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Peroxisomal Multifunctional Protein-2 Deficiency Causes Motor Deficits and Glial Lesions in the Adult Central Nervous System

2006; Elsevier BV; Volume: 168; Issue: 4 Linguagem: Inglês

10.2353/ajpath.2006.041220

1525-2191

Steven Huyghe, H. Schmalbruch, Leen Hulshagen, Paul Van Veldhoven, Myriam Baes, Dieter Hartmann,

RNA modifications and cancer

In humans, mutations inactivating multifunctional protein-2 (MFP-2), and thus peroxisomal β-oxidation, cause neuronal heterotopia and demyelination, which is clinically reflected by hypotonia, seizures, and death within the first year of life. In contrast, our recently generated MFP-2-deficient mice did not show neurodevelopmental abnormalities but exhibited aberrations in bile acid metabolism and one of three of them died early postnatally. In the postweaning period, all survivors developed progressive motor deficits, including abnormal cramping reflexes of the limbs and loss of mobility, with death at 6 months. Motor impairment was not accompanied by lesions of peripheral nerves or muscles. However, in the central nervous system MFP-2-deficient mice overexpressed catalase in glial cells, accumulated lipids in ependymal cells and in the molecular layer of the cerebellum, exhibited severe astrogliosis and reactive microglia predominantly within the gray matter of the brain and the spinal cord, whereas synaptic and myelin markers were not affected. This culminated in degenerative changes of astroglia cells but not in overt neuronal lesions. Neither the motor deficits nor the brain lesions were aggravated by increasing the branched-chain fatty acid concentration through dietary supplementation. These data indicate that MFP-2 deficiency in mice causes a neurological phenotype in adulthood that is manifested primarily by astroglial damage. In humans, mutations inactivating multifunctional protein-2 (MFP-2), and thus peroxisomal β-oxidation, cause neuronal heterotopia and demyelination, which is clinically reflected by hypotonia, seizures, and death within the first year of life. In contrast, our recently generated MFP-2-deficient mice did not show neurodevelopmental abnormalities but exhibited aberrations in bile acid metabolism and one of three of them died early postnatally. In the postweaning period, all survivors developed progressive motor deficits, including abnormal cramping reflexes of the limbs and loss of mobility, with death at 6 months. Motor impairment was not accompanied by lesions of peripheral nerves or muscles. However, in the central nervous system MFP-2-deficient mice overexpressed catalase in glial cells, accumulated lipids in ependymal cells and in the molecular layer of the cerebellum, exhibited severe astrogliosis and reactive microglia predominantly within the gray matter of the brain and the spinal cord, whereas synaptic and myelin markers were not affected. This culminated in degenerative changes of astroglia cells but not in overt neuronal lesions. Neither the motor deficits nor the brain lesions were aggravated by increasing the branched-chain fatty acid concentration through dietary supplementation. These data indicate that MFP-2 deficiency in mice causes a neurological phenotype in adulthood that is manifested primarily by astroglial damage. During the last decades it has been clearly established that peroxisomes are ubiquitously present in mammalian tissues. These organelles are involved in multiple processes such as the catabolism and synthesis of lipids and sterols and the degradation of hydrogen peroxide. Peroxisomal β-oxidation, in which multifunctional protein-2 (MFP-2) plays a pivotal role, is crucial for the breakdown of very-long-chain fatty acids, branched chain fatty acids, and cholesterol.1Van Veldhoven PP Casteels M Mannaerts GP Baes M Further insights into peroxisomal lipid breakdown via α- and β-oxidation.Biochem Soc Trans. 2001; 29: 292-298Crossref PubMed Google Scholar, 2Wanders RJA Vreken P Ferdinandusse S Jansen GA Waterham HR Van Roermund CWT van Grunsven EG Peroxisomal fatty acid α- and β-oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases.Biochem Soc Trans. 2001; 29: 250-267Crossref PubMed Scopus (0) Google Scholar Synthesis of docosahexaenoic acid (DHA), a major lipid compound of the central nervous system (CNS), and degradation of leukotrienes, important signaling molecules, also need peroxisomal β-oxidation.3Sprecher H Metabolism of highly unsaturated n-3 and n-6 fatty acids.Biochim Biophys Acta. 2000; 1486: 219-231Crossref PubMed Scopus (656) Google Scholar, 4Ferdinandusse S Meissner T Wanders RJA Mayatepek E Identification of the peroxisomal β-oxidation enzymes involved in the degradation of leukotrienes.Biochem Biophys Res Commun. 2002; 293: 269-273Crossref PubMed Scopus (30) Google Scholar Most of the current knowledge on peroxisomal metabolism has been gathered from studying peroxisomal function in liver. In contrast, clinical and pathological observations in patients with various peroxisomal defects point toward an especially important role of peroxisomes in nervous tissue during development and in adulthood.5Powers JM The pathology of peroxisomal disorders with pathogenetic considerations.J Neuropathol Exp Neurol. 1995; 54: 710-719Crossref PubMed Scopus (78) Google Scholar, 6Clayton PT Clinical consequences of defects in peroxisomal β-oxidation.Biochem Soc Trans. 2001; 29: 298-305Crossref PubMed Google Scholar Peroxisomes have been identified in both neurons and glia by immunocytochemical and electron optical methods,7Moreno S Nardacci R Cimini A Ceru MP Immunocytochemical localization of D-amino acid oxidase in rat brain.J Neurocytol. 1999; 28: 169-185Crossref PubMed Scopus (93) Google Scholar, 8Farioli-Vecchioli S Moreno S Ceru MP Immunocytochemical localization of acyl-CoA oxidase in the rat central nervous system.J Neurocytol. 2001; 30: 21-33Crossref PubMed Scopus (37) Google Scholar, 9Itoh M Suzuki Y Takashima S A novel peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein: its expression in the developing human brain.Microsc Res Tech. 1999; 45: 383-388Crossref PubMed Scopus (13) Google Scholar, 10Moreno S Mugnaini E Ceru MP Immunocytochemical localization of catalase in the central nervous system of the rat.J Histochem Cytochem. 1995; 43: 1253-1267Crossref PubMed Scopus (95) Google Scholar, 11Holtzman E Peroxisomes in nervous tissue.Ann NY Acad Sci. 1982; 386: 523-525Crossref Scopus (49) Google Scholar, 12Arnold G Holtzman E Microperoxisomes in the central nervous system of the postnatal rat.Brain Res. 1978; 155: 1-17Crossref PubMed Scopus (81) Google Scholar but surprisingly little is known about their precise role in the different neural cell types. Generalized impairment of peroxisomal function, as encountered in peroxisome biogenesis disorders such as Zellweger syndrome, causes severe developmental abnormalities including a very characteristic neuronal migration defect, facial dysmorphisms, and severe hypotonia. It should be kept in mind that besides the neurodevelopmental abnormalities also postdevelopmental degenerative neuronal lesions including demyelination, photoreceptor degeneration, cerebellar atrophy, and peripheral neuro-pathies are well documented in peroxisome biogenesis disorders and/or peroxisomal disorders with a single protein deficiency such as X-linked adrenoleukodystrophy, α-methylacyl-CoA racemase deficiency, or Refsum disease.13Wanders RJA Barth PG Heymans HSA Single peroxisomal enzyme deficiencies.in: Scriver CR Beaudet AL Valle D Sly WS The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3219-3256Google Scholar, 14Gould SJ Raymond GV Valle D The peroxisome biogenesis disorders.in: Scriver CR Beaudet AL Valle D Sly WS The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3181-3217Google Scholar, 15Wanders RJA Jakobs C Skjeldal OH Refsum disease.in: Scriver CR Beaudet AL Valle D Sly WS The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3303-3322Google Scholar, 16Moser HW Smith KD Watkins PA Powers JM Moser AB X-linked adrenoleukodystrophy.in: Scriver CR Beaudet AL Valle D Sly WS The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3257-3301Google Scholar, 17Ferdinandusse S Denis S Clayton PT Graham A Rees JE Allen JT McLean BN Brown AY Vreken P Waterham HR Wanders RJA Mutations in the gene encoding peroxisomal α-methylacyl-CoA racemase cause adult-onset sensory motor neuropathy.Nat Genet. 2000; 24: 188-191Crossref PubMed Scopus (214) Google Scholar Humans with MFP-2 deficiency strongly resemble Zellweger patients.13Wanders RJA Barth PG Heymans HSA Single peroxisomal enzyme deficiencies.in: Scriver CR Beaudet AL Valle D Sly WS The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3219-3256Google Scholar, 14Gould SJ Raymond GV Valle D The peroxisome biogenesis disorders.in: Scriver CR Beaudet AL Valle D Sly WS The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3181-3217Google Scholar Key constant features are a severe neonatal hypotonia and seizures and absent or at least severely delayed development resulting in death within the first year of life. Neuronal migration defects, giving rise to focal cortical heterotopia and polymicrogyria are documented for ∼80% of the cases. We recently generated mouse models with peroxisome assembly defects (Pex5 knockout)18Baes M Gressens P Baumgart E Carmeliet P Casteels M Fransen M Evrard P Fahimi D Declercq PE Collen D Van Veldhoven PP Mannaerts GP A mouse model for Zellweger syndrome.Nat Genet. 1997; 17: 49-57Crossref PubMed Scopus (223) Google Scholar or with peroxisomal β-oxidation defects (MFP-219Baes M Huyghe S Carmeliet P Declercq PE Collen D Mannaerts GP Van Veldhoven PP Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl branched fatty acids and bile acid intermediates but also of very long chain fatty acids.J Biol Chem. 2000; 275: 16329-16336Crossref PubMed Scopus (165) Google Scholar and MFP-1/MFP-2 knockout20Baes M Gressens P Huyghe S De Nys K Qi C Jia Y Mannaerts GP Evrard P Van Veldhoven PP Declercq PE Reddy JK The neuronal migration defect in mice with Zellweger syndrome (Pex5 knockout) is not caused by the inactivity of peroxisomal β-oxidation.J Neuropathol Exp Neurol. 2002; 61: 368-374Crossref PubMed Scopus (43) Google Scholar) to investigate the pathogenesis of peroxisomal disorders and to further explore the role of peroxisomes in brain. In contrast to the situation in humans, a Zellweger-like pathomorphology was only seen in the model with peroxisome assembly defects (Pex5 knockout)18Baes M Gressens P Baumgart E Carmeliet P Casteels M Fransen M Evrard P Fahimi D Declercq PE Collen D Van Veldhoven PP Mannaerts GP A mouse model for Zellweger syndrome.Nat Genet. 1997; 17: 49-57Crossref PubMed Scopus (223) Google Scholar but not in mice with peroxisomal β-oxidation defects. The phenotype of MFP-2 knockout mice significantly diverged from the clinical presentation and pathology in MFP-2-deficient patients13Wanders RJA Barth PG Heymans HSA Single peroxisomal enzyme deficiencies.in: Scriver CR Beaudet AL Valle D Sly WS The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3219-3256Google Scholar, 14Gould SJ Raymond GV Valle D The peroxisome biogenesis disorders.in: Scriver CR Beaudet AL Valle D Sly WS The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3181-3217Google Scholar, 18Baes M Gressens P Baumgart E Carmeliet P Casteels M Fransen M Evrard P Fahimi D Declercq PE Collen D Van Veldhoven PP Mannaerts GP A mouse model for Zellweger syndrome.Nat Genet. 1997; 17: 49-57Crossref PubMed Scopus (223) Google Scholar, 19Baes M Huyghe S Carmeliet P Declercq PE Collen D Mannaerts GP Van Veldhoven PP Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl branched fatty acids and bile acid intermediates but also of very long chain fatty acids.J Biol Chem. 2000; 275: 16329-16336Crossref PubMed Scopus (165) Google Scholar; ie, the MFP-2 knockout mice were not hypotonic at birth and did not display a neuronal migration defect.19Baes M Huyghe S Carmeliet P Declercq PE Collen D Mannaerts GP Van Veldhoven PP Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl branched fatty acids and bile acid intermediates but also of very long chain fatty acids.J Biol Chem. 2000; 275: 16329-16336Crossref PubMed Scopus (165) Google Scholar However, from day 2 on they were severely growth retarded, which correlated with major abnormalities in bile acid synthesis,21Dirkx R Vanhorebeek I Martens K Schad A Grabenbauer M Fahimi D Declercq P Van Veldhoven PP Baes M Absence of peroxisomes in hepatocytes causes mitochondrial and ER abnormalities.Hepatology. 2005; 41: 868-878Crossref PubMed Scopus (138) Google Scholar resulting in a generalized failure to thrive and the premature death of approximately one-third of the knockouts within the first 3 weeks. After weaning, the surviving MFP-2 knockout mice resumed normal weight gain but remained smaller than wild-type (WT) littermates.19Baes M Huyghe S Carmeliet P Declercq PE Collen D Mannaerts GP Van Veldhoven PP Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl branched fatty acids and bile acid intermediates but also of very long chain fatty acids.J Biol Chem. 2000; 275: 16329-16336Crossref PubMed Scopus (165) Google Scholar Metabolic alterations observed in MFP-2 knockout mice, including accumulations of very-long-chain fatty acids and phytanic and pristanic acids, were similar to the changes seen in patients and correspond to a severe defect in peroxisomal β-oxidation. The present study focuses on a progressive neuromotor phenotype that develops in all MFP-2-deficient mice surviving into adulthood and which is lethal at the age of 6 months, ie, at a time schedule quite distinct from that of the initial early postnatal metabolic problems. No abnormalities in the neuromuscular system were found, but we observed lipid accumulations in ependyma and Bergmann glia and a generalized astrogliosis and microglia activation throughout the gray matter, which in sharp contrast to human peroxisomal diseases spared myelinated fiber tracts. Even more surprising, this was not associated with overt neuronal damage, but resulted in the stainability by a degeneration marker, Fluoro-Jade, of astroglial cells, pointing toward an unexpected novel target of MFP-2 deficiency in the murine brain. Homozygous MFP-2-deficient mice were obtained in the offspring of heterozygous MFP-2+/− breeding pairs, which were in a mixed Swiss [Tac:(Sw)fBR]/sv129 background. Mice were bred in the animal housing facility of the University of Leuven under conventional conditions. They had unlimited access to standard rodent food chow (Muracon-G; Carfil Quality-Pavan Services, Oud-Turnhout, Belgium) and water and were kept on a 12-hour light/dark cycle. All animal experiments were approved by the Institutional Animal Ethical Committee of the University of Leuven. A rotarod (Ugo Basile Biological Research Apparatus, Varese, Italy) with a constant speed of 17 rpm was used. Two days before the test, mice were trained for two periods of 3 minutes. The test consisted of two trials of 3 minutes with a 1-hour interval. The latency time for the mouse to drop from the rod was recorded. For light and electron microscopical analysis of plastic-embedded sections, mice were deeply anesthetized with Hypnorm (fentanyl/fluanizone) and midazolam (Janssen Pharmaceutica, Beerse, Belgium). The transcardial perfusion with Ringer's solution containing 0.1% procain and heparin 5 units/ml tissues (2 minutes) was followed by an in situ fixation with buffered 2.5% glutaraldehyde and 20 mg/ml sodium cacodylate (10 minutes). Specimens were postfixed for another 24 hours with glutaraldehyde, osmicated, and embedded in Embed 812 (Electron Microsocopy Sciences, Fort Washington, PA). Sections 3 μm thick were stained with p-phenylenediamine and studied by light microscopy. Thin sections for electron microscopy were stained with lead citrate and uranyl acetate and investigated in a Philips CM 100 microscope (Eindhoren, The Netherlands). Three or four mice of each genotype, derived from different litters were used for this analysis. The procedure for immunohistochemical analysis of paraffin material was described in detail.22Hartmann D De Strooper B Saftig P Presenilin-1 deficiency leads to loss of Cajal-Retzius neurons and cortical dysplasia similar to human type 2 lissencephaly.Curr Biol. 1999; 15: 719-727Abstract Full Text Full Text PDF Scopus (131) Google Scholar Briefly, Nembutal-anesthetized mice were perfused with modified Bouin's solution. Brain and spinal cord were postfixed overnight and embedded in paraffin following routine procedures. Three to four different mice of each genotype aged 3 weeks, 5 weeks, 8 weeks, 3 months, and 5 months were used. Sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol and rinsed in 0.1 mol/L phosphate-buffered saline. After antigen retrieval by limited proteolysis or heating in citrate buffer, and blocking of endogenous peroxidase, sections were subsequently incubated for 1 hour with the primary antibody diluted in blocking buffer with normal goat serum (2%), and after washing for 1 hour, with a secondary antibody in the same buffer. All incubations were done in parallel and photograph exposures were equal for control and MFP-2 knockout mouse sections. The following primary antibodies were used: polyclonal rabbit antibodies against bovine glial fibrillary acidic protein (GFAP) (DAKO, Heverlee, Belgium) and bovine catalase (Rockland, Gilbertsville, PA), monoclonal rat antibodies against F4/80 (American Type Culture Collection, LGC Promochem, Teddington, UK) and against rat lysosome-associated membrane protein-1 (LAMP-1) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), monoclonal mouse antibodies against bovine Calbindin-D-28K (Sigma-Aldrich, Bornem, Belgium), bovine myelin basic protein (MBP) (Sigma-Aldrich), and porcine GFAP (Sigma-Aldrich). The secondary antibodies (goat anti-rabbit IgGs and goat anti-mouse IgGs, Sigma-Aldrich) were conjugated with peroxidase, and tyramide signal amplification kits (PerkinElmer Life Sciences, Inc., Boston, MA), using tyramide-conjugated fluorochromes, were used for antibody detection. For double immunostainings one of the secondary antibodies was labeled with Alexa-568 or Alexa-546 (Molecular Probes Europe BV, Leiden, The Netherlands). The biotin-tagged lectin Ricinus communis agglutinin-1 (RCA-1) (Vector Laboratories, Burlingame, CA) was used as a marker for activated microglia, the Vectastain ABC kit (Vector Laboratories) was used for visualization. Animals were perfusion fixed with 10% neutral buffered formalin (Prosan, Germany) and embedded in paraffin as described above. Dewaxed sections were stained with Fluoro-Jade B (Chemicon International) to screen for degenerating neurons and glia. Parallel sections were incubated with antibodies against cleaved (activated) caspase 3 to detect apoptotic cells. It should be noted that although the Fluoro-Jade assay was originally developed to identify early (precell death) stages of neuronal degeneration, it has more recently been shown that it also identifies degenerating glial cells.23Ye X Carp RI Schmued LC Scallet AC Fluoro-Jade and silver methods: application to the neuropathology of scrapie, a transmissible spongiform encephalopathy.Brain Res Brain Res Protoc. 2001; 8: 104-112Crossref PubMed Scopus (42) Google Scholar Free-floating frontal and sagittal vibratome sections of the brain were stained with the lipid-soluble dye Oil Red O (C.I. no. 26125; BDH Laboratory Supplies, UK) 0.24% w/v in isopropanol/water (3:2) for 18 minutes and counterstained with methyl green (Vector Laboratories). A parallel series of sections was additionally double-stained by antibodies against GFAP as described above. Three to four different mice of each genotype aged 4 weeks, 8 weeks, 3 months, and 5 months were analyzed. Brain homogenates were analyzed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels as previously described,21Dirkx R Vanhorebeek I Martens K Schad A Grabenbauer M Fahimi D Declercq P Van Veldhoven PP Baes M Absence of peroxisomes in hepatocytes causes mitochondrial and ER abnormalities.Hepatology. 2005; 41: 868-878Crossref PubMed Scopus (138) Google Scholar using the same antibodies as those used for immunocytochemical staining. The mice were anesthetized with Hypnorm (fentanyl/fluanizone) and midazolam and the sciatic nerve was crushed for 2 minutes with a microsurgical needle holder with smooth branches. The wound was closed with one suture. Nerve regeneration was monitored by testing the toe-spreading reflex. The mice were kept in superficial anesthesia [Hypnorm (fentanyl/fluanizone) and midazolam]. The sensory conduction velocity was measured along the tail nerve with two proximal stimulating and two distal recording electrodes. A ground electrode was placed between these two sets. The gastrocnemius muscle was stimulated with the cathode at the sciatic notch and the anode in the skin of the lateral abdomen. Stimulus strength was supramaximal. The different recording electrode was placed inside the gastrocnemius muscle and the indifferent one in the skin of the hind paw. All solvents were of the highest quality commercially available (Biosolve, Valkenswaard, The Netherlands). Butylated hydroxytoluene (0.05%, w/v) was added at all stages of the extraction to minimize auto-oxidation of polyunsaturated fatty acids (PUFAs). Lipids were extracted from tissues, homogenized in 3.8 ml of CH3OH/CHCl3/H2O (2:1:0.8), using a Polytron tissue homogenizer,24Van Veldhoven P Bell RM Effect of harvesting methods, growth conditions and growth phase on diacylglycerol levels in cultured human adherent cells.Biochim Biophys Acta. 1988; 959: 185-196Crossref PubMed Scopus (137) Google Scholar and separated into neutral lipids, fatty acids, and phospholipids by solid phase extraction (Bond Elut NH2 column, 500 mg; Varian Benelux, Sint-Katelijne-Waver, Belgium).20Baes M Gressens P Huyghe S De Nys K Qi C Jia Y Mannaerts GP Evrard P Van Veldhoven PP Declercq PE Reddy JK The neuronal migration defect in mice with Zellweger syndrome (Pex5 knockout) is not caused by the inactivity of peroxisomal β-oxidation.J Neuropathol Exp Neurol. 2002; 61: 368-374Crossref PubMed Scopus (43) Google Scholar, 25Kaluzny MA Duncan LA Merritt MV Epps DE Rapid separation of lipid classes in high yield and purity using bonded phase columns.J Lipid Res. 1985; 26: 135-140Abstract Full Text PDF PubMed Google Scholar Cholesterol,26Van Veldhoven PP Meyhi E Mannaerts GP Enzymatic quantitation of cholesterol esters in lipid extracts.Anal Biochem. 1998; 258: 152-155Crossref PubMed Scopus (27) Google Scholar cholesteryl esters,26Van Veldhoven PP Meyhi E Mannaerts GP Enzymatic quantitation of cholesterol esters in lipid extracts.Anal Biochem. 1998; 258: 152-155Crossref PubMed Scopus (27) Google Scholar neutral glycerolipids,27Van Veldhoven PP Swinnen JV Esquenet M Verhoeven G Lipase-based quantitation of triacylglycerols in cellular lipid extracts: requirement for presence of detergent and prior separation by thin-layer chromatography.Lipids. 1997; 32: 1297-1300Crossref PubMed Scopus (35) Google Scholar and phosphorus content of the phospholipid fraction28Van Veldhoven P Mannaerts G Inorganic and organic phosphate measurement in the nanomolar range.Anal Biochem. 1987; 161: 45-48Crossref PubMed Scopus (629) Google Scholar were determined as previously described. The content of DHA in the phospholipid fraction was determined by GC analysis as previously described.29Janssen A Baes M Gressens P Mannaerts GP Declercq P Van Veldhoven PP Docosahexaenoic acid deficit is not a major pathogenic factor in peroxisome-deficient mice.Lab Invest. 2000; 80: 31-35Crossref PubMed Scopus (49) Google Scholar Phytanic, pristanic acid, and C26:0 were quantified by GC-MS analysis in phospholipids and neutral lipids.20Baes M Gressens P Huyghe S De Nys K Qi C Jia Y Mannaerts GP Evrard P Van Veldhoven PP Declercq PE Reddy JK The neuronal migration defect in mice with Zellweger syndrome (Pex5 knockout) is not caused by the inactivity of peroxisomal β-oxidation.J Neuropathol Exp Neurol. 2002; 61: 368-374Crossref PubMed Scopus (43) Google Scholar MFP-2-deficient mice developed a dyskinesia of the limbs, the first signs of which were visible at the age of 3 weeks. On lifting mice by their tail they either overstretched and cramped the hind legs or contracted them to the trunk often together with the front legs whereas WT mice spread their limbs and struggled (Figure 1, A and B). These abnormalities became much more prominent after 3 months. The walking pattern of MFP-2 knockout mice was characterized by an unsteady gait. Rotarod testing revealed that motor coordination and equilibrium were already affected at the age of 8 weeks (Figure 1C). Instead of walking on the rotating rod, MFP-2-deficient mice often gripped the rod and made passive rotations before dropping off during the 3-minute test period. During the fifth to sixth month the mobility of the MFP-2 knockout mice was severely reduced. Because they had difficulties in standing up and reaching the chow, food pellets were placed into the cage. Nevertheless, they progressively lost weight down to only 35 to 40% of the body weight of their WT or heterozygous littermates. The mice died at the age of 5 to 6 months after dramatic wasting, so that in the final stage of their disease they lay immobile on their side. At the time of death, the major organs appeared macroscopically normal, except for a severe shrinkage of the testicles (Huyghe et al, unpublished observations) and the absence of white adipose tissue. The morphology of the CNS of MFP-2 knockout mice in preterminal stage (5 months old, as compared to a life-span of 5 to 6 months) appeared normal on hematoxylin and eosin-stained sections (data not shown). However, Oil Red O-positive lipid droplets were seen in specific regions of the CNS whereas no lipid droplets were found in any region of the CNS of the WT or heterozygous littermates. Lipid storage was most impressive within ependymal cells along the entire ventricular system and within selected regions of the gray matter. The ependymal lining of the four ventricles and the spinal central canal (Figure 2, A–D) showed very prominent fatty inclusions that unambiguously distinguished sections from knockout and WT mice. It was thereby puzzling to observe that in the lateral ventricles the size of the inclusions consistently correlated to the position within the medial, dorsal, or lateral walls, respectively, pointing toward functional differences in lipid turnover between ependymal cells in these different locations (Figure 2A, insets). These inclusions were globular or near globular, membrane-bound, and heavily osmiophilic except oblong osmiophobic inclusions suggestive of remnants of ciliae (Figure 2D). However, scanning electron microscopical analysis of the ependymal cells of the lateral ventricle revealed no abnormal ciliae (data not shown). No lipid inclusions were found in the choroid plexus epithelium, despite its close developmental and topographic relationship to ependymal cells. Strikingly, development of lipid inclusions abruptly ceases at the juncture between ependyma and plexus epithelium (Figure 2B, d and e, arrows in insets). Next to the ventricular system, lipid inclusions were especially prominent in the molecular layer of the cerebellar cortex, where they were predominantly present in radial Bergmann glial fibers (Figure 2, E–H). Besides their highly characteristic distribution pattern, this was confirmed by co-staining with Oil Red O and anti-GFAP (Figure 2H). Smaller inclusions, albeit rarely, occurred in Purkinje cell dendrites (data not shown). Lipid inclusions in both ependyma and cerebellar molecular layer develop between the fourth and eighth postnatal week, ie, clearly after the period during which the first cohort of MFP-2-deficient mice dies (shown for Bergmann glia in Figure 2, I–K) and further increase thereafter (Figure 2, compare K with F). Within Bergmann glia, accumulation of lipid droplets follows a striking gradient, affecting cell body and (to a low degree) distal fiber segments first, while the pearl chain pattern of droplet-filled fibers becomes fully visible only from ∼8 weeks onward. Lipid accumulations were only rarely found in the cerebral cortex in fibrous astrocytes close to blood vessels (data not shown). Furthermore, lipid droplets were found in the hypothalamus and in the inner segment of the pallidum, where they were always present in the gray matter and no accumulating lipids were seen in the white matter. To clarify the biochemical nature of the lipid droplets, lipid analysis of the cerebellum, the brain region with the highest amount of lipid droplets, was performed. Cholesterylester levels were found to be fivefold increased but levels of triglycerides and phospholipids were unaltered (Table 1). Analysis of the fatty acid composition revealed that the C26:0 levels were at least 10-fold increased in the phospholipid fraction but not in the neutral lipids (including cholesterylesters) (Table 1). Surprisingly, the concentration of DHA, a PUFA that depends on peroxisomal β-oxidation for its synthesis, was the same in phospholipids of MFP-2 knockout and control mice. Because this was an unexpected finding, food pellets were analyzed for the presence of DHA. This PUFA could indeed be detected (0.09 μmol/g pelleted chow), but was substantially lower than the main fatty acids C18:2, C16:0, C18:0, C18:1, and C18:3, respectively 28.80, 9.84, 4.98, 3.47, and 3.04 μmol/g chow (mean of