Cerebral inflammation in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disease that affects approximately 1 in 25 000 males. It is characterized by elevated levels of saturated very long chain fatty acids (VLCFA), i.e., >C22:0, particularly in ganglioside and cholesterol ester fractions of brain white matter and adrenal cortex. Failure of peroxisomal very long chain fatty acyl-CoA synthetase (VLCS) to activate these VLCFA prevents their degradation by peroxisomal beta-oxidation. X-ALD maps to Xq28 and the gene encodes a peroxisomal membrane protein and not the gene for VLCS. The two most common forms of X-ALD are the cerebral (CER) form, with an inflammatory demyelinating reaction that resembles multiple sclerosis (MS), and adrenomyeloneuropathy (AMN), which involves the spinal cord and in which the inflammatory reaction is mild or absent. Investigations into the nature of the cerebral inflammatory demyelinating reaction in X-ALD will be the subject of this review.     

ABCD1 mutations and the X‐linked adrenoleukodystrophy mutation database: Role in diagnosis and clinical correlations

X‐linked adrenoleukodystrophy (X‐ALD) is caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC half‐transporter (ALDP) involved in the import of very long‐chain fatty acids (VLCFA) into the peroxisome. The disease is characterized by a striking and unpredictable variation in phenotypic expression. Phenotypes include the rapidly progressive childhood cerebral form (CCALD), the milder adult form, adrenomyeloneuropathy (AMN), and variants without neurologic involvement. There is no apparent correlation between genotype and phenotype. In males, unambiguous diagnosis can be achieved by demonstration of elevated levels of VLCFA in plasma. In 15 to 20% of obligate heterozygotes, however, test results are false–negative. Therefore, mutation analysis is the only reliable method for the identification of heterozygotes. Since most X‐ALD kindreds have a unique mutation, a great number of mutations have been identified in the ABCD1 gene in the last seven years. In order to catalog and facilitate the analysis of these mutations, we have established a mutation database for X‐ALD ( http://www.x‐ald.nl ). In this review we report a detailed analysis of all 406 X‐ALD mutations currently included in the database. Also, we present 47 novel mutations. In addition, we review the various X‐ALD phenotypes, the different diagnostic tools, and the need for extended family screening for the identification of new patients. Hum Mutat 18:499–515, 2001. © 2001 Wiley‐Liss, Inc.     

Decreased expression of ABCD4 and BG1 genes early in the pathogenesis of X-linked adrenoleukodystrophy

Childhood cerebral adrenoleukodystrophy (CCER), adrenomyeloneuropathy (AMN) and AMN with cerebral demyelination (AMN-C) are the main phenotypic variants of X-linked adrenoleukodystrophy (ALD). It is caused by mutations in the ABCD1 gene encoding a half-size peroxisomal transporter that has to dimerize to become functional. The biochemical hallmark of ALD is the accumulation of very-long-chain fatty acids (VLCFA) in plasma and tissues. However, there is no correlation between the ALD phenotype and the ABCD1 gene mutations or the accumulation of VLCFA in plasma and fibroblast from ALD patients. The absence of genotype-phenotype correlation suggests the existence of modifier genes. To elucidate the mechanisms underlying the phenotypic variability of ALD, we studied the expression of ABCD1, three other peroxisomal transporter genes of the same family (ABCD2, ABCD3 and ABCD4) and two VLCFA synthetase genes (VLCS and BG1) involved in VLCFA metabolism, as well as the VLCFA concentrations in the normal white matter (WM) from ALD patients with CCER, AMN-C and AMN phenotypes. This study shows that: (1) ABCD1 gene mutations leading to truncated ALD protein are unlikely to cause variation in the ALD phenotype; (2) accumulation of saturated VLCFA in normal-appearing WM correlates with ALD phenotype and (3) expression of the ABCD4 and BG1, but not of the ABCD2, ABCD3 and VLCS genes, tends to be correlated with the severity of the disease, acting early in the pathogenesis of ALD.     

Evaluation of pharmacological induction of fatty acid beta-oxidation in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurometabolic disorder associated with elevated levels of saturated unbranched very-long-chain fatty acids (VLCFA; C > 22:0) in plasma and tissues, and reduced VLCFA beta-oxidation in fibroblasts, white blood cells, and amniocytes from X-ALD patients. The X-ALD gene (ABCD1) at Xq28 encodes the adrenoleukodystrophy protein (ALDP) that is related to the peroxisomal ATP-binding cassette (ABCD) transmembrane half-transporter proteins. The function of ALDP is unknown and its role in VLCFA accumulation unresolved. Previously, our laboratory has shown that sodium 4-phenylbutyrate (4PBA) treatment of X-ALD fibroblasts results in increased peroxisomal VLCFA beta-oxidation activity and increased expression of the X-ALD-related protein, ALDRP, encoded by the ABCD2 gene. In this study, the effect of various pharmacological agents on VLCFA beta-oxidation in ALD mouse fibroblasts is tested. 4PBA, styrylacetate and benzyloxyacetate (structurally related to 4PBA), and trichostatin A (functionally related to 4PBA) increase both VLCFA (peroxisomal) and long-chain fatty acid [LCFA (peroxisomal and mitochondrial)] beta-oxidation. Isobutyrate, zaprinast, hydroxyurea, and 5-azacytidine had no effect on VLCFA or LCFA beta-oxidation. Lovastatin had no effect on fatty acid beta-oxidation under normal tissue culture conditions but did result in an increase in both VLCFA and LCFA beta-oxidation when ALD mouse fibroblasts were cultured in the absence of cholesterol. The effect of trichostatin A on peroxisomal VLCFA beta-oxidation is shown to be independent of an increase in ALDRP expression, suggesting that correction of the biochemical abnormality in X-ALD is not dependent on pharmacological induction of a redundant gene (ABCD2). These studies contribute to a better understanding of the role of ALDP in VLCFA accumulation and may lead to the development of more effective pharmacological therapies.     

Epilepsy and genetic malformations of the cerebral cortex

Malformations of the cerebral cortex are an important cause of developmental disabilities and epilepsy. Here we review those malformations for which a genetic basis has been elucidated or is suspected and the types of associated epilepsy. Schizencephaly (cleft brain) has a wide anatomo-clinical spectrum, including partial epilepsy in most patients. Familial occurrence is rare. Heterozygous mutations in the EMX2 gene were reported in 13 patients. X-linked bilateral periventricular nodular heterotopia (BPNH) consists of typical BPNH with epilepsy in females and prenatal lethality in males. About 88% of patients have partial epilepsy. Filamin A mutations, all leading to a truncated protein, have been reported in three families and in sporadic patients. The most frequent forms of lissencephaly (agyria-pachygyria) are caused by mutations of LIS1. XLIS mutations cause classical lissencephaly in hemizygous males and subcortical band heterotopia (SBH) in heterozygous females. The thickness of the heterotopic band and the degree of pachygyria correlate with the likelihood of developing Lennox-Gastaut syndrome. Mutations of the coding region of XLIS were found in all reported pedigrees and in 38-91% of sporadic female patients with SBH. With few exceptions, children with LIS1 mutations have isolated lissencephaly, with severe developmental delay and infantile spasms. Autosomal recessive lissencephaly with cerebellar hypoplasia, accompanied by severe developmental delay, seizures, and hypotonia has been associated with mutations of the reelin gene. Fukuyama congenital muscular dystrophy is due to mutations of the fukutin gene and is accompanied by polymicrogyria. Febrile seizures and epilepsy with generalized tonic-convulsions appear in about 50% of children but are usually not severe. Tuberous sclerosis (TS) is caused by mutations in at least two genes, TSC1 and TSC2; 75% of cases are sporadic; 60% of patients have epilepsy, manifested in 50% of them as infantile spasms. TSC1 mutations seem to cause a milder disease with fewer cortical tubers and lower frequency of seizures. Among several syndromes featuring polymicrogyria, bilateral perisylvian polymicrogyria had familial occurrence on several occasions. Genetic heterogeneity is likely, including autosomal recessive, X-linked dominant, X-linked recessive inheritance, and association with 22q11.2 deletions. About 65% of patients have severe epilepsy, often Lennox-Gastaut syndrome.     

X-linked adrenoleukodystrophy: role of very long-chain acyl-CoA synthetases

The principal biochemical abnormality in the neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD) is elevated plasma and tissue levels of very long-chain fatty acids (VLCFA). Enzymes with very long-chain acyl-CoA synthetase (VLACS) activity are required for VLCFA metabolism, including degradation by peroxisomal beta-oxidation or incorporation into complex lipids, and may also participate in VLCFA synthesis. Two enzymes with VLACS activity, ACSVL1 and BG1, were investigated for their potential role in X-ALD biochemical pathology. Skin fibroblast mRNA levels for ACSVL1, an enzyme previously shown to be in peroxisomes and to participate in VLCFA beta-oxidation, were not significantly different between normal controls, patients with childhood cerebral X-ALD, and patients with adrenomyeloneuropathy. Similar results were obtained with mRNA for BG1, a non-peroxisomal enzyme that is highly expressed in nervous system, adrenal gland, and testis, the principal tissues pathologically affected in X-ALD. No significant differences in the immunohistochemical staining patterns of tissues expressing either ACSVL1 or BG1 were observed when wild-type and X-ALD mice were compared. Western blot analysis of BG1 protein levels showed no differences between fibroblasts from controls, cerebral X-ALD, or adrenomyeloneuropathy patients. BG1 protein levels were similar in wild-type and X-ALD mouse brain, spinal cord, testis, and adrenal gland. We hypothesized that one function of BG1 was to direct VLCFA into the cholesterol ester synthesis pathway. However, BG1 depletion in Neuro2a cells using RNA interference did not decrease incorporation of labeled VLCFA into cholesterol esters. We conclude that the role, if any, of ACSVL1 and BG1 in X-ALD biochemical pathology is indirect.     

Biochemical Aspects of X‐Linked Adrenoleukodystrophy

X‐linked adrenoleukodystrophy (X‐ALD) is the most common peroxisomal disorder. The disease is characterized by the accumulation of very long‐chain fatty acids (VLCFA; >C22) in plasma and tissues. X‐ALD is caused by mutations in the ABCD1 gene encoding ALDP, an adenosine triphosphate (ATP)‐binding‐cassette (ABC) transporter located in the peroxisomal membrane. In this paper, we describe the current knowledge on the function of ALDP, its role in peroxisomal VLCFA beta‐oxidation and the consequences of a defect in ALDP on VLCFA metabolism. Furthermore, we pay special attention to the role of the VLCFA elongation system in VLCFA homeostasis, with elongation of very long‐chain fatty acids like‐1 (ELOVL1) as key player, and its relevance to X‐ALD.     

Elongation of very long-chain fatty acids is enhanced in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a progressive neurodegenerative disorder characterized by the accumulation of saturated and mono-unsaturated very long-chain fatty acids (VLCFA) and reduced peroxisomal VLCFA beta-oxidation activity. In this study, we investigated the role of VLCFA biosynthesis in X-ALD fibroblasts. Our data demonstrate that elongation of both saturated and mono-unsaturated VLCFAs is enhanced in fibroblasts from patients with peroxisomal beta-oxidation defects including X-ALD, and peroxisome biogenesis disorders. These data indicate that enhanced VLCFA elongation is a general phenomenon associated with an impairment in peroxisomal beta-oxidation, and not specific for X-ALD alone. Analysis of plasma samples from patients with X-ALD and different peroxisomal beta-oxidation deficiencies revealed increased concentrations of VLCFAs up to 32 carbons. We infer that enhanced elongation does not result from impaired peroxisomal beta-oxidation alone, but is due to the additional effect of unchecked chain elongation. We demonstrate that elongated VLCFAs are incorporated into complex lipids. The role of chain elongation was also studied retrospectively in samples from patients with X-ALD previously treated with "Lorenzo's oil." We found that the decrease in plasma C26:0 previously found is offset by the increase of mono-unsaturated VLCFAs, not measured previously during the trial. We conclude that evaluation of treatment protocols for disorders of peroxisomal beta-oxidation making use of plasma samples should include the measurement of saturated and unsaturated VLCFAs of chain lengths above 26 carbon atoms. We also conclude that chain elongation offers an interesting target to be studied as a possible mode of treatment for X-ALD and other peroxisomal beta-oxidation disorders.     

A very long-chain acyl-CoA synthetase-deficient mouse and its relevance to X-linked adrenoleukodystrophy

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.     

Suppression of peroxisomal membrane protein defects by peroxisomal ATP binding cassette (ABC) proteins

X-Linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by reduced peroxisomal very long chain fatty acid (VLCFA) beta-oxidation. The X - ALD gene product (ALDP) is a peroxisomal transmembrane protein with an ATP binding cassette (ABC). ALDP and three other ABC proteins (PMP70, ALDR, P70R) localize to the peroxisomal membrane. The function of this family of peroxisomal membrane proteins is unknown. We used complementation studies to begin analysis of their role in VLCFA beta-oxidation and on the peroxisomal membrane. Expression of either ALDP or PMP70 restores VLCFA beta- oxidation in X-ALD fibroblasts, indicating overlapping functions. Their expression also restores peroxisome biogenesis in cells that are deficient in the peroxisomal membrane protein Pex2p. Thus it is likely that complex protein interactions are involved in the function and biogenesis of peroxisomal membranes that may contribute to disease heterogeneity.      

Zellweger syndrome and associated phenotypes.

Until recently, the peroxisome was considered a "reactor chamber" for H2O2 producing oxidases, and it is now recognised as a versatile organelle performing complex catabolic and biosynthetic roles in the cell. Zellweger syndrome (ZS), the paradigm of human peroxisomal disorders, is characterised by neonatal hypotonia, severe neuro-developmental delay, hepatomegaly, renal cysts, senorineural deafness, retinal dysfunction, and facial dysmorphism. It is now clear that ZS is at the severe end of a phenotypic spectrum of Zellweger-like syndromes which may present for diagnosis later in childhood and even in adult life. It is important that clinical geneticists are aware of these milder clinical variants as the availability of sensitive and specific biochemical assays of peroxisomal function (for example, serum VLCFA ratios, platelet DHAP-AT activity) makes their diagnosis relatively straightforward.     

Enoyl-CoA hydratase deficiency: identification of a new type of D-bifunctional protein deficiency.

D-bifunctional protein is involved in the peroxisomal beta-oxidation of very long chain fatty acids, branched chain fatty acids and bile acid intermediates. In line with the central role of D-bifunctional protein in the beta-oxidation of these three types of fatty acids, all patients with D-bifunctional protein deficiency so far reported in the literature show elevated levels of very long chain fatty acids, branched chain fatty acids and bile acid inter-mediates. In contrast, we now report two novel patients with D-bifunctional protein deficiency who both have normal levels of bile acid intermediates. Complementation analysis and D-bifunctional protein activity measurements revealed that both patients had an isolated defect in the enoyl-CoA hydratase domain of D-bifunctional protein. Subsequent mutation analysis showed that both patients are homozygous for a missense mutation (N457Y), which is located in the enoyl-CoA hydratase coding part of the D-bifunctional protein gene. Expression of the mutant protein in the yeast Saccharomyces cerevisiae confirmed that the N457Y mutation is the disease-causing mutation. Immunoblot analysis of patient fibroblast homogenates showed that the protein levels of full-length D-bifunctional protein were strongly reduced while the enoyl-CoA hydratase component produced after processing within the peroxisome was undetectable, which indicates that the mutation leads to an unstable protein.     

Peroxisomal very long chain fatty acid beta-oxidation activity is determined by the level of adrenodeukodystrophy protein (ALDP) expression

Impaired peroxisomal beta-oxidation of saturated very long chain fatty acids (VLCFA, >/=C22:0) results in increased VLCFA levels in the tissues and body fluids of patients with disorders of peroxisomal biogenesis (i.e., Zellweger syndrome and neonatal adrenoleukodystrophy) and single peroxisomal protein defects (i.e., X-linked adrenoleukodystrophy (X-ALD) and acyl-CoA oxidase deficiency). We show that SV40T transformation also results in impaired peroxisomal beta-oxidation and VLCFA accumulation despite the presence of abundant peroxisomes. To explore the mechanism responsible for this observation, we have examined expression of key components of peroxisomal VLCFA beta-oxidation. We found that expression of both acyl-CoA oxidase, the rate limiting enzyme of peroxisomal VLCFA beta-oxidation and the adrenoleukodystrophy protein (ALDP), the defective gene product in X-ALD, are reduced after SV40T transformation. Surprisingly, ALDP overexpression by itself restores peroxisomal VLCFA beta-oxidation in SV40T-transformed control and X-ALD cells. These results demonstrate that ALDP is a fundamental component in VLCFA peroxisomal beta-oxidation and may serve as a "gatekeeper" for VLCFA homeostasis.     

Neonatal lupus is a novel cause of positive newborn screening for X-linked adrenoleukodystrophy

We report three unrelated individuals, each exposed to maternal autoantibodies during gestation and found to have elevated very long-chain fatty acids (VLCFAs) in the newborn period after screening positive by California newborn screening (NBS) for X-linked adrenoleukodystrophy (ALD). Two probands presented with clinical and laboratory features of neonatal lupus erythematosus (NLE); the third had features suggestive of NLE and a known maternal history of Sjogren's syndrome and rheumatoid arthritis. In all three individuals, subsequent biochemical and molecular evaluation for primary and secondary peroxisomal disorders was nondiagnostic with normalization of VLCFAs by 15 months of age. These cases add to the expanding differential diagnosis to consider in newborns who screen positive for ALD via elevated C26:0-lysophosphatidylcholine. Though the pathophysiology of how transplacental maternal anti-Ro antibodies damage fetal tissue is not well-understood, we postulate that the VLCFA elevations reflect a systemic inflammatory response and secondary peroxisomal dysfunction that improves once maternal autoantibodies wane after birth. Additional evaluation of this phenomenon is warranted to better understand the intricate biochemical, clinical, and possible therapeutic overlap between autoimmunity, inflammation, peroxisomal dysfunction, and human disease.     

X-linked adrenoleukodystrophy: very long-chain fatty acid metabolism, ABC half-transporters and the complicated route to treatment

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in elevated levels of very long-chain fatty acids (VLCFA) and reduced VLCFA beta-oxidation in peroxisomes. The peroxisomal membrane harbors three additional closely related ABC half-transporters, ALDRP, PMP70 and PMP69 (PMP70R). ABC half-transporters must dimerize to form a functional full-transporter. Whether ALDP forms a homodimer or a heterodimer has not yet been resolved, but most indirect evidence favors homodimerization. The peroxisomal ABC half-transporters are functionally related. Over-expression of ALDRP can correct the biochemical defect both in X-ALD patients cells and the Abcd1 knockout mouse, providing an exciting new possibility for treatment of X-ALD patients. This paper provides an overview of current knowledge and the problems that have been encountered.     

Inhibition of peroxisomal β-oxidation by thioridazine increases the amount of VLCFAs and Aβ generation in the rat brain

Alzheimer's disease (AD) is characterized by the accumulation of the β-amyloid peptide (Aβ), which is generated from sequential cleavages of the amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase. Fatty acid alterations in AD brains have recently received substantial attention. Because increased very long chain fatty acid (VLCFA) levels in AD brains imply that peroxisomal β-oxidation dysfunction may be associated with AD pathogenesis, we investigated the effects of impaired peroxisomal β-oxidation on Aβ generation in vivo and in vitro using thioridazine, a selective peroxisomal β-oxidation inhibitor. Under the experimental conditions, thioridazine caused VLCFA accumulation and increases in Aβ₄₀ content, APP immunoreactivity and APP_751+770 mRNA expressions in the rat cerebral cortex. A correlation analysis showed that the Aβ₄₀ levels were positively correlated with the cortex C_24:0 and C_26:0 levels. Additionally, the primary cerebral cortex neurons treated with this compound showed increases in APP_751+770 mRNA, APP protein, BACE1 mRNA and protein, and secreted Aβ40 levels. This work supports an emerging viewpoint that impaired peroxisomal function may play an important role in the progression of AD pathology.     

Polymicrogyria and deletion 22q11.2 syndrome: window to the etiology of a common cortical malformation

Several brain malformations have been described in rare patients with the deletion 22q11.2 syndrome (DEL22q11) including agenesis of the corpus callosum, pachygyria or polymicrogyria (PMG), cerebellar anomalies and meningomyelocele, with PMG reported most frequently. In view of our interest in the causes of PMG, we reviewed clinical data including brain-imaging studies on 21 patients with PMG associated with deletion 22q11.2 and another 11 from the literature. We found that the cortical malformation consists of perisylvian PMG of variable severity and frequent asymmetry with a striking predisposition for the right hemisphere (P = 0.008). This and other observations suggest that the PMG may be a sequela of abnormal embryonic vascular development rather than a primary brain malformation. We also noted mild cerebellar hypoplasia or mega-cisterna magna in 8 of 24 patients. Although this was not the focus of the present study, mild cerebellar anomalies are probably the most common brain malformation associated with DEL22q11.     

Neonatal adrenoleukodystrophy: new cases, biochemical studies, and differentiation from Zellweger and related peroxisomal polydystrophy syndromes

Eight new cases of autopsy-confirmed or suspected neonatal adrenoleukodystrophy (NALD) are presented together with new biochemical data on very-long-chain fatty acids (VLCFA) and plasmalogens and a review of all previously published cases. The clinical, biochemical, and histopathologic abnormalities characteristic of this newly recognized form of adrenoleukodystrophy are analyzed in detail and compared to the principal characteristics of the similar disorder, the cerebrohepatorenal syndrome of Zellweger (ZS). Using strict pathologic criteria for the diagnosis of NALD, we find that, despite many clinical resemblances, NALD and the ZS are distinguishable on the basis of histology and peroxisomal biochemistry. Patients with NALD demonstrate adrenal atrophy, systemic infiltration by abnormal lipid-laden macrophages, and elevations of saturated VLCFA. In contrast, patients with ZS have chondrodysplasia, glomerulocystic disease of the kidney, central nervous system dysmyelination, and elevations of unsaturated as well as saturated VLCFA, but they lack adrenal atrophy. We conclude that NALD and the ZS probably represent at least two different genetic defects.     

Toxic effects of X-linked adrenoleukodystrophy-associated, very long chain fatty acids on glial cells and neurons from rat hippocampus in culture

Saturated very long chain fatty acids (VLCFAs; > or =C22:0) accumulate in X-linked adrenoleukodystrophy (X-ALD, OMIM 300100), a severe hereditary neurodegenerative disease, due to peroxisomal impairment. Previous studies analysed the development of X-ALD in humans and gene knockout animal models. However, the toxic effect of VLCFA leading to severe symptoms with progressive and multifocal demyelination, adrenal insufficiency and inflammation still remains unclear. To understand the toxic effects of VLCFA in the brain, here we exposed neural cells to VLCFA and analysed the cellular consequences. We found that oligodendrocytes and astrocytes challenged with docosanoic- (C22:0), tetracosanoic- (C24:0) and hexacosanoic acids (C24:0) die within 24 h. VLCFA-induced depolarization of mitochondria in situ and increased intracellular Ca2+ level in all three brain cell types provides indications about the mechanism of toxicity of VLCFA. Interestingly, VLCFAs affect to the largest degree the myelin-producing oligodendrocytes. In isolated mitochondria, VLCFAs exert a detrimental effect by affecting the inner mitochondrial membrane and promoting the permeability transition. In conclusion, we suggest that there is a potent toxic activity of VLCFA due to dramatic cell physiological effects with mitochondrial dysfunction and Ca2+ deregulation. This provides the first evidence for mitochondrial-based cell death mechanisms in neurodegenerative disease with peroxisomal defects and subsequent VLCFA accumulation.