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Ataxia with Loss of Purkinje Cells in a Mouse Model for Refsum Disease

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Ataxia with Loss of Purkinje Cells in a Mouse Model for Refsum Disease Ataxia with loss of Purkinje cells in a mouse model for Refsum disease Sacha Ferdinandussea,1,2, Anna W. M. Zomerb,1, Jasper C. Komena, Christina E. van den Brinkb, Melissa Thanosa, Frank P. T. Hamersc, Ronald J. A. Wandersa,d, Paul T. van der Saagb, Bwee Tien Poll-Thed, and Pedro Britesa Academic Medical Center, Departments of aClinical Chemistry (Laboratory of Genetic Metabolic Diseases) and dPediatrics, Emma’s Children Hospital, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; bHubrecht Institute, Royal Netherlands Academy of Arts and Sciences 3584 CT Utrecht, The Netherlands; and cRehabilitation Hospital ‘‘De Hoogstraat’’ Rudolf Magnus Institute of Neuroscience, 3584 CG Utrecht, The Netherlands Edited by P. Borst, The Netherlands Cancer Institute, Amsterdam, The Netherlands, and approved October 3, 2008 (received for review June 23, 2008) Refsum disease is caused by a deficiency of phytanoyl-CoA hy- Clinically, Refsum disease is characterized by cerebellar droxylase (PHYH), the first enzyme of the peroxisomal ␣-oxidation ataxia, polyneuropathy, and progressive retinitis pigmentosa, system, resulting in the accumulation of the branched-chain fatty culminating in blindness, (1, 3). The age of onset of the symptoms acid phytanic acid. The main clinical symptoms are polyneuropathy, can vary from early childhood to the third or fourth decade of cerebellar ataxia, and retinitis pigmentosa. To study the patho- life. No treatment is available for patients with Refsum disease, genesis of Refsum disease, we generated and characterized a Phyh but they benefit from a low phytanic acid diet. Phytanic acid is knockout mouse. We studied the pathological effects of phytanic derived from dietary sources only, specifically from the chloro- acid accumulation in Phyh؊/؊ mice fed a diet supplemented with phyll component phytol. When phytanic acid levels are kept low phytol, the precursor of phytanic acid. Phytanic acid accumulation via dietary reduction, a halt in the progression of the symptoms caused a reduction in body weight, hepatic steatosis, and testicular has been reported (4–7). atrophy with loss of spermatogonia. Phenotype assessment using Although the clinical symptoms of Refsum disease are well the SHIRPA protocol and subsequent automated gait analysis using known, only limited postmortem data are available on Refsum the CatWalk system revealed unsteady gait with strongly reduced patients and the pathogenesis of the disorder is poorly under- paw print area for both fore- and hindpaws and reduced base of stood. Insight herein is of crucial importance for the develop- support for the hindpaws. Histochemical analyses in the CNS ment of potential therapeutic options. Phytanic acid has been showed astrocytosis and up-regulation of calcium-binding pro- claimed to be toxic, and it has been shown to be an activating teins. In addition, a loss of Purkinje cells in the cerebellum was ligand of the nuclear receptor peroxisome proliferator receptor observed. No demyelination was present in the CNS. Motor nerve ␣ (PPAR␣). To study the pathological consequences of phytanic conduction velocity measurements revealed a peripheral neurop- acid accumulation, we generated of a mouse model for Refsum athy. Our results show that, in the mouse, high phytanic acid levels disease by targeted disruption of the Phyh gene. cause a peripheral neuropathy and ataxia with loss of Purkinje Results cells. These findings provide important insights in the pathophys- iology of Refsum disease. Disruption of the Phyh Gene. To disrupt the mouse Phyh gene, part of the gene containing exons 4–7 was replaced with the hygro- fatty acid oxidation ͉ metabolic disorder ͉ peroxisomes mycin B-resistance gene by homologous recombination in ES cells as depicted in Fig. 1A. Correct targeting of Phyh was confirmed by Southern blot analysis and PCR on genomic DNA efsum disease is an autosomal recessive lipid-storage disor- (Fig. 1 B and C). The disruption of the Phyh gene resulted in the der (MIM266500) characterized by the accumulation of the R absence of detectable mRNA (Fig. 1D), PHYH protein, and a branched-chain fatty acid phytanic acid (3,7,11,15-tetramethyl- complete loss of PHYH enzyme activity (data not shown). The hexadecanoic acid) in tissues and body fluids of patients (1). absence of the Phyh gene did not result in embryonic lethality Phytanic acid levels are elevated because of a deficiency of the nor did it affect postnatal viability. When kept under standard enzyme phytanoyl-CoA hydroxylase (PHYH). PHYH catalyzes Ϫ/Ϫ ␣ laboratory conditions, Phyh mice developed normally and the first step of the peroxisomal -oxidation pathway, i.e., the had no obvious developmental abnormalities. They were fertile, conversion of phytanoyl-CoA to 2-hydroxy-phytanoyl-CoA. This and interbreeding of PhyhϪ/Ϫ males and females led to viable ␣ -oxidation is required for the degradation of phytanic acid, progeny. which contains a methyl group at the third carbon atom of its ␤ -backbone and therefore cannot be degraded via -oxidation. As Biochemical Abnormalities in Phyh؊/؊ Mice Fed a Phytol-Supple ␣ a result of -oxidation, the terminal carboxyl-group of phytanic mented Diet. In contrast to the human diet, standard rodent chow acid is released as CO2, producing pristanic acid, a 2-methyl contains only low amounts of branched-chain fatty acids and the branched-chain fatty acid (2,6,10,14-tetramethylpentadecanoic acid), which can be degraded via ␤-oxidation in the peroxisome. The molecular basis of Refsum disease is heterogeneous, but Author contributions: S.F., A.W.M.Z., R.J.A.W., P.T.v.d.S., B.T.P.-T., and P.B. designed re- the majority of patients have mutations in the gene encoding search; S.F., A.W.M.Z., J.C.K., C.E.v.d.B., M.T., and P.B. performed research; F.P.T.H. con- Ϸ tributed new reagents/analytic tools; S.F., A.W.M.Z., J.C.K., and P.B. analyzed data; and S.F., PHYH (2). In humans, the PHYH gene is 21 kb and consists R.J.A.W., P.T.v.d.S., B.T.P.-T., and P.B. wrote the paper. of 9 exons and 8 introns. It encodes a protein with a calculated The authors declare no conflict of interest. mass of 38.6 kDa, including a cleavable peroxisomal targeting This article is a PNAS Direct Submission. signal type 2 (PTS2). This PTS2-signal is recognized by peroxin 1S.F. and A.W.M.Z. contributed equally to this work. 7, which is a receptor required for the import of newly synthe- 2To whom correspondence should be addressed at: Laboratory Genetic Metabolic Diseases, sized PTS2-containing proteins into peroxisomes. Peroxin 7 is F0–226, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. encoded by PEX7, which is the second gene that can be mutated E-mail: [email protected]. in patients with Refsum disease (2). The impaired import of This article contains supporting information online at www.pnas.org/cgi/content/full/ PHYH into the peroxisome leads to a deficient PHYH activity 0806066105/DCSupplemental. in patients with mutations in the PEX7 gene. © 2008 by The National Academy of Sciences of the USA 17712–17717 ͉ PNAS ͉ November 18, 2008 ͉ vol. 105 ͉ no. 46 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806066105 Downloaded by guest on September 29, 2021 A Control 0.1% phytol 0.25% phytol EcoRV HindIII EcoRI BamHIClaI HindIII ABC wild type allele 1 23456 789 1 kb XX Phyh+/+ targeting vector HYG DEF EcoRV HindIIIEcoRI HindIII targeted allele HYG 1 2 3 89 5’ probe Phyh-/- BC D +/+ +/- +/+ +/- -/- kb Ϫ/Ϫ +/+ +/- -/- Phyh Fig. 3. Phyh mice on a phytol diet develop hepatic lipidosis. H&E staining 12.3 bp of livers from WT (A–C) and PhyhϪ/Ϫ mice (D–F) on a control diet (A and D), on 260 a 0.1% phytol diet (B and E), and on 0.25% phytol diet (C and F). In PhyhϪ/Ϫ 201 mice on a 0.25% phytol diet (F), steatosis is clearly detected by the large lipid 10.0 β-actin vacuoles present throughout the liver parenchyma. On a 0.1% phytol diet (E), PhyhϪ/Ϫ mice showed signs of microsteatosis (small lipid vacuoles within hepatocytes). (Scale bar: 100 ␮m.) Fig. 1. Generation of targeting vector and PhyhϪ/Ϫ mice. (A) Strategy of gene targeting. The WT allele, targeting vector and the targeted allele are depicted. After homologous recombination, exons 4–7 and parts of introns 3 probably caused by lipoatrophy, because upon dissection, almost and 7 are deleted. The location of the 5Ј probe used for Southern blot analysis no white fat could be observed in the PhyhϪ/Ϫ mice after the is shown. (B) Southern blot analysis of genomic DNA isolated from ES cells, 0.25% phytol diet. Ј digested with EcoRV and BamHI, and probed with the 5 probe yielded a Because of the phytol diets, phytanic acid accumulated in 12.3-kb fragment for the WT allele (ϩ/ϩ) and a 10-kb fragment for the targeted allele (ϩ/Ϫ). (C) Genotyping by PCR on genomic DNA. Amplification plasma and tissues (Fig. 2). After 3 weeks on the 0.25% phytol from the WT allele (ϩ/ϩ) resulted in a 201-bp fragment and amplification from diet, plasma phytanic acid levels Ͼ1 mmol/L were reached. Such the targeted allele (Ϫ/Ϫ) a fragment of 260 bp. (D)(Upper) PCR analysis of liver levels have also been reported in Refsum disease patients (8). Of cDNA from WT and PhyhϪ/Ϫ mice. No amplification of exons 1–8 could be the different tissues analyzed, liver showed the highest accumu- Ϫ Ϫ detected in Phyh / mice. (Lower) A control for cDNA amplification was lation of phytanic acid, followed by kidney, testis, and cerebel- ␤ carried out with primers for -actin.
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