|Molecular Vision 2002;
Received 30 July 2002 | Accepted 20 September 2002 | Published 23 September 2002
Low docosahexaenoic acid levels in rod outer segments of rats with P23H and S334ter rhodopsin mutations
Anderson,1,2,4 Maureen B. Maude,2,3 Mark
McClellan,2,3 Michael T. Matthes,4 Douglas Yasumura,4
Matthew M. LaVail4,5
Departments of 1Cell Biology and 2Ophthalmology, University of Oklahoma Health Sciences Center; 3Dean A. McGee Eye Institute, Oklahoma City, OK, USA; 4Beckman Vision Center and 5Department of Anatomy, University of California San Francisco, San Francisco, CA, USA
Correspondence to: Robert E. Anderson, MD, PhD, Department of Ophthalmology, University of Oklahoma Health Sciences Center, 608 Stanton L. Young Blvd., Oklahoma City, OK 73104; Phone: (405) 271-8250; FAX: (405) 271-8128; email: firstname.lastname@example.org
Purpose: Previous studies have shown that the level of docosahexaenoic acid (22:6n-3, DHA) is lower in the rod outer segment (ROS) membranes of dogs and mice with inherited retinal degeneration than in ROS from appropriate controls. In the present study, we analyzed the ROS fatty composition of several lines of transgenic rats with P23H and S334ter rhodopsin mutations. Lines were chosen that have different rates of retinal degeneration.
Methods: At 21-22 days of age, animals were perfused and eyes fixed and sectioned for morphologic examination. Others were killed and retinas isolated for preparation of ROS by sucrose step-gradient centrifugation. Fatty acid composition of ROS phospholipids was determined by gas-liquid chromatography. Membrane purity was assessed by polyacrylamide gel electrophoresis.
Results: Retinas of the slow degenerating lines were indistinguishable from controls, whereas there was a 15-20% and 50-60% loss of photoreceptor cell nuclei in intermediate and fast degenerating lines, respectively. Except for the slow P23H line, all mutant lines had lower levels of 22:6n-3 and total n-3 fatty acids in ROS phospholipids, compared to wild-type controls, and the level of 22:6n-3 was lowest in those lines with the fastest rate of degeneration. The relative levels of the other fatty acid families (saturated, monoenoic, and n-6) increased proportionately. The n-6/n-3 ratio increased in the more rapidly degenerating lines, but the phospholipid/protein ratios did not change. The low levels of 22:6n-3 in the ROS membranes were not compensated for by an increase in 22:5n-6, which always occurs in the retina of animals where 22:6n-3 levels are reduced by dietary manipulation.
Conclusions: Rats that express mutant rhodopsins have lower levels of 22:6n-3 in their ROS phospholipids than wild-type animals. We propose that photoreceptor-specific mutations provoke a metabolic stress in rod photoreceptor cells that generates an oxidant stress in these cells. The retina responds to this stress by reducing the level of substrate for lipid peroxidation (22:6n-3).
Retinal rod outer segment membranes (ROS) contain a high level of docosahexaenoic acid (DHA, 22:6n-3), a long chain polyunsaturated fatty acid (PUFA) of the linolenic acid family (18:3n-3) . The role of 22:6n-3 in the retina has not been exactly defined, although dietary deprivation of this essential fatty acid or its precursors leads to changes in retinal function in rats [2-5], guinea pigs [6-8], and monkeys [9-13]. Furthermore, pre-term and term human infants fed formulas without 22:6n-3 or its precursors have a slower development of the visual system as determined by measurements of visual acuity and visual evoked responses [14-18]. Longer term effects on intelligence quotient [19,20] and stereoscopic vision [18,21] have also been shown to be related to breast-feeding, which provides 22:6n-3, or to the inclusion of 22:6n-3 in formulas of pre-term human infants. Recent studies of Litman and colleagues have shown that the equilibrium between metarhodopsin I and metarhodopsin II [22,23] and the formation of the meta II-transducin complex [24,25] may be influenced by the level of 22:6n-3 in the phospholipid bilayer. Therefore, there is ample evidence that 22:6n-3 is essential for optimal development and function of the visual system.
A number of years ago, Converse and her colleagues demonstrated that the blood levels of 22:6n-3 and other long chain PUFA were lower in patients with retinitis pigmentosa (RP) than in appropriate controls [26,27]. This finding was quickly confirmed by a number of laboratories [28-36], and it is now quite clear that patients with RP, regardless of mode of inheritance or genotype, have a significantly lower level of 22:6n-3 and other long chain PUFA in plasma and red blood cells compared to control populations. Hoffman et al. , in a careful analysis of patients with X-linked RP (XLRP), showed a dramatic reduction in plasma and RBC 22:6n-3 levels that correlated with retinal function; the patients with the lowest blood levels of 22:6n-3 had the lowest electroretinographic amplitudes and the fastest loss of photoreceptor function. In a recent study, Hoffman et al.  gave an oral dose of 13C-18:3n-3 to XLRP patients and found reduced conversion to 22:6n-3 compared to age-matched controls, suggesting that the reduced levels of 22:6n-3 in these patients could be due to a down-regulation of certain enzymes involved in synthesis of long chain PUFA from shorter chain precursors.
In parallel with the human studies, we found that the blood of dogs with progressive rod-cone degeneration (prcd) had lower blood levels of 22:6n-3 than controls . Subsequent analysis of the ROS of affected animals showed that they also had lower levels of 22:6n-3 , although, ironically, the livers of affected dogs had significantly elevated amounts of 22:6n-3 . To test the hypothesis that the retinal degeneration in these animals may be due to the reduced levels of 22:6n-3 in their ROS, affected and control animals were given supplements of fish oil or placebo oil for four months. At the end of the clinical trial, the 22:6n-3 levels in blood, ROS, and other tissues of affected animals were increased by fish oil supplementation . However, the supplementation had no effect on the rate of retinal degeneration in these animals.
To determine if the reduced level of 22:6n-3 in ROS of animals with inherited retinal degeneration occurs in other species, we analyzed the fatty acid composition of ROS membranes from mice with rds/peripherin  or P216L peripherin mutations  and two separate lines of mice with a G90D rhodopsin mutation . A consistent finding was a lower level of 22:6n-3 in isolated ROS membranes, which suggested that this might be a general phenomenon among animals with different retinal degeneration genotypes. In the present study, we analyzed the fatty acid composition of ROS from transgenic rats with P23H and S334ter rhodopsin mutations. Lines expressing different levels of the transgene had different rates of retinal degeneration. Our results show that mutant lines have lower 22:6n-3 in their ROS and that the level of 22:6n-3 is lowest in those animals with the fastest rate of retinal degeneration.
Animals and retinal phenotype
Transgenic rats with P23H or S334ter mutations in rhodopsin  were produced by Chrysalis DNX Transgenic Sciences, (Princeton, NJ) on an albino Sprague-Dawley background and maintained at UCSF in a 12 h:12 h light-dark environment at an in-cage illuminance level of approximately 120-160 lux. The P23H rats express a mouse opsin gene with a proline to histidine substitution at codon 23, and the S334ter rats express a mouse opsin gene bearing a termination codon at residue 334, resulting in a C-terminal truncated opsin protein. The rats are bred so that they carry a single copy of the transgene and two copies of the wild-type rhodopsin gene. Different lines of each type of mutation express different levels of mutant opsin compared to native rhodopsin, with correspondingly different rates of photoreceptor degeneration . We studied P23H rats from lines 2, 3, and 1, which show relatively slow, intermediate, and fast rates of degeneration, respectively, and S334ter rats from lines 9, 4, and 5, which show similar slow, intermediate, and fast rates, respectively.
All experiments were carried out on rats at 21-22 days of age. To document the status of the retina at the time of retina removal for biochemical analyses (below), some rats from all lines were euthanized by overdose of carbon dioxide, and their eyes were prepared for histological examination with epoxy resin sections as described elsewhere . All procedures involving the rats adhered to the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research and were approved by the University of California, San Francisco Committee on Animal Research.
Preparation of rod outer segment membranes
The method of preparing ROS membranes was a modification  of the discontinuous sucrose gradient centrifugation procedure originally described by Papermaster and Dryer . All solutions were saturated with argon to minimize lipid oxidation during the preparation of ROS membranes. Rats were euthanized by overdose of carbon dioxide inhalation and cervical dislocation, and the retinas were removed from the eyes by "winkling" , pooled (4 per tube), and frozen in tubes on dry ice. The tubes were then shipped from the laboratory of MML to REA on dry ice where they were homogenized in 6 ml of 1.175 g/ml sucrose buffered with 10 mM Tris-acetate (pH 7.4) containing 70 mM NaCl, 2 mM MgCl2, and 0.1 mM EGTA. The homogenate was transferred to a 17-ml centrifuge tube and overlaid sequentially with 6 ml of 1.140 g/ml sucrose, followed by 5 ml 1.115 g/ml sucrose. The tube was spun at 82000 g for 1 h at 4 °C in an ultracentrifuge equipped with a swinging bucket rotor. The sucrose interfacial band containing the ROS membranes (1.115/1.140) was removed and diluted with 4 volumes of 50 mM Tris-acetate (pH 7.4) containing 5 mM MgCl2 and 0.1 mM EDTA. The ROS membranes were pelleted at 27000 g for 1 h and washed once more with the same buffer. In general, the yield of ROS membranes at the 1.115/1.140 interface is greater than 80%, based on previous comparisons of rhodopsin recovery. Purity of membrane preparations was determined by SDS-polyacrylamide gel electrophoresis. Protein was determined by BCA reagent according to the manufacturer's instructions.
Lipid extraction and analysis
Lipids were extracted from ROS by the procedure of Bligh and Dyer . To obtain total phospholipids, the lipid extracts were spotted on a thin layer chromatoplate, which was developed in hexane:diethyl ether:glacial acetic acid (75:35:1, by volume). Phospholipids, which remained at the origin, were scraped from the plate and converted to methyl esters by heating at 100 °C for 2 h in 2% sulfuric acid in anhydrous methanol. An internal standard of 17:0 and 21:0 was added prior to methanolysis. Methyl esters were analyzed by capillary gas liquid chromatography (GLC) on a Varian 3700 gas chromatograph equipped with a DB-225 (30 m X 0.25 mm I.D., J& W Scientific, Folsom, CA) fused silica capillary column. A split injection mode of 1:30 was utilized and the esters were eluted from the column with the linear velocity of helium carrier gas at 40 cm/sec. The column temperature was programmed from 160 to 220 °C at 1 °C/min. Injector and hydrogen flame detector temperatures were maintained at 240 and 270 °C, respectively. Fatty acid methyl esters were identified by comparison of their relative retention times with authentic standards and the relative mole percentages were calculated. Results are reported as relative mole percent.
Differences between groups were determined by the Student's t-test. A p-value of <0.05 was set as our criterion for significance.
For both the P23H and S334ter transgenic rats, we chose three lines that had different rates of degeneration. The P23H-2 and S334ter-9 lines had a slow degeneration that was still not complete by one year of age (LaVail, M.M., unpublished data). At 21-22 days of age, their retinas were indistinguishable from wild-type control retinas (cf. Figure 1B and Figure 1E with Figure 1A). Those lines with fast rates of degeneration (P23H-1, Figure 1D; S334ter-5, Figure 1G) had only about 50-60% of their photoreceptors remaining at 21-22 days of age, and their photoreceptor inner and outer segments were significantly shorter than normal. The lines with intermediate rates of degeneration (P23H-3, Figure 1C; S334ter-4, Figure 1F) still had about 80-85% of their photoreceptors remaining at 21-22 days of age.
Purity of ROS membranes
Polyacrylamide gels were run on most ROS preparations and representative silver-stained gels are shown in Figure 2. In all but one line, the major protein was rhodopsin and the gels appeared to be similar for preparations of membranes from wild-type and mutant animals. However, in line 5 of the S334ter animals, the yield of ROS membranes was quite low and obviously contaminated with other proteins. Fatty acid values from these membranes are not reported.
Fatty acid composition of ROS phospholipids from S334ter rhodopsin mutant rats
The fatty acid composition of the phospholipids from wild-type and the slow and intermediate degenerating line of S334ter rhodopsin mutant rats is presented in Table 1. Two saturated fatty acids, palmitic (16:0) and stearic (18:0), and the major PUFA 22:6n-3 make up over 75% of the fatty acids of ROS phospholipids. Arachidonic acid (20:4n-6) is the major n-6 component in ROS, but is present in much lower levels than 22:6n-3. In the wild-type ROS, the n-6/n-3 ratio was 0.25, indicating four times more n-3 PUFA in these membranes than n-6 PUFA. In the slow degenerating S334ter-9 animals, there were several significant changes in fatty acid composition compared to wild-type. The level of oleic (18:1n-9) was significantly increased, as was 20:4n-6 and 22:4n-6; however, the level of 22:6n-3 was significantly lower. In line S334ter-4, which has a faster rate of retinal degeneration, the fatty acid differences were more striking and the differences from wild-type values had a higher level of significance. There were also differences between the two S334ter transgenic lines. In line S334ter-4 (intermediate), the level of saturated fatty acids was higher, while n-3 PUFA were significantly lower than in the S334ter-9 ROS. The phospholipid to protein ratios were not significantly different between wild-type and mutant groups.
Fatty acid composition of ROS phospholipids from P23H rhodopsin mutant rats
The fatty acid composition of ROS from wild-type and P23H mutant lines is shown in Table 2. There was no significant difference between values for the slowest degenerating line (P23H-2) compared to wild-type animals, although the pattern of lower n-3 PUFA was evident. However, in line P23H-3 (intermediate) and line P23H-1 (fast), differences of high significance were found compared to wild-type values. As observed for the S334ter mutant ROS, there was significant increases in 16:0, 18:0, 18:1n-9, and 20:4n-6, and reductions in 22:6n-3 and total n-3 fatty acids. There were no significant differences between line P23H-2 (slow) and line P23H-3 (intermediate). However, line P23H-1 (fast) had higher levels of saturated, monoenoic, and n-6 fatty acids, and lower levels of 22:6n-3 and total n-3 PUFA than lines 2 or 3. The n-6/n-3 ratio was significantly elevated in ROS from the intermediate and fast degenerating animals, compared to wild-type, but there were no significant differences in the phospholipid/protein ratios.
The n-6 PUFA increased and the n-3 PUFA decreased in the mutant membranes of both transgenic lines, with two notable exceptions. In the n-6 family, 24:4n-6 progressively decreased in the faster degenerating lines, while the levels of 22:5n-3 progressively increased.
We examined the 22:6n-3 levels in ROS from rats with two mutations known to cause degenerations in humans and found that animals with P23H or S334ter rhodopsin mutations have lower 22:6n-3 compared to controls, and the level is lowest in those animals with the fastest rate of retinal degeneration. In these studies, which were carried out on ROS prepared by differential sucrose gradient centrifugation, we examined the membranes by SDS polyacrylamide gel electrophoresis and determined that the level of purity was similar for all preparations, except for line S334ter-5 transgenic rats, which was not used for fatty acid analysis. Therefore, the fatty acid differences we report are due to differences within the ROS phospholipid bilayer rather than to contaminating membranes from other retinal organelles. Bicknell et al.  have also recently reported that the ROS of P23H-3 rats have lower DHA than ROS from wild-type animals.
The high levels of n-3 fatty acids in retina and brain must be derived from dietary sources . Attempts to reduce the level of these fatty acids in neural tissues is met with some resistance, as both retina  and brain  have mechanisms to conserve these fatty acids during dietary deprivation. Nevertheless, retinal changes can be made with a long-time deprivation of n-3 fatty acids . These diet-induced changes, however, are always accompanied by an increase in the levels of n-6 fatty acids, particularly 22:5n-6 [50-52]. Since 22:5n-6 did not increase in the present study or in other studies where 22:6n-3 levels were reduced in animals with inherited retinal degenerations, we conclude that the reduction in 22:6n-3 in ROS is not due to dietary or metabolic restriction of 22:6n-3 or shorter-chain n-3 precursors.
In our early studies on humans with RP, we suspected that the lower blood levels of 22:6n-3 may reflect the genotype of the patient . However, it is now clear that with the large number of genotypes and multiple mutations within the same gene (see Retinal Information Network), the reduced level of 22:6n-3 in the blood of RP patients is not directly related to the mutation. This is also true for the animals we analyzed with inherited retinal degenerations, including the prcd-affected dogs [39,53]; Abyssinian cat ; Briard dog ; mice with rds/peripherin , P216L peripherin , or G90D  mutations; and the P23H and S334ter results reported herein. Thus, we are dealing with a general phenomenon in which animals with a photoreceptor-specific mutation in a gene unrelated to fatty acid metabolism have lower levels of 22:6n-3 in their ROS membranes. Interestingly, a mutation in a gene involved in long-chain fatty acid metabolism in yeast has been identified in two forms of autosomal dominant macular dystrophy . However, serum lipid analysis of these patients has not yet been published.
It was originally thought that the reduced level of 22:6n-3 in blood and ROS membranes of humans with RP and prcd-affected dogs was causally related to the retinal degeneration. To test this hypothesis, Aguirre et al. carried out two clinical trials in prcd-affected dogs. In the first , animals were placed on fish oil or placebo supplementation at five months of age and continued until they were nine months old. ERG and histological evaluation revealed no protective effect of fish oil supplementation. In a second as yet unpublished clinical trial [Aguirre, Acland, and Anderson, unpublished data], affected and control female dogs were started on supplementation with 22:6n-3 capsules or placebo oil prior to conception, and the mothers were continued on the supplement throughout the nursing period. After weaning, pups were placed on 22:6n-3 or placebo supplements and continued through nine months of age. Although 22:6n-3 supplementation increased the level of 22:6n-3 in the ROS of affected animals above that of unaffected controls fed placebo oil, there was no effect on the rate of retinal degeneration in the 22:6n-3 supplemented animals. Thus, it is clear that, at least in the prcd-affected dogs, the rate of retinal degeneration is not dependent on the level of 22:6n-3 in the membranes, and that 22:6n-3 supplementation, even if started prior to conception, does not reduce the rate of degeneration in these animals.
Why are levels of 22:6n-3 lower in animals with an inherited retinal degeneration? We have previously proposed that expression of an abnormal protein places photoreceptor cells under a higher metabolic stress than wild-type animals [57,58]. Synthesis of mutant proteins, which may or may not be incorporated into outer segments, increases the catabolic activity in the inner segment. If this involves the ubiquitin system, which is active in the retina , there would an increased energy requirement and therefore increased probability of generating reactive oxygen species. We suggest that the metabolic stress is an oxidant stress and the reduction in 22:6n-3 is an adaptive response to reduce the level of PUFA substrates of lipid peroxidation. This adaptive response is similar to what we observed in albino rats raised in bright cyclic light [60-63], where the level of 22:6n-3 was reduced significantly compared to dim-reared animals and there was no concomitant increase in 22:5n-6 . Reduction of 22:6n-3 under these conditions would decrease the likelihood of lipid peroxidation. However, we cannot rule out the possibility that the retinas with the lowest levels of 22:6n-3 have the highest rate of lipid peroxidation. In this scenario, we would predict that these retinas would also have the fastest rate of degeneration, which is what we found in the present study. We would further predict that supplementing these animals with 22:6n-3 or other n-3 fatty acids could exacerbate the rate of degeneration by providing an abundant source of lipid peroxidation substrates. However, in our feeding experiments with prcd-affected dogs, we did not find any evidence of increased retinal degeneration in the n-3 supplemented groups .
How are the lower levels of 22:6n-3 generated and maintained in animals with inherited retinal degenerations? There are at least five possible mechanisms, including: (1) decreased synthesis of 22:6n-3 from shorter chain n-3 fatty acid precursors, (2) decreased incorporation of 22:6n-3 into retinal phospholipids, (3) selective oxidation of 22:6n-3 and other n-3 fatty acids, (4) reduced uptake of 22:6n-3 from the blood into the retina, or (5) reduced association of 22:6n-3-containing phospholipids with newly synthesized membranes destined for the outer segment. There is some support for several of these possibilities. In our study, the precursors of 22:6n-3 and 22:5n-6 (22:5n-3 and 22:4n-6, respectively) were slightly increased in the ROS phospholipids of the mutant animals, while their products (24:5n-3 and 24:4n-6, respectively) were decreased, suggesting a reduction in the elongation step that adds a 2-carbon unit (first possibilty). Hoffman et al.  drew a similar conclusion from their fatty acid analysis of blood from XLRP patients and their more recent stable isotope study described in the Introduction . However, we found lower levels of 22:6n-3 in ROS of prcd-affected dogs fed large amounts of fish oil, which contain high levels of 22:6n-3 , compared to normal controls, so other possibilities must also pertain. We found reduced synthesis of phospholipid molecular species that contain 22:6n-3 (second possibility), in rats raised in bright cyclic light (Anderson, R.E., unpublished data), a condition that leads to reduced 22:6n-3 levels in ROS phospholipids [52,60]. However, we have not yet carried out these experiments on mutant rats. In the stable isotope study of Hoffman et al. , careful measurements of expired 13CO2 did not show any increase in 18:3n-3 oxidation in affected patients (third possibility). Systemic oxidation would lead to a deficiency in these essential fatty acids and thus reduce their availability for synthesis of 22:6n-3. If this were the case, we would expect to find increased 22:5n-6, as discussed above. Since this did not occur, we do not favor the possibility of extensive systemic oxidation of n-3 PUFA. Whether selective oxidation of n-3 PUFA occurs in the retinas remains to be determined. Rodriguez de Turco et al.  found that newly synthesized membranes containing rhodopsin were enriched in 22:6n-3 in newly synthesized phosphatidylcholine and phosphatidylethanolamine, the two major phospholipids of ROS . Since Bicknell et al.  found lower rhodopsin levels in retinas of P23H rats, it is possible that the ROS membranes of transgenic rats contain a lower packing density of rhodopsin than wild-type, which could result in lower levels of 22:6n-3 being incorporated into the membranes (fifth possibility). However, Bicknell et al.  did not determine the packing density of rhodopsin in the ROS, and our measurements of protein/phospholipid ratios in purified ROS did not reveal any differences between transgenic and wild-type animals. Thus, while there may some hints about the mechanism of reduction of 22:6n-3 in the retinas of animals with inherited retinal degeneration, the actual manner by which the reduction is achieved is still a mystery.
In summary, we have found that transgenic rats expressing a mutant rhodopsin protein known to cause retinal degenerations in humans have a reduced ROS level of 22:6n-3, an essential fatty acid that is important in maintaining optimal function of the retina. While the reduction in 22:6n-3 does not appear to contribute directly to the degeneration, the consistent finding of low ROS levels suggests that this fatty acid may be involved indirectly in the process, perhaps as an indicator of increased oxidant stress in the photoreceptor cells.
This work was supported by grants from the National Institutes of Health/National Eye Institute (EY00871, EY01919, EY02162, EY04149, EY06842 and EY12190), Research to Prevent Blindness, Inc., New York, NY; The Foundation Fighting Blindness, Baltimore, MD; The Macula Vision Research Foundation, Samuel Roberts Nobel Foundation, Inc., Ardmore, OK; and Presbyterian Health Foundation, Oklahoma City, OK. MML is an RPB Senior Scientist Investigator.
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