Defining the peroxisome

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0 2 4 6 8 0 2 4 6 8 20 Defining the peroxisome PEROXISOMAL DISEASES Ronald JA Wanders Laboratory for Genetic Metabolic Disorders Department of Pediatrics & Clinical Chemistry Academic Medical Center

1 Defining the peroxisome PEROXISOMAL DISEASES Ronald JA Wanders Laboratory for Genetic Metabolic Disorders Department of Pediatrics & Clinical Chemistry Academic Medical Center University Hospital Amsterdam, enzyme activity (% of total) peroxisomes mitochondria katalase glutdh PGI B-Hex esterase 954 Ultrastructural identification of peroxisomes as small cellular bodies ( microbodies ) by Rhodin 960s Biochemical characterisation of peroxisomes by DeDuve and co-workers 976 Identification of a complete fatty acid ß-oxidation system in rat liver peroxisomes CEREBRO-HEPATO-RENAL SYNDROME OF ZELLWEGER = ZELLWEGER SYNDROME (ZS) KEY FINDINGS INDICATING THAT ZELLWEGER SYNDROME IS TRUELY A DISEASE OF THE PEROXISOME.. ACCUMULATION OF HEXACOSANOIC ACID (C26:0) IN PLASMA FROM ZELLWEGER PATIENTS Brown etal (982) Johns Hopkins Med. J. 5, 44-5 First described in 964 More than 200 patients described in literature Zellweger patients show a great number of abnormalities including craniofacial, neurological, ocular, hepatological and skeletal aberrations Patients usually die early in life within the first year Peroxisomes are absent in all body cells of Zellweger patients as first shown by Goldfischer etal. (97) Science 82, in hepatocytes and kidney tubule cells. C26:0 (µmol/l) in plasma CONTROL ZELLWEGER

2 KEY FINDINGS INDICATING THAT ZELLWEGER SYNDROME IS TRUELY A DISEASE OF THE PEROXISOME. 2.PLASMALOGEN DEFICIENCY IN ZELLWEGER SYNDROME Heijmans etal (98) Nature 06, ABNORMAL METABOLITES IN ZELLWEGER SYNDROME. Accumulation of very-long-chain fatty acids Fatty acid ß-oxidation in peroxisomes E + ppe) Percentage ppe of (P Zellweger syndrome 2. Deficiency of plasmalogens Ether-phospholipid biosynthesis 0 brain kidney liver muscle heart Zellweger Controls ABNORMAL METABOLITES IN ZELLWEGER SYNDROME ABNORMAL METABOLITES IN ZELLWEGER SYNDROME. Accumulation of very-long-chain fatty acids Fatty acid ß-oxidation in peroxisomes. Accumulation of very-long-chain fatty acids Fatty acid ß-oxidation in peroxisomes 2. Deficiency of plasmalogens Ether-phospholipid biosynthesis Zellweger syndrome 2. Deficiency of plasmalogens Ether-phospholipid biosynthesis Zellweger syndrome. Accumulation phytanic acid Fatty acid alpha-oxidation. Accumulation phytanic acid Fatty acid alpha-oxidation 4. Increased urinary excretion of oxalate and glycolate Glyoxylate detoxification 5. Increased L-pipecolic acid L-Pipecolic acid oxidation

3 METABOLIC FUNCTIONS OF PEROXISOMES IN HUMANS Fatty acid alpha-oxidation Fatty acid ß-oxidation Etherphospholipid biosynthesis Fatty acid ß-oxidation Etherphospholipid biosynthesis Metabolic abnormalities in ZS VLCFA Bile acid intermediates Pristanic acid * Erythrocyte plasmalogens METABOLIC FUNCTIONS OF PEROXISOMES IN HUMANS METABOLIC FUNCTIONS OF PEROXISOMES IN HUMANS Fatty acid alpha-oxidation Phytanic acid * L-Pipecolic acid oxidation Glyoxylate detoxification Glyoxylate detoxification Urinary oxalate and glycolate # Other functions L-pipecolic acid oxidation L-Pipecolic acid # Moderately elevated, AGT normal * Maybe normal, dependant on diet METABOLIC FUNCTIONS OF PEROXISOMES IN HUMANS Fatty acid ß-oxidation Etherphospholipid biosynthesis Fatty acid alpha-oxidation Glyoxylate detoxification L-pipecolic acid oxidation Metabolic abnormalities in ZS VLCFA Bile acid intermediates Pristanic acid * Erythrocyte plasmalogens Phytanic acid * UNIQUE SET OF PEROXISOMAL BIOMARKERS Urinary oxalate and glycolate # L-Pipecolic acid LIST OF PEROXISOMAL DISORDERS PEROXISOME BIOGENESIS DISORDERS - Zellweger syndrome (ZS) - Neonatal adrenoleukodystrophy (NALD) - Infantile Refsum disease (IRD) - Rhizomelic chondrodysplasia punctata (RCDP) SINGLE PEROXISOMAL ENZYME DEFICIENCIES - X-linked adrenoleukodystrophy - D-bifunctional protein deficiency - Acyl-CoA oxidase deficiency - 2-Methylacyl-CoA racemase deficiency - Sterol carrier protein x deficiency - DHAPAT-deficiency (RCDP Type 2) - Alkyl DHAP-synthase deficiency (RCDP Type ) - Refsum disease - Hyperoxaluria Type - Acatalasaemia # Moderately elevated, AGT normal * Maybe normal, dependant on diet

4 NEWLY IDENTIFIED PEROXISOMAL DISORDERS PEROXISOMAL DYNAMICS DISORDERS OF PEROXISOMAL DYNAMICS (FISSION, ELONGATION ETC) - DLP deficiency (Waterham etal 2007 New Engl.J.Med. 56, PEXß deficiency (Ebberink etal 202 J Med Genet. 202 May;49(5):07- ) ELONGATION & CONSTRICTION FISSION PEXB FIS DLP MITOCHONDRIAL DYNAMICS FIS DLP MFN MFN2 OPA LIST OF PEROXISOMAL DISORDERS PEROXISOME BIOGENESIS DISORDERS - Zellweger syndrome (ZS) - Neonatal adrenoleukodystrophy (NALD) - Infantile Refsum disease (IRD) - Rhizomelic chondrodysplasia punctata (RCDP) SINGLE PEROXISOMAL ENZYME DEFICIENCIES - X-linked adrenoleukodystrophy - D-bifunctional protein deficiency - Acyl-CoA oxidase deficiency - 2-Methylacyl-CoA racemase deficiency -Sterol carrier protein x deficiency - DHAPAT-deficiency (RCDP Type 2) -Alkyl DHAP-synthase deficiency (RCDP Type ) Zellweger spectrum disorders Pathway affected Beta-oxidation Plasmalogen biosynthesis Maintenance of a functional mitochondrial tubular network is the balanced result of the opposing actions of proteins promoting mitochondrial fusion and those promoting mitochondrial fission - Refsum disease -Hyperoxaluria Type - Acatalasaemia Alpha-oxidation Glyoxylate metabolism H 2 O 2 metabolism

LIST OF PEROXISOMAL DISORDERS PEROXISOME BIOGENESIS DISORDERS - Zellweger syndrome (ZS) - Neonatal adrenoleukodystrophy (NALD) - Infantile Refsum disease (IRD) - Rhizomelic chondrodysplasia punctata

protein x deficiency Zellweger spectrum disorders Pathway affected Beta-oxidation ZELLWEGER SPECTRUM DISORDERS : ZELLWEGER SYNDROME, NEONATAL ADRENOLEUKODYSTROPHY AND INFANTILE REFSUM DISEASE :

5 LIST OF PEROXISOMAL DISORDERS PEROXISOME BIOGENESIS DISORDERS - Zellweger syndrome (ZS) - Neonatal adrenoleukodystrophy (NALD) - Infantile Refsum disease (IRD) - Rhizomelic chondrodysplasia punctata (RCDP) SINGLE PEROXISOMAL ENZYME DEFICIENCIES - X-linked adrenoleukodystrophy - D-bifunctional protein deficiency - Acyl-CoA oxidase deficiency - 2-Methylacyl-CoA racemase deficiency -Sterol carrier protein x deficiency Zellweger spectrum disorders Pathway affected Beta-oxidation ZELLWEGER SPECTRUM DISORDERS : ZELLWEGER SYNDROME, NEONATAL ADRENOLEUKODYSTROPHY AND INFANTILE REFSUM DISEASE : PHENOTYPIC VARIANTS WITH OVERLAPPING CLINICAL SIGNS AND SYMPTOMS RANGING IN SEVERITY FROM ZS TO NALD AND IRD - DHAPAT-deficiency (RCDP Type 2) -Alkyl DHAP-synthase deficiency (RCDP Type ) - Refsum disease -Hyperoxaluria Type - Acatalasaemia Plasmalogen biosynthesis Alpha-oxidation Glyoxylate metabolism H 2 O 2 metabolism Common features include neurodevelopmental delay, retinopathy, perceptive deafness, and variable liver disease ZSDs IDENTIFICATION OF HUMAN PEROXISOME BIOGENESIS GENES BY SEQUENCE SIMILARITY CG Gifu Baltimore A dam Gene involved. A 8 IX PEX26/5 2. B 7/5 XI PEX0. C 4/6 III PEX6 4. D 9 X PEX6 5. E II PEX 6. F 0 V PEX2 7. G 2 VIII PEX 8. H VII PEX 9. J 4 XIII PEX IV PEX5. - VI PEX XII PEX4 RCDP NA NA NA PEX7 PTS protein Cargo binding 5s HUMAN PEROXISOME BIOGENESIS 5s Docking Recycling PTS protein Import 5s 26 6 Membrane protein 6 9

6 HUMAN PEROXISOME BIOGENESIS HUMAN PEROXISOME BIOGENESIS PTS2 protein 5L Membrane protein PTS protein 5s PTS2 protein 5L Membrane protein Cargo binding 7 5L 7 Recycling 7 5L 9 Cargo binding 5s 7 7 Recycling 9 5L Docking Import 6 Docking Import PTS2 protein PTS protein PTS2 protein COMPLEMENTATION ANALYSIS OF PBD CELLS Co-transfection of PEXx and GFP-SKL COMPLEMENTATION ANALYSIS OF PBD CELLS Co-transfection of PEXX and GFP-SKL Re-appearance of peroxisomes: GFP-SKL Defective PEXx pcdna-pexx + pegfp-skl + Patient cell CG? no peroxisomes: No defective PEXx GFP-SKL repeat with other PEXX cdnas Patient cell + pegfp-skl + ppex (2 days after transfection) PEX defective Patient cell + pegfp-skl+ ppex (2 days after transfection) PEX not defective

7 PBD GENETIC COMPLEMENTATION ANALYSIS Results after analysis of 600 PBD patients (PEG + GFP-SKL) PBD GENETIC COMPLEMENTATION ANALYSIS Results after analysis of 600 PBD patients (PEG + GFP-SKL) PEX gene Phenotype Number of cell lines Frequency (%) PEX gene Phenotype Number of cell lines Frequency (%) PEX 2 PEX2 PEX 4 PEX5 5 PEX6 6 PEX0 7 PEX2 8 PEX 9 PEX4 0 PEX6 PEX9 2 PEX26/5 ZS ZS, NALD ZS, NALD ZS ZS, NALD ZS, IRD ZS, IRD PEX 2 PEX2 PEX 4 PEX5 5 PEX6 6 PEX0 7 PEX2 8 PEX 9 PEX4 0 PEX6 PEX9 2 PEX26/5 ZS ZS, NALD ZS, NALD ZS ZS, NALD ZS, IRD ZS, IRD Data from Ebberink etal Hum Mutat. 20 Jan;2():59-69 Data from Ebberink etal Hum Mutat. 20 Jan;2():59-69 PEROXISOME BIOGENESIS DISORDERS AND MUTATION ANALYSIS OF THE PEX GENE MUTATED IN 59% OF PBD-PATIENTS Many private mutations Two frequently occurring mutations identified c.2528 G>A (p.g84d)* ZELLWEGER SPECTRUM DISORDERS : ZELLWEGER SYNDROME, NEONATAL ADRENOLEUKODYSTROPHY AND INFANTILE REFSUM DISEASE : PHENOTYPIC VARIANTS WITH OVERLAPPING CLINICAL SIGNS AND SYMPTOMS RANGING IN SEVERITY FROM ZS TO NALD AND IRD -allele l frequency in our patient t population : 6% - G84D substitution causes instability of Pexp with partial reduction in function - patients homozygous for c.2528 G A all show MILD phenotype (IRD) 2 c.2097_2098inst (p.i700fsx4)** - allele frequency in our patient population : 7% - truncated protein with full loss of Pexp function - patients homozygous for c.2097 inst all show SEVERE phenotype (ZS) * Reuber etal. (997) Nat. Genet. 7, ; Portsteffen etal (997) Nat. Genet. 7, ** Maxwell etal (999) Hum. Genet 05, 8-44; Collins etal. (999) Hum. Mutat. 4, 45-5 c.2097_2098inst / c.2097_2098inst c.2528g>a / c.2528g>a

CATALASE IMMUNOFLUORESCENCE IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX PRISTANIC ACID ß-OXIDATION IN FIBROBLASTS FROM PATIENTS WITH

2528g>a (G84D) MUTATION PRISTANIC ACID ß-OXIDATION IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX Pristanic acid ß-oxidation (%) 0 20

8 CATALASE IMMUNOFLUORESCENCE IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX PRISTANIC ACID ß-OXIDATION IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX Pristanic acid ß-oxidation (%) Control PEX p.g84d (c.2528g>a) PEX p.i700fsx4 (c.2097_2098inst) PEX p.g84d (c.2528g>a) PEX p.i700fsx4 (c.2097_2098inst) 7 C EFFECT OF A SHIFT IN TEMPERATURE (7 C 0 C) ON THE NUMBER OF PEROXISOMES IN FIBROBLASTS FROM A PATIENT HOMOZYGOUS FOR THE MILD c.2528g>a (G84D) MUTATION PRISTANIC ACID ß-OXIDATION IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX Pristanic acid ß-oxidation (%) Control PEX p.g84d (c.2528g>a) PEX p.i700fsx4 (c.2097_2098inst) 7 C Control PEX p.g84d (c.2528g>a) PEX p.i700fsx4 (c.2097_2098inst) 0 C 7 C 0 C Control PEX p.g84d (c.2528g>a) PEX p.i700fsx4 (c.2097_2098inst) 40 C Gootjes et al. HUMAN MUTATION 24:0-9 (2004)

9 CONCLUSION Zhang etal Proc Natl Acad Sci U S A. 200 Mar 2;07(2): A shift in temperature (7 C 0 C) not only induces an increase in the number of peroxisomes but also restores the functional capacity of peroxisomes QUESTION Is it possible to restore peroxisome biogenesis in fibroblasts from patients homozygous for PEXp.G84D at 7 C? Screened a large library of small compounds (2080) for their capacity to restore peroxisome formation in cells homozygous for c.2528 G>A (p.g84d) 2 new compounds identified : Ro-8220, GF0920X PEROXISOMES IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX AND THE EFFECT OF GLYCEROL AND ARGININE PEROXISOMES IN FIBROBLASTS FROM PATIENTS WITH DIFFERENT MUTATIONS IN PEX AND THE EFFECT OF INCREASING CONCENTRATIONS OF ARGININE (7 C) Untreated Arginine 40 mm Glycerol 5% (v/v)

10 CONCLUSION Glycerol and arginine but also other compounds are able to (partially) restore peroxisome formation and function (!) in cells from patients homozygous for p.g84d Practical approach :. Continue on the arginine line. 2. Test arginine in vivo (mouse model!) CURRENT RESEARCH ZELLWEGER SPECTRUM DISORDERS : ZELLWEGER SYNDROME, NEONATAL ADRENOLEUKODYSTROPHY AND INFANTILE REFSUM DISEASE : PHENOTYPIC VARIANTS WITH OVERLAPPING CLINICAL SIGNS AND SYMPTOMS RANGING IN SEVERITY FROM ZS TO NALD AND IRD Fundamental approach Resolve the folding pathway for PEX and identify which factors are involved Common features include neurodevelopmental delay, retinopathy, perceptive deafness, and variable liver disease ZELLWEGER SPECTRUM DISORDERS : MARKED HETEROGENEITY IN CLINICAL SIGNS AND SYMPTOMS EXAMPLE : PBD DEFECT IN A PATIENT WITH ISOLATED CEREBELLAR ATAXIA Third child of non-consanguineous parents Normal neurological examination at 4 years At age 8, cerebellar signs with impaired gait, dysmetria, ataxia of the trunc, dysarthria, and slow saccades without oculomotor o oto apraxia a a and hyporeflexia e ICARS score 7/00 No other neurological symptoms WISC-IV analysis : full, verbal, and performance IQ were 80, 74 and 75 (normal range) Brain MRI: isolated cerebellar atrophy at 8 years Normal electroretinogram, peripheral nerve conduction and visual, brainstem auditory, and somatosensory evoked potentials Molecular studies of cerebellar ataxia genes including frataxin and aprataxin : normal Peroxisomal parameters in plasma : abnormal! ZELLWEGER SPECTRUM DISORDERS : A MARKEDLY HETEROGENEOUS GROUP OF DISEASES EXAMPLE : PBD DEFECT IN A PATIENT WITH ISOLATED CEREBELLAR ATAXIA Sevin C, Ferdinandusse S, Waterham HR, Wanders RJ, Aubourg P. Orphanet J Rare Dis. 20 Mar 0;6:8.

11 ZELLWEGER SPECTRUM DISORDERS : A MARKEDLY HETEROGENEOUS GROUP OF DISEASES EXAMPLE : PBD DEFECT IN A PATIENT WITH ISOLATED CEREBELLAR ATAXIA Detailed studies in fibroblasts : very mild abnormalities with the virtual normal presence of peroxisomes Sequencing of candidate PEX genes revealed a homozygous c.865_866insa mutation in the PEX2 gene leading to a frameshift 7 codons upstream of the stop codon. PEX gene mutations usually result in a severe neurological phenotype (Zellweger spectrum disorders). PSEUDO ZELLWEGER SYNDROME (Goldfischer etal (986) J.Pediatr.08, 25-2) Infant girl with the full spectrum of clinical and pathological abnormalities described for Zellweger syndrome including craniofacial dysmorphia, neuronal migration defect, etc. Abnormal VLCFA s and bile acid intermediates However, abundant peroxisomes in liver parenchymal cells! PS Similar patient with isolated cerebellar ataxia identified by Regal etal Ann Neurol. 200 Aug;68(2):259-6 PSEUDO ZELLWEGER SYNDROME (Goldfischer etal (986) J.Pediatr.08, 25-2) Infant girl with the full spectrum of clinical and pathological abnormalities described for Zellweger syndrome including craniofacial dysmorphia, neuronal migration defect, etc. Abnormal VLCFA s and bile acid intermediates t However, abundant peroxisomes in liver parenchymal cells! 2002 : Identification of true defect in this patient at the level of D-bifunctional protein (Ferdinandusse etal (2002), Am.J.Hum.Genet. 70, ) PEROXISOMAL FATTY ACID ß-OXIDATION IN HUMAN PEROXISOMES AND ITS DEFICIENCIES cholesterol C26:0 CoASH pristanic acid THCA X-ALD ALDP acylcoa oxidase deficiency AOX pth ATP DBP AOX2 pth2 CoASH racemase Racemase deficiency D-bifunctional protein deficiency

12 KAPLAN-MEIER SURVIVAL ANALYSIS IN D-BIFUNCTIONAL PROTEIN DEFICIENCY D-BIFUNCTIONAL PROTEIN DEFICIENCY (Ferdinandusse etal (2006), Ann.Neurol. 59, 92-04) 26 patients with D-BP deficiency identified in our laboratory Questionnaires sent out. Outcome : Neonatal hypotonia 8/85 (98%) Seizures in first month of life 79/85 (9%) External dysmorphia 5/79 (67%) Visual system failure 40/7 (55%) Neocortical dysplasia 2/45 (27%) Liver disease 8/69 (26%) Demyelination cerebral hemispheres 9/47 (9%) Cerebellar atrophy 8/49 (6%) Early death (< 2year) 04/6 (90%) PEROXISOMAL FATTY ACID ß-OXIDATION IN HUMAN PEROXISOMES AND ITS DEFICIENCIES ATP cholesterol C26:0 CoASH pristanic acid THCA X-ALD ALDP AOX pth DBP AOX2 pth2 CoASH racemase Racemase deficiency LABORATORY DIAGNOSIS OF THE ZELLWEGER SPECTRUM DISORDERS (ZS,NALD,IRD), D-BIFUNCTIONAL PROTEIN DEFICIENCY AND ACYL-CoA OXIDASE DEFICIENCY Initial test Biogenesis defect Abnormal PLASMA VLCFA-analysis Definitely ZSD, DBP deficiency, or ACOX deficiency Full study in fibroblasts Single enzyme defect Complementation analysis DBP and ACOX activity acylcoa oxidase deficiency D-bifunctional protein deficiency DNA analysis (PEX genes) DNA analysis

13 LABORATORY DIAGNOSIS OF THE ZELLWEGER SPECTRUM DISORDERS (ZS,NALD,IRD), D-BIFUNCTIONAL PROTEIN DEFICIENCY AND ACYL-CoA OXIDASE DEFICIENCY Initial test PLASMA VLCFA-analysis PLASMA VLCFA MAY BE ENTIRELY NORMAL IN PATIENTS AFFECTED BY THE ZELLWEGER SPECTRUM DISORDERS (ZS,NALD,IRD), D-BIFUNCTIONAL PROTEIN DEFICIENCY AND ACYL-CoA OXIDASE DEFICIENCY Abnormal Normal Example : Rosewich etal (2006) Neuropediatrics 7, Biogenesis defect Complementation analysis DNA analysis (PEX genes) Definitely ZSD, DBP deficiency, or ACOX deficiency Full study in fibroblasts Single enzyme defect DBP and ACOX activity DNA analysis ZSD, DBP deficiency, or ACOX deficiency not excluded Full study in fibroblasts Abnormal Normal ZSD, DBP deficiency, or ACOX deficiency virtually excluded 2 Month old boy Dysmorphic features including high forehead, broad nasal bridge, high-arched palate, syndactyly (second and third toes on both feet) Psychomotor retardation, muscular hypotonia, clonic seizures MRI : typical pattern showing pachygyria, perisylvian polymicrogyria, cerebral and cerebellar white matter abnormalities Retinopathy Repetitive measurement of VLCFA s and other peroxisomal metabolites : NO ABNORMALITIES Detailed studies in fibroblasts : acyl-coa oxidase deficiency, splice-site mutation Other examples : Soorani-Lunsing etal (2005) J.Inher.Metab.Dis. 28, 72-74, etc. LIST OF PEROXISOMAL DISORDERS PEROXISOME BIOGENESIS DISORDERS - Zellweger syndrome (ZS) - Neonatal adrenoleukodystrophy (NALD) - Infantile Refsum disease (IRD) - Rhizomelic chondrodysplasia punctata (RCDP) SINGLE PEROXISOMAL ENZYME DEFICIENCIES - X-linked adrenoleukodystrophy - D-bifunctional protein deficiency - Acyl-CoA oxidase deficiency - 2-Methylacyl-CoA racemase deficiency - Sterol carrier protein x deficiency - DHAPAT-deficiency (RCDP Type 2) - Alkyl DHAP-synthase deficiency (RCDP Type ) - Refsum disease - Hyperoxaluria Type - Acatalasaemia Zellweger spectrum disorders X-LINKED ADRENOLEUKODYSTROPHY (XALD) Most frequent peroxisomal disorder Marked phenotypic heterogeneity Two phenotypes most frequent including :. Childhood cerebral ALD (CCALD) 2. Adrenomyeloneuropathy (AMN) In all XALD phenotypes : accumulation of very-long-chain fatty acids due to their deficient oxidation in peroxisomes Caused by mutations in the ABCD gene wich codes for the peroxisomal half-abc-transporter ALDP (Mosser etal 99, Nature 6, )

14 X-LINKED ADRENOLEUKODYSTROPHY FATTY ACID ß-OXIDATION OF C26:0 INCLUDING THE ROLE OF ALDP AND THE CROSSTALK BETWEEN MITOCHONDRIA AND PEROXISOMES Accumulation of very-long-chain fatty acids in plasma 0.5 C26:0 (µmol/l) in plasma CONTROL X-ALD Deficient oxidation of C26:0 in intact fibroblasts Activity (%) Medium chain fatty acids Peroxisome C26:0-CoA Deficient in X-ALD ALDP ALDP C26:0-CoA Peroxisomal ß-oxidation (? cycles) TE Medium chain TE Acetyl-CoA Acetate Acyl-CoA CO CA T T Medium chain Acyl-carnitine Acetyl-carnitine CACT CONTROL X-ALD C26:0 Medium chain fatty acids CoASH Mitochondrion Medium chain Acyl-carnitine Acetyl-carnitine CPT CAT 2 Medium chain Acetyl-CoA Acyl-CoA ß-oxidation CoASH Krebs cycle Acetate CO 2 CO 2 PUTATIVE SECONDARY STRUCTURE OF ALDP IN THE PEROXISOMAL MEMBRANE MOLECULAR ANALYSIS OF ABCD IN X-ALD Matrix 587 different mutations identified ( Membrane 00 (5%) missense mutations NH 2 Cytosol Walker A Walker B 66 (28%) frame shift mutations 68 (2%) nonsense mutations 5 (6%) small insertions/deletions 8 (%) larger deletions of one or more exons COOH C sequence

PUTATIVE SECONDARY STRUCTURE OF ALDP IN THE PEROXISOMAL MEMBRANE Argfs EFFECT OF TEMPERATURE ON THE AMOUNT OF ALDP PROTEIN IN FIBROBLASTS FROM PATIENTS CARRYING DIFFERENT MISSENSE MUTATIONS IN ABCD

15 PUTATIVE SECONDARY STRUCTURE OF ALDP IN THE PEROXISOMAL MEMBRANE Argfs EFFECT OF TEMPERATURE ON THE AMOUNT OF ALDP PROTEIN IN FIBROBLASTS FROM PATIENTS CARRYING DIFFERENT MISSENSE MUTATIONS IN ABCD Matrix Membrane Cytosol Arg554His Walker A NH 2 Asp94His Arg660Trp Walker B COOH Ala66Thr C sequence Glu609Gly EFFECT OF TEMPERATURE ON C26:0 BETA-OXIDATION IN FIBROBLASTS FROM PATIENTS CARRYING DIFFERENT MISSENSE MUTATIONS IN ABCD EFFECT OF TEMPERATURE ON THE C26:0/C22:0 RATIO IN FIBROBLASTS FROM PATIENTS CARRYING DIFFERENT MISSENSE MUTATIONS IN ABCD 7 C 0 C 2% 7% 0% 0% 60% 75% 2% 9% % 2% 5% 4%

16 CONCLUSIONS A shift in temperature (7 C 0 C) leads to increased amounts of ALDP in fibroblasts from some but not all patients carrying different missense mutations in ABCD Increased amounts of ALDP do not necessarily imply improved catalytic functioning of ALDP CURRENT RESEARCH Practical approach : search for compounds able to counteract misfolding of ALDP at 7 C Fundamental approach : identification of the factors involved in folding of ALDP LIST OF PEROXISOMAL DISORDERS PEROXISOME BIOGENESIS DISORDERS - Zellweger syndrome (ZS) - Neonatal adrenoleukodystrophy (NALD) - Infantile Refsum disease (IRD) - Rhizomelic chondrodysplasia punctata (RCDP) SINGLE PEROXISOMAL ENZYME DEFICIENCIES - X-linked adrenoleukodystrophy - D-bifunctional protein deficiency - Acyl-CoA oxidase deficiency - 2-Methylacyl-CoA racemase deficiency - Sterol carrier protein x deficiency - DHAPAT-deficiency (RCDP Type 2) - Alkyl DHAP-synthase deficiency (RCDP Type ) - Refsum disease - Hyperoxaluria Type - Acatalasaemia Zellweger spectrum disorders Rhizomelic chondrodysplasia punctata (RCDP) - Growth retardation, dwarfism - Facial dysmorphia - Rhizomelic shortening of upper extremities - Severe mental retardation MUTANT GENE : PEX7 CODES FOR THE PTS2 RECEPTOR REQUIRED FOR THE CORRECT TARGETING OF PEROXISOMALPROTEINS =peroxisomal thiolase.fytanoyl-coa hydroxylase 2.Alkyl-DHAP synthase.peroxisomal thiolase Cargo binding 7 5L HUMAN PEROXISOME BIOGENESIS 7 PTS2 protein 5L Recycling 7 5L Membrane protein 9 2=alkyl DHAP synthase =phytanoyl-coa hydroxylase Docking Import PLASMALOGENS PHYTANIC ACID

17 PEROXISOMES AND THEIR ROLE IN PLASMALOGEN BIOSYNTHESIS? ACYLCoA NADPH PEROXISOMES AND THEIR ROLE IN PLASMALOGEN BIOSYNTHESIS RCDP type 2? ACYLCoA NADPH ACYLCoA ACYLCoA REDUCTASE ACYLCoA ACYLCoA REDUCTASE DHAPAT DHAP NADP DHAPAT DHAP NADP ACYLDHAP CoASH? LONG-CHAIN ALCOHOL ACYLDHAP CoASH? LONG-CHAIN ALCOHOL ALKYLDHAP SYNTHASE LONG-CHAIN ALCOHOL FATTY ACID TRANSPORT TO ER ER ALKYLDHAP SYNTHASE LONG-CHAIN ALCOHOL FATTY ACID TRANSPORT TO ER ER ALKYLDHAP ALKYLDHAP PEROXISOME PLASMALOGENS RCDP type PEROXISOME PLASMALOGENS Initial test RCDP type PEX7 LABORATORY DIAGNOSIS OF PEROXISOMAL CHONDRODYSPLASIA PUNCTATA Abnormal PLASMALOGEN analysis in erythrocytes Normal Definitely RCDP type,2 or RCDP type,2 or not excluded Full study in fibroblasts RCDP type 2 RCDP type GNPAT ADHAPS Full study in fibroblasts Abnormal Normal RCDP type,2 or virtually excluded LIST OF PEROXISOMAL DISORDERS PEROXISOME BIOGENESIS DISORDERS - Zellweger syndrome (ZS) - Neonatal adrenoleukodystrophy (NALD) - Infantile Refsum disease (IRD) - Rhizomelic chondrodysplasia punctata (RCDP) SINGLE PEROXISOMAL ENZYME DEFICIENCIES Pathway affected - X-linked adrenoleukodystrophy - D-bifunctional protein deficiency - Acyl-CoA oxidase deficiency Beta-oxidation - 2-Methylacyl-CoA racemase deficiency - Sterol carrier protein x deficiency - DHAPAT-deficiency (RCDP Type 2) Plasmalogen - Alkyl DHAP-synthase deficiency (RCDP Type ) biosynthesis - Refsum disease Alpha-oxidation - Hyperoxaluria Type Glyoxylate metabolism - Acatalasaemia H 2 O 2 metabolism

18 Biogenesis Merel Ebberink Hans Waterham ACKNOWLEDGEMENTS DNA Hans Waterham Clinic Bwee-Tien Poll-The Peter Barth Metabolism Sacha Ferdinandusse Ries Duran Wim Kulik Nico Abeling Mouse models Pedro Brites (RCDP) Sacha Ferdinandusse (Refsum) Stephan Kemp (XALD)

Hereditary disorders of peroxisomal metabolism.

Hereditary disorders of peroxisomal metabolism http://www.cytochemistry.net/cell-biology/perox.jpg Peroxisomes single membrane organelles from less than 100 to more than 1000 per eucaryotic cell more than