Human Molecular Genetics, 2000, Vol. 9, No. 18 2609-2616
© 2000 Oxford University Press
Co-expression of mutated and normal adrenoleukodystrophy protein reduces protein function: implications for gene therapy of X-linked adrenoleukodystrophy
Brain Research Institute and 1Institute of Neurology, University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria
Received 19 July 2000; Revised and Accepted 13 September 2000.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Inherited defects in the X-chromosomal adrenoleukodystrophy (ALD; ABCD1) gene are the genetic cause of the severe neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD). Biochemically the accumulation of very long-chain fatty acids, caused by impaired peroxisomal ß-oxidation, is the pathognomonic characteristic of the disease. Due to the X-chromosomal inheritance of X-ALD no data are available to clarify the question whether mutated adrenoleukodystrophy proteins (ALDPs) can negatively influence normal ALDP function. Here we show that restoration of ß-oxidation in X-ALD fibroblasts following transient transfection with normal ALD cDNA is more effective in ALDP-deficient fibroblasts compared with fibroblasts expressing normal amounts of mutated ALDP. Furthermore, we utilized the HeLa Tet-on system to construct a stable HeLa cell line expressing a constant level of endogenous ALDP and doxycycline-inducible levels of mutated ALDP. The induction was doxycycline dosage-dependent and the ALDP correctly localized. Interestingly, although mutated ALDP increased >6-fold in a dosage-dependent manner the total amount of ALDP (mutated and normal) remained approximately even as demonstrated by western blot and flow cytometric analyses. Thus, apparently mutated and normal ALDP compete for integration into a limited number of sites in the peroxisomal membrane. Consequently, increased amounts of mutated ALDP resulted in decreased peroxisomal ß-oxidation and accumulation of very long-chain fatty acids. These findings have direct implications on future gene therapy approaches for treatment of X-ALD, since in some patients a non-functional endogenous protein could act in a dominant negative way or displace the introduced, normal protein.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Mutations in the adrenoleukodystrophy (ALD; ABCD1) gene cause a severe neurodegenerative disorder, X-linked adrenoleukodystrophy (X-ALD; McKusick no. 300100) (1,2), in which impaired peroxisomal ß-oxidation leads to accumulation of very long chain fatty acids (VLCFAs) in tissues and body fluids, in particular in brain white matter and adrenal glands. The encoded ALD protein (ALDP) is one of four known ABC transporters localized in the peroxisomal membrane. The mechanism by which ALDP is involved in fatty acid metabolism still remains to be elucidated. A variety of clinical phenotypes can result from the same mutation, ranging from childhood lethal cerebral ALD to the milder variants adrenomyeloneuropathy and Addison’s disease. Children affected by the cerebral variant show inflammatory demyelination and usually die within 3–5 years after onset of neurologic symptoms.
Many different treatments of X-ALD have been tried, in most cases leading to no improvements (3). Currently, the only promising approach seems to be allogeneic bone marrow transplantation, provided that it is performed at an early stage of the disease before severe neurologic involvement occurs. Within the central nervous system, microglia, astrocytes and endothelial cells are the predominant cell types expressing the ALD protein (4). The importance of microglial dysfunction in X-ALD is also supported by the observation that bone marrow transplantation can reverse demyelination in X-ALD patients (5). The improvement of demyelinating lesions after heterologous bone marrow transplantation is possibly due to the replacement of abnormal microglia function by normal hematopoietic donor-derived cells (6).
Because allogeneic bone marrow transplantation is associated with a high risk of mortality and suitable HLA-matched donors are difficult to find, ALD gene transfer into autologous hematopoietic stem cells followed by transplantation would be a valuable therapeutic alternative circumventing many problems caused by conventional bone marrow transplantation. By using retroviral-mediated ALD gene transfer Doerflinger et al. (7) demonstrated that CD34+ cells from peripheral blood or bone marrow can be efficiently transduced with ALD cDNA and that the biochemical defect of X-ALD was corrected in these cells. Expression of ALDP was also observed in CD68+ cells, suggesting that monocyte-macrophages, the target bone marrow cells in X-ALD, were produced from transduced progenitor cells. Although it is still necessary to develop vectors and techniques allowing more efficient transduction, these results indicate the feasibility of gene-therapeutic approaches for X-ALD.
More than 340 ALD mutations have been reported to date, 61% of which are non-recurrent. Based on studies of primary fibroblast cultures obtained from 73 X-ALD patients, 67% have no detectable ALDP immunoreactivity, whereas the remaining 33% show reduced or normal levels of mutated ALDP (http://www.x-ald.nl ). In vitro studies carried out using X-ALD cell lines either lacking or having reduced amounts of mutated ALDP demonstrated that correction of defective ß-oxidation and normalization of VLCFA content can be obtained by transduction or transfection with normal ALD cDNA (8–11). Due to the X-chromosomal inheritance of the disease and because the ALD gene is subject to random X inactivation in female carriers (12), no natural situation occurs to clarify whether presence of mutated ALD protein has a dominant-negative effect on normal protein. In autologous bone marrow transplantation based on ex vivo introduction of normal copies of ALD cDNA into X-ALD patients’ own bone marrow cells, normal and mutated protein would co-exist in the same cell in all patients who express mutated but stable ALDP. It has not been studied in detail whether presence of mutated ALD protein interferes with functional activity of normal protein. As dimerization of ALDP with itself and other peroxisomal ABC transporters has recently been shown in vitro (13,14), interactions between normal and mutated ALDP seem likely, perhaps leading to non-functional heterodimers. Or, if the ratio of mutated to normal protein is unfavourable, mutated ALDP could displace normal protein from the peroxisomal membrane. In either case, normalization of ß-oxidation would be less likely.
Here we clearly demonstrate dosage-dependent negative influence of mutated ALDP on normal ALDP function when co-expressed in the same cell. These findings have direct implications on future gene therapy approaches for X-ALD.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Peroxisomal ß-oxidation of X-ALD fibroblasts can be restored more efficiently by transfection when no mutated ALDP is present
Fibroblast cultures obtained from X-ALD patients described to express immunoreactive but non-functional ALDP were collected and analysed in detail. Western blot analysis comparing the amount of ALDP with that of healthy control fibroblasts revealed normal levels of ALDP in three of the cell lines (data not shown). Indirect immunofluorescence studies showed that the mutated proteins were correctly localized to peroxisomes as judged by co-localization with the peroxisomal marker protein catalase (data not shown). Previous studies have demonstrated that introduction of normal ALD cDNA into X-ALD fibroblasts lacking immunoreactive ALDP led to a significant increase in the rate of ß-oxidation comparable to that of healthy control fibroblasts (8–10). Also in fibroblasts containing reduced but detectable amounts of ALDP, VLCFA storage could be normalized after retroviral-mediated transduction with normal ALD cDNA (11). In accordance with these data, transient transfection of ALDP-deficient fibroblasts (A626T) with normal ALD cDNA resulted in restoration of ß-oxidation. However, in three different X-ALD fibroblast cell lines with normal levels of mutated ALDP, restoration of ß-oxidation was significantly lower than in ALDP-deficient cells (Fig. 1). We also transfected one of these cell lines, D194H, with an haemagglutinin (HA)-tagged normal construct to investigate the influence of mutated ALDP on correct peroxisomal targeting of normal ALDP when both are present in the same cell, a situation expected after gene transfer into autologous bone marrow cells of some X-ALD patients. For comparison ALDP-deficient fibroblasts were transfected with the same construct. The typical peroxisomal staining pattern of HA-tagged ALDP could be observed in both cell lines, indicating that the transferred protein was expressed and correctly localized, irrespective of the presence of mutated protein (data not shown). The transfection efficiency was estimated by counting HA-positive cells and was essentially the same (
20%) for both cell lines, indicating that transfection efficiency was not altered by presence of mutated ALDP. Average transfection efficiency was determined in three independent experiments and was 14% for the ALDP-deficient cell line (range 10–17%), 20% for the D194H cell line (range 15–23%), 26% for the S213C cells and 24% for the N148S cell line (range 22–30%).
|
Construction of a stable HeLa cell line expressing a constant level of endogenous ALDP and inducible levels of mutated ALDP
For further investigation of the functional relationship between mutated and normal ALDP, we used the HeLa Tet-on system (15), which allows stringent control of mutant ALDP over a wide range of expression under an inducible promoter whereas endogenous ALDP levels remain constant. To be able to further distinguish between endogenously expressed normal ALDP and mutated ALDP, a 9 amino acid motif from the influenza haemagglutinin protein was added to the C-terminus of D194H ALDP. Functional activity of ALDP was not affected by the tag, since an HA-tagged normal ALD construct restored ß-oxidation in ALDP-deficient fibroblasts to the same extent as constructs without a tag (data not shown). Furthermore, addition of the tag did not alter the subcellular localization of ALDP, as the protein expressed from the tagged construct was detected in peroxisomes by both anti-HA and anti-ALDP antibodies (data not shown). HeLa Tet-on cells were transfected with HA-tagged D194H-ALD cDNA and stable transformants were selected. PCR analysis confirmed the presence of both endogenous ALD gene and integrated D194H-ALD cDNA. Based on indirect immunofluorescence studies one clone, HeLa-D194H, which showed low background expression and high inducibility, was finally selected. The correct peroxisomal localization of overexpressed mutated HA-tagged ALDP was demonstrated by indirect immunofluorescence (Fig. 2) after induction with 2 µg/ml doxycycline for 48 h. No staining outside the peroxisomes above typical cellular background staining could be observed, indicating that overexpression of D194H-ALDP did not alter its localization. Uninduced cells were used as a control and showed no detectable HA staining.
|
Since the endogenous and the transfected ALD mRNAs differ in size, both mRNAs are detected by the ALD cDNA probe. Induction of D194H-ALD expression was verified by analysing mRNA and protein levels of both normal and mutated ALDP. Northern blot analysis of the HeLa-D194H clone at various concentrations of doxycycline showed robust induction even at the lowest concentration (Fig. 3a). Relative levels of normal and mutated ALD mRNA were compared at different doxycycline concentrations and quantified by phosphor imager analysis (Fig. 3b). Endogenous ALD mRNA, normalized to the loading control ß-actin, remained more or less constant, whereas the amount of mutated ALD mRNA increased
12-fold at the highest doxycycline concentration, thus confirming the high inducibility of the system. Concerning the ratio of normal to mutated ALD mRNA, in HeLa-D194H without doxycycline the level of mutated ALD mRNA was 30% of the level of endogenous ALD mRNA. On induction with the highest doxycycline concentration (5 µg/ml), the level of mutated to endogenous mRNA increased 6-fold over the level of endogenous ALD.
|
In order to obtain insights into the mechanism by which mutated ALDP interferes with normal protein function, we performed western blot analysis of total cell lysates using antibodies directed towards the HA tag and ALDP. The anti-HA antibody detects only mutated ALDP, whereas the anti-ALDP antibody detects normal as well as mutated ALDP. Thus, the relative expression level of normal ALDP can be measured indirectly, by detecting the overall amount of (mutated and normal) ALDP and subtracting the amount of mutated ALDP. Western blot analysis was performed with HeLa-D194H cells at varying concentrations of doxycycline (Fig. 4). Relative protein amounts were quantitated by densitometric analysis of western blots. Total ALDP amount (normal and mutated ALDP) remained approximately constant. Four independent western blot analyses showed essentially the same results and one representative blot was scanned (Fig. 4b). To correct for differences in loading and transfer, the membrane was incubated with an antibody detecting the 36 kDa glyceraldehyde-3-phosphate dehydrogenase, which is not induced by doxycycline, and the amount of ALDP was normalized to the level of this control. These results could be confirmed by flow cytometric analysis, where three independent experiments showed approximately constant levels of total ALDP amount after induction with 2 µg/ml doxycycline (data not shown). At the same time, mutated ALDP expression increased at least 6-fold, as densitometric quantitation revealed. Mutated ALDP was detected with anti-HA antibody (Fig. 4a) in three independent western blot analyses and confirmed the robust induction seen at the mRNA level (Fig. 3). As the total amount of normal plus mutated ALDP remained more or less unchanged and the amount of mutated ALDP increased, the amount of normal ALDP must as a consequence decrease. Therefore, apparently excess of mutated ALDP leads to a displacement of normal ALDP. Since it is possible that massive overexpression of mutated protein results in decreased levels of other peroxisomal ABC transporters, also the level of PMP70 was analysed by immunoblotting (Fig. 4c). Incubation with 2 µg/ml doxycycline resulted in no changes in PMP70 level. Taken together, protein analyses indicate that overexpression of mutated ALDP specifically reduces normal ALDP levels but not PMP70 levels.
|
Expression of mutated ALDP reduces the normal protein function in HeLa cells
To investigate the functional impact of mutated ALDP on normal ALDP we measured the peroxisomal ß-oxidation in the inducible HeLa system. Mutated D194H–ALDP was induced by different doxycycline concentrations (Fig. 5a). A slight decrease in ß-oxidation was observed for uninduced HeLa-D194H cells and after induction with the lowest doxycycline concentration. Compared with normal HeLa Tet-on cells, the decrease for both uninduced and slightly induced HeLa-D194H cells was not statistically significant. When expression of D194H-ALDP was further upregulated by higher doxycycline concentrations the rate of ß-oxidation was markedly reduced and this reduction was statistically significant compared with normal HeLa Tet-on cells. Thus, overexpression of mutated D194H-ALDP is sufficient to reduce ß-oxidation in HeLa Tet-on cells. In the induced HeLa-D194H cells the overall reduction in the rate of ß-oxidation was
45%, which is a less severe effect than the reduction of 80% for X-ALD fibroblasts compared with healthy fibroblasts (Fig. 5a). Also, fibroblasts that completely lack ALDP show residual ß-oxidation activity (of 20%); however, nothing is known about the extent of this residual activity in HeLa cells.
|
To establish whether the decreased rate of ß-oxidation also leads to intracellular accumulation of very long chain fatty acids, expression of D194H-ALDP was induced in HeLa-D194H cells for 1 week and the content of VLCFA was determined by gas chromatography. The increase in VLCFA accumulation measured for different expression levels of mutated ALDP (Fig. 5b) correlates very well with the observed decrease in peroxisomal ß-oxidation (Fig. 5a). The modest decrease in ß-oxidation at low expression levels was not sufficient to cause VLCFA accumulation. In contrast, more pronounced expression of mutated D194H-ALDP at 2 or 5 µg/ml doxycycline resulted in a 7-fold increase in the accumulation of VLCFA compared with untreated cells.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
During the past decades, many attempts have been made to cure X-ALD (3), yet none of the methods tried have influenced the neurodegenerative progression of this lethal disease except bone marrow transplantation of X-ALD patients with only slight neurologic involvement at the time of transplantation (3,16–19). Of 126 cerebral ALD cases who had undergone bone marrow transplantation worldwide until 1999, the 5 year actuarial probability of survival was 62% by Kaplan–Meier analysis (19). Survival data of engrafted patients were superior to those of non-transplanted (20). Thus, so far bone marrow transplantation is the only proven therapeutic intervention for cerebral ALD. However, high risks are associated with this therapy, like acute graft-versus-host disease, and HLA-matched donors are often difficult to find. X-ALD belongs to a group of storage disorders that are currently being investigated for use of gene therapy in treatment (21–23). It is a monogenetic disorder and the biochemical defect can be rescued by introduction of DNA encoding normal ALD protein into fibroblasts (8–10) and hematopoietic stem cells (7). Recently, it was demonstrated that ALDP is able to form homodimers (13,14). For these reasons, we started to investigate possible interactions between normal and mutated ALD protein when both accumulate in the same cell.
About 67% of all X-ALD patients show no immunoreactive ALDP, whereas the remaining 33% have reduced or normal levels of non-functional ALDP. ß-oxidation could also be restored in fibroblasts expressing reduced levels of mutated ALDP (11). However, our data clearly demonstrate that presence of mutated protein at normal levels interferes with restoration of ß-oxidation in X-ALD fibroblasts, indicating that complementation of ALDP function depends on the level of mutated protein expressed. Reduced levels of mutated ALDP do not seem to inhibit normal protein function, whereas normal or elevated levels of mutated protein have a negative effect on normal protein function.
To further elucidate the mechanism of interaction between normal and mutated ALDP, we constructed a HeLa Tet-on cell line with D194H mutated ALDP under a strong inducible promoter. Northern blot analysis of HeLa-D194H cells at different levels of induction revealed that the amount of endogenous ALD mRNA remains constant, independent of the level of overexpression of mutated ALD mRNA. Even after massive overexpression we did not observe any aberrantly localized ALDP, suggesting that ALDP that is not incorporated into the peroxisome is rapidly degraded. As judged by western blot analysis normal ALDP levels decreased with increasing expression levels of mutated ALDP, although the normal mRNA level was unaffected. Thus, mutated and normal ALD proteins seem to compete for integration into a limited number of sites available in the peroxisomal membrane. Since in vitro other peroxisomal ABC transporters, like ALDRP and PMP70, can replace the function of defective ALDP (10,24), we investigated the possibility that overexpression of mutated ALDP leads to displacement of PMP70. However, western blot analysis showed no reduction of PMP70. Thus, up-regulation of one of the peroxisomal ABC transporters does not seem to cause a reduction in the levels of other ABC transporters, but rather displacement seems to occur specifically between normal and mutated ALDP.
Alternatively, mutated ALDP could exert a dominant-negative effect on normal ALDP due to the formation of non-functional heterodimers. A similar mechanism of interaction was proposed for PMP70 to explain the observation that expression of mutant proteins inhibited peroxisomal ß-oxidation in CHO cells overexpressing wild-type PMP70 (25). The probability that heterodimers of mutated and normal ALDP are formed increases with increasing amounts of mutated ALDP expression. However, direct interaction between D194H mutated ALDP and normal ALDP is still hypothetical and there is no direct evidence that such heterodimers are completely non-functional. Of the three mutations analysed in transient transfection assays, N148S and S213C are located in transmembrane domains, whereas D194H is in the first cytosolic loop of the ALDP (Fig. 6). In yeast two-hybrid experiments performed with ALDP lacking the N-terminal 361 amino acids, homodimers still formed (13), arguing against the possibility that the three mutations N148S, D194H and S213C interfere with dimerization of ALDP. Whether displacement or formation of non-functional dimers lead to the reduction in ALDP function may be a matter of the ratio of the expression levels of normal and mutated protein. Presumably, a displacement mechanism would be favoured at excess levels of mutated ALDP and a negative dominant effect at lower levels of the mutated protein.
|
Our data have direct implications for future trials of gene therapy in X-ALD: in patients with normal amounts of mutated ALD protein, introduction of normal ALD cDNA may not lead to the efficient restoration of the biochemical defect expected in patients without detectable ALD protein. Thus, particular attention should be given to the actual amount of endogenous mutated ALDP in patients considered for gene therapy. However, the efficacy of restoring ß-oxidation by ALD gene transfer could be tested relatively easily on an individual basis, by transfection of fibroblast cultures from candidate patients before the transplantation procedure.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Cell lines and cell culture
Based on protein studies described in the literature we collected fibroblast cell lines expressing normal amounts of mutated stable ALDP. Three different fibroblast cell lines described as having normal levels of non-functional ALDP were obtained: fibroblasts N148S (26–29, patient 55 in ref. 26) were kindly provided by Dr Aubourg (Paris), D194H (kindred no. 12 in ref. 30) were provided by Dr Wanders (Amsterdam) and cell line S213C (31) was obtained from Drs Holzinger and Roscher (Munich). SV40-transformed X-ALD fibroblasts lacking ALDP (mutation A626T) were kindly provided by Drs Smith and Braiterman (Baltimore, MD) (32). Fibroblasts obtained from a non-diseased person were used as healthy controls. Fibroblasts were maintained in RPMI supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 U/ml penicillin and 100 ug/ml streptomycine (Biowhittaker, Walkersville, MD). HeLa Tet-on cell lines and gene expression systems were obtained from Clontech (Palo Alto, CA) (15). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin and 100 µg/ml streptomycine (Biowhittaker).
Plasmids and in vitro mutagenesis of ALD cDNA
The entire coding region of the human ALD cDNA (33) was EcoRI-inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, Groningen, The Netherlands) and vector pTRE (Clontech). To be able to distinguish between endogenously expressed normal ALDP and induced expression of mutated ALDP, an HA tag was added to the C-terminus of ALDP by PCR using upstream primer Oli 52 (corresponding to nucleotides 926–955 of the coding region, GenBank accession no. Z21876) and reverse primer Oli 237 (5'-GCTCTAGATTACTAAGCGTAGTCTGGGACGTCGTATGGGTAGGTGGAGGCACCCTGGAGGCCACCTG-3').
To introduce the point mutation D194H into the pTRE-ALD expression plasmid, in vitro mutagenesis was performed using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Briefly, complementary degenerate primers from nucleotides 950–975 of the ALD cDNA (GenBank accession no. Z21876) introducing a single point mutation 966G
C resulting in the amino acid exchange 194DH were used in a thermal cycling reaction according to the manufacturer’s recommendations. The parental, non-mutated plasmid strand was digested selectively with DpnI. Ultracompetent XL1 blue cells were transformed according to the manufacturer’s instructions. Resulting transformants were screened by restriction analysis and presence of the point mutation was confirmed by DNA sequencing.
Transfection
For transient transfection studies in fibroblasts, 20 µg of the respective plasmid DNA were transfected by electroporation at 200 V and 950 µF capacitance into 4 x 106 cells. Cells were kept on ice for 5 min and grown in RPMI for 72 h after transfection. For stable transfection of 1 x 106 HeLa Tet-on cells, 40 µg of D194H-ALD expression plasmid plus 2 µg of hygromycin resistance-conferring plasmid (pTK-Hyg; Clontech) were co-transfected at 220 V with a capacitance of 950 µF (Gene pulser and capacitance extender plus; BioRad, Hercules, CA) in 0.4 cm cuvettes. After electroporation, cells were kept at room temperature for 10 min and plated in 20 Petri dishes with DMEM. For clonal selection, 200 µg/ml hygromycin B (ICN, Costa Mesa, CA) was added to growth media. Clones were transferred into 24 well plates and DNA was isolated as previously described (10).
Identification of transfected clones by PCR
DNA from 24 HeLa Tet-on clones was subjected to PCR analysis, resulting in two fragments of different sizes: the endogenously expressed genomic ALD DNA was amplified as a 334 bp fragment (including 145 bp from intron 6) whereas clones with the D194H-ALD cDNA gave an additional fragment of 189 bp. Both fragments were obtained in a single reaction by amplification with primer Oli 68 corresponding to nucleotides 1526–1550 of the ALD cDNA (GenBank accession no. Z21876) and Oli 71 (nucleotides 1693–1715, GenBank accession no. Z21876). Of 24 clones analysed, 10 carried the mutated D194H-ALD cDNA. To test for high inducibility and low background expression of mutated ALD protein, immunofluorescence of all positive clones was compared in an uninduced state (0 µg/ml doxycycline) and induced state (2 µg/ml doxycycline) (Sigma, St Louis, MO). As the modified CMV promoter used in the HeLa Tet-on system is cell cycle dependent, the cells were synchronized by removal of FCS for 72 h prior to induction. Finally, one clone, HeLa-D194H, was selected that showed high inducibility and no background expression.
Northern blot
D194H-ALDP expression was induced by different doxycycline concentrations. Total RNA was isolated using the Qiagen RNeasy mini kit and QIAshredder according to the manufacturer’s instructions (Qiagen, Bothell, WA). RNA concentration was determined and 60 µg of total RNA were subjected to poly(A)+ selection by oligo(dT) cellulose (Roche, Basel, Switzerland) as recommended by the manufacturer. mRNA was electrophoresed on a 1.2% denaturing formaldehyde agarose gel (34) and transferred to Biodyne B nylon membrane (Pall, East Hills, NY). A 1997 bp ALD cDNA fragment (nucleotides 261–2258, GenBank accession no. Z21876) was radioactively labelled by random priming using [
-32P]dCTP (Amersham Pharmacia, Uppsala, Sweden) and used as a probe. As a control for equal loading and transfer, the ubiquitously expressed ß-actin was used. Relative intensities of mRNAs were analysed using a Storm Phosphor Imager (Molecular Dynamics/Amersham Pharmacia, Uppsala, Sweden).
ß-oxidation
The rate of ß-oxidation was determined according to Watkins et al. (35). Briefly, cells were incubated with 1–2 x 105 d.p.m. of radioactively labelled 1-[14C]lignoceric acid (American Radiolabelled Chemicals, St Louis, MO) or 1-[14C]palmitic acid (ICN) in a total of 5 nmol fatty acid for 1 h at 37°C. After alkaline hydrolysis, the amount of water soluble radioactivity was determined by Folch extraction. For each substrate, the ß-oxidation activity was expressed as nmol/h/mg of protein extract and the C24:0/C16:0 ratio was determined.
Immunofluorescence
Cells were grown on glass slides for microscopy. Indirect immunofluorescence studies were performed as described (36). After Triton X-100 treatment, cells were incubated with the following commercially available antibodies: mouse monoclonal anti-ALDP antibody (1:500 dilution, ALD 1D6; Euromedex, Mundolsheim, France), mouse monoclonal anti-HA antibody (1:40; Roche), sheep anti-human catalase antibody (1:100; The Binding Site, Birmingham, UK), biotinylated anti-mouse antibody (1:200; Amersham Pharmacia), FluoroLink Cy2 Avidin (1:50; Amersham Pharmacia) and rhodamine-conjugated anti-sheep antibody (1:100; Accurate Chemical and Scientific, Westbury, NY).
Gas chromatographic analysis of VLCFA
For VLCFA determination, a confluent 75 cm2 flask of each cell type was harvested by trypsinization. The cell pellet was dispersed in 0.5–1.0 ml of distilled water, sonicated for 30 s at 70 W efficiency and the protein content determined. As an internal standard, 2.5 µg of C23:0 fatty acid was added with 5 ml of chloroform:methanol (1:1) to 0.5 ml of fibroblast homogenate (500 µg–2 mg protein) and kept at room temperature for 1 h. The protein precipitate was removed by centrifugation and Folch partition was performed (37). Fatty acid methyl esters were prepared according to ‘procedure 1’ for plasma samples (38) and analysed by gas chromatography as previously described (39).
Western blot analysis
Pelleted cells were resuspended in homogenization buffer (0.25 M sucrose, 10 mM Tris pH 8.0, 0.1 mM EDTA) and disrupted by sonification. Protein content was determined with Protein Assay (BioRad). Equal amounts of protein extract (between 10 and 100 µg of total protein per lane) were loaded on SDS–polyacrylamide gels (40) with a prestained molecular weight standard (BioRad), electrophoresed and transferred to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). Membranes were blocked with 4% non-fat dried milk powder in TBST (20 mM Tris pH 7.5, 500 mM NaCl, 0.05% Tween 20) before incubation with primary antibodies: anti-ALDP 1D6 (1:1000; Euromedex), anti-HA (1:267; Roche) and anti-PMP70 (1:2000; kindly provided by Dr Gärtner, Düsseldorf). As a detection system, biotinylated anti-mouse antibody (1:500; Amersham Pharmacia) and avidin peroxidase conjugate (1:40 000; Sigma) were used. For visualization, chemiluminescence substrate (Super Signal Pico; Pierce, Rockford, IL) or a chromogenic substrate (for anti-ALDP) were used according to the manufacturer’s instructions. For the PMP70 antibody, a goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (Jackson Research, West Grove, PA) was used at a 1:2000 dilution and detected with a chromogenic substrate.
Flow cytometric analysis
Between 7.5 x 105 and 1 x 106 cells were harvested by trypsinization and used for flow cytometric analysis. Equal amounts of cells in a total volume of 50 µl of phosphate-buffered saline (PBS) were incubated in 100 µl of fixation solution. Fixation was stopped by addition of PBS, cells were pelleted by centrifugation and resuspended in 100 µl of permeabilization solution at room temperature for 15 min as recommended (IntraStain Fixation and Permeabilisation kit; Dako, Copenhagen, Denmark). Permeabilized cells were incubated with anti-ALDP 1D6 (Euromedex), diluted 1:10 in permeabilization solution and FITC-conjugated anti-mouse (Dako) diluted 1:20 in permeabilization solution for 1 h each. The fluorophore-conjugated secondary antibody was incubated in the dark. Analysis of fluorescent cells was performed using Galaxy flow cytometer and FloMax software (Dako). Cells which were incubated with the secondary antibody served only as a negative control. A total of 20 000 cells were counted and the number of fluorescent cells was determined and their average fluorescence was measured.
|
ACKNOWLEDGEMENTS |
|---|
We thank Christina Truppe and Martina Krammer for excellent technical assistance. We are grateful to Drs Kirby Smith, Lelita Braiterman, Patrick Aubourg, Ron Wanders, Adalbert Roscher and Andreas Holzinger for providing the cell lines used in this study. We are also thankful to Dr Jutta Gärtner for the generous gift of the PMP70 antibody. This work was supported by Austrian Science Foundation, project P-12073-MED.
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +43 1 42 77 62 812; Fax: +43 1 42 77 96 28; Email: johannes.berger@univie.ac.at
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Moser, H.W., Smith, K.D. and Moser, A.B. (1995) X-linked adrenoleukodystrophy. In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), The Metabolic and Molecular Bases of Inherited Disease. McGraw Hill, New York, NY, pp. 2325–2349.
2 Aubourg, P. (1996) X-linked adrenoleukodystrophy. In Moser, H.W. (ed.), Handbook of Clinical Neurology, Vol. 22. Elsevier, Amsterdam, The Netherlands, pp. 447–483.
3 Moser, H.W. (1997) Adrenoleukodystrophy: phenotype, genetics, pathogenesis and therapy. Brain, 120, 1485–1508.
4 Fouquet, F., Min Zhou, J.-M., Ralston, E., Murray, K., Troalen, F., Magal, E., Robain, O., Dubois-Dalcq, M. and Aubourg, P. (1997) Expression of the adrenoleukodystrophy protein in the human and mouse central nervous system. Neurobiol. Dis., 3, 271–285.[Web of Science][Medline]
5 Aubourg, P., Blanche, S., Jambaque, I., Rocchiccioli, F. Kalifa, G., Naud-Sadreau, C., Rolland, M.O., Debre, M., Chaussain, J.L., Griscelli, C. et al. (1990) Reversal of early neurologic and neuroradiologic manifestations of X-linked adrenoleukodystrophy by bone marrow transplantation. N. Engl. J. Med., 322, 1860–1866.[Web of Science][Medline]
6 Krivit, W., Sungh, J.H., Shapiro, E.G. and Lockman, L.A. (1995) Microglia: the effector cell for reconstitution of the central nervous system following bone marrow transplantation for lysosomal and peroxisomal storage diseases. Cell. Transplant., 4, 385–392.[Web of Science][Medline]
7 Doerflinger, N., Miclea, J.-M., Lopez, J., Chomienne, C., Bougneres, P., Aubourg, P. and Cartier, N. (1998) Retroviral transfer and long-term expression of the adrenoleukodystrophy gene in human CD34+ cells. Hum. Gene Ther., 9, 1025–1036.[Web of Science][Medline]
8 Cartier, N., Lopez, J., Moullier, P., Rocchiccioli, F., Rolland, M.O., Jorge, P., Mosser, J., Mandel, J.L., Bougneres, P.F., Danos, O. et al. (1995) Retroviral-mediated gene transfer corrects very-long-chain fatty acid metabolism in adrenoleukodystrophy fibroblasts. Proc. Natl Acad. Sci. USA, 92, 1674–1678.
9 Shinnoh, N., Yamada, T., Yoshimura, T., Furuya, H., Yoshida, Y., Suzuki, Y., Shimozawa, Y., Orii, T. and Kobayashi, T. (1995) Adrenoleukodystrophy: the restoration of peroxisomal ß-oxidation by transfection of normal cDNA. Biochem. Biophys. Res. Commun., 210, 830–836.[Web of Science][Medline]
10 Netik, A., Forss-Petter, S., Holzinger, A., Molzer, B., Unterrainer, G. and Berger, J. (1999) Adrenoleukodystrophy-related protein can compensate functionally for adrenoleukodystrophy protein deficiency (X-ALD): implications for therapy. Hum. Mol. Genet., 8, 907–913.
11 Flavigny, E., Sanhaj, A., Aubourg, P. and Cartier, N. (1999) Retroviral-mediated adrenoleukodystrophy-related gene transfer corrects very long chain fatty acid metabolism in adrenoleukodystrophy fibroblasts: implications for therapy. FEBS Lett., 448, 261–264.[Web of Science][Medline]
12 Migeon, B.R., Moser, H.W., Moser, A.B., Axelman, J., Silence, D. and Norum, R.A. (1981) Adrenoleukodystrophy: evidence for X-linkage, inactivation and selection favoring the mutant allele in heterozygous cells. Proc. Natl Acad. Sci. USA, 78, 5066–5070.
13 Liu, L.X., Janvier, K., Berteaux-Lecellier, V., Cartier, N., Benarous, R. and Aubourg, P. (1999) Homo- and heterodimerization of peroxisomal ATP-binding cassette half-transporters. J. Biol. Chem., 274, 32738–32743.
14 Smith, K.D., Kemp, S., Braiterman, L.T., Lu, J.F., Wei, H.M., Geraghty, M., Stetten, G., Bergin, J.S., Pevsner, J. and Watkins, P.A. (1999) X-linked adrenoleukodystrophy: genes, mutations and phenotypes. Neurochem. Res., 24, 521–535.[Web of Science][Medline]
15 Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W. and Bujard, H. (1995) Transcriptional activation by tetracyclines in mammalian cells. Science, 268, 1766–1769.
16 Moser, H.W., Tutschka, P.J., Brown, F.R., Moser, A.B., Yeager, A.M., Singh, I., Mark, S.A., Kumar, A.A., McDonnell, J.M., White, C.L. et al. (1984) Bone marrow transplant in adrenoleukodystrophy. Neurology, 34, 1410–1417.
17 Malm, G., Ringden, O., Anvret, M., von Döbeln, U., Hagenfeldt, L., Isberg, B., Knuutila, S., Nennesmo, I., Winiarski, J. and Marcus, C. (1997) Treatment of adrenoleukodystrophy with bone marrow transplantation. Acta Paediatr., 86, 482–492.
18 Krivit, W., Peters, C. and Shapiro, E. (1999) Bone marrow transplantation as effective treatment of central nervous system disease in globoid cell leukodystrophy, metachromatic leukodystrophy, adrenoleukodystrophy, mannosidosis, fucosidosis, aspartylglucosaminuria, Hurler, Maroteaux-Lamy, and Sly syndromes, and Gaucher disease type III. Curr. Opin. Neurol., 12, 167–176.[Medline]
19 Krivit, W., Aubourg, P., Shapiro, E. and Peters, C. (1999) Bone marrow transplantation for globoid cell leukodystrophy, adrenoleukodystrophy, metachromatic leukodystrophy, and Hurler syndrome. Curr. Opin. Hematol., 6, 377–382.[Medline]
20 Krivit, W., Lockman, L.A., Watkins, P.A., Hirsch, J. and Shapiro, E.G. (1995) The future for treatment by bone marrow transplantation for adrenoleukodystrophy, metachromic leukodystrophy, globoid cell leukodystrophy and Hurler syndrome. J. Inher. Metab. Dis., 18, 398–412.[Web of Science][Medline]
21 Mazurier, F., Moreau-Gaudry, F., Salesse, S., Barbot, C., Ged, C., Reiffers, J. and de Verneuil, H. (1997) Gene transfer of the uroporphyrinogen III synthase cDNA into haematopoietic progenitor cells in view of a future gene therapy in congenital erythropoietic porphyria. J. Inherit. Metab. Dis., 20, 247–257.[Web of Science][Medline]
22 Hoogerbrugge, P.M., van Beusechem, V.W., Fischer, A., Debree, M., le Deist, F., Perignon, J.L., Morgan, G., Gaspar, B., Fairbanks, L.D., Skeoch, C.H. et al. (1996) Bone marrow gene transfer in three patients with adenosine deaminase deficiency. Gene Ther., 3, 179–183.[Web of Science][Medline]
23 Fairbairn, L.J. Lashford, L.S., Spooncer, E., McDermott, R.H., Lebens, G., Arrand, J.E., Arrand, J.R., Bellantuono, I., Holt, R., Hatton, C.E. et al. (1996) Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow. Proc. Natl Acad. Sci. USA, 93, 2025–2030.
24 Braiterman, L.T., Zheng, S., Watkins, P.A., Geraghty, M.T., Johnson, G., McGuiness, M.C., Moser, A.B. and Smith, K.D. (1998) Suppression of peroxisomal membrane protein defects by peroxisomal ATP binding cassette (ABC) proteins. Hum. Mol. Genet., 7, 239–247.
25 Imanaka, T., Aihara, K., Takano, T., Yamashita, A., Sato, R., Suzuki, Y., Yokota, S. and Osumi, T. (1999) Characterization of the 70 kDa peroxisomal membrane protein, an ATP binding cassette transporter. J. Biol. Chem., 274, 11968–11976.
26 Feigenbaum, V., Lombard-Platet, G., Guidoux, S., Sarde, C.O., Mandel, J.L. and Aubourg, P. (1996) Mutational and protein analysis of patients and heterozygous women with X-linked adrenoleukodystrophy. Am. J. Hum. Genet., 58, 1135–1144. [Web of Science][Medline]
27 Fuchs, S., Sarde, C.O., Wedemann, H., Schwinger, E., Mandel, J.L. and Gal, A. (1994) Missense mutations are frequent in the gene for X-chromosomal adrenoleukodystrophy (ALD). Hum. Mol. Genet., 3, 1903–1905.
28 Wichers, M., Kohler, W., Brennemann, W., Boese, V., Sokolowski, P., Bidlingmaier, F. and Ludwig, M. (1999) X-linked adrenomyeloneuropathy associated with 14 novel ALD-gene mutations: no correlation between type of mutation and age of onset. Hum. Genet., 105, 116–119.[Web of Science][Medline]
29 Takano, H., Koike, R., Onodera, O., Sasaki, R. and Tsuji, S. (1999) Mutational analysis and genotype-phenotype correlation of 29 unrelated Japanese patients with X-linked adrenoleukodystrophy. Arch. Neurol., 56, 295–300.
30 Ligtenberg, M.J., Kemp, S., Sarde, C.O., van Geel, B.M., Kleijer, W.J., Barth, P.G., Mandel, J.L., van Oost, B.A. and Bolhuis, P.A. (1995) Spectrum of mutations in the gene encoding the adrenoleukodystrophy. Am. J. Hum. Genet., 56, 44–50.[Web of Science][Medline]
31 Gärtner, J., Braun, A., Holzinger, A., Roerig, P., Lenard, H.G. and Roscher, A.A. (1998) Clinical and genetic aspects of X-linked adrenoleukodystrophy. Neuropediatrics, 29, 3–13.[Web of Science][Medline]
32 Watkins, P.A., Gould, S.J., Smith, M.A., Braiterman, L.T., Wei, H.M., Kok, F., Moser, A.B., Moser, H.W. and Smith, K.D. (1995) Altered expression of ALDP in X-linked adrenoleukodystrophy. Am. J. Hum. Genet., 57, 292–301.[Web of Science][Medline]
33 Mosser, J., Douar, A.M., Sarde, C.O., Kioschis, P., Feil, R., Moser, H., Poustka, A.M., Mandel, J.L. and Aubourg, P. (1993) Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature, 361, 726–730.[Medline]
34 Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl Acad. Sci. USA, 77, 5201–5205.
35 Watkins, P.A., Ferrell, E.V., Pedersen, J.I. and Hoefler, G. (1991) Peroxisomal fatty acid ß-oxidation in HepG2 cells. Arch. Biochem. Biophys., 289, 326–336.
36 Keller, G.A., Gould, S., Deluca, M. and Subramani, S. (1987) Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc. Natl Acad. Sci. USA, 84, 3264–3268.
37 Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226, 497–509.
38 Moser, H.W. and Moser, A.B. (1991) Measurement of saturated very long chain fatty acids in plasma. In Hommes, F.A. (ed.), Techniques in Diagnostic Human Biochemical Genetics. Wiley-Liss, New York, NY, pp. 117–191.
39 Forss-Petter, S., Werner, H., Berger, J., Lassmann, H., Molzer, B., Schwab, M.H., Bernheimer, H., Zimmermann F. and Nave, K.A. (1997) Targeted inactivation of the X-linked adrenoleukodystrophy gene in mice. J. Neurosci. Res., 50, 829–843.[Web of Science][Medline]
40 Laemmli, U.K. (1970) Cleavage of structural proetins during the assembly of the head of bacteriophage T4. Nature, 227, 680.[Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  I. Weinhofer, S. Forss-Petter, M. Zigman, and J. Berger Cholesterol regulates ABCD2 expression: implications for the therapy of X-linked adrenoleukodystrophy Hum. Mol. Genet., October 15, 2002; 11(22): 2701 - 2708. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. N. O'Neill, M. Aoki, and R. H. Brown Jr. ABCD1 translation-initiator mutation demonstrates genotype-phenotype correlation for AMN Neurology, December 11, 2001; 57(11): 1956 - 1962. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||