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Episodic ataxia type 2 (EA2) is an autosomal dominant disorder associated with attacks of ataxia that are typically precipitated by stress, ethanol, caffeine or exercise. EA2 is caused by loss-of-function mutations in the CACNA1A gene, which encodes the α1A subunit of the CaV2.1 voltage-gated Ca(2+) channel. To better understand the pathomechanisms of this disorder in vivo, we created the first genetic animal model of EA2 by engineering a mouse line carrying the EA2-causing c.4486T>G (p.F1406C) missense mutation in the orthologous mouse Cacna1a gene. Mice homozygous for the mutated allele exhibit a ~70% reduction in CaV2.1 current density in Purkinje cells, though surprisingly do not exhibit an overt motor phenotype. Mice hemizygous for the knockin allele (EA2/- mice) did exhibit motor dysfunction measurable by rotarod and pole test. Studies using Cre-flox conditional genetics explored the role of cerebellar Purkinje cells or cerebellar granule cells in the poor motor performance of EA2/- mice and demonstrate that manipulation of either cell type alone did not cause poor motor performance. Thus, it is possible that subtle dysfunction arising from multiple cell types is necessary for the expression of certain ataxia syndromes.

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.

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.

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.

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 beta-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 beta-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.

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 long-chain 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 mutantbubblegum (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 vector-transfected cells, COS-1 cells expressing hsBG had increased acyl-CoA synthetase activity with either long-chain 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 acyl-CoA 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 adrenoleukodystrophy (X-ALD) is a peroxisomal disorder that affects 1 in 35000 males and is marked by neurodegeneration, adrenal insufficiency and infertility. All characteristics are not always present in every X-ALD patient and the rate of progression varies. X-ALD has a variety of phenotypes, the two major phenotypes are cerebral X-ALD (CALD) and adrenomyeloneuropathy (AMN)(Moser 2000). The Other patients only have adrenal insufficiency, or rarely, remain asymptomatic. As the nature of X-ALD is progressive, these adrenal and asymptomatic patients often develop nervous system involvement as they age. CALD most commonly presents in childhood (mean age of onset, 7.2 ±1.7 years) but can also strike adolescents and adults. There are focal sites of demyelination of the cerebral white matter associated with perivascular inflammation of the affected site. This inflammatory demyelination is rapidly progressive over the course of a few years and usually ends in early death. AMN presents in adulthood (mean age of onset, 28 ± 9 years) as a peripheral myelopathy with spinal cord involvement usually with no brain disease. There is a lack of inflammation at these sites and this disease progresses much more slowly than the cerebral form. A significant fraction of AMN patients eventually develop brain disease. Adrenal insufficiency is often, but not always, present in both cerebral and AMN patients.

Peroxisomes are single membrane-bound subcellular organelles present in most eukaryotic cells (8). Peroxisomes are involved in several vital metabolic pathways, including β-oxidation of very-long-chain fatty acids (VLCFA; C>22:0), plasmalogen biosynthesis, oxidation of H2O2, α-oxidation of phytanic acid, bile acid synthesis, and cholesterol biosynthesis (40). Two major classes of peroxisomal disorders have been described. The first class, peroxisomal biogenesis disorders (PBDs; McKusick 601539), is a heterogeneous group of autosomal recessive diseases characterized by alterations in various peroxisomal proteins (called peroxins and encoded by PEX genes) involved in peroxisome biogenesis (38). PBDs include Zellweger syndrome (McKusick 214100), neonatal adrenoleukodystrophy (McKusick 202370), infantile Refsum's disease (McKusick 266510), and rhizomelic chondrodysplasia punctata (McKusick 215100). The second class of peroxisomal disorders, typified by X-linked adrenoleukodystrophy (X-ALD; McKusick 300100), includes disorders with a single peroxisomal enzyme or protein defect. X-ALD is the most common peroxisomal disorder, with an incidence of approximately 1 in 17,000 (4, 9). It is a postnatal rapidly progressive disease that affects primarily the central nervous system white matter, the adrenal cortex, and the testis (23). The biochemical signature of X-ALD is increased levels of saturated unbranched VLCFA in plasma and tissues, particularly in the cholesterol ester, ganglioside, and proteolipid fractions of the brain white matter and cholesterol esters of the adrenal cortex (23). It has been clearly established that in fibroblasts, white cells, and amniocytes from X-ALD patients, there is a decrease in peroxisomal VLCFA degradation. Reduced activity of peroxisomal very-long-chain acyl coenzyme A (acyl-CoA) synthetase (VLCS), the enzyme that activates VLCFA to initiate their degradation, has been demonstrated in fibroblasts from X-ALD patients. However, the X-ALD gene, ABCD1, identified by positional cloning (24), encodes a protein (ALDP) that is a member of the ATP binding cassette (ABC) transporter superfamily of membrane proteins (16). ALDP is located in the peroxisomal membrane (25) but has no homology to any known VLCS (34) and no demonstrable VLCS activity (36). The role of ALDP in VLCFA metabolism, the pathophysiology of X-ALD, and its relationship to VLCS activity have yet to be determined. Fatty acids are activated by thioesterification to CoA by fatty acyl-CoA synthetases before they can participate in either catabolic or anabolic pathways (41). Fatty acyl-CoA synthetases capable of activating short-chain fatty acids (SCFA; C2 to C4), medium-chain fatty acids (MCFA; C6 to C10), long-chain fatty acids (LCFA; C12 to C20), or VLCFA (C>20) have been described. The protein encoded by the VLCS gene activates both VLCFA and LCFA, in contrast to long-chain acyl-CoA synthetase, which activates only LCFA. Long-chain acyl-CoA synthetase activity is found in peroxisomes, mitochondria, and microsomes, while VLCS activity is only found in peroxisomes and microsomes. Steinberg et al. (37) reported that for VLCS, the rate of activation of LCFA is 10- to 20-fold higher than the rate of activation of VLCFA. It has been suggested that ALDP is directly involved in VLCFA β-oxidation through transport of VLCS, VLCFA, or a required cofactor across the peroxisomal membrane. It should be noted, however, that the absence of ALDP results in the reduction, but not elimination, of VLCS activity and VLCFA β-oxidation in peroxisomes, suggesting either that there are compensatory activities in the peroxisome or that the effect of ALDP on peroxisomal VLCFA β-oxidation in fibroblasts is indirect. There are an estimated 48 mammalian ABC proteins (7), located in cellular and subcellular membranes, that transport a wide variety of substrates, including ions, sugars, amino acids, proteins, and lipids (15, 16). Mammalian ABC transporter proteins typically consist of two hydrophobic transmembrane domains and two hydrophilic nucleotide-binding folds encoded by a single gene. Peroxisomal ABC transporters (7) comprise a subgroup (D) of related proteins that are encoded as half-transporters with a single transmembrane domain and a single nucleotide-binding fold. In mammals, there are four ABC subfamily D (ABCD) proteins, ALDP (encoded by the ABCD1 gene), the adrenoleukodystrophy-related protein ALDRP (encoded by the ABCD2 gene), the 70-kDa peroxisomal membrane protein PMP70 (encoded by the ABCD3 gene), and the PMP70-related protein PMP70R (encoded by the ABCD4 gene). Other mammalian ABC half-transporters identified to date dimerize to form functional transporters (33, 35). Homodimerization of ALDP and heterodimerization of ALDP with ALDR and PMP70 in vitro were demonstrated in our laboratory (34) and by Liu et al. (19). The functional significance of these dimers is unknown. However, differences in substrate transport among the various possible homo- and heterodimers could reflect the metabolic demands of various cell types since the peroxisomal ABC transporters are known to have differing tissue expression patterns in vivo (3, 12, 28, 39). This laboratory (6, 17) and others (10, 27) have demonstrated that overexpression of ALDR or PMP70, as well as ALDP, cDNA in fibroblasts from X-ALD patients improves peroxisomal β-oxidation. Thus, these peroxisomal ABC half-transporters, either specifically or nonspecifically, are able to facilitate peroxisomal VLCFA β-oxidation in fibroblasts lacking ALDP. Previously, we reported the effect of a pharmacological agent, sodium 4-phenylbutyrate (4PBA), on fibroblasts from human PBD and X-ALD patients and ALD mice and showed that 4PBA treatment corrects VLCFA β-oxidation and VLCFA levels in these cells (17, 43). Initially, correction of VLCFA β-oxidation and VLCFA levels in these cells was attributed to an observed two- to threefold increase in ALDRP expression (17). However, more recent data suggest that increased histone acetylation, unrelated to increased ALDR expression, is associated with induction of VLCFA β-oxidation in cultured skin fibroblasts and indicates that induction of peroxisomal VLCFA β-oxidation by 4PBA and other pharmacological agents was associated with induction of mitochondrial LCFA β-oxidation (22). These data imply that there is intracellular communication between mitochondria and peroxisomes in human and mouse fibroblasts. Recently, Baumgart et al. reported mitochondrial alterations caused by defective peroxisomal biogenesis in a mouse model (PEX5−/−) of Zellweger syndrome (2). These included severe alterations of mitochondrial ultrastructure, changes in the expression and activity of mitochondrial respiratory chain complexes, and an increase in the heterogeneity of the mitochondrial component in various organs and specific cell types (2). Mitochondrial abnormalities have also been reported in seminal studies documenting a peroxisomal defect in Zellweger syndrome (14) and in X-ALD (30). There are precedents, therefore, for mitochondrial involvement in peroxisomal disorders. In this study, we measured peroxisomal VLCFA β-oxidation activity and VLCFA levels in ALD mouse tissues before and after treatment with various pharmacological agents to investigate the relationship between peroxisomal VLCFA β-oxidation and VLCFA levels. The relationship between mitochondrial and peroxisomal fatty acid β-oxidation activities in cultured skin fibroblasts (i) with naturally occurring mutations in fatty acid metabolism and (ii) in the presence of drugs affecting mitochondrial activity was investigated. The role of mitochondria in the induction of peroxisomal VLCFA β-oxidation in fibroblasts by 4PBA was examined, and mitochondria in mouse tissue were examined ultrastructurally for morphological abnormalities.

Bile acids are synthesized de novo in the liver from cholesterol and conjugated to glycine or taurine via a complex series of reactions involving multiple organelles. Bile acids secreted into the small intestine are efficiently reabsorbed and reutilized. Activation by thioesterification to CoA is required at two points in bile acid metabolism. First, 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid, the 27-carbon precursor of cholic acid, must be activated to its CoA derivative before side chain cleavage via peroxisomal beta-oxidation. Second, reutilization of cholate and other C24 bile acids requires reactivation prior to re-conjugation. We reported previously that homolog 2 of very long-chain acyl-CoA synthetase (VLCS) can activate cholate (Steinberg, S. J., Mihalik, S. J., Kim, D. G., Cuebas, D. A., and Watkins, P. A. (2000) J. Biol. Chem. 275, 15605-15608). We now show that this enzyme also activates chenodeoxycholate, the secondary bile acids deoxycholate and lithocholate, and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid. In contrast, VLCS activated 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate, but did not utilize any of the C24 bile acids as substrates. We hypothesize that the primary function of homolog 2 is in the reactivation and recycling of C24 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.