Low Docosahexaenoic Acid Levels in Rod Outer Segment Membranes of Mice with rds/Peripherin and P216L Peripherin Mutations

Abstract

purpose. Humans with retinitis pigmentosa and dogs with progressive rod–cone degeneration (prcd) have lower than normal blood levels of long-chain polyunsaturated fatty acids, including docosahexaenoic acid (DHA), the major fatty acid found in retinal rod outer segments (ROS). In addition, prcd-affected dogs have lower levels of DHA in their ROS than control animals. The present study was designed to determine whether mice that are heterozygous for the rds mutation and transgenic mice heterozygous for a specific rds/peripherin mutation (P216L) have lower DHA levels in their ROS and other tissues than do control mice.

methods. Wild-type (rds ^+/+) mice, mice with the rds ^−/− (null) and rds ^+/− mutations, and mice with the P216L rds/peripherin mutation on the rds ^+/− background were maintained in the vivarium under identical husbandry conditions, and tissues were removed from each group for analysis at approximately 2 months of age. Fatty acid compositions of total lipids from plasma, red blood cells, liver, and ROS were determined by gas–liquid chromatography. ROS purity from each group was determined by SDS-PAGE with silver staining. The morphologic status of retinas representing each genotype was analyzed by light and electron microscopy.

results. There was no difference between rds ^+/−, P216L on rds ^+/−, and rds ^+/+ (control) animals in the fatty acid composition of plasma, expressed as relative mole percent or as nanomoles fatty acid per milliliter of plasma. Small but statistically significant differences were found in 18:0 and C-22 polyunsaturated fatty acids of red blood cells. In the liver, the control animals had higher levels of 20:4n-6. In contrast, the ROS of control animals had levels of DHA that were 1.4 times that of ROS from either rds ^+/− or P216L on rds ^+/− mice of the same age. The reduction in DHA was not accompanied by an increase in 22:5n-6, which always occurs in neural tissues of animals deprived of n-3 fatty acids. SDS-PAGE of the three ROS membrane preparations showed that they were of identical purity.

conclusions. Mice heterozygous for the spontaneous rds/peripherin mutation or mice carrying the P216L mutation on this heterozygous background have a statistically significant reduction of DHA in their ROS membranes. The authors propose that reduction in DHA is an adaptive response to metabolic stress caused by the mutation.

Retinal rod outer segment (ROS) membranes contain higher levels of docosahexaenoic acid (DHA, 22:6n-6) than any other membrane system thus far examined. ¹ Numerous studies have established that the high level of DHA in ROS membranes provides an optimal microenvironment for photon capture and visual excitation. Early studies demonstrated a reduction of ERG amplitudes in animals with reduced levels of DHA in ROS. ^{2 3 4 5 6} More recently, in vitro studies have suggested that the rate of the conformational change in rhodopsin from metarhodopsin I to metarhodopsin II is faster in membranes containing DHA. ^{7 8} Therefore, the ideal situation for optimal function seems to be high levels of DHA in ROS membranes. 

DHA belongs to the n-3 family of essential polyunsaturated fatty acids. ⁹ These acids, along with the n-6 family, cannot be synthesized de novo by vertebrates or invertebrates, and they or their shorter chain precursors must therefore be obtained in the diet. By their very nature, these fatty acids are quite susceptible to lipid peroxidation, because of the large number of double bonds. Therefore, conditions that may present an oxidant stress to photoreceptors, such as light and oxygen, could lead to retinal degeneration. 

Previous studies have shown that albino rats stressed by rearing in bright cyclic light (300–800 lux) have lower levels of DHA in their ROS membranes. ^{10 11} Animals returned to dim cyclic light had higher ROS DHA levels within 3 weeks. ¹² Thus, we proposed that these animals underwent a biochemical and morphologic adaptation to reduce the efficiency of photon capture and to lower the level of substrate for lipid peroxidation, to protect the retina from light-induced oxidant stress. ¹³  

Retinitis pigmentosa (RP) is an inherited retinal degeneration that may be transmitted as autosomal dominant, autosomal recessive, X-linked, and sporadic modes of inheritance. Most recently, more than 120 genes have been identified that are linked to retinal degeneration, and 60 have been cloned (available in the public domain at http://www.sph.uth.tmc.edu/Retnet/ hosted by the University of Texas Houston Health Science Center). Thus, in human retinal degenerations, a variety of mutations can ultimately result in death of photoreceptor cells. In 1983, Converse et al. ¹⁴ reported that some of her Scottish patients with RP had lower blood levels of DHA and other polyunsaturated fatty acids than unaffected family members or unrelated persons living in the same household. Since that time, many laboratories have reported similar findings in multiple genotypes of patients with RP. ^{15 16 17 18 19 20 21 22 23 24 25} The reduced DHA levels do not appear to segregate with any particular genotype, suggesting that this may be a general phenomenon in persons with RP. 

The reduced blood levels of DHA were also observed in several animal models of inherited retinal degenerations. ^{26 27 28} The most extensively studied model is the miniature poodle with progressive rod–cone degeneration (prcd), which has a retinal degeneration that resembles that in humans. ²⁹ In addition to lower blood levels of DHA, ²⁶ the ROS of affected dogs also have reduced DHA. ³⁰ The genotype of these animals has not yet been determined, although a mutation in the rds/peripherin gene has been excluded. ³¹ We sought to determine whether the reduced blood and ROS DHA levels in the prcd-affected dogs were unique to their genotype or whether this is a general phenomenon in animals with inherited retinal degeneration. To that end, we examined the fatty acid composition of several tissues and ROS from mice heterozygous for the natural occurring rds/peripherin mutation (rds ^+/−), rds ^+/− transgenic mice carrying a P216L rds/peripherin mutation known to cause RP in humans, and wild-type control animals (rds ^+/+). The results show that ROS from both heterozygous genotypes had significantly lower DHA levels in their ROS than wild-type animals. 

Materials and Methods

Animals

Control mice were derived from the Balb/c strain and were wild type at the rds locus (rds ^+/+). One of the experimental groups was also from the Balb/c strain and heterozygous for the spontaneous rds null mutation (rds ^+/−), which results from an insertion of mouse repetitive RNA into the second exon of the rds gene. ³² The second experimental group was derived from the C57BL/6 strain, heterozygous at the rds locus (rds ^+/−) and transgenic for a P216L mutation (line 1376) in which the transgenic mRNA is expressed at a level that is approximately equal to the wild-type mRNA. ³³ This mutation was chosen, because it also causes dominant RP in humans. All animals were approximately 2 months of age when the tissues were collected. 

The animals were born and raised in the University of California at Los Angeles Division of Laboratory Animal Medicine and were maintained on a normal diet (Rodent Diet; Harlan Teklad, Madison, WI) and 12-hour dark/12-hour light cyclic lighting. Lighting conditions outside the cages never exceeded 20 lux. The experiments described were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Tissue Collection

Animals were killed with CO₂ and exsanguinated by opening the abdominal cavity and withdrawing as much blood as possible from the inferior vena cava. In each case, this procedure was performed with a fresh 1.0-ml syringe and 25-g needle. Routinely, blood volumes of approximately 0.5 ml were collected. The blood from each animal was expelled from the syringe into 3.0-ml lavender-capped tubes containing EDTA (Vacutainer; Becton Dickenson, Franklin Lakes, NJ), and the contents were gently mixed by inversion to prevent clotting. The samples were then centrifuged for 10 minutes at 450g. Plasma was separated from compacted red blood cells with a Pasteur pipette and the samples were frozen on dry ice. 

After exsanguination, a fragment of liver weighing approximately 250 mg was removed from each animal, placed in an individual container, and frozen on dry ice. Finally, the eyes from each animal were enucleated, and the retinas were dissected in a droplet of cold Hanks’ balanced salt solution (4°C). After dissection, each retina was frozen in a tube precooled with dry ice. For each genotype, the retinas were pooled in groups of 16. All tissues were stored at −80°C until analysis. 

Light and Electron Microscopy

Mice were anesthetized with 50 mg/kg of Nembutal (Abbott Laboratories, Santa Clara, CA). In some cases, the animals were subsequently fixed by transcardiac perfusion with formaldehyde and glutaraldehyde (1% and 2%, respectively) in 0.1 M sodium phosphate buffer (pH 7.2), or the eyes were removed and fixed by immersion after removal of the cornea. After fixation, the posterior portion of each eye was cut into quadrants and fixed additionally for 1 hour in 1% osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.2). The tissues were then dehydrated and embedded in Araldite 502 (Pelco, Redding, CA). Sections of 0.5-μm thickness were stained with toluidine blue before light photomicrography. Ultrathin sections for electron microscopy were stained with uranium and lead salts. 

Preparation of ROS Membranes

ROS were prepared from frozen retinas on a discontinuous sucrose gradient according to the procedure of Papermaster and Dryer, ³⁴ as modified by Wiegand and Anderson. ³⁵ Purity of membrane preparations was determined by SDS-PAGE. 

Lipid Extraction and Analysis

Lipids were extracted by the procedure of Bligh and Dyer. ³⁶ Aliquots of each lipid extract were used for preparation of methyl esters, which were analyzed by gas–liquid chromatography, as previously described. ³⁷  

Data Analysis

Results are reported as relative mole percent or nanomoles fatty acid/milliliter plasma. Differences between groups were determined by the Student’s t-test. P < 0.05 was set as our criterion for significance. 

Results

Morphologic Status of Retinas

ROS of rds ^+/+ mice were normal, rod-shaped structures consisting of approximately 1000 discs per cell. Photoreceptor cell number was reduced to approximately 85% of control in rds ^+/− mice (Fig. 1A) , and their oversized ROS discs were rolled into whorls (Fig. 1B) . Photoreceptor cell number was reduced to approximately 65% in rds ^+/− animals carrying the P216L transgene (Fig. 2A) and their whorled outer segments were more dysmorphic than rds ^+/− animals (Fig. 2B) . 

Purity of ROS Membranes

Silver-stained gels of the ROS prepared from mutant and wild-type animals are shown in Figure 3 . In the three groups of animals, rhodopsin was the dominant protein in these membranes. There were no obvious differences in the purity of the preparation of ROS from the three groups of animals. Therefore, any difference in the fatty acid composition of ROS membranes among these groups of animals is not the result of contamination of membranes with non-ROS material. 

Fatty Acid Analysis of Liver, Plasma, and Red Blood Cells

Table 1 contains the fatty acid composition of red blood cells and liver total lipids from mutant and wild-type animals. Only those fatty acids showing a statistically significant difference between groups are presented. There was no statistically significant difference in fatty acid composition of the plasma from the three groups of animals. The levels of DHA and other long-chain polyunsaturated fatty acids were essentially the same among all three groups. There were small but statistically significant differences in the fatty acids from red blood cells among the groups. Stearic acid (18:0), 22:5n-3, and 22:6n-3 were lower in control animals, and 22:4n-6 and 22:5n-6 were higher, compared with mutant animals. In the liver, control animals had significantly higher levels of arachidonic acid (20:4n-6); however, there were no differences in the liver DHA levels among groups. 

In contrast, there were statistically significant differences in the fatty acid composition of ROS from mutant and wild-type animals (Table 2) . Wild-type mice had larger amounts of DHA (1.4 times) and total n-3 fatty acids (1.4 times) than either mutant group. There was no difference in levels of DHA and other fatty acids between the two mutant groups. However, ROS from both mutant groups had higher levels of 20:4n-6, stearic acid (18:0), and oleic acid (18:1n-9) than control animals, which compensated for the reduction in DHA. There was no specific increase in 22:5n-6 in the mutant animals, which occurs when DHA levels are reduced by dietary manipulations. ¹¹ In fact, the ROS level of 22:5n-6 was always less than 0.25% and was not included in Table 2 . 

Discussion

Polyunsaturated fatty acid levels in ROS membranes can be experimentally manipulated in two ways: diet and habitat illuminance. Because n-3 and n-6 fatty acids are essential and must be obtained from the diet, it is possible to change their tissue composition by selected dietary restrictions. We ^{11 37 38} and others ^{39 40 41} have shown that DHA levels in ROS can be reduced if animals are fed a diet without n-3 fatty acids. Under such conditions, the reduction in DHA and ROS is accompanied by a selective increase in 22:5n-6, so that the sum of 22:5n-6 and DHA remains constant, regardless of diet. Results of dietary manipulation suggest that maintenance of high levels of polyunsaturated fatty acids in ROS is a priority for the retina, and restriction of n-3 fatty acids stimulates the production of 22:5n-6, the most highly polyunsaturated fatty acid of the n-6 family that is found in membranes. On the other hand, the reduction in ROS DHA levels that occurs during rearing in bright cyclic light is not accompanied by an equivalent increase in 22:5n-6. ^{10 11} In these animals, the total level of polyunsaturated fatty acids in the ROS (sum of n-6 and n-3 fatty acids) is significantly lower than in ROS from animals raised in dim cyclic light. Under these conditions, it appears that the retina downregulates the level of polyunsaturated fatty acids, perhaps because of an oxidant stress due to the bright cyclic light. ^{42 43}  

In the present study, we found that the DHA levels in rds ^+/− and the P216L peripherin mutants on the rds ^+/− background were significantly lower (28%–31% less) than those of wild-type control animals. In the mutant animals, there was no concomitant increase in 22:5n-6, which rules out the possibility that the reduction was due to dietary deficiency of n-3 fatty acids in the mutant animals. Thus, the reduction seen in the mutant mice is similar to that reported for the ROS of prcd affected dogs. Although the mutation in the dog is not known, it is not in a gene encoding rds/peripherin. ³¹ Therefore, the DHA reduction is not due to a specific genotype, but rather appears to be a general phenomenon found in ROS of animals with inherited retinal degenerations. 

The reason for the reduced level of DHA in ROS of animals with inherited retinal degeneration is not known. However, the fatty acid composition of the ROS of these animals suggests that the synthesis of DHA-containing glycerolipids is downregulated in retinas of animals with inherited retinal degenerations. In the animals raised in bright cyclic light, we speculate that the reduction in DHA is in response to an oxidant stress and that the reduction reduces the availability of substrate for lipid peroxidation. ¹³ Also, the reduction in DHA in ROS would result in a less efficient photon capture and thus provide additional protection against light damage in these animals. In the mutant mice, we speculate that the mutation causes metabolic stress, which may also be an oxidant stress. In this case, reduction of ROS DHA levels would be an adaptive response of these retinas to protect against retinal degeneration. 

Although striking differences were found in DHA levels in ROS of mutant and wild-type animals, no differences were found in plasma and only small differences in red blood cell and liver fatty acids. In contrast, in numerous studies in humans ^{15 16 17 18 19 20 21 22 23 24 25} and our previous study in dogs, ²⁶ statistically significant differences were found in DHA levels in the blood. However, in these studies, the differences were usually rather small, and a relatively large sample size was required for significance to be demonstrated. In the present study, we examined three to four samples from each group of animals, and this may not have been sufficient to show significance. Similarly, the liver of prcd-affected dogs has significantly higher levels of DHA compared with control animals. Again, these differences, although statistically significant, are rather small and a large sample size was required for the differences to achieve statistical significance. Given the statistically significant difference seen in the present study in arachidonic acid levels in liver of wild-type compared with mutant animals, the slight differences seen in DHA level may have achieved significance if we had analyzed more individual liver samples from these three groups of animals. 

In summary, we found that mice with a natural or genetically engineered mutation in the rds/peripherin gene had lower levels of DHA in their ROS membranes. We speculate that this reduction is an adaptive response to a metabolic stress (perhaps oxidant) caused by the mutation. To test this hypothesis, mice with the same rds/peripherin and P216L peripherin mutations reported herein are currently being fed diets containing different levels of n-3 fatty acids to determine the effects on the chemistry, structure, and function of their retinas. 

 Supported by National Eye Institute Grants EY00871, EY04149, and EY12190 (REA); and National Eye Institute Grants EY00444 and EY00331 (DB); Research to Prevent Blindness (REA, DB); The Foundation Fighting Blindness (REA, DB); the Samuel Roberts Nobel Foundation, Ardmore, Oklahoma (REA); and the Presbyterian Health Foundation, Oklahoma City (REA). DB is the Dolly Green Professor of Ophthalmology at UCLA and REA is the Dean A. McGee Professor of Ophthalmology at the Oklahoma University Health Science Center.

 Submitted for publication January 8, 2001; revised March 7, 2001; accepted March 14, 2001.

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Figure 1.

 

Photoreceptors and outer segments from a 2-month-old Balb/c mouse that was rds ^+/−. (A) The number of photoreceptors at this age was nearly normal, as indicated by the 8 to 10 nuclei in each aligned column in the outer nuclear layer (ONL). The normal number of nuclei in an rds ^+/+ mouse at this age is approximately 10. The photoreceptor outer segments (arrows) were truncated and dysmorphic. (B) Electron micrograph of the dysmorphic, truncated outer segments (OS) shown in (A). The disc diameters were larger than normal, and the discs were arranged into whorls. Magnification, (A)× 550; (B) ×4400.

Figure 2.

 

Photoreceptors and outer segments from a 2-month-old C57BL/6 mouse that was rds ^+/− and hemizygous for a P216L mutation in an rds transgene. (A) The number of photoreceptor nuclei in the outer nuclear layer (ONL) was reduced to six to seven per column (65%) at this age. (B) Electron micrograph of the outer segments (OS) shown in (A). The discs were oversized and whorled as in Figure 1B , but more dysmorphic, due to the dominant negative effect of the P216L rds mutation. Magnification, (A) ×550; (B) × 6500.

Figure 3.

 

Representative silver-stained polyacrylamide gels of the ROS prepared from the three groups of animals. STD, weight standards; WT, control animals.

Table 1.

 

Percentages of Selected Fatty Acids of Red Blood Cells and Liver Total Lipids

Tissue	Fatty Acid	Control	P216L	rds/Peripherin
Red blood cells	18:0	15.4 ± 1.5*	18.6 ± 0.8*	17.1 ± 1.6
	18:2n-6	11.9 ± 1.6	11.6 ± 0.4, †	10.2 ± 0.5, †
	22:4n-6	1.24 ± 0.05, † *	0.95 ± 0.09, †	1.04 ± 0.10*
	22:5n-6	0.59 ± 0.12*	0.40 ± 0.03	0.42 ± 0.04*
	22:5n-3	0.97 ± 0.14*	1.28 ± 0.02*	1.25 ± 0.21
	22:6n-3	6.44 ± 0.66*	6.86 ± 0.27, †	7.56 ± 0.16* , †
Liver	20:4n-6	12.9 ± 2.6*	7.10 ± 1.7*	7.93 ± 1.1*

 Data are expressed as percentage of total nanomoles of fatty acid ± SD.

*  P < 0.05.

†  P < 0.01.

Table 2.

 

Fatty Acid Composition of Total Lipids from Rod Outer Segments

Fatty Acid	Control	P216L	rds/Peripherin	Probabilities
								Control vs. P216L	Control vs. rds	P216L vs. rds
Saturate
14:0	0.2 ± 0.0	0.5 ± 0.2	0.4 ± 0.1	0.0132	0.0001	0.4570
16:0	20.7 ± 1.4	22.4 ± 1.7	24.6 ± 2.4	0.1378	0.0188	0.1205
18:0	21.1 ± 0.7	24.6 ± 0.8	23.6 ± 1.1	0.0002	0.0033	0.1056
Subtotal	42.2 ± 1.5	48.1 ± 2.4	49.0 ± 3.3	0.0038	0.0056	0.6364
Monoenoic
16:1	0.2 ± 0.0	0.3 ± 0.1	0.3 ± 0.1	0.0666	0.0173	0.4715
18:1	5.5 ± 0.3	9.6 ± 1.4	8.8 ± 0.9	0.0007	0.0001	0.2641
20:1	0.2 ± 0.0	0.4 ± 0.2	0.2 ± 0.0	0.0950	0.0070	0.1645
22:1	0.2 ± 0.2	0.3 ± 0.1	0.5 ± 0.5	0.2140	0.3161	0.5272
Subtotal	6.2 ± 0.2	10.9 ± 1.9	10.0 ± 1.2	0.0017	0.0002	0.3178
n-6
18:2	0.7 ± 0.1	0.9 ± 0.3	0.6 ± 0.1	0.1524	0.7437	0.0684
20:3	0.3 ± 0.0	0.5 ± 0.3	0.3 ± 0.0	0.3376	0.0269	0.0935
20:4	4.6 ± 0.4	6.4 ± 0.7	6.0 ± 0.4	0.0022	0.0004	0.2774
22:4	0.6 ± 0.0	0.8 ± 0.1	0.6 ± 0.3	0.0154	0.9913	0.1834
Subtotal	6.5 ± 0.6	8.9 ± 0.9	7.9 ± 0.7	0.0025	0.0126	0.0743
n-3
20:5	0.4 ± 0.0	0.7 ± 0.3	0.6 ± 0.1	0.0778	0.0448	0.1920
22:5	0.5 ± 0.0	0.7 ± 0.1	0.5 ± 0.1	0.0001	0.3223	0.0068
22:6	42.7 ± 1.4	29.2 ± 5.1	30.9 ± 3.7	0.0014	0.0003	0.5568
24:5	0.3 ± 0.1	0.7 ± 0.2	0.4 ± 0.2	0.0231	0.3649	0.0799
24:6	1.2 ± 0.1	0.5 ± 0.3	0.7 ± 0.2	0.0026	0.0008	0.2665
Subtotal	45.1 ± 1.5	32.1 ± 4.7	33.2 ± 3.9	0.0012	0.0004	0.6896
n-6/n-3	0.15 ± 0.01	0.28 ± 0.06	0.24 ± 0.04	0.0039	0.0018	0.2153

 Fatty acids less than 0.25% are not presented but are included in the subtotals. Data are expressed as percentage total nanomoles of fatty acid ± SD.