The importance of ether-phospholipids: A view from the perspective of mouse models

Biochimica et Biophysica Acta 1822 (2012) 1501–1508

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Review

The importance of ether-phospholipids: A view from the perspective of mouse models☆ Tiago Ferreira da Silva a, b, Vera F. Sousa a, b, Ana R. Malheiro a, Pedro Brites a,⁎ a b

Nerve Regeneration Group, Instituto de Biologia Molecular e Celular, Porto, Portugal Instituto de Ciências Biomédicas Abel Salazar, University of Porto, Porto, Portugal

a r t i c l e

i n f o

Article history: Received 1 November 2011 Received in revised form 6 January 2012 Accepted 23 May 2012 Available online 31 May 2012 Keywords: Plasmalogen Peroxisome Nervous tissue Oxidative stress Knockout Disease mechanism

a b s t r a c t Ether-phospholipids represent an important group of phospholipids characterized by an alkyl or an alkenyl bond at the sn-1 position of the glycerol backbone. Plasmalogens are the most abundant form of alkenylglycerophospholipids, and their synthesis requires functional peroxisomes. Defects in the biosynthesis of plasmalogens are the biochemical hallmark of the human peroxisomal disorder Rhizomelic Chondrodysplasia Punctata (RCDP), which is characterized by defects in eye, bone and nervous tissue. The generation and characterization of mouse models with defects in plasmalogen levels have significantly advanced our understanding of the role and importance of plasmalogens as well as pathogenetic mechanisms underlying RCDP. A review of the current mouse models and the description of the combined knowledge gathered from the histopathological and biochemical studies is presented and discussed. Further characterization of the role and functions of plasmalogens will contribute to the elucidation of disease pathogenesis in peroxisomal and non-peroxisomal disorders. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The discovery of the relationship between lack of peroxisomes and the Zellweger syndrome [1,2] was the initial driving force to unravel the intricacies behind peroxisome biogenesis, protein import into peroxisomes and peroxisomal functions [3–5]. The knowledge gained in the last three decades allowed a better understanding of the Zellweger syndrome, the identification of new peroxisomal disorders and methods to perform pre- and post-natal diagnosis. Peroxisomes are mostly known for their ability to perform (1) β-oxidation of verylong-chain fatty acids, (2) α-oxidation of phytanic acid, (3) degradation of hydrogen peroxide, and (4) the biosynthesis of ether-phospholipids [6,7]. Nevertheless, the determination of the peroxisomal proteome is still unraveling new peroxisomal proteins [8], which increases the functions performed by these organelles and potentially the number of human peroxisomal disorders. The complexity of human peroxisomal disorders is emphasized by (1) the lack of a unique or specific clinical presentation, (2) the variable

☆ This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease. ⁎ Corresponding author at: Instituto de Biologia Molecular e Celular, Nerve Regeneration Group, Rua do Campo Alegre 823, 4150‐180 Porto, Portugal. Tel.: + 351 226074916; fax: + 351 226099157. E-mail address: [email protected] (P. Brites). 0925-4439/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2012.05.014

degrees of biochemical defects, (3) the multitude of genes that can affect peroxisome biogenesis, (4) the absence of genotype–phenotype correlations, and by (5) the variety of organs and tissues affected. These features combined with a general unavailability of postmortem material have in some cases, hindered a detailed characterization of the tissue pathology and the mechanisms behind disease progression. To circumvent some of these problems mouse models for human peroxisomal disorders have been generated. Mouse models have been generated for most peroxisomal disorders including the Zellweger spectrum (i.e., the Pex5, Pex2, Pex11 and Pex13 knockout mice; [9–13]), X-linked adrenoleukodystrophy (the ATP-binding cassette, sub-family D, member 1 (Abcd1) knockout mouse; [14–17]), Refsum disease (the phytanoyl-CoA 2‐hydroxylase (PHYH) knockout mouse; [18]), and D-bifunctional protein deficiency (the DBP or MFP2 knockout mouse; [19,20]). For further reading and a comprehensive listing of the current mouse models with either peroxisome biogenesis defects or β-oxidation defects see the chapter in this issue by Baes and Van Veldhoven [21]. In this review we present the combined knowledge obtained from the generation and characterization of mouse models for Rhizomelic Chondrodysplasia Punctata (RCDP). RCDP is a complex disorder in terms of clinical presentation, pathology and genetics. The disorder is caused by an impairment in the biosynthesis of ether-phospholipids of which plasmalogens play crucial roles that are further delineated in this review.

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2. Characteristics and functions of plasmalogens In mammalian cells ether-phospholipids represent an important group of phospholipids. In distinction to diacyl-glycerolphospholipids, which contain two acyl chains linked to the glycerol backbone by ester bonds, in ether-phospholipids the acyl chain in position 1 is linked to the glycerol backbone by either an alkyl- or an alkenyl-bond [22]. Plasmalogens are the most abundant form of ether-phospholipids and are alkenyl-glycerophospholipids containing either an ethanolamine or a choline moiety in the polar headgroup of the glycerol backbone [23]. Alkyl-glycerophospholipids are less abundant, although plateletactivating factor is abundant in neutrophils [24]. The biosynthesis of plasmalogens is initiated in peroxisomes through the activity of two enzymes namely, glyceronephosphate O-acyltransferase (GNPAT) and alkylglycerone phosphate synthase (AGPS), and is finalized in the endoplasmic reticulum (reviewed in [25]). The structure and composition of plasmalogens, and in particular the vinyl-ether bond at the sn-1 position, are thought to dictate their role in cellular membranes. Plasmalogens have been found to serve as (a) endogenous antioxidants, since the vinyl-ether bond is prone to attack by oxidants, (b) mediators of membrane structure and dynamics, since they exhibit a tendency to form nonbilayer lipid phases, reduce surface tension and viscosity and, (c) storages of polyunsaturated fatty acid and lipid mediators, since the sn-2 position usually contains docosahexaenoic acid and arachidonic acid, whose metabolism produces leukotrienes and prostaglandins. Recent reviews on the functions and roles of plasmalogens are recommended for further reading [26,27]. Although most experimental settings have been performed in vitro, some studies were performed in cells derived from plasmalogen-deficient patients. Based on the growing number of cells and tissues with defects caused by a plasmalogen deficiency, and the availability of mouse models with defects in plasmalogens the aim should now be to investigate the attributed roles and functions of plasmalogens in target cells and tissues from plasmalogen deficient mice. 3. Plasmalogen deficiency in humans The clinical presentation of RCDP typifies the consequences of a plasmalogen deficiency. The clinical features of RCDP patients include, shortening of femur and humerus, cataracts, contractures, hypotonia, atypical facial appearance (e.g., prominent forehead, anteverted nares, long philtrum) and mental and growth retardation [28–30]. On X-ray imaging the ectopic stippled calcifications, flared epiphyses and coronal clefts in vertebral bodies further denote the defects in endochondral ossification present in RCDP patients [28,31,32]. Magnetic resonance imaging (MRI) may reveal delayed myelination, ventricular enlargement, cerebellar atrophy, and rarely neuronal migration defects, which may contribute to the poor neurologic prognosis of RCDP patients [30,33–37]. In RCDP patients the impairment in plasmalogen biosynthesis can be caused by three different defects. These defects also denote the three forms of RCDP depending on which gene is mutated. In RCDP type 1, mutations in PEX7 [38,39], encoding the cytosolic receptor for peroxisomal proteins carrying the peroxisomal targeting signal 2 (PTS2), impair the import of three proteins (reviewed in [40,41]), of which AGPS, is the cause of the impaired plasmalogen biosynthesis. In RCDP type 2, mutations in GNPAT, encoding the first enzyme in the plasmalogen biosynthesis pathway, are responsible for the inability to synthesize plasmalogens [42]. In RCDP type 3, mutations in AGPS, encoding the second enzyme in the plasmalogen biosynthesis, are the cause of the plasmalogen defect [43–45]. Despite this genetic heterogeneity, all forms of RCDP share the same clinical presentation, which indicates that the presentation of RCDP type 1patients is primarily caused by the plasmalogen defects, although the defects in β- and α-oxidation may worsen or modulate the disease state

[46,47]. Although there is no obvious genotype–phenotype correlation in any of the RCDP types, atypical presentations (usually lacking the bone defects and the shortening of proximal bones) have been described and linked to high residual levels of plasmalogens [33,38,48–50]. In Zellweger patients the lack of functional peroxisomes also leads to a defect in the biosynthesis of plasmalogens [51]. The clinical presentation of Zellweger patients shares some features with that of RCDP patients, but the disease severity is greater (including hypotonia, cranial and facial dysmorphism, hepato- and splenomegaly) since all peroxisomal functions are impaired in Zellweger patients [52–54]. MRI finding may include dysmyelination, and a severe defect in neuronal migration seen as cerebral cortical microgyria and pachygyria, cerebral and cerebellar neuronal heterotopias and dysplasias of the inferior olive [55–58]. Given the multitude of biochemical abnormalities it becomes difficult to attribute a given symptom or tissue pathology to one of the biochemical defects. Recently, reduced levels of plasmalogens have been found in Xlinked adrenoleukodystrophy (ALD) and D-bifunctional protein (DBP) deficiency, two peroxisomal disorders etiologically unrelated to RCDP [59,60]. ALD is primarily characterized by adrenal insufficiency, testicular atrophy and inflammatory demyelination of the central nervous system [61–65]. At the biochemical level ALD is characterized by the accumulation of very-long-chain fatty acids due to impaired β-oxidation [66–68]. The molecular basis of ALD lies in mutations in the ABCD1 gene, encoding a peroxisomal transmembrane protein belonging to the ATP-binding-cassette (ABC) transporter family, which is thought to function as a transporter of fatty acids [69–71]. ALD has a complex presentation and variable onset of disease (see chapters in this issue by S. Kemp and A. Pujol). Recently, a detailed study in affected areas of the brain from ALD patients has revealed low levels of plasmalogens [59]. The deficiency in plasmalogens in active demyelinating areas may act synergistically with the accumulation of VLCFA and thereby exacerbate the pathology and accelerate disease progression. The clinical presentation of D-bifunctional protein (DBP) deficiency is characterized by neonatal hypotonia, seizures, craniofacial dysmorphism and visual and hearing impairment [72,73]. At the biochemical level the disorder is characterized by a defect in peroxisomal β-oxidation with accumulation of VLCFA, branched-chain fatty acids (e.g. pristanic acid) and bile acid intermediates (di- and trihydroxycholestanoic acids) [72,73]. Phospholipid analyses in the brain of a DBP patient revealed a decreased amount of plasmalogens in gray matter [60]. These findings warrant further analyses in other DBP patients, and the investigation if the observed defects are related to an impairment in the biosynthesis of plasmalogens or an increased degradation. Previous work has shown that acyl-CoA generated in peroxisomes through β-oxidation can be used in the biosynthesis of plasmalogens [74]. This link between two distinct peroxisomal pathways, may suggest that impairments in peroxisomal β-oxidation can affect plasmalogen levels as seen in ALD and DBP patients. Nevertheless, studies in mice with defects in peroxisomal β-oxidation did not show a correlation between impaired peroxisomal β-oxidation and abnormalities in plasmalogen levels [21,75]. Similarly to what has been observed in ALD, a plasmalogen deficiency may increase the nervous tissue pathology in DBP patients, although in DBP the plasmalogen deficiency seems to be more pronounced in gray matter, whereas in ALD the deficiency in plasmalogens is localized to affected white matter regions. Interestingly, reduced plasmalogen levels have also been found in non-peroxisomal disorders. These include Alzheimer's disease [76–80], Parkinson's disease [81], Gaucher disease [82] and Pelizaeus–Merzbacher disease (PMD) [83]. In PMD, a leukodystrophy caused by mutations in the PLP1 gene encoding the myelin proteolipid protein-1, levels of plasmalogens are reduced in fibroblasts and lymphocytes from PMD patients [83]. In Alzheimer's disease, reduced levels of

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plasmalogens have been found in affected brain areas [84] as well as in serum [85]. The investigation of plasmalogen levels in Alzheimer's disease has revealed that the deficiency is pronounced in affected brain areas, and that a secondary peroxisomal dysfunction may worsen the pathology. In addition, it has been shown that production and accumulation of amyloid β lead to reduced activity of AGPS with consequences in the levels of plasmalogens [77]. The continued study of the role of plasmalogens in these disorders, will not only contribute to a better understanding of the disease state but also to highlight the role of plasmalogens in neurodegeneration. In these disorders, and similar to what has been proposed for ALD [86], the deficiency in plasmalogens is unrelated to the primary defect which suggests that plasmalogen levels may serve as markers for a peroxisomal dysfunction and/or for increased oxidative stress. The continued study of the pathogenesis and the mechanisms behind the disease state in RCDP patients will still be of crucial importance to the understanding of the consequences of plasmalogen deficiency in other neurodegenerative diseases, since the defect in plasmalogens in RCDP is primary. The need to better understand and characterize the RCDP pathogenesis and the mechanisms behind the disease were one of the driving forces behind the endeavor that several groups have undertaken to generate mouse models for RCDP. Below we will present the current models of RCDP followed by a description and comparison of the pathology in the tissues most affected by a plasmalogen deficiency.

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phytanic acid readily observed in plasma, brain and liver [87]. Similar findings have been observed in the mouse model for Refsum's disease, i.e., the Phyh KO [18]. The levels of phytanic acid in liver, kidney and cerebellum of Phyh KO mice fed a control standard diet are low and feeding a phytol-enriched diet causes a massive accumulation of phytanic acid. Dietary restriction of phytanic acid intake in a large cohort of Refsum patients has shown the effectiveness of the Westminster diet with 30% of patients presenting normal levels of phytanic acid and 50% showing a marked decreased [88]. As presented in the following section, the Pex7 KO serves as a good mouse model for RCDP type 1 and to study the pathology caused by a plasmalogen deficiency. In addition the ability to generate a mouse model with multiple defects (e.g. plasmalogen deficiency combined with accumulation of phytanic acid) may be of added value when trying to understand the pathology and mechanisms behind the generalized loss of peroxisomal functions as seen in Zellweger patients or the Zellweger model (the Pex5 KO mouse). A similar strategy has been used to combine a generalized accumulation of VLCFA with a plasmalogen deficiency [89]. By crossing the mutant Pex7 allele with a mutant ALD allele (Abcd1 KO), the generation of the double mutant (Pex7:Abcd1 double KO) has shown that a plasmalogen deficiency modulates the pathology caused by VLCFA accumulation, and mimics some aspects of ALD, which are not present in the ALD mouse model. In contrast to the Abcd1 KO, the Pex7:Abcd1 double KO showed astrocytosis, microgliosis and demyelination [89]. 4.2. The Pex7 hypomorphic mouse

4. Mutants with defects in the biosynthesis of plasmalogens 4.1. The Pex7 knockout mouse The Pex7 knockout (KO) mouse has been generated by the deletion of exon 3 and flanking portions of introns 2 and 3 through homologous gene recombination [87]. Pex7 KO mice on a Swiss Webster background are born alive but display several features reminiscent of RCDP patients that include dwarfism and hypotonia. Around 50% of Pex7 KO mice die within the first 24 h. Although the cause of death hasn't been determined, the hypotonia combined with decreased mobility hampers feeding which will have negative consequences during this early neonatal period. The Pex7 KO pups that surpass the difficulties of this period, survive to adulthood. Tissues and cells derived from Pex7 KO mice clearly showed the role of Pex7 in PTS2-mediated import with the presence in protein extracts of the precursor form of thiolase (encoded by the ACAA1 gene) and AGPS. At the biochemical level the studies in Pex7 KO mice showed extremely reduced levels of plasmalogens (ranging from undetectable to approximately 1.6% of normal levels) in erythrocytes, eyes, kidney, heart, brain, and sciatic nerve. Studies of de novo plasmalogen biosynthesis in cultured mouse embryonic fibroblasts (MEFs) from Pex7 KO mice showed a virtual inability to form plasmalogens which is correlated with an extremely reduced amount of AGPS. Additionally, at birth the Pex7 KO mice were found to have elevated levels of VLCFA in plasma, liver and brain. Using MEFs a defect was observed in the β-oxidation of VLCFA and pristanic acid. Surprisingly, livers and brains from adult Pex7 KO mice showed normal levels of VLCFA whereas accumulation of these fatty acids is still observed in spleen and kidney. This tissue and age variability in the accumulation of VLCFA has been postulated to be due to the existence of another peroxisomal enzyme with thiolytic activity that can take over the defect of peroxisomal thiolase [87]. A third biochemical defect is caused by the defect in import of Phyh which impairs α-oxidation of phytanic. Nevertheless, under normal feeding conditions the Pex7 KO defect in α-oxidation of phytanic acid is silent, since normal rodent food only contains residual amounts of phytanic acid and phytol (the phytanic acid precursor) which are not sufficient to cause an accumulation in tissues. Only after Pex7 KO mice are fed a diet containing phytol is the accumulation of

The generation of a hypomorphic Pex7 allele was achieved by the introduction of a neomycin resistance cassette in the intron 2 of the murine Pex7 locus [90]. One of the driving forces behind generating a hypomorphic mouse was to circumvent the early lethality as seen in Pex7 KO and Gnpat KO mice, which hampers studies with large amounts of mice and requires large quantities of breeding pairs in order to obtain mice for different experimental setups. Mice heterozygous for the hypomorphic allele showed Pex7 transcripts that were between 10 and 50% of wild type levels. Mice homozygous for the hypomorphic allele on a C57BL/6:129/SvEv mixed background did not show increased lethality and had a normal life span, although similarly to Pex7 KO mice had reductions in body weight. Possibly in concordance with the hypomorphic state, the biochemical defects varied between different tissues. Plasmalogen levels were reduced between 19 and 72% in the tissues analyzed (brain, heart and liver) which were in accordance with a reduction in AGPS levels and reduced biosynthesis. Similarly to what was observed in Pex7 KO mice, homozygous hypomorphic Pex7 mice accumulated phytanic acid in plasma and tissues upon feeding a diet rich in phytol. The Pex7 hypomorphic mouse mimics some aspects of the milder forms of RCDP. These milder forms of RCDP are characterized by high residual levels of plasmalogens which denote the relationship between plasmalogen levels and disease state [91,92]. By comparison, the hypomorphic Pex7 mouse allows us to determine the levels of plasmalogens necessary to develop tissue pathology and understand which are the tissues and organs mostly dependent on plasmalogens for normal physiology and development. 4.3. The Gnpat knockout mouse The Gnpat KO mouse has been generated by the deletion of exons 5 to 7 through homologous gene recombination [93]. Gnpat KO mice on a C57BL/6 background showed increased lethality during the first weeks after birth, with reduced body weight and impaired growth. The activity of Gnpat in tissues and fibroblasts from Gnpat KO mice was not detectable, which led to the extremely reduced levels of plasmalogen in brains from KO mice. The consequences of this complete loss of plasmalogens included reduced levels of flotillin-1 and

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F3/contactin in brain-derived lipid rafts and reduced expression of connexin 43 in fibroblasts. Combined with the observation that plasmalogens are enriched in lipid rafts of a human epidermal carcinoma cell line [94], the results of reduced expression of lipid rafts markers suggest that in the absence of plasmalogens and despite a normal fractionation pattern, the composition of these domains is altered. The consequences of a plasmalogen deficiency for membrane structure and organization are also highlighted by the abnormal expression of connexins and claudins in Gnpat KO mice [95,96]. Altered expression of connexins 43 and 32 may affect the assembly of gap junctions whereas the altered expression of claudins 3 and 11 may affect the assembly of tight junctions. Combined these defects corroborate the role of plasmalogens in modulating membrane structure, and organization, which can have severe consequences in target tissues (see below). Gnpat KO mice serve as a good mouse model for RCDP and to study the pathology caused by a single plasmalogen deficiency. The complete lack of plasmalogens results in a very severe phenotype, similar to that of Pex7 KO mice and RCDP patients which sometimes hinders some studies. Nevertheless, the studies performed in mice with a virtual lack of plasmalogens may be easier to understand the straightforward relationship between the biochemistry and the pathology. The comparison of pathology and phenotype observed in Pex7 and Gnpat KO mice lacks major differences between the two mice. Although the genetic background is different between both mice, they develop the same type and the same degree of pathology (see below), indicating that in Pex7 KO mice the observed defect is possibly solely due to the impaired biosynthesis of plasmalogens. 4.4. The blind sterile 2 mouse The blind sterile 2 (bs2) mouse is a spontaneously arising mutation in intron 14 of the Agps locus [97]. The mutation is hypomorphic since it reduces expression of Agps by 85% of WT levels. At the biochemical level the consequences of Agps reduced expression are clearly seen as a decreased amount of plasmalogens in brains of bs2 mice. The bs2 mice represent a model for RCDP type 3, in which the partial deficiency in the plasmalogen biosynthesis allows, similarly to the Pex7 hypomorphic mouse, the study of the consequences of partial loss of plasmalogens as seen in the mild or atypical RCDP patients. 5. Tissue pathology caused by plasmalogen loss

The abnormal differentiation of lens fiber cells, combined with the accumulation of cytosolic content and increased vacuolization affects the formation of a translucent structure. In addition, abnormal cell– cell contacts/adhesion may worsen the pathology since in addition to the nuclear cataract, cortical lens fibers also show vacuolization, loss of cell adhesion and the formation of morgagnian globules. Combining the histological assessment of lens from plasmalogendeficient mice with the defects observed in connexin and claudin expression in Gnpat KO mice, it has been hypothesized that the development of cataracts is at least in part due to abnormalities in gap junctions that may affect the process of cell–cell contact and communication hindering the correct maturation and lens translucency. 5.2. Testis Testis and spermatozoa are extremely enriched in etherphospholipids. In seminiferous tubules peroxisomes can be detected in Leydig cells, Sertoli cells and spermatogonia but mature spermatozoa lack peroxisomes [106–108]. The testis is also very sensitive to plasmalogen loss as judged by the observation that, with the exception of Pex7 hypomorphic mice all other mouse models are infertile. The ability of Pex7 hypomorphic to reproduce may be related to a higher level of Pex7 expression that can yield higher rates of plasmalogen biosynthesis. Histological changes can be seen as early as P21 and are characterized by a degeneration of the seminiferous tubules, and the presence of multinucleated cells within the tubules [89,95]. The pathology is progressive and by 2 months of age, the seminiferous tubules of Pex7 or Gnpat KO mice are devoid of spermatogonia. The Sertolionly phenotype in adult KO mice indicates the importance of plasmalogens for the normal spermiogenesis and survival of germinal cells. A detailed study using Gnpat KO mice has revealed that a complete loss of plasmalogens leads to an arrest in spermiogenesis at the round spermatid stage [95]. The continuous degeneration of spermatocytes also affects Sertoli cells that show increased vacuolization. The blood–testis barrier (BTB) is also affected in Gnpat KO mice as judged by the leakage of intratesticular injections of a biotin tracer. In addition dysregulation in the spatio-temporal expression of claudin 9 and 11 not only modulates the BTB but also indicates that a plasmalogen deficiency affects the correct formation and function of tight junctions. It would be interesting to evaluate in testis, the abnormalities found in the expression of connexin 43 in Gnpat KOderived fibroblasts, since alterations in gap junctions also severely affect BTB and spermatogenesis and may place plasmalogens as crucial membrane components for cell–cell contact and communication.

5.1. Eye 5.3. Bone In the normal eye, the lens, the epithelial lens cells and the retina are enriched in plasmalogens [98–101]. In RCDP patients congenital cataracts are a key presentation that highlights the importance of plasmalogens for the normal development of the lens. In addition, atypical or mild RCDP patients may not present bone defects, but develop lens opacities that lead to cataract formation [102–105]. The importance of plasmalogens for the normal homeostasis of the eye is also strikingly evident in the analyses of plasmalogen deficient mice. All mouse models with defects in the biosynthesis of plasmalogens develop cataracts, regardless of having full or partial reductions in plasmalogens levels. The pathology in the eye includes (a) disruption of the polarity, number and architecture of lens epithelial cells, (b) disorganization of the anterior and posterior sutures with formation of lenticonus, (c) disorganization and swelling of lens fiber cells, which also present abnormalities in differentiation and maturation, and (d) a nuclear cataract. Surprisingly, no defects have been reported in the retina, although optic nerve hypoplasia has been described in Gnpat KO mice (see below).

Skeletal abnormalities are a hallmark in the clinical presentation of classical Zellweger and RCDP patients. The defects in proximal limbs, vertebral bodies and generalized growth suggest that impaired plasmalogen biosynthesis affects the process of endochondral ossification. Despite the fact that both Pex7 and Gnpat KO mice lack plasmalogens, at birth KO mice do not show such drastic alterations as seen in patients. Nevertheless, whole mount skeletal stainings have revealed impairments in endochondral ossification in newborn pups and during embryonic development (Brites et al., unpublished results). At birth the Pex7 hypomorphic mouse also shows defects in ossification and X-ray images of 3 months old mice also revealed impaired growth. Based on the few histological evaluations of bone in RCDP patients and the results obtained in mice, the lack of plasmalogens seems to affect the process of endochondral ossification through a delay or arrest in chondrocyte maturation at the pre-hypertrophic state. In addition Pex7 KO osteoclasts showed an increased number of vacuoles in the ruffled border (active zone of bone remodeling) which suggests

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that the lack of plasmalogens can also affect the process behind bone resorption. Nevertheless, it is still quite puzzling that in humans and during the early neonatal period the deficiency is mostly seen in proximal bone elements whereas distal bones, although ossified through the same endochondral process are not severely affected.

5.4. Nervous tissue In humans, the impact of a plasmalogen deficiency on nervous tissue and neurologic functions is highlighted not only by the alterations seen on MRI (e.g. delayed myelination, ventricular enlargements, and cerebellar atrophy), and on histological changes in a few autopsy reports (e.g. impaired neuronal migration, impaired myelination, astrocytosis) but also by the severe spasticity and mental defects observed throughout life in RCDP patients [109,110]. Nervous tissue and myelin are extremely enriched in plasmalogens. In myelin, plasmalogens have been localized to the inner leaflet of myelin and seminal work has showed their contribution to the structure and stability of myelin [111]. The usage of mouse models with defects in plasmalogens has greatly increased our knowledge as to the consequences of plasmalogen loss to the central and peripheral nervous system (CNS and PNS, respectively). In the CNS a deficiency in plasmalogens leads to (a) optic nerve hypoplasia, (b) impaired myelination, (c) astrocytosis, (d) impaired Purkinje cell innervation and may also modulate neuronal migration. In the PNS a deficiency in plasmalogens affects (a) nerve conduction, (b) myelination and (c) myelinated axons. Impaired cortical neuronal migration is readily observed in Pex5 KO mice (a model for Zellweger syndrome) and surprisingly Pex7 KO mice also showed a defect in cortical neuronal migration [9,87]. The defect in Pex7 KO mice is not as severe as in Pex5 KO mice which, suggests that the impairment in neuronal migration observed in Pex5 mutant mice and in Zellweger patients may be in part due to a combined defect in plasmalogens and accumulation of VCLFA. Recently, a double Gnpat:Mfp2 KO was generated in order to evaluate the consequences of a plasmalogen deficiency combined with that of a total defect in peroxisomal β-oxidation [75]. This Gnpat:Mfp2 double mutant showed minor defects in cortical migration and cerebellar foliation, that were milder than those present in Pex7 KO mice. Although the two models (i.e., Pex7 KO and Gnpat:Mfp2 DKO) share a defect in plasmalogens the defects in β-oxidation are different, with Pex7 KO mice having an accumulation of VLCFA and Mfp2 having an accumulation of VLCFA, bile acid intermediates and pristanic acid. In addition the results obtained may also suggest that a yet undetermined PTS2 protein, that would be deficient in Pex7 KO mice but not in Gnpat KO mice, is important during the process of neuronal migration. Recently, a comparative proteomic analysis has identified a new PTS2 candidate protein in mouse kidney [112]. Similar approaches using nervous tissue samples from WT and Pex7 KO mice may also uncover nervous tissue-specific PTS2 proteins. Optic nerve hypoplasia was first observed in Gnpat KO mice, but it is also present in Pex7 KO mice (Brites et al., unpublished results). Optic nerves from Gnpat KO mice showed a decrease in the number of large myelinated axons, and evidences of degeneration in the form of axonal swellings and increased length of the nonmyelinated portion of the optic nerve. A detailed histological and morphometric analysis should result in the deeper understanding on the consequences of plasmalogen deficits to axons and myelin in the optic nerve. Nevertheless the consequences of a plasmalogen deficiency to myelination in the CNS are readily seen in the corpus callosum and cerebellum of Pex7 and Gnpat KO mice. Thinner white matter tracks are observed in aged Pex7 KO mice indicative of demyelination [89], whereas in young Gnpat KO mice dysmyelination is observed in the cerebellar vermis and terminal regions of cerebellar lobules [96]. In the cerebellum, Purkinje cell abnormalities were also

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found in relation to innervation with abnormal spinogenesis and synaptogenesis [96]. In the PNS, a deficiency in plasmalogens affects nerve conduction and immunohistological assessment of sciatic nerves with an antibody against myelin basic protein reveals the loss of myelinated fibers and a reduction in the amount of myelin around axons [89]. Combined with the electrophysiological data, the results indicate that Pex7 KO mice develop a peripheral neuropathy, but further studies should aim at understanding the consequences of plasmalogen loss to neurons and/or to Schwann cells. 6. Mice as models for human disorders Currently there are four mouse models for RCDP. The availability of straight KOs and hypomorphic mutants will undoubtedly allow a better understanding on the consequences of a deficiency in plasmalogens, since both sets of mice mimic different aspects of the human disease. At the biochemical level all models show defects in the biosynthesis of plasmalogens that correspond with what is known for RCDP patients. The phenotype of these plasmalogendeficient mice is strikingly similar among them. As reviewed here, all mutant mice show defects in the lens (leading to cataract formation) and the testis (leading to male infertility). The detailed characterization of the mutant phenotype has also revealed that a deficiency in plasmalogens also affects ossification and myelination. Some mild variations with less pronounced pathology may be observed in the hypomorphic mutants (e.g., myelin defects in Pex7 hypomorphic mouse when compared to Gnpat KO mouse). These may be explained by the differences in expression and the residual plasmalogen levels present in hypomorphic mutant when compared to the KO mutants. The available mouse mutants serve as good models for the human disorder. In the last years, following the first publications on the generation of these mouse models it has become evident that soon their characterization will surpass that of the human disease (e.g. the defects in myelin, and in testis are poorly characterized in RCDP patients). Although these mice were in part generated to better understand the human condition and to serve as tools to test therapeutic interventions, they are allowing the determination and characterization of pathologies that have not been described in RCDP patients. Although some care must be given to some of the “new” pathologies described in plasmalogen-deficient mice, it should be emphasized that the usage of mouse models does not exclude the need to perform similar analyses in tissues or cells from RCDP patients. The inability to gain access to postmortem material hinders such studies but only with them can we ascertain the true relevance of the murine findings to human health and better management of the disease state in RCDP patients. 7. Concluding remarks Plasmalogens are a unique phospholipid that despite their obscure nature, composition and biosynthesis have been shown crucial for human health. A deficiency in plasmalogens affects multiple cells in a wide variety of tissues and organs. This myriad of presentations and defects is nevertheless confusing due to the fact that based on the proposed functions of plasmalogens no given role can unify the pathology and explain the disease mechanism. The generation and usage of mouse models with defects in the biosynthesis of plasmalogens is shedding new knowledge into the pathology and will serve as excellent tools in which we can investigate and determine the mechanisms behind the observed pathology. The strengths of these mouse mutants are also in their usage as models, in which researchers and physicians can devise and test therapeutic interventions [113,114].

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