Molecular Vision 2004; 10:199-207 <http://www.molvis.org/molvis/v10/a25/> Received 20 December 2003 | Accepted 10 March 2004 | Published 26 March 2004

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P23H and S334ter opsin mutations: Increasing photoreceptor outer segment n-3 fatty acid content does not affect the course of retinal degeneration

Rex E. Martin,^1,2,3 Isabelle Ranchon-Cole,^2,3 Richard S. Brush,^2,3 Clint R. Williamson,^2,3 Steven A. Hopkins,^2,3 Feng Li,^2,3 Robert E. Anderson^1,2,3
 
 

Departments of ¹Cell Biology and ²Ophthalmology, University of Oklahoma Health Sciences Center; ³Dean A. McGee Eye Institute, Oklahoma City, OK

Correspondence to: Rex E. Martin, PhD, Department of Cell Biology, Dean A. McGee Eye Institute, University of Oklahoma Health Sciences Center, 608 Stanton L. Young Boulevard, Oklahoma City, OK, 73104; Phone: (405) 271-7366; FAX: (405) 271-8128; email: rex-martin@ouhsc.edu

Abstract

Purpose: The n-3 polyunsaturated fatty acids (PUFA) facilitate retinal development and function. Rats carrying transgenes with P23H and S334ter rhodopsin mutations lose their photoreceptors and have lower levels of 22:6n-3 in rod photoreceptor outer segments (ROS) than wild type (WT) animals. We tested the hypothesis that the rate of retinal degeneration in these mutant animals could be sensitive to the n-3 fatty acid content of retina.

Methods: Beginning embryonic day 15, WT and heterozygous transgenic rats with P23H and S344ter rhodopsin mutations were fed semi-synthetic diets enriched in n-6 (safflower oil, SO) or n-3 (flaxseed oil, FO) PUFA. At 35 and 55 days of age, electroretinographic (ERG) response, outer nuclear layer (ONL) thickness, and fatty acid composition of plasma and ROS were determined. Student's t-tests and multivariate analysis of variance with post hoc tests determined statistical differences.

Results: Rats fed FO or SO diets had different n-6/n-3 PUFA ratios in plasma (1.3 and 62) and ROS (0.2 and 1.1, respectively). Although there were profound effects of the diets on the plasma fatty acid composition, there were only minor differences between WT and transgenic animals within each dietary regime. The ROS of FO fed rats had 70% more 22:6n-3 than those fed SO, and the WT had higher concentrations of 22:6n-3 than the transgenic animals (WT>P23H>S334ter). In contrast, there was no difference in 22:6n-3 levels in ROS of WT and transgenic rats fed the SO diet. At P55, both transgenic lines had diminished ERGs and ONL thickness relative to the WT. There was no detectable effect of ROS fatty acid enrichment on the rate of retinal degeneration in the transgenic animals. However, the FO-diet provided a modest protection of function (b-wave) in S334ter animals.

Conclusions: Feeding n-3 fatty acids to rats with mutant rhodopsin transgenes significantly increased the levels of 22:6n-3 in ROS membranes, but had no effect on the rate of retinal degeneration. Therefore, the degeneration is not the result of low (or high) 22:6n-3 in ROS and supplementation with 18:3n-3 will not rescue dying photoreceptor cells in these animal models of inherited retinal degenerations.

Introduction

Docosahexaenoic acid (DHA, 22:6n-3) is the major long-chain polyunsaturated fatty acid (PUFA) in neuronal tissues and is highly enriched in the disk membranes of photoreceptor rod outer segments (ROS) [1,2]. An essential fatty acid, 22:6n-3 or an n-3 precursor must be obtained from the diet [3]. ROS conserve 22:6n-3 during n-3 fatty acid deficiency [4] by an efficient recycling loop between the retina and the retinal pigment epithelium [5,6], which makes it difficult to deplete n-3 fatty acids in adult rats. Significant reduction in retinal DHA levels can only be achieved by depriving pregnant rats of n-3 fatty acids from the third trimester and throughout the nursing period, and maintaining the pups on an n-3 deficient diet [7,8]. Under these conditions, 22:6n-3 is significantly reduced in ROS phospholipids and is replaced by an equivalent amount of 22:5n-6 [7,8], a PUFA derived from linoleic acid (18:2n-6) and a member of the n-6 family of essential fatty acids. Normally a very minor component of neuronal membranes, 22:5n-6 becomes enriched only during n-3 fatty acid deficiency [4,7-11]. A schematic showing the elongation and desaturation pathways for n-3 and n-6 fatty acids is given in Figure 1.

It has been known for two decades that persons with retinitis pigmentosa (RP) have lower plasma levels of 22:6n-3 than appropriate controls [12-23]. These differences, although small, are consistently found among patients with all forms of RP. The largest differences occur in patients with X-linked retinitis pigmentosa (XLRP) and are correlated with the severity of the disease. Hoffman et al. [19] showed that the individuals with the lowest 22:6n-3 in blood also had the weakest electroretinographic response and the most rapid loss of vision.

Lower 22:6n-3 levels were also found in blood of dogs with progressive rod-cone degeneration (prcd) [24-26] and in the Abyssinian cat [27]. Levels of 22:6n-3 were also lower in the ROS of prcd-affected dogs compared to unaffected controls [26]. The ROS from mice with rds/peripherin [28] and G90D rhodopsin [29] mutations have lower 22:6n-3 than controls, but these differences were not found in the blood of rds/peripherin and control animals. Lower 22:6n-3 levels were found in ROS of transgenic rats with P23H rhodopsin [30,31] or S334ter rhodopsin [31] mutations. In both of these transgenic animal models of human disease, the severity of photoreceptor degeneration directly correlated with the degree of 22:6n-3 loss from photoreceptor outer segments [31]. These findings collectively suggest that inherited retinal degeneration is associated with a general phenomenon of reduced 22:6n-3 in ROS phospholipids, with the lowest levels of 22:6n-3 being present in those retinas with the fastest rate of degeneration.

The blood and ROS findings suggested that n-3 fatty acid deficiency could underlie inherited retinal degenerations. To test this hypothesis, studies were undertaken to determine if adding n-3 fatty acids to the diet could change the rate of retinal degeneration. Using the prcd dog model, Aguirre et al [26] reported that fish oil supplements, which contain high levels of n-3 PUFA, did not alter the rate of degeneration, even though the levels of 22:6n-3 increased significantly in the plasma and ROS of affected animals. Bicknell et al. [30] recently showed that P23H transgenic rats could elongate and desaturate n-3 fatty acid precursors to form 22:6n-3, and found that no substantial protection was afforded by increasing the n-3 fatty acids in the diet.

In the present study, we used two strains of transgenic rat with retinitis pigmentosa phenotype (P23H and S334ter) to test the hypothesis that dietary supplementation with n-3 fatty acids could alter the fatty acid content of their retina and thus affect the rate of retinal degeneration. The transgenic and wild type (WT) rats received semisynthetic diets containing either 10% safflower (n-6 rich) or 10% flaxseed (n-3 rich) oil as their only source of essential fatty acids. Dams were started on the diets in the third trimester of fetal development and their pups (the experimental subjects) remained on the diet throughout the study. Our results show that although the diets changed the fatty acid content of plasma and the photoreceptor outer segments, the rate of photoreceptor degeneration was not affected. Results are discussed in light of current hypotheses of neuroprotection and regulation of 22:6n-3 levels in the retina.

Methods

Diets/animals

Dr. Matthew LaVail (University of California, San Francisco) generously provided breeding pairs of homozygous transgenic rats with P23H (line 3) and S334ter (line 4) rhodopsin mutations, which made it possible to establish breeding colonies at the Dean A. McGee Eye Institute. The homozygous P23H rats carry two copies of a mouse opsin gene with a proline to histidine substitution at codon 23. The S334ter rats express a mouse opsin gene that bears a termination codon at residue 334, which encodes a C-terminal truncated opsin protein [32]. Homozygous mutant males were bred to WT female Sprague-Dawley (SDCD, Harlan Sera-Lab; Indianapolis, Indiana) rats to generate heterozygous P23H and S334ter transgenic offspring, which carried a single copy of the transgene and two copies of the wild type rhodopsin gene. Pregnant WT rats and rats carrying heterozygous litters were maintained in dim cyclic light (5 lux, 12 h on/off). From embryonic day 15 through lactation, dams were given one of two isocaloric, semisynthetic diets containing 10% (g/g) dietary fat exclusively derived from flaxseed oil (FO, n-3 enriched) or safflower oil (SO, devoid of n-3 fats; Dyets Inc., Bethlehem, PA). Litters were culled or fostered (as appropriate) to 10 pups of mixed sex. The pups (the subject of this study) were weaned at 21 days of age and remained on their respective diets for the duration of the experiments (35 or 55 days). The fatty acid composition of the diets was determined by gas chromatography (see below). All procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center and the Dean A. McGee Eye Institute approved all protocols.

Plasma and rod outer segment (ROS) preparation

Blood samples (about 1.8 ml) were obtained by cardiac puncture after CO₂ induced unconsciousness and cervical dislocation. Blood samples were centrifuged at 2000x g in EGTA-containing tubes to obtain at least 100 μl of plasma for lipid extraction. Retinas were "winkled" (dissected) from the eyes [33] and four retinas from two animals of the same litter were pooled for ROS preparation following previously described procedures [34,35]. Retinas were homogenized in 10 mM Tris acetate (pH 7.4) containing 70 mM NaCl, 2 mM MgCl₂, and 0.1 mM EGTA, and 1.17 g/ml sucrose. Discontinuous sucrose gradients containing 1.11, 1.13, and 1.17 g/ml were centrifuged at 82,000x g for 70 min. The 1.11/1.13 g/ml interfacial band containing ROS was collected; diluted with 10 volumes of 10 mM Tris acetate (pH 7.4) containing 70 mM NaCl, 5 mM MgCl₂, and 0.1 mM EGTA; and centrifuged at 27,000x g for 30 min. The pellet was resuspended in 100 μl H₂O for lipid extraction.

Lipid extraction

Plasma and ROS total lipids were extracted in chloroform:methanol; 0.33 mM diethylenetriaminepentaacetic acid (DTPA) was added to the aqueous phase as an iron chelator [36]. The purified lipid extracts were concentrated and stored at -80 °C under N₂ in a known volume of chloroform:methanol (2:1, v/v).

Fatty acid derivatization and gas chromatography

Fatty acids in total lipids from ROS and plasma were converted to methyl esters by the procedure of Morrison and Smith [37]. An internal standard mixture made with approximately equal parts of pentadecanoic acid (15:0), heptadecanoic acid (17:0), and heneicosanoic acid (21:0) was added just prior to methylation. Methyl ester products were separated from other lipids (primarily cholesterol) by thin layer chromatography on Silica gel 60 (EM Science; Gibbstown, NJ) using a solvent system of hexane:ether (80:20, v/v). The fatty acid composition of ROS and plasma total lipids was determined by gas chromatography with a DB-225 capillary column (30 m x 0.25 mm I.D.; J & W Scientific, Folsom, CA) and a Varian 3500 gas-liquid chromatograph that was equipped with a model 8100 autosampler (Walnut Creek, CA). The samples were dissolved in nonane and 3 μl of each was injected at 250 °C with the split ratio set to 22:1. The column temperature was programmed to hold at 160 °C for 1 min, then raised to 220 °C at 1 °C/min, and held at 220 °C for 10 min. Helium carrier gas flowed linearly at 42.4 cm/sec (1 ml/min). Hydrogen flame detector temperature was maintained at 270 °C. A Perkin-Elmer (Norwalk, CT) Model 970 System Interface module converted analog output to digital format and the chromatographic peaks were integrated and processed with Turbochrome Navigator® software (Perkin-Elmer). 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.

Electroretinography

Electroretinography (ERG) was used to evaluate retinal function. Rats were dark-adapted overnight, anesthetized with an intramuscular injection of a mixture of ketamine (120 mg/kg) and xylazine (6 mg/kg), and their pupils were dilated with 1% tropicamide. The ERGs were recorded using UTAS-E 2000 Visual Electrodiagnostic System (LKC Technologies, Gaithersburg, MD). Gold contact lens electrodes were placed on the eyes, a gold reference electrode was placed on the tongue and a ground electrode on the foot. The animal's eye was placed in a integrating sphere (Labsphere, North Sutton, NH). Full field scotopic ERGs were elicited with 10 ms flashes of white light starting at threshold intensity and increasing to saturation. The interval between flashes was 60 s. Sensitivity curves plotted the a-wave and b-wave amplitude of the ERG as a function of stimulus intensity (log neutral density filter). The software program Origin 6.0 (Microcal, Inc, Northampton, MA, USA) was used to fit the data of each rat, giving the saturated a-wave (A_max) and b-wave (B_max) amplitudes.

Histology and morphometry

Rats were killed with CO₂ asphyxiation. Eyes were enucleated, placed in fixative (4% paraformaldehyde, 20% isopropyl alcohol, 2% TCA, 2% ZnCl₂) for 72 h, transferred to 70% ethanol for 72 h, and embedded in paraffin. Sections of 5 μm were cut in the vertical meridian through the optic nerve and the thickness of the outer nuclear layer (ONL) was determined in 0.5 mm increments from the optic nerve to the superior and inferior ora serrata. The values were plotted to show regional effects (see Results).

Statistics

Experimental n for histology (ONL thickness) and ERG was 4 to 7 rats, where each rat was taken from a different litter. Fatty acid analysis was done on three independent ROS preparations of four pooled retinas (2 rats per litter) of 35 and 55 day old animals. The n for plasma fatty acid analysis was 4-6 rats where each rat was taken from a different litter. Multivariate analysis of variance with post hoc Newman-Keuls tests determined statistical differences for fatty acid composition. Multivariate analysis of variance with post hoc Scheffé tests determined statistical differences between maximal ERG responses. Differences in ONL thicknesses were determined with Student's t-test. In all parts of the statistical analysis, αwas chosen to be 0.05.

Results

Fatty acid content of the diet

The fatty acids in both diets were derived entirely from flaxseed oil (FO) or safflower oil (SO). FO contains about 20% 18:2n-6 and about 50% 18:3n-3, while SO has about 65% 18:2n-6 and about 0.4% 18:3n-3 (Figure 2). Thus, the FO diet is n-3 enriched and the SO diet is n-3 deficient. Since these are seed oils, they do not contain any PUFA of chain length longer than 18 carbons. The differences in PUFA in these two diets are reflected in their extreme n-6/n-3 ratios, which were 0.4 and 189.1 for FO and SO, respectively.

Plasma fatty acid profiles

Feeding FO and SO diets from the third trimester of gestation caused dramatic changes in the plasma fatty acid content in each line of rat. There were no statistically significant, age-specific differences in fatty acid content of plasma, so the values reported in (Figure 3) are the pooled results from 35 and 55 day old animals. The primary effect was attributable to diet. Rats that were fed FO had significantly lower plasma n-6/n-3 ratios than those fed SO (1.3 versus 61.7), which resulted in 4.7 times as much 22:6n-3 and 127 times as much 20:5n-3 as those fed SO. The plasma of FO fed rats had no detectable 22:5n-6, which represented 2.3% of the total in SO fed rats. Levels of other n-6 and n-3 fatty acids were shifted as expected. The FO fed rats had higher plasma 18:3n-3 (12.7% versus 0.1%, not shown) than SO fed rats, while SO fed rats had higher 18:2n-6 (27.1% versus 22.7%, not shown) and 20:4n-6 (26.2% versus 7.6%) than FO fed rats.

Relative to WT rats, there were only two strain-specific effects in plasma and these were seen only in FO fed rats: 27% more 20:5n-3 in P23H rats and 39% less 22:6n-3 in S334ter rats. Although statistically insignificant, there was also a tendency for progressive decrease in plasma 22:5n-6 content in the rats fed SO (WT>P23H>S334ter).

Rod outer segment fatty acid profiles

The fatty acid composition of ROS membranes was less sensitive to dietary manipulation than the plasma, but the trends were similar (Figure 3). There was no effect of age, so the values from 35 and 55 day old animals were averaged. When values from the three strains were averaged, ROS from rats fed FO had significantly lower n-6/n-3 ratios than those fed SO (0.2 versus 1.1, respectively). They also had 1.7-times more 22:6n-3 and 3.6 times more 20:5n-3 than those fed SO, whereas the SO fed animals had 78-times more 22:5n-6 and 1.23-times more 20:4n-6 than the FO fed groups. In all three groups fed SO, 22:6n-3 was replaced with 22:5n-6. The average 22:6n-3 level for all FO fed rats was 34.5% of the total fatty acids in the ROS membranes, compared to 20.4% in the SO fed groups, which also contained 12.5% 22:5n-6. The combined percentage of 22:6n-3 and 22:5n-6 (32.9%) is not statistically different from 22:6n-3 level in the FO fed group.

Unlike the plasma, the fatty acid composition of the ROS was profoundly affected by the P23H and S334ter transgenes (Figure 3). In the FO fed animals, ROS from S334ter rats had higher levels of 20:5n-3 and lower levels of 22:6n-3 than WT rats. The level of 22:6n-3 in P23H ROS was also lower than in WT; 20:5n-3 was higher in the P23H membranes, but the difference was not significant. There were only traces of 22:5n-6 in the ROS of the FO-diet group, but the 20:4n-6 levels were significantly higher in both transgenic groups than in the WT.

In the SO fed rats, 20:4n-6 was higher in P23H and S334ter than in WT, although 22:5n-6 was lower in both transgenic groups. Interestingly, 20:5n-3 was also higher in the ROS of the S334ter animals than in the WT and there was no difference in the level of 22:6n-3 in the three groups. Regardless of diet, there was a significant increase in precursor fatty acids (20:5n-3 and 20:4n-6) in the ROS of transgenic rats compared to WT controls, and a decrease in the products (22:6n-3 and 22:5n-6, respectively), with the S334ter animals showing the greatest effect.

Effect of diet on retinal structure

The effect of diet on retinal structure was determined by measuring ONL thickness along the vertical meridian, from the optic nerve head to superior and inferior ora serrata. The only difference relative to WT in the 35 day old rats was a decreased ONL thickness in the S334ter rats (data not shown). By 55 days of age however, differences between the WT and transgenic animals were more significant. Detailed analyses of the ONL thicknesses in 55 day old rats are reported in Figure 4. The overlap between the curves in each of these figures indicates that there were no significant effects of diet on photoreceptor cell survival, although the loss of rod nuclei in the transgenic animals is evident. Figure 5 reports the average ONL thicknesses and clearly demonstrates that both transgenic lines had significantly thinner ONLs and thus fewer photoreceptors than the WT rats. The reduction relative to WT in ONL thickness was more severe in the S334ter rats (26%) than the P23H rats (11%).

Effect of diet on retinal function

Plots of ERG responses as a function of flash intensity show clear differences between transgenic and WT animals (Figure 6). In these typical V-log I plots, there is some suggestion that the amplitudes of the b-waves, and possible the a-wave of the S334ter rats, may be affected by diet. To examine these potential differences in greater detail, the maximal ERG responses for the a-waves and b-waves were calculated (A_max and B_max, respectively), and the results are presented in (Figure 7). The transgenic animals had significantly lower ERG responses than the WT rats in every instance except one, the b-wave of FO fed P23H rats (p=0.051). There was no effect of diet within a line, except for the B_max of S334ter rats, which was 38% greater in the FO fed rats. There was only one difference between the two transgenic strains; in FO fed rats, the A_max was 40% lower in the S334ter rats.

Discussion

Retinal degenerations have many etiologies and are of many subtypes. They all however, lead eventually to photoreceptor cell death and blindness [28,29,38-44]. Numerous investigations have shown that the high levels of 22:6n-3 present in normal retinas is reduced in photoreceptor outer segments in the cells that are destined to degenerate [26,28-31,45]. There are also numerous investigations showing that levels of 22:6n-3 in the blood are diminished during retinal degeneration [12-15,17-22,46]. These results, obtained in dogs, cats, mice, and humans, led to the hypothesis that photoreceptor degeneration might be slowed or prevented if the animals were supplemented with 22:6n-3.

In the present study, we fed diets enriched with n-6 or n-3 fatty acids to rats that carried transgenes for either the P23H or the S334ter rhodopsin mutations and found that, even though the diets increased plasma and photoreceptor outer segment 22:6n-3 levels, they could not alter the course of degeneration. This finding validates the idea that dietary n-3 fatty acid supplementation will not preserve retinal structure, at least in these rhodopsin mutation induced degenerations [30] or in dogs with progressive rod-cone degeneration [26]. Whether n-3 fatty acid supplementation will protect retinas with other mutations is not known.

An interesting finding was that the B_max value for S334ter rats was greater in animals fed the FO-diet, and the A_max values in WT and P23H rats fed FO were not different. Although far from compelling, these results suggest that feeding n-3 fatty acids may provide some functional protection to these retinas. Our laboratory demonstrated over 30 years ago that n-3 fatty acids could modulate retinal function [47,48]. Whether the functional protection noted in this study is real will require additional studies.

With the possible exception of the ELOVL4 mutation [42], no primary mutation leading to a retinal degeneration has been found in any gene that encodes enzymes involved in fatty acid metabolism. Therefore, the reduction in 22:6n-3 in ROS of mutant animals occurs for still unknown reasons. Our early thoughts were that the reduction in 22:6n-3 in ROS led to the degeneration [15]. This is clearly not the case, as demonstrated in this and other studies [26,30]. Alternatively, we hypothesized that the reduction in retinal 22:6n-3 levels was a protective adaptive response to an oxidant stress caused by the mutation [49,50], similar to what we have seen in albino rats raised in bright cyclic light [8,51-54]. However, if this were the case, we would expect that supplementation with n-3 fatty acids might enhance the rate of retinal degeneration. This was not observed in the current and two other studies [26,30]. One caveat worth mentioning, however, is that the two studies in transgenic rats were done under dim cyclic lighting conditions. Whether n-3 supplementation would affect photoreceptor demise in mutant animals challenged by light stress remains to be determined.

These studies show quantitative relationships between the ratios of fatty acids in ROS. Two things are of particular interest. First, the comparative levels of 22:6n-3 and 22:5n-6 in SO fed versus FO fed rats. Second, the degeneration-associated loss of ROS 22:6n-3 in FO fed versus SO fed rats. Regarding the former, the ROS of all three strains of SO fed animals had about 20% 22:6n-3, while the WT animals had 19% and both lines of transgenic rat had about 9% 22:5n-6. Summing the 22:5n-6 and 22:6n-3 of the SO fed animals, the two fatty acids combined represent about 28-39% of the total FA in ROS, which is near their total in the FO fed animals. Thus, it seems that the quantitative replacement of 22:6n-3 by 22:5n-6 that occurs in dietary n-3 fatty acid deprivation [4,7-11] also occurs in the transgenic animals. Regarding our finding of a degeneration-associated loss of ROS 22:6n-3 in FO fed rats (significant loss) versus SO fed rats (no loss), Bicknell et al. [30] described a pool of ROS 22:6n-3 that was resistant to disease-related loss. Our data confirm their finding and suggest that there may be two pools of 22:6n-3 in the retina. The first "preferred pool" predominated in SO fed rats and was retained in the diseased, FO fed rats. The second "expendable pool" was sensitive to both dietary n-3 fatty acid deprivation and photoreceptor disease, and is likely to be the pool of 22:6n-3 that is replaced by 22:5n-6 when n-3 fatty acids are deficient in the diet.

The mechanism by which 22:6n-3 is reduced in ROS of mutant animals is not known. We have previously shown that prcd-affected dogs can synthesize 22:6n-3 from n-3 precursors [55,56]. In the current study, dietary 18:3n-3 was elongated to 22:6n-3 in P23H and S334ter rats, confirming the report of Bicknell, et al. [30] which showed that P23H rats can synthesize 22:6n-3. Furthermore, in our study, there is an indication that C-22 n-6 and C-22 n-3 PUFA metabolism is compromised in the mutant animals. Figure 3 reports a statistically significant and progressive decrease in ROS 22:6n-3 content (WT>P23H>S334ter), which was accompanied by increases in its precursor, 20:5n-3 (S334ter>P23H>WT). Similarly, the transgene-associated decrease in ROS 22:5n-6 was accompanied by an increase in its precursor 20:4n-6 (WT<P23H<S334ter). We previously noted and commented on this same pattern in ROS of P23H and S334ter rats fed rat chow [31]. Within each mutant group of that study, there was also a direct correlation between the rate of degeneration and accumulation of precursor n-3 and n-6 PUFAs (20:4n-6, 22:4n-6, and 22:5n-3). Correspondingly, there was a graded decrease in the levels 22:6n-3, 24:5n-3, and 24:4n-6 in the lines with the fastest rates of degeneration (refer to (Figure 1) for the precursor-product relationships). These results suggest that there may be a down-regulation of enzymes that convert C-20 and C-22 precursor PUFAs to C-22 products. Synthesis of 22:6n-3 and 22:5n-6 requires peroxisomal beta-oxidation of 24:5n-6 and 24:6n-3, as does retroconversion of 22:5n-6 to 20:4n-6 and 22:6n-3 to 20:5n-3, respectively [57,58]. Peroxisomal disfunction can lead to photoreceptor cell death as reported for peroxisomal-associated diseases such as Zellweger Syndrome [59]. Our results suggest that peroxisomal PUFA metabolism may be compromised in the retina of these transgenic animals. This might at once help explain the mechanism of cell death in retinal degeneration and the finding that 22:6n-3 content is reduced in the ROS of all animals examined to date with an inherited retinal degeneration. It is possible that peroxisomes may be affected in retinal degenerations and the effect we are seeing is only one of a number of important reactions that occur in these organelles, one of which compromises the cell sufficiently to eventually cause cell death [60]. Other possibilities include degeneration-associated reductions in 22:6n-3 due to reduced transport to the outer segment of nascent membranes or proteins that associate with 22:6n-3-containing phospholipids [31,61], increased turnover of DHA-containing glycerolipids in ROS membranes, or selective oxidation of DHA in photoreceptor cells.

Acknowledgements

This research was supported by NIH grants EY04149, EY00871, EY12190, and RR17703; Presbyterian Health Foundation; Research to Prevent Blindness, Inc.; and The Foundation Fighting Blindness, Inc.

References

1. Anderson RE, Maude MB. Phospholipids of bovine outer segments. Biochemistry 1970; 9:3624-8.

2. Fliesler SJ, Anderson RE. Chemistry and metabolism of lipids in the vertebrate retina. Prog Lipid Res 1983; 22:79-131.

3. Tinoco J. Dietary requirements and functions of alpha-linolenic acid in animals. Prog Lipid Res 1982; 21:1-45.

4. Anderson RE, Maude MB. Lipids of ocular tissues. 8. The effects of essential fatty acid deficiency on the phospholipids of the photoreceptor membranes of rat retina. Arch Biochem Biophys 1972; 151:270-6.

5. Stinson AM, Wiegand RD, Anderson RE. Recycling of docosahexaenoic acid in rat retinas during n-3 fatty acid deficiency. J Lipid Res 1991; 32:2009-17.

6. Bazan NG, Rodriguez de Turco EB, Gordon WC. Pathways for the uptake and conservation of docosahexaenoic acid in photoreceptors and synapses: biochemical and autoradiographic studies. Can J Physiol Pharmacol 1993; 71:690-8.

7. Wiegand RD, Koutz CA, Stinson AM, Anderson RE. Conservation of docosahexaenoic acid in rod outer segments of rat retina during n-3 and n-6 fatty acid deficiency. J Neurochem 1991; 57:1690-9.

8. Wiegand RD, Koutz CA, Chen H, Anderson RE. Effect of dietary fat and environmental lighting on the phospholipid molecular species of rat photoreceptor membranes. Exp Eye Res 1995; 60:291-306.

9. Galli C, Trzeciak HI, Paoletti R. Effects of essential fatty acid deficiency on myelin and various subcellular structures in rat brain. J Neurochem 1972; 19:1863-7.

10. Anderson GJ, Connor WE, Corliss JD, Lin DS. Rapid modulation of the n-3 docosahexaenoic acid levels in the brain and retina of the newly hatched chick. J Lipid Res 1989; 30:433-41.

11. Anderson GJ, Connor WE, Corliss JD. Docosahexaenoic acid is the preferred dietary n-3 fatty acid for the development of the brain and retina. Pediatr Res 1990; 27:89-97.

12. Converse CA, Hammer HM, Packard CJ, Shepherd J. Plasma lipid abnormalities in retinitis pigmentosa and related conditions. Trans Ophthalmol Sci U K 1983; 103:508-12.

13. Converse CA, McLachlan T, Bow AC, Packard CJ, Shepherd J. Lipid metabolism in retinitis pigmentosa. In: Hollyfield JG, Anderson RE, LaVail MM, editors. Degenerative retinal disorders: clinical and laboratory investigations. Proceedings of the Sendai Symposium on Retinal Degeneration; 1986 Sep 20-24; Sendai, Japan. New York: Alan R Liss; 1987. p. 93-101.

14. Bazan NG, Scott BL, Reddy TS, Pelias MZ. Decreased content of docosahexaenoate and arachidonate in plasma phospholipids in Usher's Syndrome. Biochem Biophys Res Commun 1986; 141:600-4.

15. Anderson RE, Maude MB, Lewis RA, Newsome DA, Fishman GA. Abnormal plasma levels of polyunsaturated fatty acid in autosomal dominant retinitis pigmentosa. Exp Eye Res 1987; 44:155-9.

16. Birch DG, Birch EE, Hoffman DR, Uauy RD. Retinal development in very-low-birth-weight infants fed diets differing in omega-3 fatty acids. Invest Ophthalmol Vis Sci 1992; 33:2365-76.

17. Gong J, Rosner B, Rees DG, Berson EL, Weigel-DiFranco CA, Schaefer EJ. Plasma docosahexaenoic acid levels in various genetic forms of retinitis pigmentosa. Invest Ophthalmol Vis Sci 1992; 33:2596-602.

18. Hoffman DR, Uauy R, Birch DG. Red blood cell fatty acid levels in patients with autosomal dominant retinitis pigmentosa. Exp Eye Res 1993; 57:359-68.

19. Hoffman DR, Birch DG. Docosahexaenoic acid in red blood cells of patients with X-linked retinitis pigmentosa. Invest Ophthalmol Vis Sci 1995; 36:1009-18.

20. Maude MB, Anderson EO, Anderson RE. Polyunsaturated fatty acids are lower in blood lipids of Usher's type I but not Usher's type II. Invest Ophthalmol Vis Sci 1998; 39:2164-6.

21. Hoffman DR, DeMar JC, Heird WC, Birch DG, Anderson RE. Impaired synthesis of DHA in patients with X-linked retinitis pigmentosa. J Lipid Res 2001; 42:1395-401.

22. Schaefer EJ, Robins SJ, Patton GM, Sandberg MA, Weigel-DiFranco CA, Rosner B, Berson EL. Red blood cell membrane phosphatidylethanolamine fatty acid content in various forms of retinitis pigmentosa. J Lipid Res 1995; 36:1427-33.

23. Holman RT, Bibus DM, Jeffrey GH, Smethurst P, Crofts JW. Abnormal plasma lipids of patients with Retinitis pigmentosa. Lipids 1994; 29:61-5.

24. Anderson RE, Maude MB, Alvarez RA, Acland GM, Aguirre GD. Plasma lipid abnormalities in the miniature poodle with progressive rod-cone degeneration. Exp Eye Res 1991; 52:349-55.

25. Anderson RE, Maude MB, Acland G, Aguirre GD. Plasma lipid changes in PRCD-affected and normal miniature poodles given oral supplements of linseed oil. Indications for the involvement of n-3 fatty acids in inherited retinal degenerations. Exp Eye Res 1994; 58:129-37.

26. Aguirre GD, Acland GM, Maude MB, Anderson RE. Diets enriched in docosahexaenoic acid fail to correct progressive rod-cone degeneration (prcd) phenotype. Invest Ophthalmol Vis Sci 1997; 38:2387-407.

27. Anderson RE, Maude MB, Nilsson SE, Narfstrom K. Plasma lipid abnormalities in the abyssinian cat with a hereditary rod-cone degeneration. Exp Eye Res 1991; 53:415-7.

28. Anderson RE, Maude MB, Bok D. Low docosahexaenoic acid levels in rod outer segment membranes of mice with rds/peripherin and P216L peripherin mutations. Invest Ophthalmol Vis Sci 2001; 42:1715-20.

29. Anderson RE, Sieving PA, Maude MB, Naash MI. Mice with G90D rhodopsin mutations have lower DHA in rod outer segment membranes than control mice. In: Anderson RE, Hollyfield JG, LaVail MM, editors. New insights into retinal degenerative diseases. New York: Plenum; 2001. p. 235-45.

30. Bicknell IR, Darrow R, Barsalou L, Fliesler SJ, Organisciak DT. Alterations in retinal rod outer segment fatty acids and light-damage susceptibility in P23H rats. Mol Vis 2002; 8:333-40 <http://www.molvis.org/molvis/v8/a40/>.

31. Anderson RE, Maude MB, McClellan M, Matthes MT, Yasumura D, LaVail MM. Low docosahexaenoic acid levels in rod outer segments of rats with P23H and S334ter rhodopsin mutations. Mol Vis 2002; 8:351-8 <http://www.molvis.org/molvis/v8/a42/>.

32. Steinberg RH, Flannery JG, Naash M, Oh P, Matthes MT, Yasumura D, Lau-Villacorta C, Chen J, LaVail MM. Transgenic rat models of inherited retinal degeneration caused by mutant opsin genes. Invest Ophthalmol Vis Sci 1996; 37:S698.

33. Winkler BS. The electroretinogram of the isolated rat retina. Vision Res 1972; 12:1183-98.

34. Papermaster DS, Dreyer WJ. Rhodopsin content in the outer segment membranes of bovine and frog retinal rods. Biochemistry 1974; 13:2438-44.

35. Wiegand RD, Giusto NM, Rapp LM, Anderson RE. Evidence for rod outer segment lipid peroxidation following constant illumination of the rat retina. Invest Ophthalmol Vis Sci 1983; 24:1433-5.

36. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Med Sci 1959; 37:911-7.

37. Morrison WR, Smith LM. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride--methanol. J Lipid Res 1964; 5:600-8.

38. Zack DJ, Dean M, Molday RS, Nathans J, Redmond TM, Stone EM, Swaroop A, Valle D, Weber BH. What can we learn about age-related macular degeneration from other retinal diseases? Mol Vis 1999; 5:30 <http://www.molvis.org/molvis/v5/a30/>.

39. Lieth E, Gardner TW, Barber AJ, Antonetti DA, Penn State Retina Research Group. Retinal neurodegeneration: early pathology in diabetes. Clin Experiment Ophthalmol 2000; 28:3-8.

40. Bernstein PS, Tammur J, Singh N, Hutchinson A, Dixon M, Pappas CM, Zabriskie NA, Zhang K, Petrukhin K, Leppert M, Allikmets R. Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene. Invest Ophthalmol Vis Sci 2001; 42:3331-6.

41. Stitt AW. Advanced glycation: an important pathological event in diabetic and age related ocular disease. Br J Ophthalmol 2001; 85:746-53.

42. Zhang K, Kniazeva M, Han M, Li W, Yu Z, Yang Z, Li Y, Metzker ML, Allikmets R, Zack DJ, Kakuk LE, Lagali PS, Wong PW, MacDonald IM, Sieving PA, Figueroa DJ, Austin CP, Gould RJ, Ayyagari R, Petrukhin K. A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy. Nat Genet 2001; 27:89-93.

43. Chader GJ. Animal models in research on retinal degenerations: past progress and future hope. Vision Res 2002; 42:393-9.

44. Rivolta C, Sharon D, DeAngelis MM, Dryja TP. Retinitis pigmentosa and allied diseases: numerous diseases, genes, and inheritance patterns. Hum Mol Genet 2002; 11:1219-27. Erratum in: Hum Mol Genet. 2003; 12:583-4.

45. Anderson RE, Maude MB, Narfstrom K, Nilsson SE. Lipids of plasma, retina, and retinal pigment epithelium in Swedish briard dogs with a slowly progressive retinal dystrophy. Exp Eye Res 1997; 64:181-7.

46. Benolken RM, Anderson RE, Wheeler TG. Membrane fatty acids associated with the electrical response in visual excitation. Science 1973; 182:1253-4.

47. Hoffman DR, Birch DG. Omega 3 fatty acid status in patients with retinitis pigmentosa. World Rev Nutr Diet 1998; 83:52-60.

48. Wheeler TG, Benolken RM, Anderson RE. Visual membranes: specificity of fatty acid precursors for the electrical response to illumination. Science 1975; 188:1312-4.

49. Anderson RE, Maude MB, Alvarez RA, Acland G, Aguirre GD. Inherited retinal degenerations. Role of polyunsaturated fatty acids. In: Christen Y, Doly M, Droy-Lefaix MT, editors. Les Seminaires Ophthalmologiques d'IPSEN. Paris: Irvinn; 1999. p. 57-65.

50. Anderson RE, Maude MB, Alvarez RA, Acland G, Aguirre GD. A hypothesis to explain the reduced blood levels of docosahexaenoic acid in inherited retinal degenerations caused by mutations in genes encoding retina-specific proteins. Lipids 1999; 34:S235-7.

51. Penn JS, Anderson RE. Effect of light history on rod outer-segment membrane composition in the rat. Exp Eye Res 1987; 44:767-78.

52. Penn JS, Anderson RE. Effects of light history on the rat retina. In: Osborne NN, Chader G, editors. Progress in retinal research. Vol 11. New York: Pergamon Press; 1992. p. 75-98.

53. Li F, Cao W, Anderson RE. Protection of photoreceptor cells in adult rats from light-induced degeneration by adaptation to bright cyclic light. Exp Eye Res 2001; 73:569-77.

54. Li F, Cao W, Anderson RE. Alleviation of constant-light-induced photoreceptor degeneration by adaptation of adult albino rat to bright cyclic light. Invest Ophthalmol Vis Sci 2003; 44:4968-75.

55. Alvarez RA, Aguirre GD, Acland GM, Anderson RE. Docosapentaenoic acid is converted to docosahexaenoic acid in the retinas of normal and prcd-affected miniature poodle dogs. Invest Ophthalmol Vis Sci 1994; 35:402-8.

56. Chen H, Ray J, Scarpino V, Acland GM, Aguirre GD, Anderson RE. Synthesis and release of docosahexaenoic acid by the RPE cells of prcd-affected dogs. Invest Ophthalmol Vis Sci 1999; 40:2418-22.

57. Luthria DL, Mohammed BS, Sprecher H. Regulation of the biosynthesis of 4,7,10,13,16,19-docosahexaenoic acid. J Biol Chem 1996; 271:16020-5.

58. Sprecher H. Metabolism of highly unsaturated n-3 and n-6 fatty acids. Biochim Biophys Acta 2000; 1486:219-31.

59. Martinez M. Abnormal profiles of polyunsaturated fatty acids in the brain, liver, kidney and retina of patients with peroxisomal disorders. Brain Res 1992; 583:171-82.

60. Depreter M, Espeel M, Roels F. Human peroxisomal disorders. Microsc Res Tech 2003; 61:203-23.

61. Rodriguez de Turco EB, Deretic D, Bazan NG, Papermaster DS. Post-Golgi vesicles cotransport docosahexaenoyl-phospholipids and rhodopsin during frog photoreceptor membrane biogenesis. J Biol Chem 1997; 272:10491-7.