Restoration of biochemical function of the peroxisome in the temperature-sensitive mild forms of peroxisome biogenesis disorder in humans

Brain & Development 22 (2000) 8±12

Original article

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Restoration of biochemical function of the peroxisome in the temperaturesensitive mild forms of peroxisome biogenesis disorder in humans Atsushi Imamura a,b,*, Nobuyuki Shimozawa a, Yasuyuki Suzuki a, Zhongyi Zhang a, Toshiro Tsukamoto b, Yukio Fujiki c, Tadao Orii d, Takashi Osumi b, Naomi Kondo a a Department of Pediatrics, Gifu University School of Medicine, Gifu 500-8705, Japan Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo 678-1297, Japan c Department of Biology, Graduate School of Science, Kyushu University, Fukuoka 812-8581, Japan d Faculty of Human Welfare, Chubu Gakuin University, Seki, Gifu 501-3936, Japan. b

Received 20 April 1999; received in revised form 9 July 1999; accepted 12 July 1999

Abstract We have found that peroxisome assembly is temperature-sensitive (ts) in mild forms of peroxisome biogenesis disorders (PBDs), that is all infantile Refsum disease (IRD) patients and a few neonatal adrenoleukodystrophy patients of several complementation groups. The number of peroxisomes increased daily in incubation at 308C in the ts cells. Oxidation of very long-chain fatty acids, processing of acyl-CoA oxidase and dihydroxyacetonephosphate acyltransferase activity also improved after 8 days incubation at 308C in the IRD ®broblasts. These biochemical functions of the peroxisome did not change at 308C in Zellweger ®broblasts. Number of peroxisomes gradually decreased after 4 days when the temperature shifted from 30 to 378C in the ts cells. These results indicate that the biochemical functions of peroxisome are also restored by incubation at 308C in the mild and ts phenotype of PBDs, and the results will aid to predict the severity and the prognosis of affected children. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Peroxisome biogenesis disorders; Temperature-sensitivity; Zellweger syndrome; Neonatal adrenoleukodystrophy; Infantile Refsum disease; Betaoxidation; Dihydroxyacetonephosphate acyltransferase activity

1. Introduction The peroxisome is often an encountered organelle involved in vital metabolic functions, such as the oxidative processes involving H2O2, beta-oxidation of fatty acids, and biosynthesis of plasmalogens. Peroxisome biogenesis disorders (PBDs) are lethal diseases characterized by multiple defects in peroxisomal functions. PBDs are genetically classi®ed into at least 11 complementation groups (CGs) [1±3], and each CG contains various clinical phenotypes, such as Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD). ZS represents the severest form of PBD, and is characterized by severe neurologic, hepatic, and renal abnormalities, mental retardation, and death in early infancy. NALD, the next severe, whereas IRD is the mildest, with signi®cantly different in clinical features such as age of death and severity of neurological abnormalities. Notwithstanding the cloning of the causal genes and identi®cation of the mutations for several * Corresponding author. Tel.:181-58-265-1241; fax:181-58-265-9011. E-mail address: [email protected] (A. Imamura)

CGs [4±16], it is unknown at the molecular level why such diverse phenotypes occur in the same complementation groups. We have found that peroxisome assembly is temperaturesensitive (ts) in the ®broblasts from mild forms of PBDs [17]. In these cells, peroxisomes were formed, and the number of peroxisomes was increased at 308C after several days but not at 378C, whereas virtually no peroxisomes were seen in ZS cells even at 308C. The biochemical activities of peroxisomes were greatly elevated after incubation at 308C. In this paper, we dicussed the correlation between morphological change and biochemical functions of peroxisome using a time course trial in these cells.

2. Materials and methods 2.1. Cell lines and strains Skin ®broblasts from normal controls and patients with PBDs were cultured in Ham's F12 medium supplemented with 10% fetal calf serum. F-01 and E-14 were the ®bro-

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A. Imamura et al. / Brain & Development 22 (2000) 8±12

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blasts of ZS in CG-F and CG-E, respectively. F-05 and E-06 were the ®broblasts of IRD in each CG, revealed ts as described [17]. All strains were classi®ed by complementation analysis of cell fusion as described [18,19]. 2.2. Immuno¯uorescence study For the detection of peroxisomes, cell lines were ®xed, permeabilized with 0.1% Triton X-100 and processed for indirect immuno¯uorescence [20]. The ®rst antibodies we used were rabbit antibodies to human catalase, and in double immuno¯uorescence, rabbit anti-rat PMP70 antibodies was used. 2.3. Time course trial of ts ®broblasts The ts cells (F-05, E-06) and ZS cells (F-01, E-14) were cultured at 308C, ®xed after 1, 2, 4, and 8 days and immuno¯uorence study was performed using antibody to human catalase. The cells preincubated at 308C for 8 days, were ®xed after 1, 2, and 4 days after the temperature was shifted from 30 to 378C, and immuno¯uorence study was done in the same way. 2.4. Other methods Continuous cell labeling with 35S-methionine and immunoprecipitation of acyl-CoA oxidase (AOX) with rabbit anti-rat AOX antibody as performed, as described [20]. The activity of lignoceric acid and palmitic acid oxidation was measured by published methods [21]. Dihydroxyacetonephosphate acyltransferase (DHAP-AT) activity was measured as described [22], using 14C-labeled DHAP as a substrate. 3. Results In the ®broblasts of the IRD patient (F-05) revealing ts, peroxisomes were gradually formed at 308C and the number of peroxisome increased daily (Fig. 1). However, in those of ZS patient (F-01, E-14), no peroxisomes were found at either 30 or 378C (data not shown). Catalase and 70 kDa peroxisomal membrane protein (PMP70) were co-localized in the ts cells after 4 days incubation at 308C (Figs. 1C and 2), hereby con®rming the identity of these catalase-positive granules as peroxisomes. The number of formed peroxisomes was gradually reduced after 4 days when the temperature shifted from 30 to 378C in the ts cells (Fig. 1). The time course trial was performed also in the ®broblasts of IRD patient (E-06), and it revealed the same morphological appearance of peroxisomes as F-05 (data not shown). The beta-oxidation activity of very-long-chain fatty acids (VLCFA) (represented by the relative beta-oxidation activities to lignocerate and palmitate [7]: C24:C16) and the activity of dihydroxyacetonephosphate acyltransferase (DHAP-AT) were measured using control and PBD ®bro-

Fig. 1. Time trial course of ts ®broblasts of the patient with IRD(F-05). Cells were cultured for 1, 2, 4, and 8 days (panels A±D) under 308C, then stained with antibodies to human catalase. Cells were preincubated above 8 days under 308C, and cultured for 1,2, and 4 days (E±G) under 378C, then stained with antibodies to human catalase. Scale bar, 10 mm.

blasts. The ZS cells (F-01, E-14) had 10% or less VLCFA oxidation and DHAP-AT activities, as compared to those of control cells at both temperatures. In ZS (F-01, E-14) and control cells, these activities showed no remarkable exchange between 30 and 378C (Table 1). In contrast, in the IRD cells (F-05, E-06), the two activities were signi®cantly higher at 30 than at 378C (Table 1). These activities were higher after 8 days incubation at 308C than after 4 days in the ts cells. Acyl-CoA oxidase (AOX), an enzyme of the peroxisomal beta-oxidation system, is processed into two smaller polypeptides in peroxisomes, and this processing is disturbed in patients with PBDs [23]. On continuous labeling of the IRD cells (F-05, E-06) at 308C but not 378C, the processed form of AOX (subunit B: 53 kDa) was detected in addition to the unprocessed form of AOX (subunit A: 75 kDa) (Fig. 3), as observed for control cells at both temperatures. In the ZS cells, subunit B was not detected, whereas subunit A occurred at a reduced amount,

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A. Imamura et al. / Brain & Development 22 (2000) 8±12

Fig. 2. Control (left) and F-05 cells (right) were cultured for 4 days under 308C, then stained with antibodies to human catalase and rat PMP70, respectively. Scale bar, 10 mm.

at both temperatures (Fig. 3). Based on these established criteria, the peroxisomes formed in the ts cells at 308C had signi®cantly improved biochemical functions.

thiolase were hardly detectable in PBD patients. We have found that peroxisome assembly is temperature-sensitive (ts) in the ®broblasts from mild forms of PBDs, such as all patients of IRD and some of NALD who belonged to different complementation groups (CGs) [17]. In the cells revealing ts phenotype (F-05, E-06), peroxisomes were formed from 48 h, and AOX processing was normalized after 72 h incubation at 308C. C24:C16 ratio and DHAPAT activities were more increased in parallel with the number of peroxisome-positive cells after 8 days than after 4 days incubation at 308C in the ts cells. Although C24:C16 in F-05, E-06 and DHAP-AT in E-06 at 308C for 8 days were still lower than those in controls, these activities might be nearly normal after more than 8 days because they were increased daily in incubation at 308C in the ts cells. These results suggested that peroxisomal biochemical functions were restored under the lower temperature in mildform PBD patients.

4. Discussion Peroxisomal biogenesis disorders (PBDs) are a group of lethal autosomal recessive diseases caused by defects in peroxisomal matrix protein import with the accompanying loss of multiple peroxisomal enzyme activities. In spite of the variations in the clinical features, the ®broblasts from the patients of all the three PBD phenotypes mostly lack peroxisomes. The oxidation of very-long-chain fatty acids (VLCFA), such as lignoceric acids (C24), was severely disturbed, and then, the accumulation of VLCFA was seen in these patients of PBDs. The activity of DHAP-AT and enzyme proteins of peroxisomal beta-oxidation, acyl-CoA oxidase (AOX), bifunctional protein, and 3-ketoacyl-CoA Table 1 Oxidation capacity of VLCFA and peroxisomal DHAP-AT activity a

F-01(ZS) F-05(IRD) E-14(ZS) E-06(IRD) Control A Control B

Peroxisome-postive cells (%) b

C24:C16 (%) c

378C

378C

308C

DHAP-AT d 308C

378C

308C

4 days

8 days

4 days

8 days

4 days

8 days

4 days

8 days

4 days

8 days

4 days

8 days

0 0 0 5 100 100

0 0 0 5 100 100

0 70 0 90 100 100

0 90 0 100 100 100

2 4 4 7 61 68

2 3 1 6 42 63

2 8 2 17 48 72

2 25 3 23 43 59

0.23 0.49 0.20 0.15 1.28 2.13

0.11 0.20 0.19 0.16 1.69 2.01

0.13 0.69 0.20 0.61 1.85 1.96

0.13 1.12 0.27 0.87 1.82 1.24

a Cells were cultured for 4 or 8 days either at 37 or 308C, and then assayed for beta-oxidation activities of lignoceric and plamitic acids at the restrictive temperatures, and DHAP-AT activity at 378C. b Catalase-postive cells among 20 cells are counted in each 5 view ®elds at X1000, and data are averages of these indicated as %. c The relative VLCFA oxidation capasity is expressed as a ratio of lignoceric acid (C24)/palmitic acid (C16) oxidation activity of the cell line, indicated as %. Using this assay, lower ratio of C24:C16 shows lower peroxisomal VLCFA oxidation activity. The method and the mean range are described in Ref. [21]. d Plasmalogen biosynthesis capacity is expressed as the activity of dihydroxyacetonephosphate acyltransferase (DHAP-AT), ®rst enzyme in the pathway leading to plasmalogen biosynthesis. Values in nmol/120 min/mg protein. The method and the mean range are described in Ref. [22].

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Fig. 3. Biosynthesis and processing of AOX in the cultured ®broblasts. Cells were cultured for 72 h and then incubated for further 24 h in the presence of 35Smethionine. Throughout these procedures, temperatures were kept at 378C (lanes 1, 3, 5, 7 and 9), or 308C (lanes 2, 4, 6, 8 and 10). AOX as recovered from the cell extracts with anti-rat AOX antibody, and subjected to SDS-PAGE followed by ¯uorography. Lanes 1 and 2, control ®broblasts; 3 and 4, ZS (F-01) ®broblasts; 5 and 6, IRD (F-05) ®broblasts; 7 and 8, ZS (E-14) ®broblasts; and 9 and 10, IRD (E-06) ®broblasts. A and B were indicated subunit A and B of AOX, respectively.

The ts phenotype of PBD patients was detected in CG-A, CG-E, and CG-F [17]. The responsible gene for CG-E and CG-F was isolated, Pex1, and Pex2, respectively whereas that of CG-A was still unknown [4,11±13]. The E55K mutation in Pex2 was identi®ed as a temperature-sensitive mutation in an IRD patient of CG-F (F-05). The Pex2 E55K gene was transfected to a Pex2-de®cient Chinese-hamster-ovary cell (CHO) mutant (Z65) [24]. The transfectant revealed the morphological appearance of peroxisome under 308C incubation as the ts phenotype of peroxisome biogenesis of the IRD ®broblasts [17]. The biochemical analysis of peroxisome was performed in the Z65 and the Pex2 E55K gene transfectant. C24 and DHAP-AT activities were more elevated in the transfectant than in Z65 under the 308C incubation (data not shown). Recently, G843D in Pex1 was identi®ed as a common and ts mutation in milder types of CG-E patients (a patient with NALD and several patients with IRD). The IRD patient of CG-E (E-06) was G843D homozygote. Pex1-de®cient CHO mutant (ZP101 and ZP107) transfected with Pex1 G843D also revealed temperature sensitivity of peroxisome biogenesis as Pex2 E55K gene transfectant of Z65 [25]. The present results suggest that the ts peroxisome assembly process is closely linked to the responsible Pex genes having the function of matrix protein import. The Pex1 product revealed ATPases associated with diverse cellular activities (AAA) protein including two ATP binding domains required for both peroxisomal targeting signal 1 (PTS1) and PTS2. The G843D mutation is at the beginning of the second AAA cassette of this protein, a position that is conserved between human and yeast, and is likely to disturb the structure of the second AAA domain and the import of peroxisome matrix proteins [11±13]. Pex2 product has cysteine-rich RING segment in the C-terminal region, which is similar to the zinc-®nger DNA-binding protein; however, the correlation between the function of the Pex2 product and E55K mutation is still unclear [26]. Peroxisomal membrane vesicles (peroxisomal ghosts) expressed Pex1 G843D or Pex2 E55K may be formed into normal structure of peroxisome under 308C, and therefore, the biochemical function of peroxisome is improved in the ts cells. Our results indicate that ®broblasts have the reversible function of peroxisome in milder type of PBDs, and that

the character is correlated with ts mutation in the putative Pex genes. Peroxisomes exist abundantly in liver or kidney, while most other organs, including ®broblasts and nervous tissue, contain only microperoxisomes [27]. According to the previous report, liver cells reveal the diversity of peroxisome in a mild, long-survival patients with PBD [28]. If ts is observed also in other cells as ®broblasts of patients with ts phenotype, ts will be useful to predict the severity and prognosis of affected children with PBDs including prenatal diagnosis. References [1] Moser AB, Rasmussen M, Naidu S, Watkins PA, McGuniness M, Hajra AK, et al. Phenotype of patients with peroxisomal disorders subdivided into sixteen complementation groups. J Pediatr 1995;127:13±22. [2] Poulos A, Chiristodoulou J, Chow CW, Goldblatt J, Paton BC, Orii T, et al. Peroxisomal assembly defects: clinical, pathologic, and biochemical ®ndings in two patients in a newly identi®ed complementation group. J Pediatr 1995;127:596±599. [3] Shimozawa N, Suzuki Y, Orii T, Moser A, Moser HW, Wanders RJ. Standardization of complementation grouping of peroxisome-de®cient disorders and the second Zellweger patient with peroxisomal assembly factor-1 (PAF-1) defect (letter). Am J Hum Genet 1993;52:843±844. [4] Shimozawa N, Tsukamoto T, Suzuki Y, Orii T, Shirayoshi Y, Mori T, et al. A human gene responsible for Zellweger syndrome that affects peroxisome assembly. Science 1992;255:1132±1134. [5] Dodt G, Braverman N, Wong C, Moser A, Moser HW, Watkins P, et al. Mutations in the PTS1 receptor gene. PXR1, de®ne complementation group 2 of the peroxisome biogenesis disorders. Nat Genet 1995;9:115±125. [6] Wiemer E, Nuttley WM, Bertolaet BL, Li X, Francke U, Wheelock MJ, et al. Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders. J Cell Biol 1995;130:51±65. [7] Fukuda S, Shimozawa N, Suzuki Y, Zhang Z, Tomatsu S, Tsukamoto T, et al. Human peroxisome assembly factor-2 (PAF-2): a gene responsible for group C peroxisome biogenesis disorder in humans. Am J Hum Genet 1996;59:1210±1220. [8] Yahraus T, Braverman N, Dodt G, Kalish JE, Morrell JC, Moser HW, et al. The peroxisome biogenesis disorder group 4 gene. PXAAA1, encodes a cytoplasmic ATPase required for stability of the PTS1 receptor. EMBO J 1996;15:2914±2923. [9] Chang CC, Lee WH, Moser HW, Valle D, Gould SJ. Isolation of the human PEX12 gene, mutated in group 3 of the peroxisome biogenesis disorders. Nat Genet 1997;15:385±388.

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A. Imamura et al. / Brain & Development 22 (2000) 8±12

[10] Okumoto K, Shimozawa N, Kawai A, Tamura S, Tsukamoto T, Osumi T, et al. PEX12, the pathogenic gene of group III Zellweger syndrome: cDNA cloning by functional complementation on a CHO cell mutant, patient analysis, and characterization of PEX12p. Mol Cell Biol 1998;18:4324±4336. [11] Reuber BE, Germain-Lee E, Collins CS, Morrell JC, Ameritunga R, Moser HW, et al. Mutations in PEX1 are the most common cause of peroxisome biogenesis disorders. Nat Genet 1997;17:445±448. [12] Portsteffen H, Beyer A, Becker E, Epplen C, Pawlak A, Kunau WH, et al. Human PEX1 is mutated in complementation group 1 of the peroxisome biogenesis disorders. Nat Genet 1997;17:449±452. [13] Tamura S, Okumoto K, Toyama R, Shimozawa N, Tsukamoto T, Suzuki Y, et al. Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-de®cient Zellweger syndrome of complementation group I. Proc Natl Acad Sci USA 1998;95:4350±4355. [14] Warren DS, Morrell JC, Moser HW, Valle D, Gould SJ. Identi®cation of PEX10, the gene defective in complementation group 7 of the peroxisome-biogenesis disorders. Am J Hum Genet 1998;63:347± 359. [15] Okumoto K, Itoh R, Shimozawa N, Suzuki Y, Tamura S, Kondo N, et al. Mutation in PEX10 is the cause of Zellweger peroxisome de®ciency syndrome of complementation group B. Hum Mol Genet 1998;7:1399±1405. [16] Honsho M, Tamura S, Shimozawa N, Suzuki Y, Kondo N, Fujiki Y. Mutation in PEX16 is causal in the peroxisome-de®cient Zellweger syndrome of complementation group D. Am J Hum Genet 1998;63:1622±1630. [17] Imamura A, Tsukamoto T, Shimozawa N, Suzuki Y, Zhang Z, Fujiki Y, et al. Temperature-sensitive phenotype of peroxisome-assembly processes represent the milder forms of human peroxisome-biogenesis disorders. Am J Hum Genet 1998;62:1539±1543. [18] Shimozawa N, Tsukamoto T, Suzuki Y, Orii T, Fujiki Y. Animal cell mutants represent two complementation groups of peroxisome-defective Zellweger syndrome. J Clin Invest 1992;90:1864±1870.

[19] Tsukamoto T, Yokota S, Fujiki Y. Isolation and characterization of Chinese hamster ovary cell mutants defective in assembly of peroxisomes. J Cell Biol 1990;110:651±660. [20] Tsukamoto T, Miura S, Nakai T, Yokota S, Shimozawa N, Suzuki Y, et al. Peroxisome assembly factor-2, a putative ATPase cloned by functional complementation on a peroxisome-de®cient mammalian cell mutant. Nat Genet 1995;11:395±401. [21] Suzuki Y, Shimazawa N, Yajima S, Yamaguchi S, Orii T, Hashimoto T. Effects of sodium 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) on fatty acid oxidation in ®broblasts from patients with peroxisomal diseases. Biochem Pharmacol 1991;41:453±456. [22] Shimozawa N, Suzuki Y, Orii T, Yokota S, Hashimoto T. Biochemical and morphologic aspects of peroxisomes in the human rectal mucosa: diagnosis of Zellweger syndrome simpli®ed by rectal biopsy. Pediatr Res 1988;24:723±727. [23] Suzuki Y, Shimozawa N, Orii T, Igarashi N, Kono N, Hashimoto T. Molecular analysis of peroxisomal beta-oxidation enzymes in infants with Zellweger syndrome and Zellweger-like syndrome: further heterogeneity of the peroxisomal disorder. Clin Chim Acta 1988;172:65±76. [24] Tsukamoto T, Shimozawa N, Fujiki Y. Peroxisome assembly factor 1: nonsense mutation in a peroxisome-de®cient Chinese hamster ovary cell mutant and deletion analysis. Mol Cell Biol 1994;14:5458±5465. [25] Imamura A, Tamura S, Shimozawa N, Suzuki Y, Zhang Z, Tsukamoto T, et al. Temperature-sensitive mutation in PEX1 moderates the phenotypes of peroxisome de®ciency disorders. Hum Mol Genet 1998;7:2089±2094. [26] Tsukamoto T, Miura S, Fujiki Y. Restoration by a 35K membrane protein of peroxisome assembly in a peroxisome-de®cient mammalian cell mutant. Nature 1991;350:77±81. [27] McKenna O, Arnold G, Holtzman E. Microperoxisome distribution in the central nervous system of the rat. Brain Res 1976;117:181±194. [28] Giros M, Roels F, Prats J, Ruiz M, Ribes A, Espeel M, et al. Long survival in a case of peroxisomal biogenesis disorder with peroxisome mosaicism in the liver. Ann NY Acad Sci 1996;804:747±749.