodes for peripherim. Acknowledgments Supported by NIH EY-6891 and EY-6892. Our thanks are extended to Nancy Ransom for excellent technical assistance, to Tammy Lowe for preparing the manuscript and to Z. D. Sharp for assistance in obtaining the /Cactin probe.
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