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Biochimica et Biophysica Acta 1763 (2006) 1511–1526 www.elsevier.com/locate/bbamcr

Review The ether lipid-deficient mouse: Tracking down plasmalogen functions ⁎ Karin Gorgas a, Andre Teigler b, Dorde Komljenovic a, Wilhelm W. Just b,

a Institut für Anatomie und Zellbiologie, Abteilung Medizinische Zellbiologie, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany b Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany

Received 29 June 2006; received in revised form 15 August 2006; accepted 23 August 2006 Available online 30 August 2006

Abstract

Chemical and physico-chemical properties as well as physiological functions of major mammalian ether-linked glycerolipids, including plasmalogens were reviewed. Their chemical structures were described and their effect on membrane fluidity and membrane fusion discussed. The recent generation of mouse models with ether lipid deficiency offered the possibility to study ether lipid and particularly plasmalogen functions in vivo. Ether lipid-deficient mice revealed severe phenotypic alterations, including arrest of spermatogenesis, development of cataract and defects in central nervous system myelination. In several cell culture systems lack of plasmalogens impaired intracellular cholesterol distribution affecting plasma membrane functions and structural changes of ER and Golgi cisternae. Based on these phenotypic anomalies that were accurately described conclusions were drawn on putative functions of plasmalogens. These functions were related to cell–cell or cell–extracellular matrix interactions, formation of lipid raft microdomains and intracellular cholesterol homeostasis. There are several human disorders, such as Zellweger syndrome, rhizomelic chondrodysplasia punctata, Alzheimer’s disease, Down syndrome, and Niemann–Pick type C disease that are distinguished by altered tissue plasmalogen concentrations. The role plasmalogens might play in the pathology of these disorders is discussed. © 2006 Elsevier B.V. All rights reserved.

Keywords: Ether lipid; Plasmalogen; Sulfogalactolipid; Spermatogenesis; Lens; Neurodegenerative disease; Myelination

1. Introduction peroxisomal enzymes dihydroxyacetonephosphate acyltrans- ferase (DHAPAT) and alkyldihydroxyacetonephosphate syn- Ether-linked glycerolipids are widely distributed in nature thase (ADAPS) [1]. Accordingly, plasmalogen levels are serving important structural and functional aspects of biological considerably reduced in disorders based on defects in peroxi- membranes. They are found in bacteria, primitive protozoa, some assembly, such as the cerebro-hepato-renal (Zellweger) fungi and higher plants and are present in mammals including syndrome (ZS) and rhizomelic chondrodysplasia punctata men. In mammals synthesis of the ether bond requires the (RCDP) [2–4]. RCDP type 2 and type 3 are both distinguished by an isolated defect in ether lipid biosynthesis. The severe Abbreviations: AAPH, 2,2′-azobis-(2-amidinopropane HCl); Aβ, β amy- pathology observed in these patients indicates the physiological loid; AD, Alzheimer’s disease; ADAPS, alkyldihydroxyacetonephosphate importance of ether lipids for normal human life. Although much synthase; BACE, beta-site amyloid precursor protein cleaving enzyme; CDR, has been learned about ether lipid functions within the last clinical dementia ratings; CGT, UDP-galactose:ceramide galactosyltransferase; decade [2,5–8], it seems that we are still far away from a CNS, central nervous system; CST, cerebroside sulfotransferase; DHA, comprehensive understanding of their physiological role. docosahexaenoic acid; DHAPAT, dihydroxyacetonephosphate acyltransferase; DS, Down syndrome; FGSLs, fucosylated glycosphingolipids; GPI, glycosyl- In this review we mainly focus on mammalian ether lipids phosphatidylinositol; LRMs, lipid raft microdomains; NPC, Niemann–Pick type particularly plasmalogens, since they represent the major C disease; OMgp, oligodendrocyte myelin glycoprotein; PAF, platelet-activating constituents in most tissues and the phenotypes connected factor; PTS, peroxisomal targeting signal; PPAR, peroxisome proliferator- with impaired ether lipids are mainly caused by impaired activated receptor; RCDP, rhizomelic chondrodysplasia punctata; ROS, reactive plasmalogen functions. However, we also consider the oxygen species; SGalAAG, 1-O-alkyl-2-O-acyl-3-O-β-D-(3′-sulfo)galactosyl- sn-glycerol; SOD, superoxide dismutase; ZS, Zellweger syndrome particular situation in testis where both ether phospholipids ⁎ Corresponding author. Tel.: +49 6221 544151; fax: +49 6221 544366. and seminolipid, a sulfogalactosyl alkylacylglycerol, contribute E-mail address: [email protected] (W.W. Just). to the phenotype of ether lipid deficiency. First, we will briefly

0167-4889/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2006.08.038 1512 K. Gorgas et al. / Biochimica et Biophysica Acta 1763 (2006) 1511–1526 summarize the chemical peculiarities of plasmalogens and their concentration after the age of 22days, thus being the more biological functions so far as they were currently discussed. stable component of myelin. Subsequently, we will focus on recent studies dealing with the GPI-anchoring of proteins is a posttranslational modification phenotypic changes seen in ether lipid-deficient mouse models that occurs in the ER and attaches proteins to the luminal side of primarily the model based on isolated DHAPAT deficiency the membrane. Thus, following vectorial transport to the plasma [9,10]. In this model DHAPAT, the key enzyme in ether lipid membrane GPI-anchored proteins are exposed at the extracel- biosynthesis, is eliminated by targeted deletion resulting in the lular site of the plasma membrane. As shown in Fig. 1G the core complete absence of ether lipids. Major phenotypes observed in structure of the GPI-anchor consists of phosphatidylinositol these animals are described representing the basis for a containing either diacyl- or 1-alkyl-2-acyl glycerol in addition discussion of ether lipid functions in mammalian cell types to glucosamine, trimannoside and phosphoethanolamine. The and organ systems. latter provides the link to the C-terminus of the GPI-anchored ectoprotein. 2. Chemical aspects of mammalian ether lipids 2.2. Chemistry of the plasmalogen-specific vinyl ether bond 2.1. Structure of ether lipids Plasmalogens are distinguished by two characteristic The major constituents of the class of mammalian ether chemical reactivities. Firstly, their vinyl ether group is acid phospholipids contain a single alkyl chain at the sn-1 position labile and secondly, plasmalogens are classified as efficient in linked to the glycerol backbone via an ether bond. Although vitro antioxidants being targets of a variety of oxidants, such as species with phosphoinositol or phosphoserine as a head group singlet oxygen, peroxy radicals and metal ions. have been described, the most abundant species belong to the The mechanism of the acid-catalyzed hydrolysis of general class of glycerophosphoethanolamine and glycerophosphocho- vinyl ethers as model compounds of plasmalogens has been line phospholipids [8]. Among these ether-bonded glyceropho- investigated in detail [16]. The reaction proceeds in several spholipids those bearing an enol ether (vinyl ether) are called steps. The first one consisting in the addition of a proton to the plasmalogens and are by far the most abundant molecules in double bond is considered to be rate limiting. Once formed, the mammalian tissues. According to a widely used nomenclature, carbonium ion is rapidly converted to the α-hydroxy-compound these plasmalogens are called plasmenylethanolamines and that in the third step easily splits into aldehyde and alcohol. plasmenylcholines, whereas their saturated counterparts are Lysoplasmenylcholine is somewhat more rapidly hydrolyzed called plasmanylethanolamines and plasmanylcholines (Fig. 1A than lysoplasmenylethanolamine and the cis-oriented double and B). The alkyl chains at the sn-1 position of plasmalogens bond, as it is present in natural plasmalogens, is much more usually have chain lengths of C16 or C18 and the vinyl ether reactive than the corresponding trans-double bond. double bond represents the cis-isomer. At the sn-2 position There is a long lasting discussion on the antioxidative plasmalogens preferably contain a long chain polyunsaturated character of plasmalogens. Within the last decade a large fatty acid both of the n-6 or n-3 class. In that sense platelet- number of articles appeared dealing with various aspects of the activating factor (PAF) bearing an acetyl residue at the sn-2 antioxidative activity of plasmalogens. Again their vinyl ether position is exceptional among the ether lipids (Fig. 1C). Recent bond is target of oxidative attack by reactive oxygen species studies in mice carrying the targeted deletion of the PAF receptor (ROS), e.g. singlet oxygen, superoxide anion, hydrogen provided evidence for a beneficial attenuation of symptoms in peroxide and peroxy radicals. Metal ion-catalyzed reactions chemical lung injury, bronchial asthma, sponge-induced subcu- particularly Fe3+/Fe2+ may strongly support these reactions. taneous granuloma formation and folic acid-induced renal The most important aspects of the scavanger functions of inflammation mediated by infiltrating inflammatory cells [11]. plasmalogens are briefly summarized, as they might be essential In this review we will not further discuss PAF and its biological for understanding plasmalogen functions. functions. For this the reader is referred to a number of recent The reactions of olefins, unsaturated hydrocarbons, e.g. sub- publications [12–14]. stituted acetylenes, ketenes or allenes, with singlet oxygen have Understanding the phenotypic changes seen in the been thoroughly reviewed and may follow mechanisms of either DHAPAT-deficient mouse model requires consideration of 1,2 addition forming dioxethanes or 1,3 addition on olefins two other ether-bonded lipid species, seminolipid (Fig. 1D) containing at least one allylic hydrogen [17]. Upon decompo- and the glycosylphosphatidylinositol (GPI)-anchor of GPI- sition dioxethanes readily cleave into two carbonyl-containing anchored proteins that in mammalian tissues frequently fragments, whereas 1,3 addition yields allylic hydroperoxides contain a 1-O-alkyl-2-O-acyl-sn-2-glycerol residue within the including a shift of the double bond to the adjacent carbon GPI anchor moiety (Fig. 1E). Seminolipid is an 1-O-alkyl-2- atoms. The oxidation of vinyl ethers with hydrogen peroxide O-acyl-3-O-β-D-(3′-sulfo)galactosyl-sn-glycerol (SGalAAG) in aqueous solution and trace amounts of catalysts was shown that predominantly occurs in testes, but may also be present to predominantly yield a mixture of α-hydroxyaldehydes and in brain [15]. Interestingly, in brain the relative molar α-hydroxyketones together with aldehydes derived from the concentrations of the diacyl and alkylacyl types of sulfoga- cleavage of the vinyl ether bond [18]. Using ethyl hexadec-1- lactosylglycerols change with age. Both types predominate in enyl ether as plasmalogen model and air oxygen as the oxidant, the myelin fraction, the alkylacyl form still increasing in disappearance of the vinyl ether and concomitant generation of K. Gorgas et al. / Biochimica et Biophysica Acta 1763 (2006) 1511–1526 1513

Fig. 1. Structure of major mammalian ether-linked membrane lipids. (A) plasmenylethanolamine (1-O-hexadecenyl-2-oleoyl-sn-glycerophosphoethanolamine, phosphatidylethanolamine plasmalogen); (B) plasmanylethanolamine (1-O-hexadecyl-2-oleoyl-sn-glycerophosphoethanolamine); (C) platelet-activating factor (PAF, 1-O-hexadecyl-2-acetyl-sn-glycerophosphocholine); (D) seminolipid (1-O-hexadecyl-2-oleoyl-3-O-β-D-(3′-sulfo)galactopyranosyl-sn-glycerol); (E) typical glycosylphosphatidyl (GPI) anchor containing an ether-linked alkyl chain.

aldehyde products was reported [19].Thisstudyalso fact that plasmalogens frequently have esterified polyunsatu- demonstrated that alk-1-enyl ether underwent slow but rated fatty acids, preferably arachidonic acid, to the sn-2 measurable oxidation. However, in the presence of polyun- hydroxyl group of the glycerol, these results might be saturated compounds, such as linoleate or linolenate, disap- important discussing the antioxidative function of plasmalo- pearance of the vinyl ether was greatly accelerated proceeding gens. Other studies using 2,2′-azobis-(2-amidinopropane HCl) at a rate comparable to that of the polyunsaturated compound (AAPH), a water-soluble peroxyl radical generator, also itself. These data suggest that the oxidation products of the reported the slow rate of oxidation of lysoplasmenylethano- polyunsaturates attack the vinyl ether rather than the vinyl lamine and a delayed oxidative degradation of sn-2 arachido- ether protects the unsaturates from oxidation. Considering the noyl diacyl glycerolipids by the presence of plasmalogens 1514 K. Gorgas et al. / Biochimica et Biophysica Acta 1763 (2006) 1511–1526 suggesting a protective role of the vinyl ether in radical- 2.3. Effect of plasmalogens on membrane fluidity generated oxidation processes [20]. More recent studies on the oxidation of model glyceropho- Model membranes composed of plasmanyl- and plasmeny- spholipids including arachidonate-containing plasmenylcholine lethanolamines were shown to exhibit a somewhat lower gel-to- utilizing different strategies of oxidation, e.g. t-butylhydroper- liquid phase transition compared with diacylglycerophos- oxide, hydrogen peroxide/Cu2+, hydrogen peroxide/Fe2+ or phoethanolamine [35]. On the other hand measurements of AAPH noted (i) qualitative and quantitative differences in the relative membrane lipid mobility as determined by the oxidation products dependent on the mechanism of free radical fluorescence anisotropy of diphenylhexatrienyl derivatives in oxidation, (ii) carbon-5 of the arachidonate as the major site of control cells and cells deficient in plasmalogens revealed a primary oxidation, (iii) plasmalogens themselves not to stop correlation between fluorescence anisotropy and cellular oxidative events, although they form peroxidized products more plasmalogen content. Hence, the plasmalogen-containing cell rapidly than their corresponding plasmanyl and diacyl glycer- membranes were less fluid than the plasmalogen-deficient ophosphocholines and (iv) the peroxidation mechanism of membranes [36]. plasmalogens intimately involves the oxidation of the arachi- The abundant appearance of polyunsaturated fatty acids at donate. Thus, attack of plasmalogens by oxidants always is the sn-2 position of plasmalogens suggests these phospholipids distinguished by an intramolecular competition between the to act as a membrane store from where polyunsaturated fatty double bonds of the vinyl ether and the polyunsaturated fatty acids particularly arachidonic acid might be released through acid [21–28]. the activity of a plasmalogen-specific phospholipase A2 [8,37– The major products and fragments generated by the 39]. However, investigations on the phospholipase A2-mediated oxidation of plasmalogens are eicosatetraenoic acid hydroxy- release of arachidonate from control and plasmalogen-deficient lated at the 5 (most abundant), 8, 9, 11, 12 and 15 position of macrophage-like cells did not show any difference in the the arachidonic acid. In addition 5,12-dihydroxylated eicosa- kinetics of arachidonic acid release indicating that in vitro data tetraenoic acid, 2-monoacylglycerophosphoethanolamine, pen- need to be carefully examined in appropriate in vivo model tadecanol, formic acid, α-hydroxyaldehydes of various chain systems in order to assess their physiological relevance [8]. lengths derived from plasmalogen epoxides, 1-formyl-2- arachidonoyl glycerophospholipid and lysophospholipid were 2.4. Plasmalogens favor membrane fusion identified [22–28]. Among the oxidation products many aldehydic compounds were detected. Malondialdehyde a Plasmenylethanolamines undergo transition from the lamel- common decomposition product of peroxides derived from lar liquid crystalline (Lα) phase to the HII inverted hexagonal polyunsaturated fatty acids is frequently used to monitor lipid phase at distinctly lower temperature than diacyl phosphatidy- peroxidation in biological material. Thiobarbituric acid reacts lethanolamines [36,40]. This propensity to form nonlamellar with some specificity with malondialdehyde and thus, lipid structures seems to predispose plasmenylethanolamines to thiobarbituric acid-reactive substances expressed in malon- play an important role in physiological processes, such as dialdehyde equivalents are a convenient measure to express balancing between bilayer and non-lamellar phases. Recent lipid peroxidation [29]. ROS have also been postulated to models on protein-free membrane fusion, such as the stalk contribute to aging-related alterations. This free radical theory hypothesis, relate on the formation of distinct intermediate of aging was recently tested in senescence-accelerated mice stages, including approach of the membranes to small distances showing abnormal aging characteristics [30]. However, no followed by local perturbations of the lipid structure, merger of change was noted in brain plasmalogen concentrations and proximal monolayers, stalk formation as well as expansion and aldehyde and α-hydroxyaldehyde levels were unaltered in final pore formation [41–43]. The low transition temperature of these mice compared with age-matched controls and remained plasmalogens may particularly facilitate formation of the initial constant during aging [31]. More recently, the isoprostanes, stages of fusion that seem to be promoted by the intrinsic nature products of arachidonic acid oxidation, were found to be of the sn-1 vinyl ether chain, as the unsaturation index of the accurate markers of lipid peroxidation in animal models of sn-1 aliphatic chain plays a dominant role in determining the oxidative stress illuminating oxidant injury in association with transition temperature [44,45]. The role of plasmalogens in a number of human diseases [32]. membrane fusion is further illuminated by observations that Strongest indication for a role of plasmalogens as endoge- show high plasmalogen concentrations in subcellular mem- nous antioxidants came from experiments with pyrene-labeled branes implicated in rapid membrane fusion, e.g. synaptic fatty acids, 12-(1′-pyrene) dodecanoic acid, fed to plasmalogen- vesicles [46]. Direct evidence for a fusogenic activity of deficient CHO cells with otherwise normal peroxisomal plasmenylethanolamine was obtained by measuring Ca2+- functions. ROS were generated by long-wavelength UV light induced fusion rates of phosphatidylserine liposomes with (>300nm) irradiation and were shown to be more cytotoxic in liposomes comprised of equimolar mixtures of phosphatidyl- mutant cells than in controls. Using mutant variants of a murine choline and either plasmenylethanolamine or phosphatidyleth- macrophage cell line or mutant human fibroblast lines from anolamine [44,45]. Highest fusion rates were measured with patients with peroxisomal disorders, evidence was provided for plasmenylethanolamine liposomes containing arachidonic acid the vinyl ether bond to function as the crucial element in this at the sn-2 position of the glycerol moiety. These data are activity protecting against ROS [8,33,34]. particularly interesting, as they attribute to the lipids involved K. Gorgas et al. / Biochimica et Biophysica Acta 1763 (2006) 1511–1526 1515 in fusion important functions. Exploring the mechanisms deficient in Zellweger patients [59]. When administered daily to implicated in the targeted movements of lipids to and within the pregnant Pex5-deficient mother it normalized brain DHA membranes might be an important future challenge. Protein- levels in the offsprings. However, restoration of brain DHA dependent fusion of membranes requires a complementary set concentrations had no effect on the pathological abnormalities, of SNARE proteins assembling into the trans-SNARE including defects in myelination, visual functions, neuronal complex. Thus, SNARE proteins bringing membranes into migration and plasmalogen biosynthesis [54]. close contact are essential determinants for fusion specificity The third Zellweger mouse model was generated by Cre- [47–49]. However, it is still a matter of debate whether loxP disruption of the Pex13 gene [53]. Pex13p is a pero- SNAREs are required for later subsequent stages of fusion, xisomal integral membrane protein involved in both PTS1 and such as for merger of membranes. Although there are data PTS2 protein import. Together with Pex14 and Pex17, Pex13 indicating a role ether lipids may have in membrane fusion in forms the docking complex that appeared to be linked via vitro, their functional implication in vivo remains to be Pex8 to the RING-finger import complex comprised of Pex2, established. Pex10 and Pex12 [60]. Similar to Pex5 and Pex2 knockout mice, Pex13 knockout mice exhibit many of the clinical fea- 3. Mouse knockout models with ether lipid deficiency tures observed in ZS. The brain of these mice shows disor- dered cerebral cortex lamination consistent with a neuronal Two peroxisomal enzymes, DHAPAT and ADAPS, are migration defect. While visceral organs of these animals were essential for the formation of the sn-1 alkylglycerol ether bond unremarkable by light microscopy, ultrastructural examination characteristic for all ether lipids [1]. Mutations in these enzymes of the liver revealed abundant large lipid droplets, mitochon- or factors involved in their posttranslational import result in dria with abnormal morphology of cristae and in kidney a altered cellular levels of ether lipids. The peroxisome biogenesis delayed differentiation of glomeruli including paucity of disorders, such as ZS or RCDP, are paradigmatic for disorders to podocyte foot processes [53]. which plasmalogen deficiency significantly contributes The phenotypic similarities in Zellweger patients and in [3,4,50]. Pex5-, Pex2-, and Pex13-deficient mice clearly indicates that these mouse models are useful systems to explore the 3.1. Models exhibiting a Zellweger phenotype pathogenic mechanisms involved in ZS. However, as nearly all of the peroxisomal functions, including hydrogen peroxide Currently, three Zellweger mouse models were described. metabolism, peroxisomal α− and β-oxidation and synthesis of They were based on the targeted deletion of either Pex5, the ether lipids were impaired, a complex phenotype results. peroxisomal targeting signal 1 (PTS1) receptor or Pex2, an Plasmalogen levels were decreased, however, how and in as integral peroxisomal membrane protein containing a RING much this contributes to the overall phenotype may be difficult finger domain and being involved in peroxisomal membrane to establish. In this context the Pex11β-deficient mouse model protein import or Pex13, a component of the receptor docking is worthwhile to be mentioned, as Pex11β-deficient mice complex [51–53]. Although differences were reported, these display several congenital anomalies shared by ZS, such as mice show many phenotypes similar to that observed in developmental delay, hypotonia, neuronal migration defects, Zellweger patients. Pex5 and Pex2 knockout mice are growth- enhanced neuronal apoptosis and neonatal lethality. However, retarded, severely hypotonic, have a disturbed food intake and these mice do not exhibit defects in peroxisomal protein import most of them die within 24h after birth. In these mice cellular and alterations in mitochondrial structure [61] and are abnormalities were found in the central nervous system (CNS), characterized by only partial defects in peroxisomal fatty acid particularly the cortical plate and the inferior olive indicating β-oxidation and ether lipid biosynthesis. Therefore, it was neuronal migration defects to occur [54]. In Pex2 knockout suggested that accumulation of peroxisomal substrates or lack mice cerebellar alterations, such as a defective foliation of peroxisomal products might not account for the symptoms patterning, were accompanied by reduced dendritic arborization caused by Pex11β deficiency. Due to the absence of severe of Purkinje cells [55]. metabolic disturbances, altered physical properties of the Ultrastructural analysis of Pex5 knockout mice revealed peroxisomal membrane affecting different metabolite transpor- alterations of mitochondrial morphology predominantly in ters were proposed to activate or inactivate one or more hepatocytes [56,57] similar to those described in early reports signaling pathways including the nuclear hormone receptors of Zellweger patients [58]. The morphological changes in the RXR, PPARα and PPARγ [62]. inner mitochondrial membrane, such as patterning of cristae, correlated with functional impairments including decreased 3.2. The Pex7 knockout mouse model activities of complex I and complex V. These impaired mitochondrial functions, however, did not affect cellular levels ADAPS, the second enzyme implicated in ether bond of ATP. Importantly, no evidence was found indicating formation is imported into peroxisomes via the PTS2 pathway oxidative damage of proteins and lipids and there were no involving Pex7, the PTS2 receptor [63]. Thus, genetic defects in signs for an elevated oxidative stress response [57]. Docosa- Pex7 result in cytosolic mislocalization and proteolytic hexaenoic acid (DHA), a major polyunsaturated fatty acid in degradation of ADAPS and consequently generate RCDP mammalian CNS and retina was reported to be profoundly type 1, a distinct human autosomal recessive peroxisomal 1516 K. Gorgas et al. / Biochimica et Biophysica Acta 1763 (2006) 1511–1526 disorder characterized by skeletal, eye and brain abnormalities cholesterol to intracellular sites of oxysterol synthesis [72]. [4,64]. Biochemically, RCDP patients show a severe depletion Cholesterol overload in NPC1 and NPC2 mutants, thus, of plasmalogens and additionally impaired α-oxidation of results from a failure to both suppress sterol regulatory phytanic acid and β-oxidation of very-long-chain fatty acids. A element-binding protein-dependent gene expression and similar phenotype is observed in Pex7 knockout mice that are activate liver X receptor functions. The defects in cholesterol born with severe hypotonia and growth retardation. Although trafficking observed in all cell types may predominantly affect most animals die during the perinatal period, some survive even neurons. NPC1 was found in recycling endosomes of beyond 18 months. The brain of these mice displayed defects in presynaptic nerve terminals, a topology suggesting NPC1 to neuronal migration. However, the abnormalities seen in the play a role in the endosomal pathway of synaptic vesicle Pex7-deleted animals were less pronounced than those in the recycling [65,67]. Pex5 knockouts [2]. In addition to brain abnormalities, Pex7 Plasmalogens might be essential for distinct steps in choles- knockout mice revealed a delayed endochondral ossification. terol transport. Evidence for this is derived from observations Defects were described in the skull, the inner ear and the axial demonstrating reduced plasmalogen concentrations in the brain and appendicular skeleton. Histological examination of the of NPC mice and accumulation of free cholesterol in an femurs further revealed reduced mineralization. In cultured endosomal/lysosomal compartment of SF with isolated plasma- fibroblasts of the Pex7 knockouts plasmalogen biosynthesis was logen deficiency [50,73]. Moreover, plasmalogen-deficient drastically reduced by 85–90% and a marked reduction in the CHO cells show a significantly reduced activity in LDL- level of plasmalogens in fibroblasts, erythrocytes and brain was stimulated cholesterol esterification by acyl-CoA cholesterol noted. These alterations in plasmalogens were accompanied by acyltransferase in the ER [74]. Although the mechanisms by an about 60% decrease in the activity of C26:0 and pristanic which plasmalogens interfere with intracellular cholesterol acid β-oxidation and an impairment of phytanic acid degrada- movements are not clear yet, the data obtained so far indicate tion by peroxisomal α-oxidation [2]. Thus, Pex7 knockout mice a requirement of plasmalogens for cholesterol trafficking from closely mimic human RCDP type 1 and represent a suitable both plasma membrane and lysosomes to the ER. model to study the defects particularly those in endochondral Post mortem analyses of phospholipids in the brain of AD ossification on a molecular level. patients revealed a significant and selective deficiency of plasmenylethanolamine that was more pronounced in the 3.3. Niemann–Pick type C disease, Down syndrome and hippocampus, in mid-temporal and frontal cortex, regions Alzheimer’s disease are distinguished by altered known to be more seriously affected in AD than the caudate concentrations of plasmalogens nucleus, substantia nigra and cerebellum [75–77].No alterations in plasmalogen content were seen in cerebral In addition to ZS and RCDP a number of other disorders white matter [76]. A more recent study investigated by ESI- are known that also are connected to altered concentrations of MS post mortem brain tissue lipid extracts of AD patients that plasmalogens. These disorders include Niemann–Pick type C were correlated to known clinical dementia ratings (CDR) (NPC) disease, Down syndrome (DS) and Alzheimer’s [78]. Plasmalogen content was determined in four different disease (AD). NPC is a rare neurovisceral disorder charac- brain regions, frontal, temporal and parietal cortex as well as terized by progressive hepatomegaly and CNS neurodegen- cerebellum. The ESI-MS analyses showed (i) significant eration [65]. The disease is further distinguished by the plasmalogen deficiency of up to 40mol% of total plasmalogen accumulation of free cholesterol, sphingolipids and other in white matter from all brain regions examined at an early lipids within the endosomal/lysosomal compartment, a stage of AD (CDR 0.5), (ii) plasmalogen deficiency in gray phenotype that is also observed in human and mouse skin matter from all cerebral regions studied and correlating with fibroblasts (SFs) with isolated ether lipid deficiency CDRs, and (iii) normal plasmalogen levels as expected in [50,66,67]. NPC is caused by mutations in either the NPC1 cerebellar gray matter at all examined CDRs. Moreover, or NPC2 protein, though NPC1 mutations account for 95% of reduced concentrations of plasmalogens of up to 10mol% of the reported NPC cases. NPC1 encodes a glycoprotein of total plasmenylethanolamine were also observed in two AD 170–190kDa with 13 putative transmembrane spans including transgenic mouse models, APPV717F and APPsw [46]. Plasma- a conserved sterol-sensing domain. NPC2, on the other hand, logen deficiency was noted in cerebral cortex, but was also is a soluble lysosomal protein with high affinity cholesterol absent in cerebellum. However, sulfatides, a class of myelin- binding and lower affinity fatty acid binding [68]. The specific sphingolipids, were depleted by up to 93% in gray functions of NPC1 and NPC2 are not clear yet. In cells matter and by up to 58% in white matter of all human brain lacking a functional NPC1 gene transport of cholesterol from regions examined [79]. In contrast, the content of ceramide, late endosomes to various intracellular destinations including potential sulfatide hydrolysis products, was elevated more than the plasma membrane is severely disturbed [69] and in 3-fold in white but not gray matter and peaked at the stage of addition esterification of low density lipoprotein cholesterol is very mild dementia (CDR 0.5). Similarly, the marked decrease impaired [70,71]. The findings that production of 25- in sulfatides already occurred in the earliest stages of clinically hydroxycholestrol and 27-hydroxycholesterol is reduced in identifiable AD, i.e. at CDR 0.5. NPC fibroblasts in response to LDL cholesterol indicate a In interpreting these data one major question arises. Is role the NPC proteins play in channeling excess LDL plasmalogen deficiency a primary cause for AD or rather does it K. Gorgas et al. / Biochimica et Biophysica Acta 1763 (2006) 1511–1526 1517 support the pathogenesis of the disease? Based on the results 4. The DHAPAT-deficient mouse model obtained from the AD transgenic mouse models indicating plasmalogen deficiency most likely to be caused by the 4.1. Isolated plasmalogen deficiency causes defects in CNS accumulation of abnormal β amyloid (Aβ) peptide species, it myelination was suggested that plasmalogen deficiency might not be the initial cause of AD and other factors additional to Aβ Initiation of axon myelination requires association between accumulation might favor progression of the disease. This myelin-forming glia and the axon to be ensheathed. This view may be further supported by the fact that absence in these process involves the formation of a large cell–cell adhesion AD mouse models of apoE that also participates in AD complex comprising the paranodal axo-glial junction [99,100]. pathogenesis [80], prevents both Aβ deposition and plasmalo- Its formation is the result of complex intra- and intercellular gen deficiency [81]. ApoE is a 299 amino acid glycoprotein. It activities providing the targeted delivery of key molecules to the is the major protein of VLDL and the major apolipoprotein in nodal domain. Two other processes following assembly of the brain. The identification of the ε4 variant of apoE as a risk factor nodes of Ranvier are related to the regulation of myelin for late-onset AD indicates cholesterol to play a direct role in thickness and the longitudinal extension of myelin segments in AD pathogenesis [82,83]. As on the one hand plasmalogen response to axon lengthening [100]. Once the nodal proteins deficiency affects intracellular cholesterol distribution and on arrived at the nascent node, they might recruit other proteins to the other hand impaired cholesterol homeostasis strongly favors complete the nodal architecture. Selection of axons and AD pathogenesis, brain plasmalogens may be linked to AD by initiation of axo-glial contacts might involve cell adhesion their effects on cholesterol [10,50,74]. molecules, such as NCAM and L1, integrins for oligodendro- Another aspect relates to the antioxidative capacity of cyte sensing of axonal molecules, laminins, the ligands for some plasmalogens mentioned above. There is some evidence for an integrins and neuregulins, neuronal effector molecules influ- increased oxidative brain damage in AD. Several factors encing glial cells to myelinate [101–104]. Although most of the including mitochondrial dysfunction, glutamatergic neurotrans- cell–cell interactions were controlled by proteins, lipid raft mission and Aβ accumulation were proposed to contribute to it microdomains (LRMs) might be a way cells deliver lipid– [84,85]. In particular, Aβ accumulation seems to produce protein complexes to distinct destinations in the plasma enhanced oxidative stress by inducing both protein and lipid membrane. The cell–cell interactions are required for the peroxidation [86–88]. Although an attractive hypothesis, the formation of tissue-specific junctional complexes prerequisite antioxidative character of plasmalogens in vivo remains to be to functional synchronization. established. Compared with plasma membranes of other somatic cells the The existence of a link between brain plasmalogens and myelin membrane contains particularly high concentrations of AD is also suggested by studies of DS patients. The cholesterol, galactolipids and plasmalogens [6,8]. Mice lacking prevalence of dementia in patients between 50 and 59years the ability to synthesize cholesterol in the oligodendrocyte show of age is about 55% and in those over 60years of age it is 75% a delay in myelination, a defect that, however, might be at least [89]. In adult patients loss of neurons is dramatic and partially compensated for by enhanced cholesterol uptake [105]. neurofibrillary tangles and neurite Aβ plaques are prominent Furthermore, mice that cannot synthesize galactolipids show [90]. The reason for these neuropathological findings may myelin defects in the CNS, including disrupted axo-glial implicate triplication of both the APP gene and the beta-site junctions and paranodes [106,107]. Thus, cholesterol and amyloid precursor protein cleaving enzyme (BACE) 2 gene glycosphingolipids are essential constituents of myelin raising that both are located on chromosome 21 [91,92]. Accordingly, the question as to their proposed function. Central to BACE-2 protein and plasma Aβ levels of DS patients were myelinogenesis is the assembly of the myelinating process to significantly increased compared with controls [93–95]. the axonal plasma membrane and its extension during axon Furthermore, the lifelong increase in ROS caused by growth. How this might be accomplished is not clear yet, mitochondrial dysfunction also may affect abnormal APP however, the concept of LRMs might offer an explanation for processing [96]. Superoxide dismutase (SOD) having anti- the underlying mechanism. LRMs are membrane microdomains oxidative properties is also present on chromosome 21 and is enriched in cholesterol and glycosphingolipids and often serve found in higher activity in the fetal brain of DS patients. as platforms that are able to accumulate distinct proteins Although deactivating superoxide, SOD may led to higher particularly those that are bound to membranes by a GPI-anchor concentrations of hydrogen peroxide and hence may contrib- [108–111]. These protein–lipid complexes were already ute to enhanced oxidative stress [97]. In this context it may be assembled in the ER and subsequently targeted to the growing worthwhile to mention that post mortem analyses of brain plasma membrane representing sites of adhesion to terminal phospholipids in DS patients between 38 and 68years of age loops of myelin creating juxtaparanodal and paranodal areas showed a significant decrease in the concentrations of [112]. The junctional complexes determining these nodal zones phosphatidylinositol and plasmenylethanolamine in frontal are essential for the correct topology of the nodal ion channels. cortex and cerebellar gray matter by 37% and 35%, Recent studies on factors inhibitory to neurite outgrowth respectively [98]. The cholesterol/phospholipid ratios, howev- identified oligodendrocyte myelin glycoprotein (OMgp). In rat er, were not significantly changed indicating compensatory spinal cord, OMgp was localized to distinct oligodendrocyte- mechanisms to maintain these ratios. like cells whose processes converged at the node. Since OMgp 1518 K. Gorgas et al. / Biochimica et Biophysica Acta 1763 (2006) 1511–1526 was detected at most of the nodes examined, it was suggested that it might function in suppressing collateral sprouting [113]. Similarly, in rat optic nerve NG2 expressing oligodendrocyte precursor-like cells have been proposed to tightly ensheath the nodal axon, thus preventing axonal sprouting [113,114]. NG2 positive cells are abundantly expressed in the adult CNS and NG2 proteoglycans have also been shown to inhibit neurite outgrowth [115]. Future aspects, therefore, might also consider the plasmalogen content of heterogeneous oligodendrocyte-like glial cell populations and the role plasmalogens play in the process of nodal ensheathment. The concept that ether lipids might play a role in building up the nodal architecture is supported by several observations. (i) Plasmalogens are the dominating glycerophosphoethanolamine species in LRMs isolated from myelin [10], (ii) plasmalogens were found to accumulate in LRMs isolated by a non-detergent technique [116], (iii) seminolipid (SGalAAG) is enriched in Triton X-100-isolated LRMs of sperm cell membranes [117], (iv) anti-tumor ether lipids, such as edelfosine (1-O-octadecyl-2- O-methyl-sn-glycerophosphocholine) and miltefosine (1-hexa- decylphosphocholine), although by themselves do not form sterol-rich domains in fluid model membranes, stabilized palmitoyl-sphingomyelin/cholesterol-rich domains against tem- perature-induced melting suggesting that they affect LRMs, (v) many GPI-anchored proteins, although not thoroughly analyzed yet, contain in their lipid anchor a 1-alkyl-2-acylglycerol moiety (see Section 2.1) the synthesis of which should require functional peroxisomes [108,118] and (vi) plasmalogen levels increase in concentration in parallel to myelination in human cerebrum, cerebellum and brain stem [119]. Finally, the optic Fig. 2. Semithin cross-sections of the orbital, myelinated portion of the optic nerve showing a significant reduction in the number of large axon profiles in the nerve and the corpus callosum of plasmalogen-deficient adult DHAPAT knockout mouse (−/−) compared with the control (+/+). Scale knockout mice show distinct hypomyelination (Fig. 2A and B) bar, 10μm. and nerve conductivity measured in the corpus callosum was significantly reduced (Teigler et al., unpublished). Thus, lack of plasmalogens that in myelin account for about 80% of total position of the glycerol moiety. In sperm plasma membranes myelin glycerophosphoethanolamines [46] has both structural approximately 2/3 of both phosphocholine and phosphoetha- and functional consequences for the myelinated axon. nolamine glycerolipids are ether-linked and contain aliphatic alkyl and alkenyl chains, whereas diacyl species account for 4.2. Ether lipids in male reproduction about 30% [120,121]. The alkyl chains at the sn-1 position are almost exclusively comprised of C16:0 and the fatty acid Cells of the non-stem germ cell lineage in testis synthesize bound at the sn-2 position is polyunsaturated, such as in ether phospholipids including seminolipid at unusually high docosapentaenoic acid or DHA. As a consequence of specific concentrations. Although much has been learned within the last lipid remodelling during sperm epididymal maturation a years, the physiological role these lipids play in male relative increase in plasmalogens in sperm plasma membranes reproduction is not known. In this section new data on was observed. This increase was accompanied by enhanced plasmalogens and seminolipid are summarized based on the levels of C22:5 and C22:6 and reduced concentrations of structural alterations in testis of the ether lipid-deficient C18:1 and C20:4 most likely prerequisite for the fertilizing DHAPAT knockout mouse and the phenotypes seen in related capacity of spermatozoa acquired during epididymal transit mouse models. Main emphasis is the impact of ether lipid [121–125]. Considering the high concentration of ether deficiency on germ cell and somatic cell maturation, cell cycle phospholipids in the germ cell membrane and the metabolic synchronization and progression of spermiogenesis in the stability of the ether linkage, ether lipids were suggested to mouse seminiferous epithelium. contribute to both the formation of macro- and microdomains required for spermatogenesis and the compartmentalization of 4.2.1. Ether phospholipids in spermatozoa the highly polarized sperm membrane, including the head Spermatozoa are unique among mammalian cells in that and its subdomains, the midpiece and the flagellum. Further- they contain high levels of ether- linked phospholipids fre- more, these ether lipids might be responsible for mainte- quently carrying polyunsaturated fatty acid chains at the sn-2 nance of the sperm’s fertilizing competence during passage K. Gorgas et al. / Biochimica et Biophysica Acta 1763 (2006) 1511–1526 1519 through the female reproductive tract initiating sperm capacita- 4.2.3. Arrest of spermatogenesis in ether lipid-deficient mice tion [123,125]. Disruption of the DHAPAT gene in mice causes infertility and arrest of the spermatogenic cycle between late pachytene 4.2.2. Synthesis and functional role of seminolipid in testis and and the spermatid stage. Seminiferous tubules are completely spermatozoa devoid of elongated spermatids and mature spermatozoa As mentioned above (Fig. 1), seminolipid (SGalAAG) is the indicating complete block of normal spermiogenesis and major sulfogalactolipid in differentiating mammalian sperma- azoospermia (Fig. 3). Depletion of germ cells, predominantly tocytes and spermatozoa accounting for 5–8mol% of total primary and secondary spermatocytes, and marked Sertoli cell lipids and more than 90% of the total glycolipids in mature vacuolization were additional signs of a disturbed synchroni- gametes. As a minor component (0.2mol%), SGalAAG was zation of the germ cell cycle. There is a gradual loss of also found in white matter of the adult mammalian brain, but intercellular bridges stabilizing the clonal syncytia of sperma- seems to be absent from most somatic cells underlining its tocytes leading to formation of multinucleated giant cells, the crucial role in spermatogenesis and fertilization. In testis, more majority of which has completed meiosis (Fig. 3C). In testes of than 95% of SGalAAG contain hexadecanol ether-linked to the older animals numerous tubules displayed the phenotype of sn-1-position and hexadecanoic acid esterified to the sn-2- partial or complete Sertoli cell-only syndrome indicating position [15,122,126,127]. The molecule is inserted into the gradual loss of spermatogonia also to occur. outer leaflet of the sperm plasma membrane. Using an anti- These observations provided first experimental evidence for sulfogalactolipid antibody, SGalAAG was exclusively detected the essential role ether phospholipids and seminolipid play in in the germ cell lineage of the adult mouse testis decorating the spermatogenesis. Ether lipids might be less important for the plasma membranes of differentiating type B spermatogonia, amplification of stem cells and the differentiation of spermato- spermatocytes, spermatids, and spermatozoa [128]. gonia in the abluminal compartment of the seminiferous SGalAAG and sulfatide contain the same sulfated galactose epithelium and might not be required for completion of meiotic head group, and the last steps of synthesis of both were karyokinesis (Fig. 3B and C). They are, however, critical catalyzed by the sequential activities of UDP-galactose: determinants for (i) the transit along somatic Sertoli cells of ceramide galactosyltransferase (CGT) located in the ER and syncytial late pachytene germ cells to the correct adluminal cerebroside sulfotransferase (CST) present in Golgi membranes compartment thereby mediating specific cellular junctions, (ii) [129,130]. SGalAAG synthesis is developmentally regulated the final steps of spermatid cytodifferentiation, and (iii) and initiated in the mouse testis at P8. Whereas a strong focal establishing and maintaining the integrity of stable intercellular patterning of seminolipid was reported at the initiation of bridges constituting syncytial clones of adluminal spermato- meiosis at P10–P12 [128,131], all seminiferous tubules were cytes and spermatids. Both complex Sertoli cell–spermatid intensely immunostained at P14–P18 coinciding with (i) junctions and stable intercellular bridges are essential for SGalAAG concentration peak at around P17, (ii) entrance of maintenance of spermiogenesis and regulated spermiation germ cells into the pachytene stage, (iii) their transit to the [138–140]. The intercellular bridges represent highly special- abluminal compartment of the seminiferous epithelium, and (iv) ized plasma membrane regions stabilized by an actin filament functional maturation of Sertoli cells forming tight junction complex. Disruption of the actin ring by cytochalasin D resulted complexes that constitute the blood–testis-barrier. The bulk of in formation of multinucleated spermatids and spermatogenic SGalAAG is synthesized in late zygotene and early pachytene arrest [141] similar to the DHAPAT knockout phenotype. spermatocytes [15,126,128,129]. In rat testis it was shown that Spermatogenesis in CST knockout mice lacking the sulfo- the activity in pachytene spermatocytes is tightly controlled and galactolipids, sulfatide and seminolipid progresses through restricted to a 3-day period of the entire 55-day spermatogenic spermatogonial amplification and differentiation, but is arrested cycle [132]. at late pachytene stage before completion of the first meiotic The sulphated galactose moiety of SGalAAG is preserved division [130]. A similar phenotype has been described in CGT throughout spermatogenesis, sperm maturation, and initial in knockout mice [129]. In DHAPAT knockout mice, however, the vitro sperm capacitation [133,134]. Using confocal fluorescence majority of syncytial germ cell clones completed meiotic microscopy the majority of SGalAAG was found in LRMs karyogenesis I and II and most likely reached the stage of localized in a globular pattern to the apical subdomain of the multinucleated spermatids. Thus, in CST and CGT knockout sperm head [117], the expected site of sperm recruitment to the mice accumulation of precursors of sulfogalactolipid synthesis egg coat [122,135,136]. Interestingly, after in vitro capacitation seemed to be more detrimental to the meiotic program than the or sperm binding to zona pellucida-coated coverslips SGalAAG complete absence of ether lipids in DHAPAT knockout mice. relocalized from the apical ridge domain to the equatorial However, detailed studies on gene expression as well as lipid and subdomain of the sperm surface. This reorganization occurs immunocytochemical analyses are required to precisely deter- prior to the acrosome reaction and concentrates SGalAAG at the mine the block in germ cell development and to delineate the predestined site implicated in lateral egg binding and subse- functionality of Sertoli cells in ether lipid-deficient mice. quent fertilizing fusion [122,137]. These studies paradigmati- Searching for mutant mouse strains impaired in spermioge- cally indicate the involvement in the dynamics of sperm head nesis and exhibiting the phenotype of multinucleated spermatids polarity of SGalAAG and its requirement for cell–cell and cell– similar to that seen in DHAPAT knockout mice revealed a extracellular matrix interactions leading to fusion. number of specific candidate genes. Interestingly, one of these 1520 K. Gorgas et al. / Biochimica et Biophysica Acta 1763 (2006) 1511–1526

such as plasmalogens, seminolipid and FGSLs seems to initiate disruption of the domain structure and breakdown of the actin cytoskeleton leading to multinucleated spermatids and apoptosis of the germ cell syncytium. Maintenance of membrane domains appears to be under the paracrine control of mature Sertoli cells. Recent data demonstrate that in the absence of Sertoli cells spermatogonia proliferate and differentiate to spermatocytes and round haploid spermatids. However, stem cell factor, a secretion product of Sertoli cells, could not induce sperm tail elongation and generation of intercellular bridges [143]. Another important aspect relates to the androgen receptor present in functionally mature Sertoli cells, but not germ cells. Androgen receptor expression is developmentally regulated and asynchronous progression of spermatogenesis and block in spermiogenesis, as observed in the ether lipid-deficient mouse, may well reflect disturbance in the androgen- dependent cross talk between germ cells and the somatic compartment of the seminiferous epithelium caused by impaired Sertoli cell maturation. The present results on the role of ether lipids in spermio- genesis were mainly obtained by structural and immunocyto- chemical analysis of the ether lipid-deficient testis. Determining as close as possible the time point of the spermatogenic arrest it appeared that ether lipids developmentally regulate in a stage- specific manner the organization and distinct compartmental- ization of the plasma membrane crucial for the integrity and maintenance of the clonal spermatid syncytium and the progression of spermiogenesis.

4.3. Role of ether lipids in lens organization and transparency

The avascular lens is built up by a single cell type forming two compartments, the anterior epithelium and the highly differentiated fiber cell mass both surrounded by a specialized basement membrane, the lens capsule. Maintenance of lens organization and transparency requires synchronized interac- tions between the capsular extracellular matrix and the lens epithelial cell membrane as well as gap junction-mediated Fig. 3. Semithin sections of wt (A) and DAPAT knockout (B, C) testes. In the intercellular communication that both guarantee life-long first wave of spermatogenesis the multilayered seminiferous epithelium of a 30- coordinated cell proliferation, differentiation and precisely day-old wt-mouse contains the germ cell set typical for stage VI of the spermatogenic cycle [169,170]; spermatogonia (1), midpachytene spermato- ordered apposition of lens fibers to the lens core [144]. cytes (2) and round spermatids in the initial phase of acrosome formation (3, Plasmalogens, in particular plasmenylethanolamines, have been arrowhead). The seminiferous epithelium of 6months old DHAPAT knockout identified as major lens phospholipids in several mammalian mice is strikingly disorganized and elongated spermatids typical for stage VI of species including mice and humans [145–150], and DHAPAT adult testis epithelium are completely absent. Opening of intercellular bridges knockout mice exhibit early postnatal bilateral cataractogenesis (B, arrows) and the initial development of multinucleated spermatid cells (B) exhibiting initial acrosome formation (C, arrowheads) are seen. In 6-month-old [9,10]. Thus, the lens represents a valuable model system to mutant mice elongated spermatids corresponding to stage VI are completely define the putative functions plasmalogens play in epithelial cell absent. S=Sertoli cell, methylene-azur II stain. Scale bar, 100μm. polarity, cell migration and differentiation as well as in integrity of cell–cell and cell–matrix adhesions. genes encodes the enzyme Gg3Cer/GM2/GD2-synthase the lack of which causes infertility by loss of a unique subset of neutral 4.3.1. Ether lipids of the lens fucosylated glycosphingolipids (FGSLs) [142]. None of these The relative amounts of plasmalogens and sphingomyelins polyunsaturated FGSLs exclusively synthesized in germ cells vary in different regions of the mammalian lens. Plasmeny- floated up with detergent-insoluble lipid rafts suggesting that the lethanolamines are enriched in the epithelial cell layer and are germ cell membrane contains functionally distinct glyco- most abundant in the outer cortical zone, but significantly sphingolipid domains required for the spermatid differentiation decrease toward the center of the lens indicating maximal program. At this checkpoint lack of major lipid constituents, synthesis of plasmalogens during lens fiber differentiation and K. Gorgas et al. / Biochimica et Biophysica Acta 1763 (2006) 1511–1526 1521 elongation. In contrast to that, sphingomyelins are enriched in a lenticonus anterior (Fig. 4A and B). Detailed histological the inner cortical zone and in lens nuclear fibers [151]. analysis of the ether lipid-deficient lens revealed (i) focal Correspondingly, enzymatic activities of both DHAPAT and disruption of the anterior monolayer of lens epithelial cells and ADAPS are 7-fold higher in cultured lens epithelial cells than in invasion of unpolarized epithelial cells into the thickened lens lens homogenates. Both enzymes co-localize with catalase in capsule, (ii) loss of cell–cell adhesion sites resulting in massive peroxisomes [150] that are particularly abundant in differenti- vacuolization and abnormal layering of secondary fiber cells in ating lens epithelial cells and elongating fiber cells. Interestingly, the outer cortical zone and (iii) aggregation at the bow area of peak levels of HMG-CoA reductase have been demonstrated in poorly differentiated epithelial cells extending as a multilayered the outer cortical lens region (outer 5% of the lens radius) sheath toward the disorganized and frequently vascularized indicating maximal cholesterol synthesis during fiber elongation posterior suture [10]. These data indicate plasmalogens to be [152]. In the avascular lens both endogenous ether lipid and essential for postnatal lens growth, integrity of cell–cell and cholesterol biosynthesis are crucial for developmental fiber cell–matrix interactions, anterior lens epithelial cell polarity and elongation as an estimated 1000-fold increase in lens fiber barrier function and maintenance of metabolic homeostasis and plasma membranes has to be guaranteed. In addition, a steep lens transparency. Under normal conditions postmitotic fiber gradient of the molar cholesterol to phospholipid ratio ranging cells at the equatorial region elongate by migration of their from 0.8 in lens epithelium to 3.5 in the nucleus is maintained in leading edges both along the epithelium and the capsule to reach lens cell membranes [153,154]. Gap junctions playing an the anterior and posterior suture, respectively. As in ether lipid- important role in cell–cell coupling required for maintenance deficient mice lens epithelial cells continue to migrate to the of overall lens transparency, were found to be highly enriched in posterior pole (Fig. 4C), plasmalogens may specifically cholesterol. In the chicken lens, for example, gap junctions contribute to the control of migration and elongation of lens exhibiting a ratio of cholesterol to phospholipid of 3.1, contain cortical fiber cells. about 57% of total fiber cholesterol [155]. Cataract similar in phenotype to that observed in the plas- malogen-deficient lens is also generated by disrupting genes 4.3.2. Plasmalogen deficiency causes cataract involved in cell–cell and cell–matrix adhesion, cell migration DHAPAT deficiency causes a number of distinct develop- and gap junction-mediated cell–cell coupling [156–161]. The mental eye defects including microphthalmia, dysgenesis of the related proteins are frequently GPI-anchored and sort to LRMs. anterior eye chamber, persistence of the hyaloid artery, Therefore, it is interesting that human inborn defects in cho- (Bergmeister papilla), and optic nerve hypoplasia. The most lesterol biosynthesis, such as X-linked dominant chondrodys- conspicuous anomalies, however, affect the adult lens display- plasia punctata (Conradi–Hunermann–Happle syndrome), ing bilateral cataract, keratolenticular adhesion and formation of mevalonic aciduria, lathosterolosis, Smith–Lemli–Opitz syndrome

Fig. 4. Major structural alterations in the lens of the DHAPAT knockout mouse. In contrast to controls (A) the adult mutant lens shows disruption of the lens epithelial cell monolayer resulting in loss of epithelial cell polarity, altered architecture of the lens capsule (arrow), disorganization of the anterior suture and the formation of a lenticonus anterior (B). Epithelial cell layers (arrowheads), lens fiber swelling and clefts at the posterior suture (star in panel C) point to the overall severe defects in cell–cell and cell–matrix interactions as well as altered fiber cell migration and elongation in the cataractous lens. Scale bar, 50μm. 1522 K. Gorgas et al. / Biochimica et Biophysica Acta 1763 (2006) 1511–1526 and cholesterol-lowering medication in laboratory animals are Acknowledgements associated with development of cataract [153,162–166]. 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