X-linked adreno-leukodystrophy is a progressive, systemic peroxisomal disorder that affects primarily nervous system myelin and axons as well as the adrenal cortex. Several divergent clinical phenotypes can occur in the same family; thus, there is no correlation between the clinical phenotype and the mutation in the ABCD1 gene in this disease. The most urgent and unresolved clinical issue is the fulminant inflammatory (immune) demyelination of the central nervous system in which a variety of cellular participants, cytokines, and chemokines are noted. A knockout mouse model exhibits mitochondrial deficits and axonal degeneration, but not inflammatory demyelination. To determine whether oxidative stress and damage might play a pathogenic role, we assessed standard biochemical and immunohistochemical markers of such activity both in our knockout mouse model and patients. We find that oxidative stress, as judged by increased immunoreactivity for the mitochondrial manganese-superoxide dismutase, is present in the knockout mouse liver, adrenal cortex, and renal cortex, tissues that normally express high levels of ABCD1 but no evidence of oxidative damage. The brain does not exhibit either oxidative stress or damage. On the other hand, both the human adrenal cortex and brain show evidence of oxidative stress (e.g. hemoxygenase-1 and manganese-superoxide dismutase) and oxidative damage, particularly from lipid peroxidation (4-hydroxynonenal and malondialdehyde). The presence of nitrotyrosylated proteins is strong circumstantial evidence for the participation of the highly toxic peroxynitrite molecule, whereas the demonstration of interferon gamma and interleukin-12 is indicative of a TH1 response in the inflammatory demyelinative lesions of the cerebral phenotype. These differences between the adreno-leukodystrophy mouse and human patients are intriguing and may provide a clue to the phenotypic divergence in this disease.

275, 15605-15608). We now show that this enzyme also activates chenodeoxycholate, the secondary bile acids deoxycholate and lithocholate, and 3␣,7␣,12␣-trihydroxy-5␤-cholestanoic acid. In contrast, VLCS activated 3␣,7␣,12␣-trihydroxy-5␤-cholestanoate, but did not utilize any of the C 24 bile acids as substrates. We hypothesize that the primary function of homolog 2 is in the reactivation and recycling of C 24 bile acids, whereas VLCS participates in the de novo synthesis pathway. Results of in situ hybridization, topographic orientation, and inhibition studies are consistent with the proposed roles of these enzymes in bile acid metabolism.

Peroxisomal disorders have been associated with malfunction of peroxisomal metabolic pathways, but the pathogenesis of these disorders is largely unknown. X-linked adrenoleukodystrophy (X-ALD) is associated with elevated levels of very-long-chain fatty acids (VLCFA; C >22:0 ) that have been attributed to reduced peroxisomal VLCFA ␤-oxidation activity. Previously, our laboratory and others have reported elevated VLCFA levels and reduced peroxisomal VLCFA ␤-oxidation in human and mouse X-ALD fibroblasts. In this study, we found normal levels of peroxisomal VLCFA ␤-oxidation in tissues from ALD mice with elevated VLCFA levels. Treatment of ALD mice with pharmacological agents resulted in decreased VLCFA levels without a change in VLCFA ␤-oxidation activity. These data indicate that ALDP does not determine the rate of VLCFA ␤-oxidation and that VLCFA levels are not determined by the rate of VLCFA ␤-oxidation. The rate of peroxisomal VLCFA ␤-oxidation in human and mouse fibroblasts in vitro is affected by the rate of mitochondrial long-chain fatty acid ␤-oxidation. We hypothesize that ALDP facilitates the interaction between peroxisomes and mitochondria, resulting, when ALDP is deficient in X-ALD, in increased VLCFA accumulation despite normal peroxisomal VLCFA ␤-oxidation in ALD mouse tissues. In support of this hypothesis, mitochondrial structural abnormalities were observed in adrenal cortical cells of ALD mice.

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative and endocrine disorder resulting from mutations in ABCD1 which encodes a peroxisomal membrane protein in the ATP binding cassette superfamily. The biochemical signature of X-ALD is increased levels of saturated very long-chain fatty acids (VLCFA; carbon chains of 22 or more) in tissues and plasma that has been associated with decreased peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity and decreased peroxisomal VLCFA beta-oxidation. It has been hypothesized that ABCD1, which has no demonstrable VLCS activity itself, has an indirect effect on peroxisomal VLCS activity and VLCFA beta-oxidation by transporting fatty acid substrates, VLCS protein or some required co-factor into peroxisomes. Here we report the characterization of a Vlcs knockout mouse that exhibits decreased peroxisomal VLCS activity and VLCFA beta-oxidation but does not accumulate VLCFA. The XALD/Vlcs double knockout mouse has the biochemical abnormalities observed in the individual knockout mice but does not display a more severe X-ALD phenotype. These data lead us to conclude that (1) VLCFA levels are independent of peroxisomal fatty acid beta-oxidation, (2) there is no ABCD1/VLCS interaction and (3) the common severe forms of X-ALD cannot be modeled by decreasing peroxisomal VLCS activity in the XALD mouse.

Abnormal dopamine neurotransmission is associated with many different genetic and acquired dystonic disorders. For instance, mutations in genes critical for the synthesis of dopamine, including GCH1 and TH cause l-DOPA-responsive dystonia. Despite evidence that implicates abnormal dopamine neurotransmission in dystonia, the precise nature of the pre- and postsynaptic defects that result in dystonia are not known. To better understand these defects, we generated a knock-in mouse model of l-DOPA-responsive dystonia (DRD) mice that recapitulates the human p.381Q>K TH mutation (c.1141C>A). Mice homozygous for this mutation displayed the core features of the human disorder, including reduced TH activity, dystonia that worsened throughout the course of the active phase, and improvement in the dystonia in response to both l-DOPA and trihexyphenidyl. Although the gross anatomy of the nigrostriatal dopaminergic neurons was normal in DRD mice, the microstructure of striatal synapses was affected whereby the ratio of axo-spinous to axo-dendritic corticostriatal synaptic contacts was reduced. Microinjection of l-DOPA directly into the striatum ameliorated the dystonic movements but cerebellar microinjections of l-DOPA had no effect. Surprisingly, the striatal dopamine concentration was reduced to ∼1% of normal, a concentration more typically associated with akinesia, suggesting that (mal)adaptive postsynaptic responses may also play a role in the development of dystonia. Administration of D1- or D2-like dopamine receptor agonists to enhance dopamine signalling reduced the dystonic movements, whereas administration of D1- or D2-like dopamine receptor antagonists to further reduce dopamine signalling worsened the dystonia, suggesting that both receptors mediate the abnormal movements. Further, D1-dopamine receptors were supersensitive; adenylate cyclase activity, locomotor activity and stereotypy were exaggerated in DRD mice in response to the D1-dopamine receptor agonist SKF 81297. D2-dopamine receptors exhibited a change in the valence in DRD mice with an increase in adenylate cyclase activity and blunted behavioural responses after challenge with the D2-dopamine receptor agonist quinpirole. Together, our findings suggest that the development of dystonia may depend on a reduction in dopamine in combination with specific abnormal receptor responses.

Activation by thioesterification to coenzyme A is a prerequisite for most reactions involving fatty acids. Enzymes catalyzing activation, acyl-CoA synthetases, have been classified by their chain length specificities. The most recently identified family is the very longchain acyl-CoA synthetases (VLCS). Although several members of this group are capable of activating very long-chain fatty acids (VLCFA), one is a bile acid-CoA synthetase, and others have been characterized as fatty acid transport proteins. It was reported that the Drosophila melanogaster mutant bubblegum (BGM) had elevated VLCFA and that the product of the defective gene had sequence homology to acyl-CoA synthetases. Therefore, we cloned full-length cDNA for a human homolog of BGM, and we investigated the properties of its protein product, hsBG, to determine whether it had VLCS activity. Northern blot analysis showed that hsBG is expressed primarily in brain. Compared with vectortransfected cells, COS-1 cells expressing hsBG had increased acyl-CoA synthetase activity with either longchain fatty acid (2.4-fold) or VLCFA (2.6-fold) substrates. Despite this increased VLCFA activation, hsBG-expressing cells did not have increased rates of VLCFA degradation. Confocal microscopy showed that hsBG had a cytoplasmic localization in some COS-1 cells expressing the protein, whereas it appeared to associate with plasma membrane in others. Fractionation of these cells revealed that most of the hsBG-dependent acyl-CoA synthetase activity was soluble and not membrane-bound. Immunoaffinity-purified hsBG from transfected COS-1 cells was enzymatically active. hsBG and hsVLCS are only 15% identical, and comparison with sequences of two conserved motifs from all known families of acylCoA synthetases revealed that hsBG along with the D. melanogaster and murine homologs comprise a new family of acyl-CoA synthetases. Thus, two protein families are now known that contain enzymes capable of activating VLCFA. Because hsBG is expressed in brain but previously described VLCSs were not highly expressed in this organ, hsBG may play a central role in brain VLCFA metabolism and myelinogenesis.

X-linked adreno-leukodystrophy is a progressive, systemic peroxisomal disorder that primarily affects the adrenal cortex, as well as myelin and axons of the central nervous system. Marked phenotypic heterogeneity does not correlate with disease-causing mutations in ABCD1, which encodes a peroxisomal membrane protein that is a member of the ABC transmembrane transporter proteins. The precise physiological functions of ABCD1 and ABCD2, a closely related peroxisomal membrane half-transporter, are unknown. The abcd1 knockout mouse does not develop the inflammatory demyelination so typical and devastating in adreno-leukodystrophy, but it does display the same lamellae and lipid profiles in adrenocortical cells under the electron microscope as the human patients. The adrenocortical cells in the mouse also exhibit immunohistochemical evidence of oxidative stress at 12 weeks but no evidence of oxidative damage. To better understand the pathogenesis of this complex disease, we evaluate the adrenal lesion of the abcd1 knockout mouse as a function of normal aging, dietary or therapeutic manipulations, and abcd genotype. The loss of abcd2 causes oxidative stress in the adrenal at 12 weeks, as judged by increased immunoreactivity for the mitochondrial manganese superoxide dismutase, in both the inner cortex and medulla. The loss of abcd2 (n ¼ 20), but not abcd1 (n ¼ 27), results in the spontaneous and premature deposition of ceroid, a known end-product of oxidative damage, predominantly in adrenal medullary cells. These data indicate that the loss of abcd2 results in greater oxidative stress in murine adrenal cells than the loss of abcd1, providing a clue to its cellular function. We also find that the adrenocortical lesion of the abcd1 knockout mouse does not produce functional impairment at ten to nineteen months or overt hypocortisolism at any age, nor does it progress histologically; these and other data align this mouse model closer to human female heterozygotes than to male ALD or AMN hemizygotes.

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by accumulation of very long-chain fatty acids (VLCFA). This accumulation has been attributed to decreased VLCFA ␤-oxidation and peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity. The X-ALD gene, ABCD1, encodes a peroxisomal membrane ATP binding cassette transporter, ALDP, that is hypothesized to affect VLCS activity in peroxisomes by direct interaction with the VLCS enzyme. Recently, a VLCS gene that encodes a protein with significant sequence identity to known rat and human peroxisomal VLCS protein has been identified in mice. We find that the mouse VLCS gene (Vlcs) encodes an enzyme (Vlcs) with VLCS activity that localizes to peroxisomes and is expressed in X-ALD target tissues. We show that the expression of Vlcs in the peroxisomes of X-ALD mouse fibroblasts improves VLCFA ␤-oxidation in these cells, implying a role for this enzyme in the biochemical abnormality of X-ALD. X-ALD mice, which accumulate VLCFA in tissues, show no change in the expression of Vlcs, the subcellular localization of Vlcs, or general peroxisomal VLCS activity. These observations imply that ALDP is not necessary for the proper expression or localization of Vlcs protein, and the control of VLCFA levels does not depend on the direct interaction of Vlcs and ALDP.