Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders.

The peroxisome biogenesis disorders (PBDs) are lethal recessive diseases caused by defects in peroxisome assembly. We have isolated PXR1, a human homologue of the yeast P. pastoris PAS8 (peroxisome assembly) gene. PXR1, like PAS8, encodes a receptor for proteins with the type-1 peroxisomal targeting signal (PTS1). Mutations in PXR1 define complementation group 2 of PBDs and expression of PXR1 rescues the PTS1 import defect of fibroblasts from these patients. Based on the observation that PXR1 exists both in the cytosol and in association with peroxisomes, we propose that PXR1 protein recognizes PTS1-containing proteins in the cytosol and directs them to the peroxisome.

Mutations in PEX1 are the most common cause of peroxisome biogenesis disorders.

The peroxisome biogenesis disorders (PBDs) are a group of lethal autosomal-recessive diseases caused by defects in peroxisomal matrix protein import, with the concomitant loss of multiple peroxisomal enzyme activities. Ten complementation groups (CGs) have been identified for the PBDs, with CG1 accounting for 51% of all PBD patients. We identified the human orthologue of yeast PEX1, a gene required for peroxisomal matrix protein import. Expression of human PEX1 restored peroxisomal protein import in fibroblasts from 30 CG1 patients, and PEX1 mutations were detected in multiple CG1 probands. A common PEX1 allele, G843D, is present in approximately half of CG1 patients and has a deleterious effect on PEX1 activity. Phenotypic analysis of PEX1-deficient cells revealed severe defects in peroxisomal matrix protein import and destabilization of PEX5, the receptor for the type-1 peroxisomal targetting signal, even though peroxisomes were present in these cells and capable of importing peroxisomal membrane proteins. These data demonstrate an important role for PEX1 in peroxisome biogenesis and suggest that mutations in this gene are the most common cause of the PBDs.

Ceramidase deficiency in Farber's disease (lipogranulomatosis).

Ceramidase activity could not be demonstrated in the kidney and cerebellum from a deceased patient with Farber's disease, whereas the activities of six control acid hydrolase enzymes appeared normal. This enzyme defect presumably accounts for the accumulation that has been described in two patients and may represent the biochemical basis of this disorder.

Prolonged survival and remyelination after hematopoietic cell transplantation in the twitcher mouse.

The twitcher mouse is an animal model of galactosylceramidase deficiency (Krabbe's disease), a human sphingolipidosis. The effects of hematopoietic cell transplantation as potential enzyme replacement therapy were examined in the twitcher mouse. Survival in twitcher mice with transplants was significantly prolonged and was associated with gradual repair of demyelination in peripheral nerves. In contrast, there was no improvement in the neurodegenerative process in the central nervous system after transplantation. These observations indicate that cellular transplantation may effectively provide in vivo enzyme replacement for the peripheral manifestations of genetic storage diseases. Strategies to perturb the blood-brain barrier may be necessary for enzyme replacement to be therapeutic in diseases with central nervous system manifestations.

Peroxisomal defects in neonatal-onset and X-linked adrenoleukodystrophies.

Accumulation of very long chain fatty acids in X-linked and neonatal forms of adrenoleukodystrophy (ALD) appears to be a consequence of deficient peroxisomal oxidation of very long chain fatty acids. Peroxisomes were readily identified in liver biopsies taken from a patient having the X-linked disorder. However, in liver biopsies from a patient having neonatal-onset ALD, hepatocellular peroxisomes were greatly reduced in size and number, and sedimentable catalase was markedly diminished. The presence of increased concentrations of serum pipecolic acid and the bile acid intermediate, trihydroxycoprostanic acid, in the neonatal ALD patient are associated with a generalized diminution of peroxisomal activities that was not observed in the patient with X-linked ALD.

A PEX10 defect in a patient with no detectable defect in peroxisome assembly or metabolism in cultured fibroblasts.

Zellweger spectrum disorders (ZSD) are diagnosed by biochemical assay in blood, urine and cultured fibroblasts and PEX gene mutation identification. In most cases studies in fibroblasts corroborate results obtained in body fluids. In 1996 Clayton and colleagues described a 10-year old girl with evidence of a peroxisome disorder, based on elevated bile acid metabolites and phytanate. At the time it was not possible to distinguish whether she had a ZSD or a single peroxisomal protein defect. Studies in our laboratory showed that she also had elevated plasma pipecolate, supporting the former diagnosis. Despite the abnormal metabolites detected in blood (phytanate, bile acid intermediates and pipecolate), analysis of multiple peroxisomal pathways in fibroblasts yielded normal results. In addition, she had a milder clinical phenotype than usually associated with ZSD. Since complementation analysis to determine the gene defect was not possible, we screened this patient following the PEX Gene Screen algorithm (PGS). The PGS provides a template for sequencing PEX gene exons independent of complementation analysis. Two mutations in PEX10 were identified, a frameshift mutation inherited from her father and a de novo missense mutation in a conserved functional domain on the other allele. This case highlights that molecular analysis may be essential to the diagnosis of patients at the milder end of the ZSD spectrum. Furthermore, it supports the concept that some tissues are less affected by certain PEX gene defects than brain and liver.

Photosensitized killing of cultured fibroblasts from patients with peroxisomal disorders due to pyrene fatty acid-mediated ultraviolet damage.

The influence of pyrene-fatty acids on the resistance of cells to ultraviolet (UV) radiation was investigated in cultured fibroblasts from patients with five types of peroxisomal disorders. All showed reduced survival compared to control. The effect varied with the biochemical defect involved and the chain length of the pyrene fatty acid. Reduced survival was observed in cells deficient in plasmalogens (rhizomelic chondrodysplasia punctata) and in cells deficient in peroxisomal fatty acid oxidation (bifunctional enzyme deficiency), which accumulated pyrene-fatty acids. X-linked adrenoleukodystrophy fibroblasts accumulated pyrene-fatty acids and showed increased UV sensitivity only when exposed to longer-chain pyrene fatty acids. UV radiation resistance was lowest in cells with combined impairment of plasmalogen synthesis and fatty acid oxidation (Zellweger syndrome, neonatal adrenoleukodystrophy), suggesting that UV sensitivity correlates inversely with the ratio of plasmalogens to radical producing substances. Fibroblasts deficient in plasmalogens gained normal UV resistance when their plasmalogen levels were normalized by hexadecylglycerol. UV resistance increased when Zellweger cells were fused with X-linked adrenoleukodystrophy cells, and also when Zellweger cells belonging to different complementation groups were fused. The results provide leads to the pathogenesis of the multiple malformations associated with peroxisomal disorders and a method for the selection of cells in which the metabolic defect has been corrected.

Cholesterol sulfate in rat tissues. Tissue distribution, developmental change and brain subcellular localization.

1. A reliable micromethod for the determination of the tissue level of cholesterol sulfate has been developed. Cholesterol sulfate was separated from the bulk of the free cholesterol by silica gel column chromatography, and the cholesterol sulfate fraction subjected to benzoylation. A small amount of contaminating free cholesterol and other lipids remaining in this fraction were converted to benzoyl esters while the cholesterol sulfate remained unreacted. The cholesterol sulfate was then separated from the benzoylated contaminants by a second silica gel chromatography column and subjected to solvolysis. The liberated cholesterol was determined by gas-liquid chromatography. 2. The cholesterol sulfate contents of the visceral organs of 43-day-old rats were determined. Every tissue examined contained small amounts of this sulfate. Kidney contained the highest concentration of cholesterol sulfate (250-300 mug/g dry tissue weight) followed by spleen (77 mug/g), adrenal gland (50-70 mug/g) and lung (50-57 mug/g). 3. In brain, cholesterol sulfate level rises sharply from 17 mug/g dry weight in 7-day-old rats to more than 50 mug/g in 15-day-olds, then it declines rapidly to 15 mug/g in the 40-day-olds and this level is maintained to adulthood. The developmental pattern in the liver resembles that in the brain, except that the peak is somewhat flatter with the highest value (60 mug/g dry weight) occurring in the 21-day-old animal. In contrast to the above two tissues, the level of kidney cholesterol sulfate increases steadily from 15 mug/g in 7-day-olds and reaches the adult level of approx. 200 mug/g in 50-day-olds. 4. The highest level of cholesterol sulfate in subcellular fractions of rat brain occurred in a fraction rich in nerve endings. The level here was 10 times higher than that in the mitochondrial fraction, which contained the lowest levels of this steroid sulfate.

3-Ketosphingolipids: application to the determination of sphingolipids which contain 4-sphingenine.

1. Ceramides and cerebrosides, both with 2-hydroxy or non-hydroxy fatty acids, and sphingomyelins were converted quantitatively to the corresponding 3-keto derivatives with the oxidative reagent 2,3-dichloro-5,6-dicyanobenzoquinone. Sulfatides were converted to 3-ketocerebrosides. 2. The conversion of these sphingolipids to the alpha,beta-unsaturated ketone permits three methods of quantitation: in Method I, the absorption of the reaction product is measured at 230 nm; Method II utilizes high performance liquid chromatography and an ultraviolet light detector; in Method III, the product is reduced with NaB3H4 and the radioactivity of the reduced products is determined. All three methods can be used to measure sphingolipids in nanomole quantities. 3. Methods I and II have been applied to the determination of cerebroside and sulfatide content of normal and metachromatic leukodystrophy brains. In the white matter of the pathological brains, there was a greater accumulation of sulfatides containing non-hydroxy fatty acids than of those containing 2-hydroxy fatty acids. The ceramide levels of the cerebellum in a Farber's disease patient and a control were determined by Method II. Method III was used to determine the content of free ceramides in human sera.

Ceramidase and ceramide synthesis in human kidney and cerebellum. Description of a new alkaline ceramidase.

It has been shown that tissues of patients with Farber's disease characteristically lack acid (pH 4.0) ceramidase. In normal cerebellum, however, ceramide cleavage and the reverse reaction, free fatty acid-dependent ceramide synthesis, both occur not only at pH 4.0 but also at pH 9.0, although normal kidney exhibits these activities only at pH 4.0. Both tissues are capable of snythesizing ceramide via an acyl-COA-dependent pathway at neutral pH. The synthetic analog of ceramide, N-oleoyl-ethanolamine, is a potent inhibitor of ceramidase.

Arachidonic acid metabolism in fibroblasts from patients with peroxisomal diseases: response to interleukin 1.

Prostaglandin E2 synthesis and eicosanoid biosynthetic enzyme activities (arachidonyl CoA synthetase, cyclooxygenase and phospholipase A2) were measured in dermal fibroblasts from patients with metabolic disorders of peroxisomal origin and compared to those from normal subjects and patients with other metabolic disorders of lipid metabolism. Basal- as well as interleukin 1-stimulated prostaglandin E2 syntheses were higher in fibroblasts from patients with X-linked adrenoleukodystrophy, the Zellweger cerebrohepatorenal syndrome and rhizomelic chondrodysplasia punctata than in normals. Basal cyclooxygenase and phospholipase A2 activities were elevated in most of the peroxisomal disease cells. Cells from patients with adrenomyeloneuropathy, however, had significantly lower cytokine-stimulated cyclooxygenase and phospholipase A2 activities than normals, as well as lower prostaglandin E2 synthesis in response to interleukin 1. The peroxisomal disease lines exhibited dose-response curves to interleukin 1 similar to controls. Receptor-binding analysis indicated that cells from patients with rhizomelic chondrodysplasia punctata expressed 5-times fewer interleukin 1 receptors than normals and the other disease lines. Exaggerated arachidonic acid metabolism in response to interleukin 1 suggests that cells from patients with peroxisomal enzyme defects may be useful in elucidating pathways for arachidonate release and eicosanoid synthesis.

Properties of acid ceramidase from human spleen.

We have characterised ceramidase activity in extracts of human spleen from control subjects and from patients with Gaucher disease. In Triton X-100 extracts of control spleens, a broad pH optimum of pH 3.5-5.0 was found; no ceramidase activity was detectable at neutral or alkaline pH. About 45-60% of acid ceramidase could be extracted from spleen without detergents, but for complete extraction, Triton X-100 was required. For the radiolabelled substrate oleoylsphingosine, a Km of 0.22 +/- 0.09 mM and a Vmax of 57 +/- 11 nmol/h per mg protein was calculated in spleen from a control subject. Flat-bed isoelectric focussing in the presence of Triton X-100 revealed a pI of 6.0-7.0 for acid ceramidase; similar values were found for sphingomyelinase and glucerebrosidase. HPLC-gel filtration indicated that in the presence of Triton X-100, acid ceramidase has an Mr of about 100 kDa. In the absence of detergents, the enzyme forms high-molecular-weight aggregates. Similar aggregation behaviour was observed for sphingomyelinase, while the elution of beta-hexosaminidase was not affected by detergents. The elution profile of glucocerebrosidase was only slightly altered by Triton X-100. There was no difference in the properties of acid ceramidase present in spleen from control subjects and from patients with type I Gaucher disease.

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.

Clinical and therapeutic aspects of adrenoleukodystrophy and adrenomyeloneuropathy.

Adrenoleukodystrophy (ALD) and its adult variant adrenomyeloneuropathy (AMN) are X-linked diseases in which a deficiency of lignoceroyl-CoA ligase, a peroxisomal enzyme needed for the degradation of very long chain fatty acids (VLCFA), has been reported. The responsible gene recently has been cloned; it codes for a peroxisomal membrane protein, ALDP, which is a member of the ABC (ATP binding cassette) transporter superfamily. Elevations in VLCFA, particularly C24 and C26, have proven useful in the diagnosis of the childhood, adolescent and adult cerebral forms and AMN. ALD and AMN commonly coexist in the same families; the same VLCFA elevations and gene mutations have been recognized in both ALD and AMN. This phenotypic heterogeneity suggests the influence of an autosomal modifier gene. Dietary manipulation using glyceryl trioleate-trieurucate oil (Lorenzo's oil) has been highly successful in lowering VLCFA, but not in affecting the rate of neurologic deterioration in symptomatic ALD boys or AMN adults. Dietary pretreatment of neurologically asymptomatic ALD patients may have some benefit and is advisable at the present time. Currently, we recommend bone marrow transplantation for those patients who show evidence of early cerebral involvement and for whom a well-matched donor is available. A drug therapy trial utilizing beta interferon and thalidomide is underway.

The inflammatory myelinopathy of adreno-leukodystrophy: cells, effector molecules, and pathogenetic implications.

Prominent inflammation in the demyelinative lesion of adreno-leukodystrophy (ALD) has suggested an immune-mediated pathogenetic component. Commercially available antibodies to T cells, B cells, macrophages, class I and II molecules, complement, IgG, IgM, IgA, interleukin-1 (IL-1), intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor-alpha (TNF) were applied to paraffin sections of formaldehyde-fixed postmortem samples. Twenty-five primary demyelinative lesions from five juvenile ALD, three adult ALD, and three adrenomyeloneuropathic patients were evaluated with appropriate positive and negative controls. Macrophages and astrocytes were the predominant cells detected at the active edge; T lymphocytes, including T4 and CD45R subsets, were nearly as numerous but usually located around vessels within the lesion. B cells and plasma cells, usually containing IgG, were uncommon. The expression of class II molecules, restricted to one adult, was problematic; class I expression was increased in microvascular and other cells. Degraded myelin was labeled with antibodies to C3d and IL-1; IL-1 and ICAM-1 immunoreactivity was seen on microvessels and astrocytes. Tumor necrosis factor-alpha immunoreactivity was detected in macrophages, but more prominently in astrocytes. These data support a natural immune response in the demyelinative lesion of ALD, consisting predominantly of reactive astrocytes, macrophages, T cells and cytokines. A two-stage pathogenetic theory is discussed. The postulated roles of TNF and reactive astrocytes, in concert with a fundamental myelinolytic biochemical defect, suggest a different pathogenetic mechanism and raise novel therapeutic possibilities.

Adrenomyeloneuropathy: a neuropathologic review featuring its noninflammatory myelopathy.

The neuropathologic features of adrenomyeloneuropathy (AMN) are reviewed by supplementing those few previously published cases with 5 additional cases collected over the years. The endocrine involvement in AMN is briefly presented to serve as a pathogenetic backdrop and to emphasize that most of the lesions in AMN, as in adreno-leukodystrophy (ALD), are noninflammatory in the traditional sense of the word. The myeloneuropathy is emphasized, but the dysmyelinative/inflammatory demyelinative lesions also are presented. The preponderance of available data indicates that the myeloneuropathy of AMN is a central-peripheral distal (dying-back) axonopathy, as was originally proposed. The severity of the myeloneuropathy does not appear to correlate with the duration or severity of endocrine dysfunction. Microglia are the dominant participating cells in the noninflammatory myelopathy. Abnormalities in the ALD gene, which encodes a peroxisomal ABC half-transporter, do not correlate with clinical phenotypes. The relationship of the gene product, ALDP, to the peroxisomal very long chain fatty acid (VLCFA) synthetase, the activity of which is deficient in ALD/AMN, is unclear. An ALD-knockout mouse model has developed axonal degeneration, particularly in spinal cord, and is therefore more reminiscent of AMN than ALD. We continue to postulate that the fundamental defect in the myeloneuropathy of AMN is an axonal or neuronal membrane abnormality perhaps due to the incorporation of VLCFA-gangliosides, which perturbs the membrane's microenvironment and leads to dysfunction and atrophy.

Solvent vapor abuse leukoencephalopathy. Comparison to adrenoleukodystrophy.

Chronic organic solvent vapor inhalation can cause permanent damage to the central nervous system. Clinical features and radiologic abnormalities are well known, but pathology has not been definitely established. This study describes the gross, microscopic and ultrastructural changes and fatty acid composition of cholesterol esters in the brain of two chronic paint sniffers as well as the electron microscopic findings from a third, all with permanent neurological impairment. The abnormalities which were the same in all cases consisted of a demyelinating process which grossly manifested itself as brain atrophy and subtle discoloration of the cerebral and cerebellar white matter. Periodic acid-Schiff-positive macrophages in the absence of foamy macrophages were the histological hallmark of this process. Electron microscopy revealed oval membrane-bound cytoplasmic bodies filled with bundles of trilaminar inclusions composed of 3 nm paired dense leaflets separated by a space 3-7 nm wide in macrophages. Biochemical analysis showed an increase of very long chain fatty acids in the white matter cholesterol esters. This study defines the morphologic substrate of solvent vapor abuse leukoencephalopathy. The novel ultrastructural observations in conjunction with biochemical findings provide a link with adrenoleukodystrophy and raise the possibility of similar mechanisms of myelin degradation in both.

Structural and chemical alterations in the cerebral maldevelopment of fetal cerebro-hepato-renal (Zellweger) syndrome.

The cerebra of four abortuses (estimated gestational age 14-22 weeks), diagnosed as cerebro-hepato-renal (Zellweger) syndrome in utero, were examined morphologically with light microscopic, immunocytochemical and ultrastructural techniques and biochemically with gas liquid chromatographic assays for cholesterol ester fatty acids and plasmalogens. Centrosylvian architectonic abnormalities consisting, in part, of thin cortical plates and broad subcortical heterotopic zones were found in all abortuses. Astrocytes, neuroblasts, immature neurons and radial glia contained abnormal pleomorphic cytosomes, presumably of variable lipid composition. The same areas exhibited increases in cholesterol ester very long chain fatty acids and decreased plasmalogens. A pathogenetic hypothesis, proposing that regional tissue constraints act in concert with a peroxisomal-derived biochemical abnormality to impede centrosylvian neuronal migration, is discussed.

Potential environmental and host participants in the early white matter lesion of adreno-leukodystrophy: morphologic evidence for CD8 cytotoxic T cells, cytolysis of oligodendrocytes, and CD1-mediated lipid antigen presentation.

The 2 most common forms of X-linked adreno-leukodystrophy (ALD) are the juvenile or childhood cerebral form with inflammatory demyelination and the adult adrenomyeloneuropathy (AMN) involving spinal cord tracts without significant inflammation. Modifier genes or environmental factors may contribute to the phenotypic variability. We performed immunohistochemical, an in situ polymerase chain reaction, and TUNEL analyses to identify several viruses, lymphocyte subpopulations, apoptotic cells, and effector molecules, focusing on morphologically normal white matter, dysmyelinative and acute demyelinative lesions. No distinguishing viral antigens were detected. Most lymphocytes were CD8 cytotoxic T cells (CTLs) with the alpha/beta TCR, and they infiltrated morphologically unaffected white matter. Only a few oligodendrocytes were immunoreactive for caspase-3. MHC class II- and TGF-beta-positive microglia were present. CD44, which can mediate MHC-unrestricted target cell death, was seen on many lymphocytes and white matter elements. CD1 molecules, which play major roles in MHC-unrestricted lipid antigen presentation, were noted. Our data indicate that unconventional CD8 CTLs are operative in the early stages of dysmyelination/demyelination and that cytolysis of oligodendrocytes, rather than apoptosis, appears to be the major mode of oligodendrocytic death. The presentation of lipid antigens may be a key pathogenetic element in ALD and AMN-ALD.