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Biochimica et Biophysica Acta 1763 (2006) 1785–1793 www.elsevier.com/locate/bbamcr

Review Generalised and conditional inactivation of Pex in mice ⁎ Myriam Baes a, , Paul P. Van Veldhoven b

a Laboratory for Cell Metabolism, Campus Gasthuisberg Onderwijs en Navorsing II, bus 823 Herestraat 49 B-3000, Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium b Department of Molecular Cell Biology, Division Pharmacology, Katholieke Universiteit Leuven, Leuven, Belgium Received 4 May 2006; received in revised form 17 August 2006; accepted 18 August 2006 Available online 25 August 2006

Abstract

During the past 10 years, several Pex genes have been knocked out in the mouse with the purpose to generate models to study the pathogenesis of biogenesis disorders and/or to investigate the physiological importance of the Pex . More recently, mice with selective inactivation of a Pex in particular cell types were created. The metabolic abnormalities in peroxisome deficient mice paralleled to a large extent those of Zellweger patients. Several but not all of the clinical and histological features reported in patients also occurred in peroxisome deficient mice as for example hypotonia, cortical and cerebellar malformations, endochondral ossification defects, hepatomegaly, liver fibrosis and ultrastructural abnormalities of mitochondria in hepatocytes. Although the molecular origins of the observed pathologies have not yet been resolved, several new insights on the importance of in different tissues have emerged. © 2006 Elsevier B.V. All rights reserved.

Keywords: Pex gene; Peroxisome; Knockout; Mouse model; ; Conditional gene inactivation

1. Introduction mechanism of peroxisomal matrix import offered a unique opportunity to apply gene targeting techniques. De- The metabolic role of peroxisomes has been extensively pending on the mutated gene, mice were created in which studied during the last 30 years mostly by using the rat liver as a either PTS1- and PTS2-dependent protein import (Pex5, Pex2, source of peroxisomes [1]. The identification of human diseases Pex13), or only PTS2-dependent import (Pex7), or peroxisome that are due to peroxisomal dysfunction underscored the proliferation (Pex11) were affected. More recently, mice with physiological importance of peroxisomal metabolism in differ- conditional inactivation of peroxisome biogenesis in specific ent tissues. However, the molecular mechanisms leading to cell types became available i.e. mice with selective inactivation anomalies such as neuronal migration defects, hypotonia, dys- in hepatocytes and in Sertoli cells. and demyelination, kidney, eye and testicular defects, abnor- malities in bone development and facial dysmorphism remain 2. Generalised inactivation of Pex genes involved in unresolved. Since naturally occurring peroxisomal disease mo- PTS1- and PTS2-dependent protein import: models for dels are lacking, it was necessary to generate such models by Zellweger syndrome? gene manipulation to study the pathogenesis of these diseases. The newly discovered Pex genes and the novel insights in the 2.1. Phenotype of the mice

Abbreviations: Acox1, acyl-CoA oxidase 1; ALD, ; Three different Pex genes were inactivated in the mouse i.e. DHAPAT, dihydroxyacetonephosphate acyltransferase; DHAP, dihydroxyace- Pex5, Pex2 and Pex13 [2–4]. In view of the complete block of tonephosphate; DHA, docosahexaenoic acid; MFP, multifunctional protein; PTS1- and PTS2-dependent matrix enzyme import in each case, NMDA, N-methyl-D-aspartate; PTS, peroxisome targeting signal; PUFA, the three knockout lines were expected to constitute a model for polyunsaturated fatty acid; SCP, sterol carrier protein; VLCFA, very long chain fatty acid Zellweger syndrome, the most severe peroxisome biogenesis ⁎ Corresponding author. Tel.: +32 16 34 72 83; fax: +32 16 34 72 91. disorder [5]. All knockout mice displayed severe hypotonia and E-mail address: [email protected] (M. Baes). growth retardation at birth and they died within a few days. A

0167-4889/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2006.08.018 1786 M. Baes, P.P. Van Veldhoven / Biochimica et Biophysica Acta 1763 (2006) 1785–1793 notable exception were the Pex2 knockout mice bred in a increased in plasma (Pex2) [3]) in liver and brain tissue lipids specific genetic background i.e. Swiss Webster×129Sv Ev a (Pex13) [4] and in liver and brain phospholipids (Pex5) [2]. fraction of which was less hypotonic and survived 1, 2 or rarely 3 More detailed investigations documented that C26:0 was also weeks [6]. As discussed below, all knockout strains displayed 10-fold increased in brain sphingolipids (ceramide and sphin- abnormalities in the formation of the brain. However, other gomyelin) in Pex5 knockout mice [15]. The concentration of the typical hallmarks present in fetal Zellweger patients were not polyunsaturated fatty acid, docosahexaenoic acid (DHA) which found, including renal cysts and facial dysmorphism. At birth the depends on peroxisomal β-oxidation for its synthesis was mice did not display major liver pathology, a feature that only reduced by 30% in newborn Pex5 knockout mice [16]. The develops in the postnatal period in patients. branched chain fatty acids, phytanic and pristanic acid, and bile At the subcellular level, embryonic fibroblast cultures de- acids have thus far not been measured in newborn Pex knockout rived from Pex5, Pex2 and Pex13 knockout mice contained mice. Plasmalogens, a subclass of ether phospholipids, were peroxisomal remnants which were reduced in number but 20- to 100-fold reduced in liver and brain (Pex5, Pex13) and increased in size as compared to regular peroxisomes [2–4]. This erythrocytes (Pex2) of newborn mice due to the inactivity of is very similar to findings in Zellweger patient fibroblasts. the peroxisomal enzymes DHAPAT and alkyl DHAP synthase. Recently, it was demonstrated that in cultured Pex13 null mouse Newborn Pex5 knockout mice contained normal levels of fibroblasts (like in Pex1 deficient human fibroblasts), remnant cholesterol in plasma, liver and brain [17] and the activity of peroxisomes displayed an altered distribution as compared to cholesterol synthesizing enzymes was either normal or slightly normal peroxisomes in wild type fibroblasts (Fig. 1A, B) [7]. increased [18]. This is not consistent with the 40% reduction of The peroxisomal ghosts were only present in the cell centre but plasma cholesterol described in newborn Pex2 knockout mice not in the periphery [7]. However, the peroxisomal ghosts were [10]. In 10-day-old Pex2 knockout mice, cholesterol levels of still associated with microtubuli, as previously shown for normal plasma and liver were also 40% reduced but they were normal in peroxisomes in different cell lines like HepG2 and Cos cells [8]. brain and other tissues. The reduced cholesterol levels were Likewise, in cultured Pex13 null neurons and astrocytes accompanied by a concerted increase in expression and activity peroxisomes were clustered in the cell soma. of cholesterol synthesizing enzymes in liver homogenates Ultrastructural examination of the liver of the three Pex probably mediated by the induction of SREBP2. Because it is knockout models revealed severe abnormalities of the mito- well known that the brain is self sufficient with regard to chondria in hepatocytes, in particular at the level of the inner cholesterol synthesis, the normal cholesterol content in brain of mitochondrial membrane [2,4,9,10] (see also 5.1). Such Pex5 and Pex2 knockout mice strongly suggests that the absence mitochondrial anomalies in hepatocytes were also described in of import competent peroxisomes has no direct negative effects some early papers on Zellweger patients [11–13]. on the cholesterogenesis. The aberrant cholesterol homeostasis Overall, it can be concluded that several but not all features of in liver of Pex2 knockout mice might be a consequence of the human Zellweger patients are mimicked in the mouse models. metabolic perturbations that occur in the liver as exemplified by This might be related to the shorter timeframe of murine as the development of steatosis and cholestasis [10]. compared to human fetal development. It is in fact quite remark- able that within the short period in which the mouse brain is 2.3. Cortical neuronal migration formed, the absence of functional peroxisomes has such an important impact, as will be further discussed below. The disturbed lamination of the cortical plate associated with medial pachygyria and lateral polymicrogyria is a major charac- 2.2. Metabolic changes teristic of Zellweger patients [19–21]. It is ascribed to a unique defect of the neuronal migration process that is clearly Most of the major metabolic changes observed in Zellweger distinguishable from other migration disorders such as those patients were recapitulated in the mouse models. An important seen in lissencephaly or double cortex syndrome. In all three Pex advantage of the latter models is that metabolite levels can be knockout models an altered distribution of cortical neurons was measured not only in body fluids but also in the diseased tissues observed [2–4] (Fig. 1C, D). For Pex5 and Pex2 knockout mice, at several stages. It was striking that C26:0 was already 2-fold a migration disorder was demonstrated by performing 5′,3′- increased at E16.5 in Pex5 knockout mouse brain and liver bromo-2′-deoxyuridine (BrdU) birthdating experiments. Fur- indicating that peroxisomal β-oxidation was already active at thermore, a delay in the differentiation of neurons, increased this stage in wildtype mice [14]. In newborn Pex knockout mice, apoptotic cell death but a normal distribution of radial glial cells this very long chain fatty acid (VLCFA) was about 10-fold were reported for Pex5 knockout pups.

Fig. 1. Pathology in generalised Pex knockout mice. (A–B) Immunofluorescent staining of α-tubulin (red) and Pex14p (green) to visualise microtubuli and peroxisomal membranes in cultured fibroblasts. In wild type fibroblasts (A) peroxisomes are associated with microtubuli throughout the cell whereas in Pex13−/− fibroblasts (B) peroxisomes are clustered in the cell centre but still aligned with microtubuli. (C–D) Cresyl violet staining of frontal sections of the cortex of newborn wild type (C) and Pex5−/− mice (D) reveals increased cell densities in the intermediate zone (IZ) of the knockout, the prospective white matter. CP=cortical plate, GZ=germinative zone. (E–F) Immunofluorescent staining of Purkinje cells in the cerebellum of 7-day-old wild type (E) and Pex2−/− mice (F) using the anti-calbindin

D28K antibody. In the knockout, these cells remain polydendritic and the size of the dendritic arbor and the degree of branching are markedly reduced. (G–H) Whole mount skeletal staining of cartilage (blue) and bone (red) of newborn wild type (G) and Pex7−/− pups (H). A lack of ossification in the middle phalanges of the hindpaws of the knockout is visible (arrows). Reprinted from [7,2,6,29] with permission from the respective publishers. M. Baes, P.P. Van Veldhoven / Biochimica et Biophysica Acta 1763 (2006) 1785–1793 1787 1788 M. Baes, P.P. Van Veldhoven / Biochimica et Biophysica Acta 1763 (2006) 1785–1793

It was further investigated whether reduced levels of DHA or but there is still no clear view on the molecular mechanism increased levels of VLCFA could play a causative role in these underlying the migration defect. defects. Supplementation of pregnant dams with DHA normal- ised the levels of this PUFA in brain but did neither improve 2.4. Formation of cerebellum hypotonia, nor the migration disorder [16]. In mice with a selective defect of peroxisomal β-oxidation by inactivating In contrast to the cortex, the formation of the cerebellum multifunctional protein 2 (MFP-2), levels of C26:0 were in- extends into the postnatal period of the mouse. Therefore, the creased to the same extent as in Pex5 knockout mice but no early death of Pex5 and Pex13 knockout mice precluded the migration defect could be observed [22]. This is at variance with analysis of cerebellar development but the longer survival of the human situation where MFP-2 deficiency is often associated some Pex2 knockout mice allowed an extensive analysis of with brain malformations which are very similar to those present cerebellar histogenesis [6]. Abnormalities were reported at the in Zellweger syndrome [23]. These observations rule out that level of granule cells with slower migration from the external alterations in fatty acid levels on their own induce cytoarchitec- granular layer (EGL) to the internal granular layer (IGL) and tonic abnormalities in mouse brain. increased cell death in the EGL. These migratory abnormalities The possible involvement of glutamatergic neurotransmis- in the postnatally developing cerebellum were more severe than sion via the NMDA receptor, known to control the speed of those seen in the prenatally developing cortex [6]. This was migration was also examined [24]. Treatment of Pex5−/− attributed to postnatal malnutrition problems, increasing hepatic embryos with NMDA antagonists induced embryonic death or renal dysfunction after birth and/or to the fact that maternal whereas NMDA agonists partially reversed the migration defect metabolism could clear toxic substances from the embryonic [25]. A deficit in NMDA signal transduction was demonstrated circulation [6]. The majority of Purkinje cells aligned under the in neuronal cultures derived from Pex5 knockout mice by cerebellar surface and only rare cells mislocalised in the IGL monitoring calcium influx in response to NMDA. Pex5−/− cells which is very different from the extensive Purkinje cell hetero- were less sensitive to NMDA than wild type cells but sensitivity topias in human Zellweger patients [19,20]. However, the could be restored by preincubation with the ether phospholipid Purkinje cells displayed stunted dendritic trees with abnormal platelet activating factor (PAF) [25]. Attempts to prove that the arborization (Fig. 1E, F). A delayed maturation of the olivary concentration of PAF is reduced in brain of Pex5−/− mice did not climbing fibers and their defective translocation from the succeed probably due to the very low levels and instability of perisomatic to the dendritic compartment of Purkinje cells re- PAF but the content of the degradation product lysoPAF was sulted in numerous spines on the soma and dendrites of Purkinje 3-fold reduced (H. Van Overloop, M. Baes and P. Van cells. Preliminary studies by Faust et al. with mixed cerebellar Veldhoven, unpublished observations). Although PAF is a well cultures further suggested that the abnormal maturation of known regulator of neuronal migration [24] and thus a very good Purkinje cells might be due to the inactivity of intrinsic pero- candidate to link peroxisome deficiency to migration, the xisomal metabolism as well as to the elimination of hepatic absence of clearcut cortical migration defects in Rhizomelic peroxisomal metabolism [28]. Indeed, also in cultured Pex2 Chondrodysplasia Punctata (RCDP) patients, who have a knockout Purkinje cells a deficient branching was observed selective and severe deficiency of ether lipids, contradicts such whereas on the other hand, supplementation of pups with mature a primary role of PAF. To further document a potential involve- bile acids not only improved survival of the mice but also ment of PAF in the neuronal migration defect associated with arborization of Purkinje cells. peroxisome deficiency, it will be of importance to investigate cortical lamination in DHAPAT knockout mice [26], a model 3. Inactivation of Pex7 involved in PTS-2 dependent with a selective depletion of ether lipids. protein import: a model for RCDP type 1? A major question is whether the brain malformations of peroxisome deficient mice are caused by the local absence of In mammals, the task of Pex7p is restricted to the import peroxisomes in the brain or by extraneural deletion of peroxi- of a few PTS2 containing proteins. Thus far only alkyl somal metabolic activity. Pex5−/− mice with a selective recon- DHAPsynthase, phytanoyl CoA hydroxylase and 3-oxoacyl- stitution of peroxisomal function in brain or in liver both CoA thiolase were unequivocally shown to possess such a exhibited a significant correction of the neuronal migration signal but they take part in three major peroxisomal pathways defect despite an incomplete reconstitution of peroxisomal i.e. ether phospholipid synthesis, α-oxidation and β-oxidation, function in the targeted tissue [27]. These data suggest that respectively. peroxisomal metabolism in brain but also in extraneural tissues In Pex7 knockout mice [29] the inactivity of mislocalised is necessary for the normal development of the mouse neocortex. alkyl DHAP synthase causes the expected depletion of plasma- Interestingly, despite the improvements of the neuronal logens in brain and erythrocytes. Due to the mislocalisation of migration, both Pex5 rescue models were as hypotonic as the phytanoyl-CoA hydroxylase a slight accumulation of phytanic generalised Pex5 knockout mice and died on the day of birth. acid was seen in newborn mice. Supplementation of adult Pex7 Overall, these investigations with the Pex knockout mouse knockout mice with phytol triggered a massive increase in models were important to prove that peroxisome deficiency leads plasma and tissue levels of this branched chain fatty acid. Based to cortical malformations in another species besides humans. A on data of RCDP type 1 patients in which straight chain pero- number of metabolites were excluded as single causative factors xisomal β-oxidation substrates are not accumulating [5,30], M. Baes, P.P. Van Veldhoven / Biochimica et Biophysica Acta 1763 (2006) 1785–1793 1789 probably because of the alternative thiolytic activity exerted by cytes have a reduced peroxisome abundance and increased SCPx, it was surprising to find that C26:0 levels were elevated in clustering and elongation of peroxisomes in common with plasma and tissues of newborn Pex7 knockout mice. Pex13 knockout fibroblasts and neurons [7].ForPex13 Given this impaired functioning of all major peroxisomal knockout fibroblasts and neurons it was shown that the remnant pathways, it is not surprising that the phenotype of Pex7 knock- peroxisomes were associated only with centrally located micro- out mice is very similar to that of Pex5 knockout mice although tubuli, and they were apparently not able to travel to the cell the penetrance is very variable [29]. About 50% of the Pex7 periphery (see also 2.1). It is not unlikely that the abnormal knockout mice were very hypotonic at birth and died postnatally, distribution of peroxisomes on these microtubuli hinders the 20% died before weaning and the others survived past 18 dynamic activity of the cytoskeleton which is necessary for months. All knockout mice displayed a neuronal migration normal migration to occur [37,38]. defect that was less severe than in Pex5 knockout mice. At birth several cartilage based structures that depend on endochondral 5. Conditional Pex5 KO mice ossification for their formation were not ossified or showed a marked delay in ossification e.g. distal bone elements of the Because of their early postnatal death, the generalised Pex2, limbs, parts of the skull and the vertebrae (Fig. 1G, H) [29]. Pex5 and Pex13 knockout mice do not allow to study the Since abnormalities in bone formation are a major hallmark functional importance of peroxisomes in adult tissues. Fortu- of RCDP patients, this mouse model should allow for the further nately, thanks to a second wave of technological developments a investigation of the precise role of peroxisomes in the ossifi- gene can now be conditionally inactivated in the mouse. The cation process. Other features observed in Pex7 knockout mice technique is based on the use of two mouse lines, one in which that need further study are cataract and male infertility [29]. two LoxP sites are introduced by homologous recombination in introns flanking an essential part of the gene of interest and 4. Inactivation of Pex11α and β another line expressing Cre recombinase. In the presence of Cre recombinase, the gene fragment encompassed by the two loxP In contrast to the other knocked out , Pex11 proteins, sites will be deleted, given that these sites have the same encoded by three different genes in mammals, do not have a orientation [39]. By mating the loxP containing mice with mice function in the import of peroxisomal matrix proteins [31]. They expressing Cre recombinase under the control of a cell type are peroxisomal membrane proteins that seem to be involved in specific promotor, the target gene will be inactivated from the the division of peroxisomes since overexpression of Pex11β moment that the promotor driving Cre expression becomes leads to peroxisome proliferation whereas inactivation causes active in the target cells. At this point two mouse lines with reduced peroxisome abundance [32,33]. Pex11αp seems to be floxed Pex genes are available i.e. Pex5-loxP [40] and Pex13- responsible for peroxisome proliferation in response to external loxP mice [41]. stimuli whereas Pex11βp is required for constitutive peroxisome biogenesis [31]. A recently reported function of Pex11βpisto 5.1. Mice with selective inactivation of peroxisomes in recruit the dynamin like protein DLP1 to the peroxisomal hepatocytes in the postnatal period membrane [34]. Pex11α knockout mice did not have an obvious phenotype As already mentioned, the only defect observed in liver of suggesting that its function can be taken over by other Pex11 newborn Pex2, Pex5 and Pex13 knockout mice was a change in proteins [35]. In tissues of Pex11β knockout mice only minor the ultrastructure of the mitochondrial inner membrane. In order metabolic alterations were found i.e. a 20% depletion of plasma- to investigate hepatic changes developing at later ages, Pex5- logen levels in brain and a 1.4-fold elevation of C26:0 in liver loxP mice were bred with mice expressing Cre under the control [36]. Moreover, studies in fibroblasts documented normal of the albumin promotor [42]. The resulting mice with hepa- import of PTS1 and PTS2 containing proteins. It was therefore tocyte specific elimination of peroxisomes will be denoted unexpected that the phenotype of Pex11β deficient mice was further as L-Pex5 knockout mice [43]. very similar to that of Pex5, Pex2 and Pex13 knockout mice [36] Functional peroxisomes were eliminated from the liver i.e. these mice were all very hypotonic and growth retarded at between the first and second postnatal week, as shown by a birth, they displayed a neuronal migration defect and died in the number of biochemical parameters. Electronmicroscopic anal- postnatal period. However, no alterations of mitochondrial ysis of 10-week-old mice further confirmed the virtual absence structure in hepatocytes were noted. Although it could be of containing peroxisomes from hepatocytes but not envisaged that the migration defect and hypotonia are caused by from endothelial cells and Kuppfer cells [43]. Only a few small metabolic abnormalities different from peroxisomal β-oxidation clusters of hepatocytes with catalase-positive peroxisomes were or ether phospholipid synthesis defects, it seems inexplicable found. Surprisingly, plasmalogen and C26:0 levels were normal that such a severe phenotype is present despite intact import of in the peroxisome deficient livers suggesting that peroxisomes PTS1 and PTS2 proteins. In order to reconcile these discre- present in other tissues provided precursors for plasmalogens pancies an alternative model of pathogenesis was proposed in and degraded C26:0. Other parameters i.e. the concentration of which the disease phenotype correlates with the abundance and branched chain fatty acids phytanic acid and pristanic acid and distribution of peroxisomes rather than with their metabolic the ratio of C27/C24 bile acids were increased in L-Pex5 knock- activity [7]. Indeed, Pex11β deficient fibroblasts and hepato- out livers, as expected. 1790 M. Baes, P.P. Van Veldhoven / Biochimica et Biophysica Acta 1763 (2006) 1785–1793

Two features that are common in postnatal Zellweger patients Testicular abnormalities were reported in patients with other also occurred in L-Pex5 knockout mice. First, they displayed a peroxisomal diseases with longer survival such as X-linked severe hepatomegaly which was due to hypertrophic and adrenoleukodystrophy [44,45] or X-linked adrenomyeloneuro- hyperplastic hepatocytes, particularly in the pericentral region pathy [46]. Male infertility was reported in three different mouse (Fig. 2A, B). Furthermore, fibrosis developed in these livers models with single peroxisomal enzyme deficiencies i.e. acyl- from the age of 20 weeks on (Fig. 2C, D). Throughout the CoA oxidase [47], MFP-2 [48] and DHAPAT [26] knockout lifetime of the mice (>15 months), they looked healthy, were mice. In acyl-CoA oxidase deficient mice a reduction in the fertile and hepatocellular integrity was unaffected as judged by Leydig cell population and hypospermatogenesis were seen normal plasma values of alanine and aspartate aminotransferase. whereas in DHAPAT knockout mice an arrest of spermatogenesis However, all the L-Pex5 knockout mice developed extensive before the stage of round spermatids resulting in the complete liver tumours from 12 months on [43]. absence of spermatozoa was described. In contrast, infertility in Severe changes in mitochondrial ultrastructure were ob- MFP-2 knockout mice [48] was associated with the accumula- served in 60–70% of the mitochondria in peroxisome deficient tion of huge lipid droplets in Sertoli cells followed by a complete hepatocytes. The most common finding was the proliferation of fatty degeneration of the tubuli seminiferi while Leydig cell pleomorphic mitochondria with rarefication of cristae or with function was preserved. In fact, until recently it was thought that abnormally curled or stacked cristae. Strikingly, mitochondria peroxisomes were present only in the interstitial cells of Leydig were normal in the few hepatocytes in which catalase-positive but according to new data [48,49] they occur also in sperma- peroxisomes were present, indicating that this is a cell autono- togonia and in Sertoli cells, both located in the basal compart- mous phenomenon. Additional ultrastructural changes in ment of the seminiferous epithelium. hepatocytes lacking perixosomes were the proliferation of The Cre-loxP technology was recently applied to demon- smooth endoplasmic reticulum (sER) and the appearance of strate the crucial role of peroxisomes in Sertoli cells. Therefore, lipid droplets and large groups of lysosomes with electron-dense Pex5-loxP mice were bred with mice expressing Cre under the deposits around dilated bile canaliculi. The ER proliferation control of the Anti-Mullerian hormone (AMH) promoter was associated with the induction of the cytochrome P450 [48,50]. In the testis extensive accumulations of neutral lipids ω-hydroxylation enzyme CYP4A1 [43]. were observed in Sertoli cells (Fig. 2G, H), beginning in pre- Analyses of mitochondrial functions demonstrated that the pubertal mice and evolving in complete testicular atrophy by the complexes I, III and Vof the respiratory chain that are embedded age of 4 months. Spermatogenesis was already severely affected in the inner membrane were severely inactivated whereas several at the age of 7 weeks and pre- and postmeiotic germ cells matrix enzymes including citrate synthase and mitochondrial gradually disappeared from the tubuli seminiferi. The AMH- β-oxidation were induced. Given the importance of the com- Pex5 knockout mice were completely infertile. Together with plexes to generate ATP, it was surprising that no significant analogous Sertoli cell abnormalities in MFP-2 knockout mice depletion of ATP levels were found [43]. However, a compen- [48], these data strongly indicate that peroxisomal β-oxidation is satory increase in glycolysis as an alternative source of ATP was essential to maintain the lipid balance in Sertoli cells which in demonstrated. Another consequence of the impaired functioning turn is crucial for male fertility. It needs to be further investigated of the respiratory chain was a collapse of the inner mitochondrial how the fatty degeneration of the testis relates to the loss of membrane potential (Fig. 2E, F). Because dysfunction of the peroxisomal function in Sertoli cells. electron transport chain is often accompanied by the generation of oxygen radicals that in turn can cause mitochondrial damage, 6. Conclusions the hypothesis that reactive oxygen species were increased in the peroxisome deficient livers was investigated. However, neither The phenotypic analyses of mouse models with peroxisome oxidative damage to proteins or lipids, nor increased peroxide biogenesis disorders have taught us that several but not all production in cultured hepatocytes or elevation of oxidative changes overlap with those occurring in human diseases. The stress defence mechanisms were found, indicating that the discrepancies may relate to the huge time difference in develop- mitochondrial alterations were not related to an excessive pro- mental period between mice and men and to differences in duction of oxidative radicals [43]. dietary composition. Altogether, it can be concluded that peroxisomes are essential In order to understand the importance of peroxisomes for the for the maintenance of other subcellular compartments in adult functioning of different tissues, these animal models still need to hepatocytes although their absence is not detrimental for the be investigated in more detail, including and functioning of the cell. The molecular interrelationship between more extensive lipidomic analyses. The latter will require peroxisomes and mitochondria remains to be investigated. sensitive techniques because several of the substrates or reaction products of the peroxisomal pathways are present in very low 5.2. Mice with selective inactivation of peroxisomes in Sertoli amounts. It will be further instrumental to compare the metabolic cells and histological abnormalities of the Pex knockouts with those of single peroxisomal enzyme or transporter knockouts that are In view of the early postnatal death of patients with pero- already available or being generated (Acox1 [47], MFP-1[51], xisome biogenesis disorders, not much is known about the MFP-2 [48,52,53], SCPx [54], phytanoyl-CoA hydroxylase, α- importance of peroxisomes for testicular function and fertility. methylacyl-CoA racemase [55], DHAPAT [26], ALD [56–58], M. Baes, P.P. Van Veldhoven / Biochimica et Biophysica Acta 1763 (2006) 1785–1793 1791

Fig. 2. Pathology in tissue selective Pex5 knockout mice. (A–B) Hematoxylin–eosin staining of livers of 10-week-old control (A) and L-Pex5 knockout mice (B) reveals hypertrophic and hyperplastic hepatocytes in the knockout, in particular in the pericentral region. (C–D) Sirius red stains collagen fibers in 20-week-old L-Pex5 knockout mice (D), compatible with fibrosis, but not in control mice (C). (E–F) Hepatocyte cultures were incubated with the mitotracker JC-1. Mitochondria from control mice (E) fluoresce orange corresponding with a normal inner mitochondrial membrane potential whereas those of L-Pex5 knockout mice fluoresce green indicative of a reduced potential, n=nucleus. (G–H) Oil red O stains neutral lipid droplets in the outer layer of tubuli seminiferi of 9-week-old AMH-Pex5 knockout mice (H) (arrowheads) but not in control mice (G). This is compatible with a Sertoli cell localisation. Leydig cells (arrows) stain in both genotypes because of their high steroid content. (A–F) Reprinted from [43] with permission from the publisher. 1792 M. Baes, P.P. Van Veldhoven / Biochimica et Biophysica Acta 1763 (2006) 1785–1793

ALDR [59], PMP34,…). The possibility that some of the ob- the cerebro-hepato-renal syndrome: defective bile acid synthesis and – served changes are not related to metabolic disturbances but to abnormal mitochondria, Gastroenterology 79 (1980) 1311 1317. [14] S. Huyghe, M. Casteels, A. Janssen, L. Meulders, G.P. Mannaerts, P.E. an altered distribution of peroxisomes or peroxisomal remnants Declercq, P.P. Van Veldhoven, M. Baes, Prenatal and postnatal develop- will also need to be further substantiated. ment of peroxisomal lipid-metabolizing pathways in the mouse, Biochem. Finally, the breeding of Pex-loxP mice with appropriate Cre J. 353 (2001) 673–680. expressing mice may reveal more unsuspected roles of peroxi- [15] B.J. Pettus, M. Baes, M. Busman, Y.A. Hannun, P.P. Van Veldhoven, Mass somes in certain cell types as already exemplified by the Sertoli spectrometric analysis of ceramide perturbations in brain and fibroblasts of mice and human patients with peroxisomal disorders, Rapid Commun. cell selective inactivation of Pex5. Mass. Spectrom. 18 (2004) 1569–1574. [16] A. Janssen, M. Baes, P. Gressens, G.P. Mannaerts, P. Declercq, P.P. Acknowledgements Van Veldhoven, Docosahexaenoic acid deficit is not a major pathogenic factor in peroxisome-deficient mice, Lab. Invest. 80 (2000) 31–35. This work was supported by grants from Fonds Wetenschap- [17] I. Vanhorebeek, M. Baes, P.E. Declercq, Isoprenoid biosynthesis is not compromised in a Zellweger syndrome mouse model, Biochim. Biophys. pelijk Onderzoek Vlaanderen (G.0235.01 and G.0385.05), Acta 1532 (2001) 28–36. Geconcerteerde Onderzoeksacties (99/09 and 2004/08) and the [18] S. Hogenboom, G.J. Romeijn, S.M. Houten, M. Baes, R.J.A. Wanders, European Union (“MMPD”, QLG1-CT2001-01277, FP5 and H.R. Waterham, Absence of functional peroxisomes does not lead to “Peroxisomes”, LSHG-CT-2004-512018, FP6). The authors deficiency of enzymes involved in cholesterol biosynthesis, J. Lipid Res. – wish to thank Prof. G. Mannaerts for critically reading the 43 (2002) 90 98. [19] P. Evrard, V.S. Caviness, J. Prats-Vinas, G. Lyon, The mechanism of arrest manuscript. of neuronal migration in the Zellweger malformation: an hypothesis based upon cytoarchitectonic analysis, Acta Neuropathol. 41 (1978) 109–117. [20] J.J. Volpe, R.D. Adams, Cerebro-hepato-renal syndrome of Zellweger: an References inherited disorder of neuronal migration, Acta Neuropathol. 20 (1972) 175–198. [1] R.J.A. Wanders, H.R. Waterham, Biochemistry of mammalian peroxi- [21] J.M. Powers, H.W. Moser, Peroxisomal disorders: genotype, phenotype, somes revisited, Annu. Rev. Biochem. 75 (2006) 295–332. major neuropathologic lesions, and pathogenesis, Brain Pathol. 8 (1998) [2] M. Baes, P. Gressens, E. Baumgart, P. Carmeliet, M. Casteels, M. Fransen, 101–120. P. Evrard, D. Fahimi, P.E. Declercq, D. Collen, P.P. Van Veldhoven, G. [22] M. Baes, P. Gressens, S. Huyghe, K. De Nys, C. Qi, Y. Jia, G.P. Mannaerts, P. Mannaerts, A mouse model for Zellweger syndrome, Nat. Genet. 17 P. Evrard, P.P. Van Veldhoven, P.E. Declercq, J.K. Reddy, The neuronal (1997) 49–57. migration defect in mice with Zellweger syndrome (Pex5 knockout) is not [3] P.L. Faust, M.E. Hatten, Targeted deletion of the PEX2 peroxisome caused by the inactivity of peroxisomal b-oxidation, J. Neuropathol. Exp. assembly gene in mice provides a model for Zellweger syndrome, a human Neurol. 61 (2002) 368–374. neuronal migration disorder, J. Cell Biol. 139 (1997) 1293–1305. [23] S. Ferdinandusse, S. Denis, P.A.W. Mooyer, C. Dekker, M. Duran, R.J. [4] M. Maxwell, J. Bjorkman, T. Nguyen, P. Sharp, J. Finnie, C. Paterson, I. Soorani-Lunsing, E. Boltshauser, A. Macaya, J. Gärtner, C.B.L.M. Majoie, Tonks, B.C. Paton, G.F. Kay, D.I. Crane, Pex13 inactivation in the mouse P.G. Barth, R.J.A. Wanders, B.T. Poll, The clinical and biochemical disrupts peroxisome biogenesis and leads to a Zellweger syndrome pheno- spectrum of D-bifunctional protein deficiency, Ann. Neurol. 59 (2006) type, Mol. Cell. Biol. 23 (2003) 5947–5957. 92–104. [5] S.J. Gould, G.V. Raymond, D. Valle, The peroxisome biogenesis disorders, [24] G.J. Bix, G.D. Clark, Platelet-activating factor receptor stimulation in: C.R. Scriver, A.L. Beaudet, D. Valle, W.S. Sly (Eds.), The Metabolic disrupts neuronal migration in vitro, J. Neurosci. 18 (1998) 307–318. and Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001, [25] P. Gressens, M. Baes, P. Leroux, A. Lombet, P. Van Veldhoven, A. Janssen, pp. 3181–3217. J. Vamecq, S. Marret, P. Evrard, Neuronal migration disorder in Zellweger [6] P.L. Faust, Abnormal cerebellar histogenesis in Pex2 Zellweger mice mice is secondary to glutamate receptor dysfunction, Ann. Neurol. 48 reflects multiple neuronal defects induced by peroxisome deficiency, (2000) 336–343. J. Comp. Neurol. 461 (2003) 394–413. [26] C. Rodemer, T.P. Thai, B. Brugger, T. Kaercher, H. Werner, K.-A. Nave, F. [7] T. Nguyen, J. Bjorkman, B.C. Paton, D.I. Crane, Failure of microtubule- Wieland, K. Gorgas, W.W. Just, Inactivation of ether lipid biosynthesis mediated peroxisome division and trafficking in disorders with reduced causes male infertility, defects in eye development and optic nerve peroxisome abundance, J. Cell Sci. 119 (2006) 636–645. hypoplasia in mice, Hum. Mol. Genet. 12 (2003) 1881–1895. [8] M. Schrader, M. Thiemann, H.D. Fahimi, Peroxisomal motility and [27] A. Janssen, P. Gressens, M. Grabenbauer, E. Baumgart, A. Schad, I. interaction with microtubules, Microsc. Res. Tech. 61 (2003) 171–178. Vanhorebeek, A. Brouwers, P.E. Declercq, D. Fahimi, P. Evrard, L. [9] E. Baumgart, I. Vanhorebeek, M. Grabenbauer, M. Borgers, P. Declercq, Schoonjans, D. Collen, P. Carmeliet, G. Mannaerts, P. Van Veldhoven, M. H.D. Fahimi, M. Baes, Mitochondrial alterations caused by defective Baes, Neuronal migration depends on intact peroxisomal function in brain peroxisomal biogenesis in a mouse model for Zellweger syndrome (PEX5 and in extraneuronal tissues, J. Neurosci. 23 (2003) 9732–9741. knockout mouse), Am. J. Pathol. 159 (2001) 1477–1494. [28] P.L. Faust, D. Banka, R. Siriratsivawong, V.G. Ng, T.M. Wikander, [10] W.J. Kovacs, J.E. Shackelford, K.N. Tape, M.J. Richards, P.L. Faust, S.J. Peroxisome biogenesis disorders: the role of peroxisomes and metabolic Fliesler, S.K. Krisans, Disturbed cholesterol homeostasis in a peroxi- dysfunction in developing brain, J. Inherit. Metab. Dis. 28 (2005) 369–383. some-deficient PEX2 knockout mouse model, Mol. Cell. Biol. 24 (2004) [29] P. Brites, A.M. Motley, P. Gressens, P.A.W. Mooyer, I. Ploegaert, V. 1–13. Everts, P. Evrard, P. Carmeliet, M. Dewerchin, M. Duran, H.R. Waterham, [11] S. Goldfischer, C.L. Moore, A.B. Johnson, A.J. Spiro, M.P. Valsamis, H.K. R.J.A. Wanders, M. Baes, Impaired neuronal migration and endochondral Wisniewski, R.H. Ritch, W.T. Norton, I. Rapin, L.M. Gartner, Peroxisomal ossification in PEX7 knockout mice: a model for rhizomelic chondrodys- and mitochondrial defects in the cerebro-hepato-renal syndrome, Science plasia punctata, Hum. Mol. Genet. 12 (2003) 2255–2267. 182 (1973) 62–64. [30] P.E. Purdue, M. Skoneczny, X. Yang, J.W. Zhang, P.B. Lazarow, [12] J.M.F .Trijbels, J.A. Berden, L.A.H. Monnens, J.L. Willems, A.J.M. Rhizomelic chondrodysplasia punctata, a peroxisomal biogenesis disorder Janssen, R.B.H. Schutgens, M. Van den Broek-Van Essen, Biochemical caused by defects in Pex7p, a peroxisomal protein import receptor: a studies in the liver and muscle of patients with Zellweger syndrome, minireview, Neurochem. Res. 24 (1999) 581–586. Pediatr. Res. 17 (1983) 514–517. [31] S. Thoms, R. Erdmann, Dynamin-related proteins and Pex11 proteins in [13] R.K. Mathis, J.B. Watkins, P. Szczepanik-Van Leeuwen, I.T. Lott, Liver in peroxisome division and proliferation, FEBS J. 272 (2005) 5169–5181. M. Baes, P.P. Van Veldhoven / Biochimica et Biophysica Acta 1763 (2006) 1785–1793 1793

[32] X. Li, S.J. Gould, PEX11 promotes peroxisome division independently of peroxisomal fatty acyl-coenzyme A oxidase gene, J. Biol. Chem. 271 peroxisome metabolism, J. Cell Biol. 156 (2002) 643–651. (1996) 24698–24710. [33] M. Schrader, B.E. Reuber, J.C. Morrell, G. Jimenez-Sanchez, C. Obie, [48] S. Huyghe, H. Schmalbruch, K. De Gendt, G. Verhoeven, F. Guillou, P.P. T.A. Stroh, D. Valle, T.A. Schroer, S.J. Gould, Expression of PEX11β Van Veldhoven, M. Baes, Peroxisomal multifunctional protein 2 is mediates peroxisome proliferation in the absence of extracellular essential for lipid homeostasis in Sertoli cells and for male fertility in mice, stimuli, J. Biol. Chem. 273 (1998) 29607–29614. Endocrinology 147 (2006) 2228–2236. [34] X. Li, S.J. Gould, The dynamin-like GTPase DLP1 is essential for [49] G. Lüers, S. Thiele, A. Schad, A. Völkl, S. Yokota, J. Seitz, Peroxisomes peroxisome division and is recruited to peroxisomes in part by PEX11, are present in murine spermatogonia and disappear during the course of J. Biol. Chem. 278 (2003) 17012–17020. spermatogenesis, Histochem. Cell Biol. 30 (2005) 1–11. [35] X. Li, E. Baumgart, G.-X. Dong, J.C. Morrell, G. Jimenez-Sanchez, D. [50] C. Lecureuil, I. Fontaine, P. Crepieux, F. Guillou, Sertoli and granulosa Valle, K.D. Smith, S.J. Gould, PEX11α is required for peroxisome cell-specific Cre recombinase activity in transgenic mice, Genesis 33 proliferation in response to 4-phenylbutyrate but is dispensable for (2002) 114–118. peroxisome proliferator-activated receptor alpha-mediated peroxisome [51] C. Qi, Y. Zhu, J. Pan, N. Usuda, N. Maeda, A.V. Yeldandi, M.S. Rao, T. proliferation, Mol. Cell. Biol. 22 (2002) 8226–8240. Hashimoto, J.K. Reddy, Absence of spontaneous peroxisome proliferation [36] X. Li, E. Baumgart, J.C. Morrell, G. Jimenez-Sanchez, D. Valle, S.J. in enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase-deficient Gould, PEX11β deficiency is lethal and impairs neuronal migration but mouse liver, J. Biol. Chem. 274 (1999) 15775–15780. does not abrogate peroxisome function, Mol. Cell. Biol. 22 (2002) [52] M. Baes, S. Huyghe, P. Carmeliet, P.E. Declercq, D. Collen, G.P. 4358–4365. Mannaerts, P.P. Van Veldhoven, Inactivation of the peroxisomal [37] S. Bielas, H. Higginbotham, H. Koizumi, T. Tanaka, J.G. Gleeson, Cortical multifunctional protein-2 in mice impedes the degradation of not only neuronal migration mutants suggest separate but intersecting pathways, 2-methyl branched fatty acids and bile acid intermediates but also of very Annu. Rev. Cell Dev. Biol. 20 (2004) 593–618. long chain fatty acids, J. Biol. Chem. 275 (2000) 16329–16336. [38] P. Gressens, Pathogenesis of migration disorders, Curr. Opin. Neurol. 19 [53] S. Huyghe, H. Schmalbruch, L. Hulshagen, P.P. Van Veldhoven, M. Baes, (2006) 135–140. D. Hartmann, Peroxisomal multifunctional protein-2 deficiency causes [39] K.-M. Kwan, Conditional alleles in mice: practical considerations for motor deficits and glial lesions in the adult CNS, Am. J. Pathol. 168 (2006) tissue-specific knockouts, Genesis 32 (2002) 49–62. 1321–1334. [40] M. Baes, M. Dewerchin, A. Janssen, D. Collen, P. Carmeliet, Generation of [54] U. Seedorf, M. Raabe, P. Ellinghaus, F. Kannenberg, M. Fobker, T. Engel, Pex5-IoxP mice allowing the conditional elimination of peroxisomes, S. Denis, F. Wouters, K.W.A. Wirtz, R.J.A. Wanders, N. Maeda, G. Genesis 32 (2002) 177–178. Assmann, Defective peroxisomal catabolism of branched fatty acyl [41] J. Bjorkman, I. Tonks, M.A. Maxwell, C. Paterson, G.F. Kay, D.I. Crane, coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier Conditional inactivation of the peroxisome biogenesis Pex13 gene by protein-x gene function, Genes Dev. 12 (1998) 1189–1201. Cre-IoxP excision, Genesis 32 (2002) 179–180. [55] K. Savolainen, T.J. Kotti, W. Schmitz, T.I. Savolainen, R.T. Sormunen, M. [42] C. Postic, M. Shiota, K.D. Niswender, T.L. Jetton, Y. Chen, J.M. Moates, Ilves, S.J. Vainio, E. Conzelmann, J.K. Hiltunen, A mouse model for a- K.D. Shelton, J. Lindner, A.D. Cherrington, M.A. Magnuson, Dual roles methylacyl-CoA racemase deficiency: adjustment of bile acid synthesis for glucokinase in glucose homeostasis as determined by liver and and intolerance to dietay methyl-branched lipids, Hum. Mol. Genet. 13 pancreatic β cell-specific gene knock-outs using Cre recombinase, J. Biol. (2004) 955–965. Chem. 274 (1999) 305–315. [56] S. Forss-Petter, H. Werner, J. Berger, H. Lassmann, B. Molzer, M.H. [43] R. Dirkx, I. Vanhorebeek, K. Martens, A. Schad, M. Grabenbauer, D. Schwab, H. Bernheimer, F. Zimmermann, K.-A. Nave, Targeted inactiva- Fahimi, P. Declercq, P.P. Van Veldhoven, M. Baes, Absence of peroxi- tion of the X-linked adrenoleukodystrophy gene in mice, J. Neurosci. Res. somes in hepatocytes causes mitochondrial and ER abnormalities, 50 (1997) 829–843. Hepatology 41 (2005) 868–878. [57] T. Kobayashi, N. Shinnoh, A. Kondo, T. Yamada, Adrenoleukodystrophy [44] J.M. Powers, H.H. Schaumburg, The testis in adreno-leukodystrophy, Am. protein-deficient mice represent abnormality of very long chain fatty acid J. Pathol. 102 (1981) 90–98. metabolism, Biochem. Biophys. Res. Commun. 232 (1997) 631–636. [45] H.W. Moser, K.D. Smith, P.A. Watkins, J.M. Powers, A.B. Moser, [58] A. Pujol, C. Hindelang, N. Callizot, U. Bartsch, M. Schachner, J.L. X-linked adrenoleukodystrophy, in: C.R. Scriver, A.L. Beaudet, D. Valle, Mandel, Late onset neurological phenotype of the X-ALD gene W.S. Sly (Eds.), The Metabolic and Molecular Bases of Inherited Disease, inactivation in mice: a mouse model for adrenomyeloneuropathy, Hum. McGraw-Hill, New York, 2001, pp. 3257–3301. Mol. Genet. 11 (2002) 499–505. [46] A. Aversa, S. Palleschi, G. Cruccu, L. Silverstroni, A. Isidori, A. Fabbri, [59] I. Ferrer, J.P. Kapfhammer, C. Hindelang, S. Kemp, N. Troffer-Charlier, Rapid decline of fertility in a case of adrenoleukodystrophy, Hum. Reprod. V. Broccoli, N. Callyzot, P. Mooyer, J. Selhorst, P. Vreken, R.J.A. 13 (1998) 2474–2479. Wanders, J.-L. Mandel, A. Pujol, Inactivation of the peroxisomal ABCD2 [47] C.-Y. Fan, J. Pan, R. Chu, D. Lee, K.D. Kluckman, N. Usuda, I. transporter in the mouse leads to late-onset ataxia involving mitochondria, Singh, A.V. Yeldandi, M.S. Rao, N. Maeda, J.K. Reddy, Hepatocellular Golgi and endoplasmic reticulum damage, Hum. Mol. Genet. 14 (2005) and hepatic peroxisomal alterations in mice with a disrupted 3565–3577.