PEX11{beta} Deficiency Is Lethal and Impairs Neuronal Migration but Does Not Abrogate Peroxisome Function

Zellweger syndrome is a lethal neurological disorder characterizedby severe defects in peroxisomal protein import. The resultingdefects in peroxisome metabolism and the accumulation of peroxisomalsubstrates are thought to cause the other Zellweger syndromephenotypes, including neuronal migration defects, hypotonia,a developmental delay, and neonatal lethality. These phenotypesare also manifested in mouse models of Zellweger syndrome generatedby disruption of the PEX5 or PEX2 gene. Here we show that micelacking peroxisomal membrane protein PEX11ß displayseveral pathologic features shared by these mouse models ofZellweger syndrome, including neuronal migration defects, enhancedneuronal apoptosis, a developmental delay, hypotonia, and neonatallethality. However, PEX11ß deficiency differs significantlyfrom Zellweger syndrome and Zellweger syndrome mice in thatit is not characterized by a detectable defect in peroxisomalprotein import and displays only mild defects in peroxisomalfatty acid ß-oxidation and peroxisomal ether lipidbiosynthesis. These results demonstrate that the neurologicalpathologic features of Zellweger syndrome can occur withoutperoxisomal enzyme mislocalization and challenge current modelsof Zellweger syndrome pathogenesis.

INTRODUCTION

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Introduction

Peroxisomes are single-membrane bound metabolic organelles thatare present in all eukaryotic cells. In their lumen reside theenzyme systems responsible for a wide variety of metabolic pathways,including the ß-oxidation of very long-chain fattyacids (VLCFAs), ${alpha}$ - and ß-oxidation of branched-chainfatty acids, biosynthesis of ether linked lipids and cholesterol,synthesis of bile acids, metabolism of polyunsaturated fattyacids, and H₂O₂ metabolism (25, 36). The importance of peroxisomesfor human health is best demonstrated by the existence of Zellwegersyndrome, a lethal neurological disorder characterized by defectsin peroxisomal matrix enzyme import (30). This defect negativelyimpacts virtually all peroxisomal metabolic functions, whichleads, in turn, to the accumulation of peroxisomal ${alpha}$ - and ß-oxidationsubstrates (e.g., phytanic acid and VLCFAs, respectively) andreduced levels of ether-linked lipids (e.g., plasmalogens) (11).Zellweger syndrome is also associated with severe defects inmitochondrial structure and function (5, 10), as well as a pleiotropicset of clinical phenotypes, including a developmental delay,hypotonia, neuronal migration defects, enhanced neuronal apoptosis,and an array of hepatic and renal abnormalities (11).

There is uncertainty regarding the etiologic agent(s) and mechanismsresponsible for the neuronal migration defect and other phenotypesof Zellweger syndrome. However, the accumulation of toxic peroxisomal ${alpha}$ - and ß-oxidation substrates or depletion of peroxisomalproducts, such as ether-linked lipids, have been proposed tocause its pathologic features (28). In contrast to the uncertaintyregarding Zellweger syndrome pathogenesis, the molecular geneticsof Zellweger syndrome and its milder variants (neonatal adrenoleukodystrophyand infantile Refsum disease) are well understood (11, 30).These peroxisome biogenesis disorders are inherited in an autosomalrecessive fashion and are caused by mutations in any of at least12 distinct PEX genes (11). Approximately 20 PEX genes are requiredfor peroxisome biogenesis, and with the exception of PEX11,all are required for peroxisomal matrix enzyme import (30).PEX11 proteins are components of the peroxisomal membrane ina wide array of species, including yeast, protozoan parasites,and mammals (1, 2, 6, 19, 21, 27, 32, 34). They appear to playan important role in peroxisome division, although the natureof their role is currently the subject of debate. In one model,PEX11 proteins are thought to play a direct role in peroxisomedivision (6, 12, 21, 27, 34). Recently, another model was proposedin which PEX11 proteins play a direct role in medium-chain fattyacid oxidation and only affect peroxisome division indirectlythrough this metabolic role (35).

Mammals express at least two PEX11 genes, the inducible PEX11 ${alpha}$ gene and the constitutively expressed PEX11ß gene(1, 2, 27, 34). Here we report an analysis of mice that lackthe PEX11ß gene. Quite unexpectedly, we found thatPEX11ß^-/- mice exhibit numerous Zellweger syndromepathologic features, including a developmental delay, hypotonia,neuronal migration defects, and enhanced neuronal apoptosis,even though they have no apparent defect in peroxisomal proteinimport and have only mild defects in peroxisomal metabolic function.

MATERIALS AND METHODS

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Materials and Methods

Cloning and disruption of the murine PEX11ß gene. A bacterial artificial chromosome (BAC) clone (no. 17747; IncyteGenomics Inc., St. Louis, Mo.) with the complete PEX11ßgene was obtained by screening a BAC library of 129/svJ mousegenomic DNA with the full-length murine PEX11ß cDNA(34). EcoRI-XbaI, XbaI, or HindIII-fragments spanning a 13-kbregion containing the PEX11ß gene were subcloned intopLITMUS vectors (New England BioLabs, Beverly, Mass.) and sequenced.The targeting vector, designed to disrupt the first three exonsof the PEX11ß gene (Fig. 1A), was generated by insertionof ApaLI-SmaI (3.1-kb PEX11ß 5' untranslated region)and HindIII-NotI (3.4 kb of intron 3) fragments into pGT-N29as the 5'- and 3'-flanking regions of the pgk-Neo cassette.A 200-µg sample of targeting vector-DNA was linearizedwith SwaI and electroporated into 6.5 x 10⁶ R1 embryonic stem(ES) cells as previously described (40). After selection withG418 at 200 µg/ml, recombined ES cell clones were identifiedby mini-Southern blot hybridization analysis of EcoRI- or EcoRV-SseI-digestedES cell genomic DNA with two flanking probes. Four PEX11ß^+/-ES cell clones were injected into blastocysts of C57BL/6 hostmice. Chimeric males from three different ES cell lines wereintercrossed with C57BL/6 mice (Jackson Laboratory, Bar Harbor,Maine), and agouti offspring were tested for the presence ofthe gene disruption by Southern blotting (33). HeterozygousF₁ mice were backcrossed with C57BL/6 mice for five generations.Genotypes of mice from generation F₂ and beyond were determinedby PCR with primer 8 (5'-GTCTAGGACAGGCTTCTGCTGTTC-3'), primer9 (5'-GTTTCCCCATCTTTCCCTTGAG-3'), and primer Neo (5'-ATATTGCTGAAGAGCTTGGCGGC-3').Amplification reactions were done with 0.1 µg of DNA forthe wild-type allele (primers 8 and 9 $->$ 590 bp) or the targetedallele (primers 8 and Neo $->$ 980 bp). For RNA blots, total RNAwas isolated from mouse livers with the Purescript RNA isolationkit (Gentra Systems, Minneapolis, Minn.). RNA (10 µg/lane)was separated by electrophoresis on 1.5% formaldehyde-agarosegels, transferred to GeneScreen Plus membranes (NEN Life ScienceProducts, Boston, Mass.), and hybridized in accordance withstandard procedures (33).

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FIG. 1. Targeting of PEX11ß. (A) Schematic representation of the PEX11ß wild-type locus (top), the targeting vector (middle), and the targeted allele (bottom). Two flanking Southern blot probes, A and B, are indicated. (B) Southern blot analyses of the G418^r ES clone DNA with probes A and B and the mouse tail DNA with probe A. Probe A detects an 8.6-kb EcoRI fragment in the wild-type allele and a 5.4-kb fragment in the targeted allele. Probe B detects a 13.4-kb EcoRV/SseI fragment in the wild-type allele and a 6.8-kb fragment in the targeted allele. (C) PCR analysis of mouse tail DNA. Positions of the primers are indicated. The wild-type (WT) allele product is 590 bp, and the targeted-allele product is 980 bp. M, molecular size markers. (D) Northern blot analysis of total liver RNA from wild-type (+/+), homozygous (-/-), and heterozygous (+/-) animals. The Northern blot shown was probed with a radioactively labeled murine PEX11ß cDNA probe (top), stripped, and probed with labeled ß-actin cDNA (bottom). (E and F) PEX11ß^-/- mice display intrauterine growth retardation and are hypotonic. One litter of newborn mice with heterozygous (+/-) and homozygous (-/-) offspring is depicted either in the conscious state (E) or under ether anesthesia (F).

MEF preparation and immunofluorescence assay. Mouse embryo fibroblasts (MEF) were isolated from E14.5 embryosas previously described (13). For indirect immunofluorescenceassay, cells were fixed for 20 min in 3% formaldehyde in Dulbecco’sphosphate-buffered saline (Life Technologies), pH 7.1; permeabilizedfor 5 min in 1% Triton X-100-Dulbecco’s phosphate-bufferedsaline; and further processed as previously described (31).Affinity-purified rabbit anti-human PEX14 antibody has beendescribed previously (31), sheep anti-human catalase antibodieswere obtained from The Binding Site (Birmingham, United Kingdom),mouse monoclonal anti-myc antibodies were obtained from thesupernatant of hybridoma 1-9E10 (7), and labeled secondary antibodieswere obtained from standard commercial sources.

Histology and electron microscopy. Under ether anesthesia, mouse embryos (embryonic day 17.5 [E17.5]and E18.5) and newborn mice (postnatal day 0.5 [P0.5]) wereperfusion fixed via the heart with 4% paraformaldehyde in phosphate-bufferedsaline, pH 7.4. Whole-mouse sections were obtained followingovernight immersion fixation (OIF) at 4°C and paraffin embedding.Five-micrometer sagittal sections of whole mice were stainedwith hematoxylin and periodic acid-Schiff (PAS) for pathologicalanalysis (3). For brain analysis, OIF was performed with openedskulls and the brains were removed and embedded in paraffin.Serial coronal sections (5 µm) were cut and stained withcresyl fast violet for pathological analysis (3). More than200 carefully matched sections were examined for neuronal migrationdefects and apoptosis. For electron microscopy, cardiac perfusionfixation of newborn mice was performed with 4% paraformaldehyde-0.05%glutaraldehyde-2% sucrose-0.05% CaCl₂-0.1 M piperazine-N,N'-bis(2-ethanesulfonicacid) (PIPES) buffer, pH 7.4, followed by OIF in the same fixativewithout glutaraldehyde. Livers were removed, cut into 100-µmsections, and postfixed for 15 min with 1.0% glutaraldehydein 0.1 M PIPES buffer. Catalase cytochemistry (30 min of preincubationand 2 h of incubation at 45°C) was performed with alkalineDAB medium as described by Fahimi (8). DAB-stained sectionswere postfixed with either aqueous or reduced osmium, embeddedin Epon 812, and examined by electron microscopy.

Biochemical assays. Livers and brains (telencephalon) of newborn mice (P0.5) wereweighed and homogenized in a tissue grinder with chloroform-methanol(2:1). Total lipids were extracted with 2:1:0.8 chloroform-methanol-water,converted to their methyl esters, dissolved in hexane at a concentrationof $~$ 1 µg/µl, and then separated by gas chromatography(DB-1 and SP-2560 columns) as previously described (24). Thelevels of VLCFAs and plasmalogens were calculated as percentagesof the total fatty acids. Plasmalogen synthesis activity wasdetermined in cultured MEF with the double-substrate, double-isotopemethod (with [¹⁴C]hexadecanol and [³H]hexadecyl-glycerol assubstrates) (29). Fatty acid ß-oxidation assays werecarried out with [1-¹⁴C]palmitic acid (C_16:0) and [1-¹⁴C]lignocericacid (C_24:0) as substrates in intact MEF (41). Branched-chainfatty acid (phytanic and pristanic acids) oxidation assays (41,42) were done with cultured MEF with [2,3-³H]phytanic acid or[1-¹⁴C]pristanic acid as the substrate.

RESULTS

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Results

PEX11ß^-/- mice display neonatal lethality, intrauterine growth retardation, and hypotonia. Exons 1 to 3 of the mouse PEX11ß gene were disruptedin R1 ES cells by gene conversion with a PEX11ß/pgk-Neo^rcassette (Fig. 1A). Correctly targeted PEX11ß^+/- celllines were identified and injected into C57BL/6 blastocysts,and chimeric animals were generated. Chimeras were mated withC57BL/6 mice, and PEX11ß^+/- heterozygote animals wereobtained from several chimeras and two independently generatedPEX11ß^+/- ES cell lines (Fig. 1B and C). These micewere backcrossed to C57BL/6 mice five times to place the PEX11ßdisruption allele over a relatively homogeneous background.PEX11ß^+/+, PEX11ß^+/-, and PEX11ß^-/-fetuses were obtained in the expected Mendelian ratios in intercrossesbetween PEX11ß^+/- animals. PEX11ß^-/- animalslack detectable PEX11ß mRNA (Fig. 1D). Animals lackingPEX11ß died shortly after birth on day 1, were undersized( $~$ 80% of wild-type size at P0.5) (Fig. 1E and F), were underweight( $~$ 60% of wild-type body weight at P0.5), were hypotonic (Fig.1E), and suckled only poorly, as was evident from the reducedamounts of milk in their gastrointestinal tracts (Fig. 1F).No heterozygote phenotype was observed.

PEX11ß^-/- mice exhibit neuronal migration defects and a developmental delay. Neonatal hypotonia and neonatal lethality caused by defectsin a peroxisomal membrane protein are unique characteristicsof Zellweger syndrome. In addition, prior mouse models of Zellwegersyndrome, which reproduce virtually all of the hallmarks ofthe human disease, also display the intrauterine growth defectswe observed in PEX11ß^-/- mice (3, 9). Therefore, weinvestigated the possibility that PEX11ß-deficientmice may display other pathologic characteristics of Zellwegersyndrome. Zellweger syndrome is characterized by defective neuronalmigration, and Zellweger syndrome mice display both a neuronalmigration defect and enhanced apoptosis within the neocortex(11).

The neuronal migration defect of PEX5^-/- mice is focal in natureand can be observed only in coronal sections through the neocortex(3) (E. Baumgart, unpublished data). In PEX11ß^-/-mice, we also detected focal areas of decreased neuronal migrationin coronal sections of the neocortex. This neuronal migrationdefect is evident from the increased numbers of neurons in theintermediate zone and layer V, as well as structural alterationsand slightly reduced thickness of the cortical plate (Fig. 2Aand B). This was observed in all three PEX11ß^-/- micethat were examined and was not observed any of the three PEX11ß^+/+littermate control mice. Thus, the neuronal migration defectappeared to be a fully penetrant phenotype of PEX11ß^-/-mice. In addition, because PEX11ß^-/- mice displaygrowth retardation and a developmental delay, we examined theneuronal migration defect in four PEX11ß^-/- embryosand four size-matched PEX11ß^+/+ embryos. The neuronalmigration defect was apparent in all four PEX11ß^-/-embryos and was not detected in any of the size-matched controlembryos (data not shown).

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FIG. 2. Neuronal defects in PEX11ß^-/- mice. Carefully matched coronal (A and B; stained with cresyl fast violet) or sagittal (C to F; stained with PAS and hematoxylin) sections of control (A and C) and PEX11ß^-/- (B and D to F) mice in medial regions of the neocortex. In panel B, the neuronal migration defect is revealed by higher cell density in the intermediate zone (IZ) and layer V, as well as the altered structure and slightly lesser thickness of the cortical plate (CP). GZ, germinative zone; SP, subplate. Neuronal apoptoses of different stages are indicated by arrows (D to F). Insets 1 and 2 in panel E depict higher magnifications of early stages of chromatin condensation with typical nuclear cap structures of apoptotic cells, indicated by white arrowheads. Black arrowheads indicate macrophages (MP) (slightly more PAS positive) with phagocytosed material. Empty spaces are clearly visible where apoptotic neurons were located (D to F). In contrast, empty spaces in panel C represent endothelium-lined capillaries.

Enhanced neuronal apoptosis was also observed in PEX11ß^-/-mice (Fig. 2C to F). Zellweger syndrome mice also exhibit adelay in renal development, and this too was observed in PEX11ß^-/-mice (Fig. 3A to D). Furthermore, we detected a focal mosaicpattern of a developmental delay and decreased glycogen in PEX11ß^-/-livers (Fig. 3E to I), although this could be due to the inabilityof PEX11ß^-/- mice to suckle properly. One other importantfeature of Zellweger syndrome is facial dysmorphism (25), butthis was not observed in Zellweger syndrome mice. However, thesemice did exhibit a developmental delay in calvarium ossification(9). PEX11ß^-/- animals also lack facial dysmorphismand exhibit a delay in the ossification of calvaria (data notshown).

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FIG. 3. Developmental delay in kidneys and livers of PEX11ß^-/- mice. Histological analysis of PAS- and hematoxylin-stained kidney (A to D) and liver (E to I) sections (5 µm) of P0.5 control (A, C, E, and G) and PEX11ß^-/- (B, D, F, H, and I) mice, depicting the strong delay in the development of both organs in PEX11ß^-/- animals. Well-developed glomeruli are present in control animals (C, arrows) but absent from PEX11ß^-/- animals (D). Tubules are also reduced in panel D. Livers of PEX11ß^-/- mice show regions of underdeveloped hepatocytes (F) with reduced glycogen (reduced PAS staining). Even areas with well-developed PEX11ß^-/- hepatocytes contained less glycogen (H) than did those of control animals (G). Arrows in panel H depict underdeveloped hepatocytes in PEX11ß^-/- mouse liver, which were not observed in controls.

Peroxisomal protein import and mitochondrial structure are not affected in PEX11ß^-/- mice. Zellweger syndrome and its corresponding mouse models displaya number of cellular abnormalities. Chief among these is theinability to import peroxisomal matrix enzymes (11). However,enzyme import appears to be unaffected in PEX11ß^-/-cells. This is shown here by the cytochemical detection of theperoxisomal marker enzyme catalase (imported through the PTS1pathway) within hepatocyte peroxisomes of PEX11ß^-/-mice (Fig. 4A and B), as well as the colocalization of the peroxisomalenzyme catalase and the peroxisomal membrane protein PEX14 inPEX11ß^-/- MEF (Fig. 4C to F). An enzyme imported throughthe PTS2 pathway, phytanoyl coenzyme A hydroxylase, was alsotargeted into peroxisomes when expressed as a myc-tagged formin PEX11ß^-/- MEF (Fig. 4G and H), demonstrating thatall peroxisomal protein import pathways are functional in cellslacking PEX11ß. Nevertheless, the loss of PEX11ßdid appear to reduce peroxisome abundance and increase peroxisomeclustering (Fig. 5A) and elongation (Fig. 4F and 5B). Defectsin mitochondrial structure have also been reported in Zellwegersyndrome patients and Zellweger syndrome mice (3, 10), but thesetoo were absent from PEX11ß^-/- hepatocytes (Fig. 5C),although we did detect mitochondrial proliferation in some cells(Fig. 4B and 5C). Zellweger syndrome is also characterized byhepatic accumulation of VLCFAs (36, 38), a substrate of theperoxisomal ß-oxidation pathway, which is thoughtto explain the presence of needle-like lipid crystals on thesurface of hepatocyte lipid droplets and within phagosomes ofKupffer cells (the resident macrophages of the liver) in mousemodels of Zellweger syndrome (3). We found no evidence of suchneedle-like lipid crystals in the livers of PEX11ß^-/-mice (Fig. 5D to F).

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FIG. 4. PEX11ß deficiency does not affect peroxisomal protein import. Ultrastructural analysis of hepatocytes from P0.5 control (A) and PEX11ß^-/- mice (B) shows normal import of the peroxisomal enzyme catalase, as determined by DAB staining (post-fixation with aqueous osmium). GLY, glycogen. (C to F) Indirect immunofluorescence analysis of control (C and D) and PEX11ß^-/- (E and F) MEF with antibodies to catalase (C and E) and PEX14 (D and F), a peroxisomal membrane protein. (G to H) Phytanoyl coenzyme A hydroxylase (PAHX)-myc targets to peroxisomes in PEX11ß^-/- fibroblasts. Cells were transfected with pcDNA3-PAHXmyc (22), grown for 2 days, and then processed for indirect immunofluorescence assay with a mouse monoclonal antibody to the myc epitope and rabbit anti-PEX14 antibodies.

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FIG. 5. No mitochondrial defects or lipid crystals in PEX11ß^-/- mouse liver. Ultrastructural analysis of DAB-stained preparations (postfixation and with aqueous osmium) revealed elevated peroxisome clustering (A) and elongation (B) in some hepatocytes of PEX11ß^-/- mice. Arrowheads indicate the positions of peroxisomes, and arrows in panel B show the position of smooth endoplasmic reticulum (sER). (C) Hepatocytes of PEX11ß^-/- mice had no mitochondrial structural abnormalities, although in some cells they did appear to be more abundant than in those of controls. MIT, mitochondria; GLY, glycogen; EC, endothelial cell; BC, blood cell. (D) Hepatocytes of PEX11ß^-/- mice lacked needle-like crystals on the surface of their lipid droplets (D; arrows show where large, needle-like crystals would be seen in Zellweger syndrome patients and mice). (E and F) VLCFA crystals were also absent from phagosomes (PS) of Kupffer cells (KC). NUC, nucleus of a Kupffer cell; EC, endothelial cell; HEP, hepatocyte; PS*, red blood cell in a phagosome. Note the high level of phagocytic degradation of different blood cells in phagosomes (E) and the occasional phagocytosis of hepatocytes (F) in Kupffer cells of PEX11ß^-/- mice.

VLCFA accumulation is not responsible for neuronal abnormalities in PEX11ß^-/- mice. The diagnosis of Zellweger syndrome is typically confirmed bybiochemical tests (24, 38). In Zellweger syndrome patients andprior mouse models of Zellweger syndrome, VLCFA levels are 10times higher than normal and plasmalogen levels are 10 to 100times lower than normal (3, 9, 24). By contrast, PEX11ß^-/-mice have normal levels of brain VLCFAs, no alteration of hepaticplasmalogens, and only slight alterations of hepatic VLCFAs(1.4x) and brain plasmalogens (0.8x) (Fig. 6A and C). We alsomeasured rates of VLCFA ß-oxidation and plasmalogensynthesis in cultured fibroblasts. Relative to controls, wedetected no change in plasmalogen synthesis rates and only aslight decrease in VLCFA ß-oxidation, to 0.6x thatof normal controls (Fig. 6B and D). Some Zellweger syndromepatients accumulate the branched-chain fatty acids phytanicacid and pristanic acid, which are substrates of the peroxisomalfatty acid ${alpha}$ - and ß-oxidation pathways, respectively,and the oxidation of these substrates is typically impairedin Zellweger syndrome fibroblasts (11, 25, 37). PEX11ß^-/-animals, on the other hand, do not accumulate these fatty acidsin the liver (Fig. 6E) or other tissues (data not shown), andPEX11ß^-/- fibroblasts have normal oxidation activitiestoward both phytanic and pristanic acids (Fig. 6F).

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FIG. 6. PEX11ß^-/- mice have a mild, generalized defect in peroxisome metabolism. In all of the graphs, black bars represent average values obtained with PEX11ß^+/+, gray bars represents average values obtained with PEX11ß^-/-, and brackets represent 1 standard deviation. Abundance of VLCFAs (A) and activities of mitochondrial (C_16:0) and peroxisomal (C_24:0) fatty acid ß-oxidation (B) in cultured MEF. (C) Plasmalogens in control and PEX11ß^-/- mice, expressed as percentages of the total fatty acids. (D) Plasmalogen synthesis activities in cultured MEF, expressed as the ratio of peroxisomal incorporation of [¹⁴C]hexadecanol to endoplasmic reticulum incorporation of [³H]hexadecyl-glycerol. (E) Levels of the branched-chain fatty acids phytanic acid and pristanic acid in the liver. (F) Rates of phytanic acid and pristanic acid oxidation in cultured fibroblasts from mutant and control animals.

DISCUSSION

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Discussion

Peroxisomal enzyme import defects are the root cause of allknown instances of Zellweger syndrome and prior mouse modelsof Zellweger syndrome (11). PEX11ß deficiency hasmost of the pathological hallmarks of Zellweger syndrome, includinga neuronal migration defect, enhanced neuronal apoptosis, adevelopmental delay, neonatal hypotonia, and neonatal lethality,without the peroxisomal enzyme import defects that are the cellularhallmarks of this disease. PEX11ß deficiency alsodiffers from Zellweger syndrome with regard to peroxisomal metabolicdefects. Zellweger syndrome patients and prior mouse modelsof Zellweger syndrome show 1,000% increases in VLCFAs, a markersubstrate of the peroxisomal ß-oxidation pathway,and 90 to 99% decreases in plasmalogens, a marker product ofthe peroxisomal ether lipid synthesis pathway (3, 9, 24). Incontrast, PEX11ß^-/- mice have no alteration in brainVLCFAs or liver plasmalogens, only a slight increase ( $~$ 40%) inliver VLCFAs, a slight decrease ( $~$ 20%) in brain plasmalogens,and no accumulation of peroxisomal fatty acid ${alpha}$ -oxidation substrates.In cultured cells, PEX11ß deficiency has no effecton plasmalogen synthesis and no effect on phytanic acid andpristanic acid oxidation and causes only a slight decrease ( $~$ 40%)in VLCFA ß-oxidation. Thus, PEX11ß deficiencyrepresents a novel peroxisomal disorder that mimics major neurologicaland developmental pathologic features of Zellweger syndromebut lacks many of its cellular and biochemical features.

It is important to note that PEX11ß-deficient micelack several other features of Zellweger syndrome, such as facialdysmorphism, enlarged cranial fontanelles, and renal cysts (23).However, these pathologic features were also absent from priormouse models of Zellweger syndrome (3, 9). One reasonable explanationfor these differences is that both Zellweger syndrome mice andPEX11ß-deficient mice die very shortly after birth,precluding the development of what may be later-stage pathologicfeatures. The severe kidney and bone development delay we observedin PEX11ß-deficient mice may be an early manifestationof these deficiencies.

Although there is no firm hypothesis about exactly which substrate,product, or combination of substrates and products is the etiologicagent of Zellweger syndrome, patients who are defective in theperoxisomal ß-oxidation enzyme D-bifunctional protein(D-BP) (14, 38) have clinical phenotypes in common with Zellwegersyndrome patients. In contrast, patients with defects in etherlipid synthesis or fatty acid ${alpha}$ -oxidation are associated withdistinct phenotypes (38, 39). These and other observations haveled to the hypothesis that Zellweger syndrome pathologic featuresmight be induced primarily by their severe fatty acid ß-oxidationdefect and perhaps by VLCFA toxicity (28). With regard to VLCFAtoxicity, PEX11ß^-/- mice have only a very mild accumulationof VLCFAs ( $~$ 40%), which is far less severe than the VLCFA accumulationof Zellweger syndrome mice ( $~$ 1,000%). VLCFA accumulation in PEX11ß^-/-mice is also less severe than the 200 to 300% increase in VLCFAsobserved in ALD-deficient mice, which have no detectable pathologicfeatures (20, 26). Thus, it appears that the developmental delay,neuronal migration defects, enhanced neuronal apoptosis, neonatalhypotonia, and neonatal lethality of PEX11ß deficiencycannot be explained by VLCFA toxicity. With regard to the hypothesisthat Zellweger syndrome pathologic features are caused primarilyby defects in peroxisomal ß-oxidation, the phenotypesof PEX11ß^-/- mice are distinct from those of micelacking the peroxisomal ß-oxidation enzyme D-BP (4).In particular, PEX11ß-deficient mice do not displaythe nearly total defect in VLCFA oxidation and branched-chain(pristanic) fatty acid oxidation of D-BP-deficient mice.

The exceedingly mild metabolic defects in PEX11ß^-/-mice raise the interesting possibility that the pathologic featuresof these mice, and perhaps Zellweger syndrome mice and Zellwegersyndrome patients, are not caused by nonspecific metabolitetoxicity. Rather, it may be that the metabolic abnormalitiesof these diseases impair animal development through a subtle,yet inappropriate, activation or inactivation of one or moresignaling pathways in the body. Peroxisomal metabolic pathwaysplay critical roles in the synthesis of many signaling lipids,including ligands of nuclear hormone receptors RXR, PPAR ${alpha}$ , andPPAR ${gamma}$ (15-17). If true, this pathogenic hypothesis raises thepossibility of therapeutic interventions that could normalizethe putative signaling pathways and the development of theseanimals. Future studies of animals should allow us to test thishypothesis directly, as well as its relevance to Zellweger syndromepathogenesis.

Our results also have implications for the disparate topicsof Zellweger syndrome diagnosis and PEX11 function. Currentdiagnostic procedures for Zellweger syndrome and other peroxisomaldisorders (24, 38) would incorrectly exclude a generalized peroxisomaldysfunction in a patient with PEX11ß deficiency, eventhough these patients display defects in two unrelated peroxisomalmetabolic pathways. Therefore, we propose that current diagnosticprotocols be modified to include the possibility of PEX11ßdeficiency in the human population. With regard to the molecularrole of PEX11ß and other PEX11 proteins in peroxisomebiogenesis, there are two competing hypotheses. One predictsthat PEX11 proteins play an important role in peroxisome division(6, 12, 21), and the other holds that PEX11 proteins play adirect role in medium-chain fatty acid oxidation and affectperoxisome abundance only indirectly (35). This issue was addressedin detail in a previous report, which concluded that PEX11 proteinsact directly in peroxisome division (18). This conclusion issupported by the observation here that PEX11ß^-/- miceare defective in two unrelated peroxisomal metabolic pathways,a strong indicator that PEX11ß plays a more directrole in peroxisome biogenesis than peroxisomal metabolism.

Although the loss of PEX11ß is lethal, PEX11ß^-/-mice still contain an intact PEX11 ${alpha}$ gene and do not lack PEX11activity altogether. In fact, the loss of PEX11ß inducesa slight increase ( $~$ 1.5x) in hepatic expression of PEX11 ${alpha}$ (datanot shown). A more complete understanding of PEX11 functionin mammals requires the generation and analysis of mice lackingall PEX11 genes.

ACKNOWLEDGMENTS

We thank the Johns Hopkins University Transgenic Core Facilityfor blastocyst injections and generation of chimeric animals,Ann and Hugo Moser for the use of their laboratory for biochemicalexperiments, and Stephanie Mihalik and Paul Watkins for helpwith the peroxisomal ${alpha}$ - and ß-oxidation activity measurements.

This work was supported by grants from the National Institutesof Health to S.J.G. (DK59479) and D.V. (HD10981). D.V. is aninvestigator of the Howard Hughes Medical Institute. E.B. wassupported by a Max-Kade Scholarship (Max-Kade Foundation, NewYork, N.Y., and Deutsche Forschungsgemeinschaft).

FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Phone: (410) 955-3424. Fax: (410) 955-0215. E-mail: sgould{at}jhmi.edu.

REFERENCES

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