Differential composition of DHA and very-long-chain PUFAs in rod and cone photoreceptors

Abstract

Long-chain PUFAs (LC-PUFAs; C20-C22; e.g., DHA and arachidonic acid) are highly enriched in vertebrate retina, where they are elongated to very-long-chain PUFAs (VLC-PUFAs; C 28) by the elongation of very-long-chain fatty acids-4 (ELOVL4) enzyme. These fatty acids play essential roles in modulating neuronal function and health. The relevance of different lipid requirements in rods and cones to disease processes, such as age-related macular degeneration, however, remains unclear. To better understand the role of LC-PUFAs and VLC-PUFAs in the retina, we investigated the lipid compositions of whole retinas or photoreceptor outer segment (OS) membranes in rodents with rod- or cone-dominant retinas. We analyzed fatty acid methyl esters and the molecular species of glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) by GC-MS/GC-flame ionization detection and ESI-MS/MS, respectively. We found that whole retinas and OS membranes in rod-dominant animals compared with cone-dominant animals had higher amounts of LC-PUFAs and VLC-PUFAs. Compared with those of rod-dominant animals, retinas and OS membranes from cone-dominant animals also had about 2-fold lower levels of di-DHA (22:6/22:6) molecular species of glycerophospholipids. Because PUFAs are necessary for optimal G protein-coupled receptor signaling in rods, these findings suggest that cones may not have the same lipid requirements as rods.

Keywords: docosahexaenoic acid; glycerophospholipids; macular degeneration; polyunsaturated fatty acids; rod- and cone-dominant retinas; supraenoic lipids.

Fig. 1.

Determination of purity of prepared retinal OS membranes by immunoblotting. A, B: Retinal OS membranes were prepared by discontinuous sucrose gradient centrifugation and analyzed by silver nitrate staining and Western blotting for OS purity using photoreceptor-specific antibodies. Opsin and transducin immunoblotting assays confirmed enrichment of OS membranes in band I OS membranes relative to band II and pellet fractions.

Fig. 2.

Quantification of PUFAs in cone-dominant Nrl^−/− and TLGS retinas compared with rod-dominant Nrl^+/− and WT mouse retinas. Cone-dominant Nrl^−/− and TLGS whole retinas have significantly reduced 22:6n3 compared with rod-dominant Nrl^+/− and WT mice. The decreased 22:6n3 is associated with increased 18:0, 18:1, and 20:4n6 in the cone-dominant retinas. However, there is no significant increase in 22:5n6 as occurs in n3 PUFA deficiency. Statistically significant differences are indicated as: ^BWT versus Nrl^−/−; ^CWT versus TLGS; ^DNrl^+/− versus Nrl^−/−; ^ENrl^+/− versus TLGS; and ^FNrl^−/− versus TLGS for P < 0.05 to P < 0.001. Error bars represent mean ± SD.

Fig. 3.

The 22:6n3 in Nrl^−/− and TLGS OS membranes compared with Nrl^+/− and WT OS membranes. Relative mole percent of fatty acids from Nrl^−/− and TLGS OS membranes show significantly reduced 22:6n3 levels, but increased 18:0, 18:1, and 20:4n6 without significantly increased 22:5n6 levels, which is consistent with decreased 22:6n3 in whole-retina fatty acids. Statistically significant differences are indicated as: ^BWT versus Nrl^−/−; ^CWT versus TLGS; ^DNrl^+/− versus Nrl^−/−; ^ENrl^+/− versus TLGS; and ^FNrl^−/− versus TLGS; P < 0.05 to P < 0.001; mean ± SD.

Fig. 4.

Quantification of retinal VLC-PUFAs in Nrl^−/− retina compared with Nrl^+/− and WT retina. Relative mole percent of whole retinal VLC-PUFAs is significantly decreased in Nrl^−/− mice compared with Nrl^+/− and WT mice. The major VLC-PUFAs in the Nrl^+/− and WT retina include 32:6n3, 34:5n3, and 34:6n3, which are reduced in the Nrl^−/− retina. However, the Nrl^−/− retina is enriched in 36:5n3 and 36:6n3. Statistically significant differences are indicated as: ^BWT versus Nrl^−/−; and ^DNrl^+/− versus Nrl^−/−; P < 0.05 to P < 0.001; mean ± SD.

Fig. 5.

Comparison of di-poly-DHA-PC and VLC-PUFA-PC in Nrl^−/−, Nrl^+/−, and WT retina. Consistent with decreased retinal DHA and VLC-PUFAs, di-poly-DHA-PC and VLC-PUFA-PC and the sum of retinal VLC-PUFA-PC are significantly decreased in Nrl^−/− retina relative to Nrl^+/− and WT retinal VLC-PUFA-PC. The most abundant VLC-PUFA-PC species in the Nrl^+/− and WT retina include 32:6/22:6, 34:5/22:6, 34:6/22:6, and 36:6/22:6. Statistically significant differences are indicated as: ^AWT versus Nrl^+/−; ^BWT versus Nrl^−/−; and ^DNrl^+/− versus Nrl^−/−; P < 0.05 to P < 0.001; mean ± SD.

Fig. 6.

Retinal LC-PUFA-PE levels in rod-dominant retinas compared with cone-dominant retinas. The most abundant LC-PUFA-PE in both rod- and cone-rich retinas was 18:0/22:6 (A, B). The 22:6/22:6-PUFA-PE is significantly decreased in cone-dominant retinas compared with rod-dominant WT and Nrl^+/− retinas (A). However, among the cone-rich retinas, there was no significant difference in 22:6/22:6-PUFA-PE levels. No VLC-PUFA-PEs were detected. Statistically significant differences are indicated as: ^AWT versus Nrl^+/−; ^BWT versus Nrl−/− ^DNrl^+/− versus Nrl−/− ^FNrl^−/− versus TLGS; ^GNrl^−/− versus tree squirrel; ^HNrl^−/− versus tree shrew; ^Itree squirrel versus TLGS; ^Jtree squirrel versus tree shrew; and ^KTLGS versus tree shrew; P < 0.05 to P < 0.001; mean ± SD.

Fig. 7.

Retinal PUFA-PS species in rod-dominant retinas compared with cone-dominant retinas. The 18:0/22:6-PS presents the major PS in both rod- and cone-rich retinas (A, B). Consistent with PC and PE data, 22:6/22:6-PS is significantly decreased in the cone-dominant retinas compared with rod-dominant WT and Nrl^+/− retinas, without any differences detected in the cone-rich retina, except for Nrl^−/− and TLGS that were significantly different at P < 0.05. Statistically significant differences are indicated as: ^BWT versus Nrl^−/−; ^DNrl^+/− versus Nrl^−/−; ^FNrl^−/− versus TLGS; ^GNrl^−/− versus tree squirrel; ^HNrl^−/− versus tree shrew; ^Itree squirrel versus TLGS; ^Jtree squirrel versus tree shrew; and ^KTLGS versus tree shrew; P < 0.05 to P < 0.001; mean ± SD.

Fig. 8.

Comparison of retinal VLC-PUFA-PC in cone-dominant versus rod-dominant retinas. Compared with the rod-dominant rat retinas (n = 4), the cone-dominant retinas of Nrl^−/− mice (n = 5), tree squirrels (n = 3), TLGSs (n = 4), and tree shrews (n = 6) all have significantly less VLC-PUFA-PC. Tree shrew retinas have much higher 34:6/22:6 and 36:6/22:6 compared with the other cone-dominant species. Statistically significant differences are indicated as: ^FNrl^−/− versus TLGS; ^GNrl^−/− versus tree squirrel; ^HNrl^−/− versus tree shrew; ^Itree squirrel versus TLGS; ^Jtree squirrel versus tree shrew; ^KTLGS versus tree shrew; and ^LSprague-Dawley rats (SD rats) versus (Nrl^−/−, tree squirrel, TLGS, tree shrew); P < 0.05 to P < 0.001; mean ± SD.