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Human Molecular Genetics Advance Access originally published online on October 15, 2004
Human Molecular Genetics 2004 13(23):2997-3006; doi:10.1093/hmg/ddh323
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Human Molecular Genetics, Vol. 13, No. 23 © Oxford University Press 2004; all rights reserved

Functional overlap between ABCD1 (ALD) and ABCD2 (ALDR) transporters: a therapeutic target for X-adrenoleukodystrophy

Aurora Pujol1,*, Isidre Ferrer2, Carme Camps1,3, Elisabeth Metzger1, Colette Hindelang1, Noëlle Callizot4, Montse Ruiz3, Teresa Pàmpols3, Marisa Giròs3 and Jean Louis Mandel1

1Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP and Collège de France, BP 10142, 67404 Illkirch Cedex, CU de Strasbourg, France, 2Institut de Neuropatologia, Hospital Universitari de Bellvitge, 08907 L'Hospitalet de Llobregat, Barcelona, Spain, 3Institut de Bioquimica Clinica, c/Mejia Lequerica, s/n 08028 Barcelona, Spain and 4Societé Neurofit, SA 67404 Illkirch Cedex, CU de Strasbourg, France

Received August 15, 2004; Accepted October 1, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disease caused by loss of function of the peroxisomal transporter ABCD1 (ALD), which results in accumulation of very long chain fatty acids (VLCFAs) in organs and serum, central demyelination and peripheral axonopathy and Addison's disease. Knockout of the ALD gene in the mouse (ALD–) results in an adrenomyeloneuropathy-like disease (a late onset form of X-ALD). In the present study, we demonstrate that axonal damage occurs as first pathological event in this model, followed by myelin degeneration. We show that this phenotype can be modulated through expression levels of an ALD-related gene (ALDR/ABCD2), its closest paralogue and a target of PPAR{alpha} and SREBP transcription factors. Overexpression of ALDR in ALD– mice prevents both VLCFAs accumulation and the neurodegenerative features, whereas double mutants for ALD and ALDR exhibit an earlier onset and more severe disease (including signs of inflammatory reaction) when compared with ALD single mutants. Thus, our results provide direct evidence for functional redundancy/overlap between both transporters in vivo and highlight ALDR as therapeutic target for treatment of X-ALD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Functional gene redundancy is a widespread mechanism in vertebrate evolution, thought to increase robustness against deleterious mutations by ‘backing-up’ vital functions for the cell or the organism (1,2). Evidence for overlapping function within gene families has been found among transcription factors (3), homeotic genes (4), signal transduction proteins (5) and metabolic pathway genes (6). In a human genetic disease, a related gene whose function overlaps with the defective gene function may be considered as therapeutic target. Strategies aiming at pharmacologically inducing functionally related genes can circumvent many hurdles of gene- and cell-based therapy (such as adverse immunological response against a neoantigen, or targeting to the relevant tissue). Well-known examples for such approaches include the compensatory drug-mediated induction of fetal haemoglobin in ß-globin disorders (7,8), the upregulation of utrophin in the dystrophin-deficient muscles of Duchenne's muscular dystrophy (9,10) or very recently, the stimulation of SMN2 transcription by valproic acid as potential therapy for spinal muscular atrophy (11). The work presented here aims at elucidating whether a similar scenario could be applied to X-linked adrenoleukodystrophy (X-ALD).

X-ALD (OMIM number 300100) is a severe neurological disorder presenting with central or peripheral demyelination and impaired function of adrenals, and the most common peroxisomal disease, with a minimum incidence of 1 in 20 000 in USA (12) and 1 in 15 000 in France (13). X-ALD patients accumulate very long chain fatty acids (VLCFA) in plasma and tissues, notably in the adrenal cortex and nervous system. The two main neurological phenotypes are the severe childhood cerebral form (CCALD), which is rapidly progressing and associated with an inflammatory response in the brain white matter, and the slowly progressive adult adrenomyeloneuropathy (AMN), which presents with distal axonopathy in spinal cord and peripheral neuropathy (reviewed in 14). The disease is caused by point mutations or deletions in the ALD (ABCD1) gene that inactivate the ALD protein (ALDP). However, the various forms of the disease may occur associated to the same mutation, even within the same family (13,14). This striking absence of phenotype–genotype correlations suggests the existence of modifier genes, modifying environmental conditions and/or stochastic factors. A mouse model of the disease (Abcd1 knockout mouse, here called ALD–) also accumulates VLCFAs in target organs, and has recently been shown to develop an AMN-like phenotype (15).

The ALD gene encodes a peroxisomal ABC transporter protein (ALDP), which belongs to a small family of peroxisomal transporters that includes three other members: ABCD2 or ALDR (ALD-related protein) (16), ABCD3 or PMP70 (17) and ABCD4 or PMP70R (18). Because of the biochemical phenotype of patients, and of yeast lacking one or both of the two yeast ABCD transporters (Pxa1p and Pxa2p), it is proposed that ABCD1 is implicated in the import of VLCFAs into the peroxisome (19,20). The functions of the three other transporters in higher eukaryotes are unknown, but their sequence homology indicate that they might have related or overlapping function(s) in peroxisomal fatty acid metabolism. For instance, PMP70 has recently been reported to be involved in the catabolism of medium chain fatty acids, as well as branched fatty acids like pristanic and phytanic acid (21). The possible functional equivalence of ALD and ALDR has been repeatedly suggested on the basis of their high sequence similarity (88% similarity and 67% identity at the protein level) (16), and the ability of ALDR to complement the biochemical defect in X-ALD fibroblasts when overexpressed (22). The two paralogues are differentially expressed in a mirror-like pattern, especially in brain (23). Interestingly, expression of the ALDR gene can be induced by fenofibrate (a PPAR{alpha} agonist) (24,25), thyroid hormone (26) and 4-phenylbutyrate (4PBA) (27) in the mouse and by cholesterol depletion via SREBP in the human monocytes/macrophages cell line THP-1 (28), which makes the ALDR gene a good pharmacological target for X-ALD therapy. Dietary treatment with 4PBA in ALD– mice has been shown to partially correct the accumulation of VLCFAs in brain and adrenal glands, correlating with ALDR protein induction and increased peroxisome proliferation (27). However, this study fell short of discriminating between whether the effect on VLCFAs was due to increased levels of ALDR, and/or to the concomitant peroxisome proliferation, or other pleiotropic effects caused by the drug, such as the correction of mitochondrial dysfunction (29).

To investigate the postulated functional redundancy of ALD and ALDR transporters in vivo, we undertook a dual strategy: on one hand, a gain-of-function approach by transgenesis in the mouse using a strong ubiquitous promoter, the chicken ß-actin, to drive the expression of ALDR, and on the other hand, the inactivation of ALDR by homologous recombination. In the current work, we describe that stable overexpression of ALDRP in the ALD– mouse background leads to full correction of VLCFA levels in target organs. This normalization of the biochemical phenotype correlates with a striking improvement of the neurological AMN-like phenotype presented by ALD– mice, demonstrating that ALDR can compensate for the absence of ALD. In parallel experiments, we have created an ALDR–/– mouse and double ALD–/ALDR–/– mutants. Our results demonstrate an accelerated and more severe neurological phenotype in the double KO mice, consistent with the concept of a high degree of overlapping functions between the two transporters. Thus, our data strengthen the potential of stimulating ALDR expression as feasible approach to X-ALD treatment.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of ALDR overexpressing and double ALD/ALDR mutant mice
The inactivation of the murine ALDR gene by gene targeting (classical homologous recombination), as described elsewhere (A. Pujol et al., unpublished data), leads to total absence of the protein as shown by western blotting (Fig. 1A). ALDR–/– animals were viable, fertile and showed no overt pathology or behavioural abnormalities until at least 6 months of age. By crossing the ALDR–/– to ALD–, we generated double mutants. In parallel experiments, we chose the ubiquitous chicken ß-actin promoter to direct transgene expression of the murine ALDR cDNA in a broad range of tissues including the main sites of pathology, such as brain, spinal cord, adrenal gland and sciatic nerve. After Southern analysis, transgene expression was assessed by western blotting. Consistent with previous reports (30), transgene expression was detected in all organs analysed (brain, spinal cord, adrenal gland, kidney, sciatic nerve, skeletal muscle, intestine and skin). One line was selected on the basis of the higher expression levels of the ALDR protein in the main target organs of interest: spinal cord, sciatic nerve and adrenal gland (5–10-fold overexpression when compared with wild-type levels, Fig. 1A). These transgenic mice are referred to as ‘tg ALDR’, and were then crossed with ALD– mice to produce ALD–/tgALDR and their control littermates, ALD+/tgALDR. All mice described were born at expected Mendelian ratios and developed to maturity without showing any abnormal clinical signs.



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  		Figure 1. (A) Expression and functional verification of the ALDR transgene and knock-out lines. Protein extracts from adrenal gland, sciatic nerve and spinal cord were assayed for ALDRP expression by western blot analysis with ALDR antibodies. Each lane was loaded with 100 µg protein concentration from tissues, as verified with anti-{gamma} tubulin antibodies. No expression of ALDR protein is detected in ALDR–/– mice, whereas the transgenic line (tg) shows expression levels of ALDR 5–10-fold higher than wild-type littermates. (B–G) Histopathology of adrenal gland at 12 months. Araldite–Epon sections through adrenal cortex. (B) Wild-type mouse. (C) ALD mutants show fibrosis at the corticomedular junction (arrows) that is prevented through ALDR overexpression in (D). (E) Double mutants exhibit a greater accumulation of lipid droplets in fascicular and reticular cell layers. (F) Cholesterol needles are a characteristic feature found in lysosomes of ALD mutants. (G) In double mutants, the cholesterol needles are not restricted to lysosomes, but are scattered all over the cytoplasm and are much more abundant. Magnification: B,C,D,E: 300x; F: 18 000x; G: 12 000x.

 
ALDR expression levels modulate the ALD phenotype in the mouse
VLCFA levels.
We analysed levels of VLCFAs in whole tissue homogenates of the main organs of pathology and serum, for the different genotypes (Table 1). In 8-month-old mice, we found that ALD inactivation leads to elevated levels of C26:0 in spinal cord and adrenal gland in agreement with previous reports (31–33). Accumulation of VLCFAs in ALD– deficient peripheral nerves had not yet been described; we found it to be in a similar range than in spinal cord (around 6-fold).


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  		Table 1. VLCFA content in mouse tissues at 8 months of age
 
The absence of ALDR protein in central or peripheral nervous system did not seem to affect levels of VLCFAs. However, in the adrenal gland, we observed a 2-fold accumulation of C24:0 and C26:0 for ALDR–/–, whereas the double mutants showed even higher levels of C24:0 and C26:0 than for ALD– or ALDR–/–. Interestingly, the C22:0 metabolite accumulated specifically in ALDR–/– adrenals and reached a 4-fold increase in double mutants. In serum, double mutants showed a significantly higher accumulation of C26:0 levels than single ALD or ALDR mutants (P<0.01).

Most importantly, overexpression of the ALDR protein in the ALD– background normalized VLCFA levels in all organs analysed. Furthermore, we noted a marked tendency to lowering levels of C24:0 and C26:0 upon ALDR overexpression in a ALD+ background. Thus, the two transporters share overlapping functions with respect to accumulation of C24:0 and C26:0 fatty acids.

Adrenal gland histopathology.
Adrenal glands were subjected to standard histological examination and electron microscopy. On semi-thin sections, adrenals of 18-month-old ALD– mice exhibited pronounced fibrosis of the reticular and fascicular cell layers (Fig. 1C, arrows). Overexpression of the ALDR protein prevented the onset of this pathological feature (Fig. 1D), whereas double knockout presented a dramatic accumulation of lipid droplets across the adrenal cortex, encompassing glomerular, fascicular and reticular cell layers (Fig. 1E). At the ultrastructural level, we observed in the ALD– mice (in particular in the fascicular cell layer of cortex and cortico-medullary junction), intralysosomal needle-like cholesterol inclusions, which are also seen in macrophages of ALD patients (34) (Fig. 1F). In ALD–/ALDR–/– mice, however, these inclusions were spread all over the cytoplasm, and are encountered ~10 times more often than in ALD– mice (Fig. 1G). These needle-like structures were not found in 12- or 20-month-old ALD–/tgALDR animals.

Electrophysiology of the PNS.
We studied two electromyographic parameters: the compound muscle action potential representative of fast conductive, mainly motor fibres (CMAP) and the sensitive nerve conduction velocities (SNCV). At 12 months of age, and consistent with our previous results (15), we found that ALD– mice had no altered SNCV or CMAP waves. On the contrary, the latency of motor wave and the SNCV were slowed down in double mutants (Table 2). ALDR mutants showed no detectable abnormality at this precise age. At 20 months, ALD mutants exhibit slowing of both motor and sensitive conduction velocities. The absence of ALDR expression does not seem to influence these parameters, since ALDR–/– mice show normal waves pattern, and double mutants do not have more important abnormalities than ALD– mice at this age. Overexpression of ALDR protein prevented the phenotype due to the lack of functional ALDP, regarding both motor and sensitive waves. Mice overexpressing the ALDR protein on wild-type background (ALD+/tgALDR) were analysed and no difference with respect to control littermates was detected in 20-month-old animals (Table 2).


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  		Table 2. Peripheral nerve conduction studies
 
Spinal cord histopathology.
We had previously detected myelin and axonal degeneration in spinal cord parenchyma of ALD– ageing animals (15). Here, we addressed the question whether myelin disturbances precede or are secondary to the axonal pathology, by studying our various models at two different time points: 12 and 22 months of age (Table 3). At 22 months, ALD– mice present signs of microglia and astrocyte activation, numerous lectin-positive cells with morphological characteristics of ramified microglia/macrophages, and glial fibrillary acidic protein (GFAP) positive cells (Fig. 2A–H). Myelin debris is a prevalent feature as revealed with Sudan black staining (Fig. 2M–P). Immunoreactivity against amyloid precursor protein (APP, Fig. 2I–L) and synaptophysin (Fig. 2Q–T) is often found along axonal swellings, strongly suggesting axonal damage. Those lesions are ubiquitinated (Fig. 2U–X), but they do not colocalize with myelin lesions as shown by serial sections analysis. A very similar type of pathology is found in ALDR–/– (Table 3), and in a more severe manner, in double mutant mice. Remarkably, all pathological features are prevented upon ALDR overexpression (ALD–/tgALDR mice at 22 months of age, n=4 mice out of 5 analysed). However, one animal did show mild lesions, similar to those found in double mutants at 12 months of age (Table 3). No abnormalities were found in 22-month-old mice overexpressing ALDR (ALD+/tgALDR).


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  		Table 3. Summary of pathological findings in spinal cord
 


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  		Figure 2. Gliosis, myelin and axonal pathologies in spinal cord. Longitudinal sections of the dorsal spinal cord in wild-type (wt, ALD–, ALD–/ALDR–/– and ALD–/tgALDR) mice at the age of 22 months processed for GFAP, lectin Lycopericon esculentum, APP, Sudan black and synaptophysin. Reactive astrogliosis is observed in ALD– and ALD–/ALDR–/– mice when compared with wt (A–C and E–g). APP deposits are seen in axons in ALD– and ALD–/ALDR–/– mice, arrows in (J) and (K). This is accompanied by lipidic debris of myelin, as revealed with Sudan black, arrows in (N) and (O). No similar findings are found in WT mice (I, M). Abnormal fibres filled with synaptophysin are found in ALD– and 2KO mice (R, S). APP and synaptophysin deposits are ubiquitinated (V, W). Prevention of lesions occurs in ALD–/tgALDR mice (D, H, L, P, T, X). For semi-quantitative analysis, see Table 3. Paraffin sections, slight haematoxylin counterstaining. (A–H), bar in (H)=25 µm. (I–X), bar in (X)=10 µm.

 
Despite the degenerative changes and astrogliosis, there was no evidence of increased apoptosis, as estimated by the biotinylated UTP nick end labelling method and caspase-3 (17 kDa) immunohistochemistry.

Analysis of 12-month-old sections revealed that the first and most reliable marker of pathology was found to be synaptophysin-immunoreactive deposits in axonal swellings of ALD– and ALDR–/– mice, and to a larger extent in double mutant mice. Thus, axonal degeneration precedes myelin disturbances in our mouse models, resembling findings in AMN patients (35).

Furthermore, by routine haematoxylin–eosin and tolu-idin blue stainings on semi-thin sections, we detected a few inflammatory cell infiltrates in double mutant animals, which were not found in either single ALD– or ALDR–/– mice. Those infiltrates were typically in small clusters (10–20 cells) adjacent to blood vessels and perimeningeal areas, and were composed mainly of T lymphocytes (Fig. 3A–C).



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  		Figure 3. Inflammatory infiltrates of cervical and lumbar spinal cord regions. (A) Toluidin blue staining of representative sections of double mutants at 20 months. Inflammatory cells are found in perivascular, perimeningeal and Virchow–Robin spaces. (B) Electron micrographs reveals a cluster of T lymphocytes at the Virchow-Robin space. (C) In a perivascular infiltrate, the majority of cells are CD3+ positive T cells. Behaviour analysis testing. Motor performance of the different mouse lines in a rotarod test, at 15 months in (D) and 20 months of age in (E). (F) Number of rearings at 20 months (*P<0.01 when compared with wt). (G) Locomotor activity of ALD– mice during an open field test of 15 min of duration, expressed as the means of crossed squares/min at 20 months. ALD– are different from wt (P<0.001), ALD–/tgALDR and ALD–/ALDR–/– (P<0.01). Groups (n=9–16 mice) are compared statistically by ANOVA and Scheffe's tests. Error bars represent the SD from the mean.

 
Neurological phenotype.
At 20 months of age, ALD– mice presented an impairment of their locomotor coordination in the rotarod test (Fig. 3E) and exploratory abilities in the open field test (Fig. 3G). Upon ALDR overexpression, these deficits were significantly prevented (Fig. 3E and G), to almost normal levels. Performance in the rotarod was considerably worsened for double mutant animals, which exhibited severe impairment already at 15 months of age (Fig. 3D). In the open field, double mutants behaved even less explorative than ALD– mice (P<0.01), and had a marked tendency to freezing (long latency before starting the movement) (Fig. 3G). Two sets of ALD+/tgALDR mice (at 12 and 20 months of age, n=8 and 9, respectively) were tested in the open field and rotarod, scoring similar than their wild-type littermates (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We had recently uncovered a late onset and progressive neurodegenerative phenotype in ALD– mice resembling AMN in patients (15). In the present study, we have gained insight into the physiopathology of the AMN-like phenotype in ALD– mice by showing evidence of early axonal damage in these mutants using ß-APP and synaptophysin as markers. The APP gene is differentially expressed in axotomized sensory and motor systems, and the transmembrane protein product APP is accumulated in damaged axons in a wide variety of lesions of the central nervous system (36–39). The synaptic vesicle-associated protein synaptophysin is accumulated in axonal swellings in acute traumatic injury and in dystrophic axons, probably as a result of impaired axonal transport (40). These lesions precede the appearance of myelin debris and residual bodies in axonal swellings, thus indicating that myelin degeneration occurs as a con-sequence or, at least, after axonal damage in these mutants. This is consistent with findings in AMN patients, which suggested that the fundamental lesion is an axonopathy (35).

The work presented here takes benefit of this model to address the question of functional redundancy between ALD and ALDR in vivo. Our results unequivocally demonstrate that overexpression of ALDR can fully correct the ALD-dependent accumulation of VLCFAs in the mouse tissues analysed. This rescue of the biochemical phenotype is translated into a striking long-term prevention of the neurodegenerative features due to ALD loss of function, up to 20–22 months of age. Thus, the functional redundancy of ALD and ALDR that was observed in cell culture systems is indeed operating in vivo. ALDR overexpression is thus a plausible therapeutic strategy, as it prevents the whole panoplies of alterations caused by ALD loss in the mouse, from the biochemical abnormalities to the pathological and neurological manifestations. Furthermore, we show that 5–10-fold overexpression of ALDR in a broad range of tissues is not detrimental to the mouse as far as we could analyse. Indeed, ALD+/tgALDR and wt/tgALDR mice behaved like control littermates in all neurological, electrophysiological and histopathological tests described in the present manuscript. This fact suggests that precise tissue-specific upregulation may not be necessary for therapeutic benefit to patients.

The correlation between biochemical and pathological features is not a straightforward issue in X-ALD. Plasma VLCFA levels in patients are not related to phenotype onset or severity, nor does their normalization through a dietary treatment (Lorenzo's oil, a mixture of erucic and oleic acids) seems to be curative or prevents disease onset (14,41). Definitive proof pointing at VLCFA accumulation as pathogenesis-causative in CNS is lacking at present, although elevated levels of VLCFA have been shown to be detrimental to adrenocortical cells in culture (34,42) and to model membranes (43). In the nervous system of our models, a complex correlation between biochemical and pathological findings was observed. ALDR fully compensates for ALD loss upon overexpression, but its inactivation does not cause VLCFA accumulation in whole homogenates of CNS (brain and spinal cord) or PNS. Double mutants exhibit the same range of VLCFA accumulation than ALD mutants (at least at 8 months of age) in spite of a more severe and earlier onset neurological phenotype at the histological, behavioural and electrophysiological levels. The more severe pathology in the CNS and PNS of the double KO mouse contrast with the finding of equivalent levels of VLCFAs in the nervous system, compared with the single KO mice. This raises the question whether accumulation of VLCFAs represents the major cause of pathology or whether accumulation of unknown substrates of ALD or ALDR may also play a role in the disease mechanism in the mouse. In contrast, in adrenals, ALDR inactivation results in an increase of C22:0 (unlike ALD knockouts) and C24:0 fatty acids (similar levels than in ALD– mice). Consistently, in double knockouts we found a synergistic accumulation of C22:0, C24:0 and C26:0, which correlated with a very marked increase in numbers of speckles and cholesterol needles, together with a more dramatic accumulation of lipid droplets in virtually all adrenal cortex cell layers (fascicular, reticular and glomerular), indicating a relevant functional interaction between the two transporters. The fact that we specifically found accumulation of VLCFAs in adrenals of ALDR knockouts but not in other organs may indicate a more important role of ALDR in the ß-oxidation of these compounds in adrenals than for instance, in sciatic nerve or spinal cord. This would be in agreement with higher expression levels of ALDR in adrenals [Fig. 1A and quantitative (Q)-PCR, data not shown]. A synergistic effect between ALD and ALDR is also seen in serum, since only double mutants show significantly higher accumulation of C26:0.

It is remarkable that, in spite of a high degree of functional redundancy between ALD and ALDR, ALD-mice (that express ALDR at normal, endogenous levels) do show phenotype. Many reasons can account for this fact, for instance that the endogenous ALDR gene is 5–10 times less expressed in physiological conditions than in the transgenic mice, and its normal expression pattern is not ubiquitous, being predominantly expressed in neurons in the CNS. We have indeed searched (by Q-PCR) for an eventual compensatory upregulation of ALDR in ALD-mice but found no differences with respect to wild-type littermates. Alternatively and most likely, specificity of transported substrates is overlapping to some degree, but not identical between the two transporters. Thus, ALDR would be less efficient at substituting for ALD's function, and its overexpression would therefore be required to reach therapeutic benefit.

One useful aspect of the double knockouts is that the AMN-like pathology is more severe with an earlier onset (~12 months rather than 20 months in the ALD KO). This should facilitate experimental approaches towards therapeutic intervention (for instance by delivery of neurotrophic factors) and analysis of pathogenic mechanisms. Furthermore, double knockouts present inflammatory infiltrates in the spinal cord composed mainly of T lymphocytes, pathological features that are not present in single ALD or ALDR mutants. Infiltrating T lymphocytes (CD8+) are often found in unaffected white matter of ALD and AMN/ALD patients, and also in acute demyelinative lesions together with macrophages (44). Thus, this suggests that double knockouts may be useful to analyse the mechanisms of the inflammatory reaction that plays a major role in CCALD pathology in humans. So far, the only proven therapy available for X-ALD, at least the CCALD phenotype, is bone marrow transplantation (45,46). Our results provide an attractive target for pharmacological treatment of ALD (all types of phenotypes, from CCALD to AMN), since upregulation of ALDR would substitute for the defective function of the ALD gene right at the first step of the pathological cascade, the biochemical dysfunction. In addition and since 75% of the mutations in the ALD gene lead to lack of protein, gene therapy approaches using the ALD cDNA might induce an immune reaction against the ALDP. Gene transfer of ALDR could also circumvent this inconvenience, as is the case for utrophin in mdx (dystrophin deficient) muscles (47). Altogether, and within the limits of potential interspecies differences, our findings warrant further efforts aiming at upregulation of ALDR as a therapy for X-ALD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of transgenic mice
The full-length cDNA of murine ALDR gene was introduced into a pCAGGS expression vector, downstream of the chicken ß-actin promoter, cytomegalovirus enhancer, ß-actin intron and bovine globin poly-adenylation signal (48). The entire insert with the promoter and coding sequence was excised with BamHI and SalI and gel-purified through sucrose gradient. Transgenic mouse clones were produced by microinjecting the purified fragment into C57BL/6J/129Sv fertilized eggs, and transferred into oviducts of foster mothers (Charles River laboratory, France). Identification of transgenic founders was conducted by Southern blot and by PCR analysis on mouse tail genomic DNA with the following primers: XF207 5'-GAAATACCACACTCATCTAT-3' and WY51 5'-ATTGGCCACACCAGCCACCA-3' (on the rabbit ß-globin enhancer). These primers amplify a 402 bp segment of the transgene, under the following PCR conditions: 95°C for 10 s, 55°C for 30 s, 68°C for 60 s for 30 cycles. A total of seven founders carrying the transgene were generated. Transgene expression was assessed by western blotting and compared with wild-type levels (Fig. 1A).

Mouse breeding
The generation of ALD-deficient mice and their genotyping has been described (32). The ALDR–/– was inactivated by homologous recombination as described (A. Pujol et al., unpublished data). To obtain double heterozygous mutants, we crossed ALD–/+ females with ALDR–/– mice. Double heterozygous were intercrossed to obtain double knockout mice and wild-type littermate controls; the offspring obeyed mendelian ratios. All mice used were on a mixed C57BL/6J/129Sv background (~87% C57BL/6J/13%129Sv). Crucial experiments (rotarod, open field tests and EMG) were confirmed with pure C57BL/6J background animals.

All methods employed in this work are in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications No. 85-23, revised 1996). Histology, morphometry, behavioural testing and electrophysiological experiments were performed in a blind way with respect to the animal's genotype.

Behavioural testing
The rotarod and locomotor activity in open field tests were performed essentially as described (15) with naive male mutant and wild-type litter mate controls. We used a rotarod apparatus from Bioseb, Paris, France; diameter of the axis: 3 cm. The animals were trained 2 days before the day of the test. They were placed on the rotarod with an initial speed of rotation of 4 rpm, which was progressively increased to 10 rpm (in 2 min). When the rotarod speed reached 10 rpm, we recorded the time until the mice fell off the rotarod. The test was stopped arbitrarily at 180 s. Three trials per animal were recorded and the mean was taken as characteristic value. Statistical comparisons between groups were done using ANOVA followed by Scheffe's test (Statview5 package). Significance was set at P<0.01.

ALDR transgene expression
Tissues were removed from euthanized mice and flash-frozen on liquid nitrogen. Frozen tissues were homogenized in extraction buffer (75 mM Tris, pH 6.8, 3.8% SDS, 4 M urea, 20% glycerol, 5% ß-mercaptoethanol), boiled for 5 min and centrifugated. We measured protein concentration of the supernatant with a Protein Assay Kit (Biorad Life Science: 100 mg of total protein was quantified). In total, 100 µg were loaded onto each lane of 8% polyacrylamide gels for 90 min at 100 mV. Resolved proteins were transferred to nitrocellulose. ALDR protein expression was detected using a 1:1000 dilution of a rabbit polyclonal antibody against the C-terminal peptide of the murine ALDR protein and made visible with anti-rabbit IgG linked to horseradish peroxidase, and ECL western blotting analysis system (Amersham Biosciences). {gamma}-Tubulin was used as internal standard and detected using monoclonal antibody from Sigma.

Lipid analysis
VLCFA (C22:0) determination was done in the lipidic lower phase obtained after chloroform/methanol extraction, from different mouse tissues. A volume of 0.1 ml of lower phase or serum was subjected to a direct one-step transmethylation reaction. Subsequently, VLCFA methyl esters (VLCFAME) were quantified by capillary gas–liquid chromatography, on a high-resolution gas chromatograph 8000 Mega series (Fisons Instruments) equipped with split/splitless injector system and flame ionization detector. The column used was a DB-1 (J&B) (30 mx0.25 mm) with 0.25 µm film thickness. Quantification and identification of each compound was made with the respective VLCFAME standarization curve using C19:0 and C27:0 as internal standards.

Electrophysiological recordings
Electromyographical recordings were performed using a Neuromatic 2000M electromyograph (Dantec, Les Ulis, France). Mice were anaesthetized with intraperitoneal injection of 60 mg/kg ketamine chlorhydrate (Imalgène 500®, Rhône Mérieux, Lyon, France).

Compound muscle action potential (CMAP) and distal latency were recorded in the gastrocnemius muscle after stimulation of the sciatic nerve. A reference electrode and an active needle were placed in the hindpaw. A ground needle was inserted on the lower back of the mouse. The sciatic nerve was stimulated with a single 0.2 ms2 square pulse at a supramaximal intensity (12.8 mA). The amplitude (mV) and the latency of the motor wave were recorded. SNCV of the caudal nerve of the tail was also recorded. The caudal nerve was stimulated with a series of 20 pulses (for 0.2 ms) with an intensity of 12.8 mA.

Electron microscopy
Animals were anaesthetized as described above and perfused with a 4% PFA and 3% of glutaraldehyde (Fluka) in phosphate buffer (pH 7.4, 0.1 M) solution. Spinal cord and adrenals were dissected and fixed overnight. Tissue was post-fixed for 1 h in 1% osmium tetroxide in phosphate buffer; dehydrated in serial ethanol solutions and embedded in an Araldite–Epon mixture. Embedded tissues were then placed at +60°C for 2 days to polymerize. Transverse semi thin sections, 1 µm in thickness, were prepared with an ultramicrotome and stained with PAS and toluidine blue. Ultrathin sections (50 nm) were observed on Philips C12-208 electron microscope.

Immunohistochemistry
Mice were perfused with a 4% PFA solution. Spinal cords were embedded in paraffin and serial sections, 5 µm thick, were cut in the longitudinal plane with a sliding microtome. The sections were stained with haematoxylin and eosin, luxol fast blue-Klüver–Barrera and Sudan black, or processed for immunohistochemistry to glial fibrillary acidic protein (GFAP, Dako, rabbit polyclonal, 1:500), ubiquitin (Dako, rabbit polyclonal, 1:500), APP (Boehringer, 1:10), synaptophysin (Dako, monoclonal, 1:500), Ki67 (Dako, monoclonal, 1:20), cleaved caspase-3 (Cell Signalling, rabbit polyclonal, 1:25) and lectin Lycopericon esculentum (Sigma, L-0651, 1:200) used as a marker of microglial cells. The sections were incubated with the modified labelled streptavidin (LSAB) technique (DAKO LSAB2 System Peroxidase). Some sections in every case were processed for the method of in situ end-labelling of nuclear DNA fragmentation (Apoptag, Oncor) following the instructions of the supplier. The number of abnormal specific profiles was counted in every 10 section for each particular stain. At least three sections corresponding to the dorsal columns of the spinal cord were analysed per animal and per stain. To identify T lymphocytes, we used a monoclonal anti-CD-3 antibody from SantaCruz as marker, at 1:500 dilution.


   ACKNOWLEDGEMENTS
 
We are indebted to Drs Patrick Aubourg, James Powers, Stéphane Fourcade, Sander Houten and Anne-Sophie Korganow for fruitful discussions. ALD mutant mice were a kind gift of Dr Kirby Smith (KKI, Baltimore, MD, USA). We thank C. Kretz, L. Reutenauer, E. Vega, R. Blanco and M. Carmona for priceless technical assistance, and Drs Marianne Lemeur and Andrée Dierich and the staff at the IGBMC animal facility for mouse care. This study was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg (HUS), the Concerted Action of the European Commission ‘Peroxisomal Leukodystrophy’ (EU contract no. BMH4-CT96-1621), the Association Française contre les Myopathies, the European Leukodystrophy Association, the Fondo de Investigacion Sanitaria (FIS, Ministerio de Sanidad project no. 01/1667) and the Spanish research network REDEMETH.


   FOOTNOTES
 
* To whom correspondence should be addressed. Email: apujol{at}igbmc.u-strasbg.fr


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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