Plasmalogens in the retina: In situ hybridization of dihydroxyacetone phosphate acyltransferase (DHAP-AT) – the first enzyme involved in their biosynthesis – and comparative study of retinal and retinal pigment epithelial lipid composition

Experimental Eye Research 84 (2007) 143e151 www.elsevier.com/locate/yexer

Plasmalogens in the retina: In situ hybridization of dihydroxyacetone phosphate acyltransferase (DHAP-AT) e the first enzyme involved in their biosynthesis e and comparative study of retinal and retinal pigment epithelial lipid composition Niyazi Acar a,*, Stephane Gregoire a, Agnes Andre a, Pierre Juaneda a, Corinne Joffre a, Alain M. Bron a,b, Catherine P. Creuzot-Garcher a,b, Lionel Bretillon a a

National Institute for Research on Agronomy, UMR FLAVIC, Eye and Nutrition Research Group, 17, rue Sully, BP86510, 21065 Dijon Cedex, France b Department of Ophthalmology, University Hospital, Dijon, France Received 3 July 2006; accepted in revised form 14 September 2006 Available online 1 November 2006

Abstract Plasmalogens (Pls) are phospholipids containing a vinyl-ether bond in the sn-1 position of the glycerol backbone. The physiological role of Pls is still enigmatic, especially within the eye where their deficiency leads to developmental abnormalities. In order to learn more about the functions of Pls in the posterior eye, we evaluated retinal Pl content as well as the expression of the first enzyme involved in Pls biosynthesis, dihydroxyacetone phosphate acyltransferase (DHAP-AT) in the retina. In situ hybridization of DHAP-AT mRNA was performed on rat eye sections. The Pl contents of calf retina and retinal pigment epithelium (RPE) samples were determined by high-performance liquid chromatography, thin-layer chromatography, and gas chromatography. DHAP-AT was highly expressed in the inner segment of photoreceptors and in the RPE, suggesting two distinct sites for Pl biosynthesis. Plasmenyl-ethanolamine was the prominent class of Pls in both neural retina and RPE (28e29% of the total phospho-ethanolamine-glycerides). According to the nature of the alkenyl residue linked to the sn-1 position of Pls, the most striking finding was the greater proportion of octadecanal-aldehyde in the sn-1 position of plasmenyl-ethanolamine of the neural retina compared to all the other classes of Pls in the neural retina and the RPE. These findings might be relevant to the biological functions of Pls against oxidative stress and in the formation of lipid rafts. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: ether-lipids; plasmalogens; in situ hybridization; dihydroxyacetone phosphate acyltransferase (DHAP-AT); retina; retinal pigment epithelium

1. Introduction Plasmalogens (Pls) constitute a specific subclass of phospholipids characterized by the presence of a vinyl-ether bond at the sn-1 position of the glycerol backbone, rather than an ester bond as in diacylglycerophospholipids (Nagan and Zoeller, 2001). In Pls, the aliphatic moieties at the sn-1 position consist of hexadecanal- (16:0), octadecanal- (18:0),

* Corresponding author. Tel.: þ33 03 80 69 32 69; fax: þ33 03 80 69 32 23. E-mail address: [email protected] (N. Acar). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2006.09.009

9-octadecanal- (18:1n-9), and 7-octadecanal-aldehydes (18:1n-7), whereas polyunsaturated fatty acids (PUFAs) are esterified at the sn-2 position. The head group is usually either ethanolamine or choline. Different tissues, and possibly even cell types within one tissue, may have variable amounts of Pls. Brain myelin possesses the highest content of ethanolamine Pls (plasmenylethanolamine) (Hack and Helmy, 1977), whereas the heart muscle has a higher content of choline Pls (plasmenyl-choline) (Panganamala et al., 1971). Moderate amounts of Pls are found in kidney, skeletal muscle, spleen, and blood cells, whereas liver is known for its low content (Forbes et al., 1953; Horrocks and Sharma, 1982). Two peroxisomal enzymes, dihydroxyacetone

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phosphate acyltransferase (DHAP-AT) and alkyl dihydroxyacetone phosphate synthase (ADAPS) are involved in the formation of the vinyl-ether double bond (Hajra, 1995). The cellular level of Pls is affected in both peroxisomal assembly disorders and single peroxisomal enzyme deficiencies: e.g., Zellweger syndrome and rhizomelic chondrodysplasia punctata (RCDP) types 1e3 (Moser, 1999, 2000). RCDP is an autosomal recessive disorder of peroxisome metabolism characterized by the presence of morphologically distinguishable peroxisomes and multiple, but not generalized, loss of peroxisomal functions. RCDP type 2 and type 3 are characterized by the isolated deficiencies of DHAP-AT and ADAPS, respectively, and result in clinical manifestations such as bone abnormalities (namely shortening of proximal long bones, calcific stippling of epiphysis, and vertebral clefts in vertebrae), contractures, cataract, and severe growth and mental retardations (Sanchez et al., 1997; Moser, 2000). The ocular phenotype related to Pls deficiency was more precisely described by using a mouse model for RCDP type 2 deficient in DHAP-AT (Rodemer et al., 2003). The lack of Pls led to ocular developmental defects resulting in microphthalmia, dysgenesis of the anterior eye chamber, and bilateral central dense cataract. In the posterior pole of the eye, the most striking abnormalities were optic nerve hypoplasia, Bruch’s membrane thickening, and persistence of hyaloid arteries. In the retina, the alterations were shown to be confined to the retinal pigment epithelium (RPE) and consisted of vacuolization, hypo- and hyper-plasia, hypo- and hyper-pigmentation, and accumulation of photoreceptor degradation products. The variety of ocular abnormalities in DHAP-AT-deficient mice provides suggestive evidence that Pls play a crucial role in ocular development and function. Since one of the physiological functions of Pls is to protect animal cell membranes against oxidative stress (Morand et al., 1988; Zoeller et al., 1988; Morandat et al., 2003) and considering that light is a risk factor or cofactor for many types of retinal degenerations (Simons, 1993; Chen et al., 1999a,b; Cruickshanks et al., 2001; Wenzel et al., 2005), it is highly conceivable that the importance of Pls is crucial within the retina and/or the RPE. To our knowledge, the metabolism of Pls in the retinal and RPE cells has not been previously described. In this work, we used in situ hybridization to localize DHAP-AT expression within rat eyes and chromatographic methods to identify and quantify the Pls species according to their aliphatic moieties at the sn-1 position of Pls in calf retina and RPE.

2. Materials and methods 2.1. Animals and tissue collection Experiments on rats were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and were conducted according to the French legislation (authorization A21200 for animal housing

and personal authorization 21CAE086 for N. Acar for experimentation on animals). Adult male Wistar rats (Janvier’s breeding, Le GenestSt-Isle, France) were used for in situ hybridization experiments. Animals were housed in animal quarters under controlled temperature (21  1  C), hygrometry (55e60%), and light conditions (12-h dark/12-h light cycle, 20 lux). They had free access to a standard chow diet and water. Rats were sacrificed by decapitation, the eyes were removed, embedded in Cryomount (Thermo-Shandon, Cergy-Pontoise, France), and frozen in cold isopentane. Cryo-sections, 8 mm thick, were mounted on SuperFrost Plus slides and stored at 80  C until further experiments. Brains were isolated, snap frozen, and stored at 80  C for riboprobe preparation. For biochemical determination of Pl levels, calf eyes were collected (Abattoirs de Beaune, Beaune, France), stored on ice, and dissected within 2 h after slaughter in order to isolate the retina and the RPE. Samples were stored at 80  C until further analyses. 2.2. In situ hybridization of DHAP-AT mRNA In situ hybridization of DHAP-AT mRNA was performed according to a procedure derived from Andre et al. (2005). 2.2.1. Brain RNA preparation Total RNA from brain samples was prepared using TRIzolÒ Reagent (Invitrogen, Cergy-Pontoise, France) according to the manufacturer’s instructions. Briefly, total RNA was extracted with chloroform followed by 2-propanol precipitation. The pellet was washed in 75% ethanol, dried, and dissolved in diethylpyrocarbonate (DEPC) water. The RNA obtained was quantified spectrophotometrically by measuring its absorbance at 260 nm. 2.2.2. Generation of cDNA from brain RNA Specific PCR primers (forward primer 50 -CGT GTT TGC CCG TCC TT-30 and reverse primer 50 -AGC GCG TCG TAA CAC TGA-30 ) were selected using the NCBI primer program for amplification of a 405-bp sequence of the rat DHAPAT gene (GenBank accession no. NM053410). They were purchased from MWG-Biotech AG (Ebersberg, Germany). RT-PCR was performed with a thermal cycler (iCycler, Bio-Rad, Marnes la Coquette, France) using 1 mg of total RNA in 25 mL of reaction mixture consisting of 2.5 units of AMV reverse transcriptase, 225 pmol of each primer, 0.5 mL of dNTP mix (10 mM), 1 mL of MgSO4 (25 mM), 5 mL of AMV 5 reaction buffer, and 2.5 units of Tfl DNA polymerase. RNA was reverse transcripted for 45 min at 48  C. After initial denaturation at 94  C for 2 min, the reaction mixture was subjected to 40 cycles of 1 min denaturation at 94  C, 1 min annealing at 53.1  C, and 1.5 min extension at 72  C, followed by a final 10-min extension step at 72  C. PCRderived fragments were separated by agarose gel electrophoresis (0.7%) and stained with ethidium bromide. The fragment of interest was excised, extracted from the agarose gel, and purified (gel extraction kit, Qiagen, Courtboeuf, France). PCR

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products were subcloned into the pGEMÒ-T easy vector (3015 bp, Promega, Charbonnieres, France). JM 109-competent cells were then transformed with this vector. A positive clone was selected for a last amplification. Plasmids were then purified using QIAfilter Plasmid Maxi Kits (Qiagen, Courtboeuf, France) and sequenced using a ceq 2000 Beckman Coulter (Beckman Coulter, Villepinte, France). 2.2.3. Riboprobe synthesis The plasmids containing the 405-bp rat cDNA fragment were linearized with BstX I and Apa I for the antisense and sense probes, respectively. Radioactive cRNA copies were synthesized using the Riboprobe in vitro transcription system (Promega, Charbonnieres, France). The mixture consisted 4 mL of transcription optimized 5 buffer, 0.5 mg of linearized plasmid, 2 mL of 100 mM DTT, 1.5 mL of 10 mM ATP, GTP, and CTP, 50 mCi of a-35S-UTP, 20 units of recombinant Rnasin ribonuclease inhibitor, and 20 units of either T7 (antisense probe) or SP6 (sense probe) RNA polymerase. This mixture was incubated for 45 min at 37  C and then 6 min after adding 1 unit RQ1 Dnase. Radioactive RNA was extracted with 100 mL of an HENS buffer containing 20 mM HEPES (pH 7.0), 5 mM EDTA, 50 mM NaCl, 0.1% SDS, and tRNA (1 mg/mL). This last compound removed unincorporated nucleotides. The RNA was precipitated for 30 min at 80  C by adding 65 mL of 7.5 M ammonium acetate and 500 mL of cold absolute ethanol. After centrifugation (20 min, 15,000 g), the pellet was dried, dissolved in HENS buffer, and then heated at 65  C for 1 min. This final solution of radioactive RNA was counted. 2.2.4. Hybridization Slides were placed in paraformaldehyde for 15 min on ice, and then rinsed twice in PBS for 5 min. They were dipped in 0.25% anhydric acetic acid/0.1 M TEA for 10 min and rinsed in 2 SSC (0.3 M NaCl, 30 mM sodium citrate) for 20 s. Slides were dehydrated with ethanol and were successively put in 5 mM MgCl2ePBS for 10 min, in 0.25 M Trise0.1 M glycine for 10 min and in 50% formamidee2 SET (0.3 M NaCl, 60 mM TriseHCl, 4 mM EDTA, pH 8.0) at 37  C for 10 min. The hybridization mixture consisting of 7.25 mL sterile water, 2.5 mL 20 SET, 0.1 g PVP (0.1 mM), 0.1 g Ficoll (0.01 M), 0.1 g BSA, 12.5 mL formamide (13 M), 4 g dextran sulfate, 50 mg/mL tRNA, and 500 mL DTT (1 M) was prepared with a-35S-labeled sense or antisense probes to obtain a concentration of 2  107 cpm/mL. These solutions (sense or antisense) were spotted on slides, and a coverslip was placed on the sections. Slides were incubated at 45  C for 4 h in a humid hybridization chamber. After incubation, the slides were washed rapidly by submersion in 4 SSC, then in 50% formamidee2 SETe0.04% mercaptoethanol for 15 min at 60  C and lastly in 4 SSC. Slides were incubated in Rnase A solution (10 mg/mL)e3 SET at 37  C for 20 min and rinsed in 1 SSC at room temperature for 30 min with a light agitation. The last wash consisted of 0.1% mercaptoethanol in 0.2 SET at 50  C for 30 min. Slides were

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dehydrated in 30%, 50%, and 70% ethanol containing 0.3 M ammonium acetate, and after in 85%, 95%, and 100% ethanol. 2.2.5. Autoradiography The hybridized sections were dried and autoradiographed. Slides were dipped in Hypercoat LM-1 emulsion (Amersham, Saclay, France) and exposed for 30 days at 4  C. At developing time, slides were put in a D-19 developer (Kodak, Chalon sur Saone, France) for 2.5 min at 15  C, rinsed, fixed (Kodak fixer) for 5 min, and placed in water for 10 min. Slides were counterstained with Harris’ hematoxylin and aqueous eosin. 2.3. Determination of retinal and RPE lipid composition with focus on Pl levels All chemical reagents were purchased from Sigmae Aldrich (St Quentin Fallavier, France) and methanol from SDS (Peypin, France). 2.3.1. Total lipid extraction and quantification of lipid classes Total lipids from calf retina and RPE samples were extracted according to the Folch procedure (Folch et al., 1957) using chloroformemethanol (2:1, v:v) and washed with 0.73% NaCl in water. The composition of total lipids was then further analyzed by determining the levels of phospholipids, triacylglycerols, free fatty acids, cholesterol, and cholesteryl esters using a combination of thin-layer chromatography (TLC) on silica gel-coated quartz rods coupled with a flame ionization detector (FID) (IatroscanÒ system, Iatron, Tokyo, Japan) according to the technique published by Ackman (1981). 2.3.2. Isolation of total phospholipids and quantification and separation of individual phospholipid classes Total phospholipids were separated from neutral lipids on silica cartridges (25 mm  10 mm i.d.; Sep-pack, Waters S.A., Framingham, MA, USA) according to Juaneda and Rocquelin (1985). Individual phospholipid classes were quantified using high-performance liquid chromatography (HPLC). The HPLC system was equipped with a silica column and a light scattering detector model 11 (Cunow, France) according to the method developed by Juaneda et al. (1990). Individual phospholipid classes were isolated by thin-layer chromatography according to the method developed by Gilfillan et al. (1983). Briefly, silica plates (20  20-cm K6 silica gel plates; Whatman, Clifton, NJ, USA) were used after activation at 100  C for 90 min. Phospholipids were separated in the first dimension using a chloroformemethanolepetroleum ethereacetic acide boric acid mixture (40:20:30:10:1.8; v:v:v:v:w). After drying under a stream of N2, lipids were visualized by brief exposure to iodine vapor. The gel areas containing total ethanolamine phospholipids (PE; including phosphatidyl-ethanolamine, plasmanyl-ethanolamine and plasmenyl-ethanolamine), total choline phospholipids (PC; including phosphatidyl-choline, plasmanyl-choline and plasmenyl-choline), total serine phospholipids (PS; including phosphatidyl-serine, plasmanyl-serine and plasmenyl-serine) plus total inositol phospholipids (PI;

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including phosphatidyl-inositol, plasmanyl-inositol and plasmenyl-inositol) were scraped from the plate for derivation before gas chromatography. 2.3.3. Derivatization of fatty acids and aldehyde aliphatic groups for gas chromatography analyses Fatty acids from aldehyde aliphatic groups linked to PE, PC, PS, and PI were transesterified with boron trifluoride in methanol (7% w/v) according to Morrison and Smith (1964). The fatty acid methyl esters (FAMEs; formed from fatty acids from the sn-1 and sn-2 positions of diacylglycerophospholipids and the sn-2 position of Pls) and dimethyl acetals (DMAs; formed from aldehyde aliphatic groups from the sn1 position of Pls) were analyzed on a Hewlett-Packard (Palo Alto, CA, USA) 5890 series II gas chromatograph equipped with a split/splitless injector, a flame ionization detector, and a BPX 70-silica capillary column (120 m  0.5 mm i.d. film thickness, 0.25 mm; SGE, Melbourne, Australia). The injector and the detector were maintained at 250  C and 280  C, respectively. Hydrogen was used as a carrier gas (inlet pressure, 300 kPa). The oven temperature was fixed at 60  C for 1 min, then increased from 60 to 200  C at a rate of 20  C/min and left at this temperature until the end of the analysis. FAME and DMA derivatives were identified by comparison with commercial or synthetic standards and quantified using the DIAMIR software (JMBS Ins., Portage, MI, USA). Within one subclass of phospholipids, the relative proportions of diacylglycerophospholipids versus Pls were determined using the fatty acid composition data. For example, for PE the proportions of plasmenyl-ethanolamine and phosphatidyl-ethanolamine were calculated as follows:

3.2. Characterization of retinal and RPE lipids focusing on Pl levels 3.2.1. Proportions of lipid classes The composition of lipid classes from calf retinal and RPE total lipids is presented in Fig. 2. In both tissues, phospholipids were the major lipid class since they accounted for approximately 85% of the total lipids. Cholesterol and triglyceride levels were similar in both the retina and the RPE with roughly 10% and less than 1% of total lipids, respectively. Only the proportions of cholesteryl esters and free fatty acids differed between retinal and RPE lipids. Cholesteryl esters accounted for 3.2% of total lipids in the retina compared to 1.4% in the RPE; free fatty acids accounted for 1.6% and 2.7% in the retina and the RPE, respectively. 3.2.2. Proportions of phospholipid species The distribution of the retinal and RPE phospholipids within classes is presented in Fig. 3. The phospholipid compositions of retinal and RPE lipids were quite similar. In both tissues, PC and PE made up approximately 40% of total phospholipids despite minor fluctuations, whereas PS þ PI accounted for 11% of total phospholipids.

3.1. Expression of DHAP-AT mRNA in the retina and the RPE

3.2.3. Proportions of Pls in individual phospholipid classes In both retinal and RPE phospholipids, Pls were mostly represented in PE (Fig. 4). In the retina, plasmenyl-ethanolamine made up 29.1% of total phosphatidyl-ethanolamine þ plasmenyl-ethanolamine whereas the levels of plasmenyl-choline and plasmenyl-serine þ plasmenyl-inositol were 1.9% and 2.2% of total phosphatidyl-choline þ plasmenyl-choline and total phosphatidyl-serine þ plasmenyl-serine þ total phosphatidyl-inositol þ plasmenyl-inositol, respectively. In the RPE, the proportions of plasmenyl-ethanolamine were 27.9% of phosphatidyl-ethanolamine þ plasmenyl-ethanolamine. In this tissue, the levels of plasmenyl-choline and plasmenyl-serine þ plasmenyl-inositol were higher than those of the retina since they were 3.5% and 10.2% of total phosphatidylcholine þ plasmenyl-choline and total phosphatidyl-serine þ plasmenyl-serine þ total phosphatidyl-inositol þ plasmenylinositol, respectively.

The expression of DHAP-AT was localized on rat retina sections by in situ hybridization using a synthetic riboprobe. In accordance with the study conducted by Andre et al. (2005), the riboprobe shared 100% homology with part of the coding sequence of Rattus norvegicus acyl-CoA DHAPAT cDNA (GenBank accession no., NM053410). The radioactive signal of the sense probe, characterizing the background, was very weak and even nonexistent (Fig. 1a). The specific expression of DHAP-AT was detected using the antisense probe (Fig. 1b). The RPE was labeled very intensively as were the photoreceptor’s inner segments. A weaker signal was observed in the outer plexiform layer of the retina. No expression of DHAP-AT mRNA was detected in the other retinal layers.

3.2.4. Characterization of the alkenyl residues linked to the sn-1 position of Pls The levels of Pl subtypes within a phospholipid family were determined according to the relative proportions of DMAs. The main Pl subtypes found in both tissues were Pls containing hexadecanal-, octadecanal-, 9-octadecanal-, and 7-octadanal-aldehydes identified and quantified by GC as 16:0DMAs, 18:0DMAs, 18:1n-9DMAs, and 18:1n-7DMAs, respectively (Fig. 5). No differences were observed in the total DMA content within PE, PC, and PS þ PI between the retina and the RPE (data not shown). The DMA profile was similar in RPE whatever the phospholipid subtype was. DMAs from RPE cells were mainly 16:0DMAs (ranging from 43.6% to 50% of total

%plasmenyl-ethanolamine ¼ 2  ð%of total DMAsÞ %phosphatidyl-ethanolamine ¼ ð%of total FAMEsÞ  ð%of total DMAsÞ

3. Results

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Fig. 1. In situ hybridization of DHAP-AT in the retina and the RPE. (a) Hybridization with the sense probe in contrast phase (left) or dark field (right). Hybridization with the sense probe for the DHAP-AT mRNA did not show any radioactive signal and thus any background. (b) Hybridization with the antisense probe in contrast phase (left) or dark field (right). The radioactive signal of the DHAP-AT mRNA-specific-antisense probe was very strong in the RPE as well as in the area of photoreceptor inner segments. A weaker radioactive signal was detected in the outer plexiform layer. RPE: retinal pigment epithelium; OS: photoreceptor outer segment; IS: photoreceptor inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. All pictures were obtained using a 100 objective on an Axiovert 25 Zeiss microscope.

Fig. 2. Top: relative proportions of lipid classes in calf retina and RPE. Results are presented as mean  SD (n ¼ 10). Bottom: representative traces of thin-layer chromatography with flame ionization detection (TLC-FID, IatroscanÒ) analyses of calf retina and RPE lipids. Phospholipids, playing a structural role in membranes, are the most represented lipids in the retina and the RPE.

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Fig. 3. Top: relative proportions of phospholipid classes in calf retina and RPE. Results are presented as mean  SD (n ¼ 10). Bottom: representative traces of high-performance liquid chromatography analyses of calf retina and RPE lipids. PC and PE made up approximately 40% of total phospholipids in the retina and in the RPE.

DMAs) and to a lesser extent 18:0DMAs (ranging from 28.3% to 38.1% of total DMAs). The 18:1n-9DMAs and 18:1n7DMAs were found in low proportions, as they ranged from 8.0% to 14.5% of the total DMAs. In the retina, only the PC profile was similar to those observed in the RPE (50.7%, 22.5%, 6.8%, and 20.0% of total DMAs for 16:0DMAs, 18:0DMAs, 18:1n-9DMAs, and 18:1n-7DMAs, respectively). In the other retinal phospholipid subclasses, the proportions of 18:0DMAs were increased, whereas those of 16:0DMAs were reduced. The mean percentage of 18:0DMAs reached 48.8% and 67.1% in PS þ PI and in PE, respectively.

4. Discussion Pls have long been considered as a biological peculiarity. However, interest in their occurrence, synthesis, and properties has increased over the past 20 years, stimulated by the implication of Pls in various degenerative diseases and the discovery of genetic disorders in which Pls are deficient. Pl deficiency is associated with the early onset of retinal abnormalities and/or retinopathy, which involve visual loss starting at early ages (Goldfischer et al., 1973; Wanders et al., 1995, 1996; Motley et al., 1997; Rodemer et al., 2003; Lyons et al., 2004). All these peroxisomal disorders

Fig. 4. Relative proportions of vinyl-ether lipids within ethanolamine, choline, serine, and inositol diacyl-phospholipids þ plasmenyl-phospholipids from calf retina and RPE. The values were calculated using the DMA and the FAME proportions (see text). Pls were most highly represented in PE from retina and RPE (29.1% and 27.9% of total phosphatidyl-ethanolamine þ plasmenyl-ethanolamine, respectively).

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Fig. 5. Levels of Pl subtypes within phospholipid families according to their aliphatic moiety in the sn-1 position. Results are presented as mean  SD (n ¼ 10). Pls from RPE cells were mainly with hexadecanal-aldehyde in the sn-1 position and to a lesser extent with octadecanal-aldehyde. The retinal content of Pls differed in PS þ PI and in PE subclasses with larger proportions of Pls containing octadecanal-aldehyde in the sn-1 position.

are caused by genetic defects in both the peroxisomal assembly and single peroxisomal enzymes and have in common that tissue levels of Pls are more or less severely decreased. Since the peroxisomal DHAP-AT and ADAPS activities are the only ones known so far that result in forming Pls in mammalians, unimpaired activities of these enzymes are essential for Pls biosynthesis. Using in situ hybridization, we localized the strongest DHAP-AT expression in the retinal layers that have the highest lipid metabolizing ability: the RPE cells and the photoreceptor inner segments are known to be key places for rhodopsin disk assembling and shedding (SanGiovanni and Chew, 2005). Photoreceptor inner segments and particularly their ellipsoid region are known to contain all the cellular organites including peroxisomes to build up rhodopsin-rich disk membranes before their export to photoreceptor outer segments. It is now accepted that protein-rich membranes (and obviously rhodopsin-rich disks) contain lipid raft microdomains (LRMs) that are different from the rest of the membrane in specific lipid and protein composition (Simons and Toomre, 2000; Nair et al., 2002). LRMs appear to be fundamental for signal transduction, which is highly conceivable for those present in rhodopsin-rich disks in terms of initiation of visual transduction. Interestingly, the recent results obtained on DHAP-ATdeficient mice by Rodemer and collaborators showed that Pls are important constituents of LRMs and that their absence affects the sequestration of proteins into LRMs, thus strongly suggesting that Pls are required for the correct assembling and function of LRMs (Rodemer et al., 2003). So the strong expression of DHAP-AT, the first enzyme involved in Pls biosynthesis in the place where rhodopsin-rich disks (and subsequently the LRMs they contain) are formed appears to be highly logical. It appears to be more difficult to explain why DHAP-AT is expressed in RPE and retinal outer plexiform layers. One logical explanation for DHAP-AT expression, and consequently of Pl formation in RPE cells could be protection against

oxidative stress. Actually, the RPE monolayer is located in a highly oxygenated environment and is exposed to high levels of visible light and therefore at risk for oxidative damage (Cai et al., 2000). RPE cells are normally well protected against oxidative damage by their enzymatic machinery (catalase, superoxide dismutase, glutathione peroxidase), by antioxidants (vitamins E and C), but also by melanin (Newsome et al., 1994). Regarding our results, another potential protective mechanism in RPE cells might involve Pls, since one of the assumed functions of these phospholipid subclasses is the protection of cell membranes against oxidative stress (Morand et al., 1988; Zoeller et al., 1988; Morandat et al., 2003). The elucidation of the presence of DHAP-AT mRNA in the inner retinal cells being more speculative, further studies are required to elucidate this question. The results we obtained on the composition of retinal lipids are in accordance with previously reported values in human, dog, calf, and chicken retina and RPE even if the trans-methylation technique we used to derivatize fatty acids and aldehydes does not take into account alkyl ether phospholipids (‘‘plasmanyl’’ type) (Anderson et al., 1970, 1997; Dorman et al., 1976; Gulcan et al., 1993). In this study, lipid analyses were done on an animal model different from the one used for in situ hybridization experiments. However, the retinal Pls composition we obtained on calf may be very close to that of rat since these animal models share huge similarities in terms of retinal phospholipid compositions (Fliesler and Anderson, 1983). Moreover, it was shown that Pls are present in the same proportions in rat and calf brain lipids (Dorman et al., 1976; Clarke and Dawson, 1981). Ethanolamine Pls were the most prominent ether-lipids, whereas choline Pls made up a very small proportion, suggesting a specific function of this Pl subtype in this tissue. Interestingly, the ethanolamine Pl content of RPE cells was very close to that of retina (28e29% of total diacylethanolamine þ plasmenyl-ethanolamine phospholipids), with only the proportions of other Pl subtypes differing

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between retina and RPE. However, when looking to the aliphatic moieties from the sn-1 position of ethanolamine Pls, we observed huge differences between RPE and retina: ethanolamine Pls with octadecanal-aldehyde (18:0) were very prominent (two-thirds of total ethanolamine Pls) in the retina, whereas ethanolamine Pls with octadecanal-aldehyde (18:0) and hexadecanal-aldehyde (16:0) were found in comparable quantities in the RPE (40% of total ethanolamine Pls for each subtype). The nature of the aliphatic moiety in the sn-1 position of Pls is determined by the nature of the fatty alcohol radical that is exchanged by ADAPS in replacement of the acyl radical first esterified by DHAP-AT (Nagan and Zoeller, 2001). The differences observed between retina and RPE in the sn-1 position of ethanolamine Pls may suggest cell-specific differences in bioavailability of the fatty alcohol radicals at the time of the biosynthesis. The physiological significance of these differences in retinal and RPE ethanolamine Pl compositions would suggest distinct functions, as suggested and discussed above. The physiological role of Pls is still unclear, although various functions have been attributed to them. Altogether, the results presented in this paper suggest the existence of different sites of biosynthesis of Pls within the retina and the RPE and reinforces some of the hypotheses attributing to Pls a protective role against oxidative stress and various functions in LRM formation and function. They contribute to understanding the molecular causes of the visual loss observed in patients suffering from peroxisomal disorders leading to abnormal Pl biosynthesis.

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