Identification and functional characterization of two missense mutations in NDRG1 associated with Charcot-Marie-Tooth disease type 4D.

Charcot-Marie-Tooth disease type 4D (CMT4D) is an autosomal-recessive demyelinating form of CMT characterized by a severe distal motor and sensory neuropathy. NDRG1 is the causative gene for CMT4D. To date, only four mutations in NDRG1 -c.442C>T (p.Arg148*), c.739delC (p.His247Thrfs*74), c.538-1G>A, and duplication of exons 6-8-have been described in CMT4D patients. Here, using targeted next-generation sequencing examination, we identified for the first time two homozygous missense variants in NDRG1, c.437T>C (p.Leu146Pro) and c.701G>A (p.Arg234Gln), in two Chinese CMT families with consanguineous histories. Further functional studies were performed to characterize the biological effects of these variants. Cell culture transfection studies showed that mutant NDRG1 carrying p.Leu146Pro, p.Arg148*, or p.Arg234Gln variant degraded faster than wild-type NDRG1, resulting in lower protein levels. Live cell confocal microscopy and coimmunoprecipitation analysis indicated that these variants did not disrupt the interaction between NDRG1 and Rab4a protein. However, NDRG1-knockdown cells expressing mutant NDRG1 displayed enlarged Rab4a-positive compartments. Moreover, mutant NDRG1 could not enhance the uptake of DiI-LDL or increase the fraction of low-density lipoprotein receptor on the cell surface. Taken together, our study described two missense mutations in NDRG1 and emphasized the important role of NDRG1 in intracellular protein trafficking.

NDRG1 functions in LDL receptor trafficking by regulating endosomal recycling and degradation.

N-myc downstream-regulated gene 1 (NDRG1) mutations cause Charcot-Marie-Tooth disease type 4D (CMT4D). However, the cellular function of NDRG1 and how it causes CMT4D are poorly understood. We report that NDRG1 silencing in epithelial cells results in decreased uptake of low-density lipoprotein (LDL) due to reduced LDL receptor (LDLR) abundance at the plasma membrane. This is accompanied by the accumulation of LDLR in enlarged EEA1-positive endosomes that contain numerous intraluminal vesicles and sequester ceramide. Concomitantly, LDLR ubiquitylation is increased but its degradation is reduced and ESCRT (endosomal sorting complex required for transport) proteins are downregulated. Co-depletion of IDOL (inducible degrader of the LDLR), which ubiquitylates the LDLR and promotes its degradation, rescues plasma membrane LDLR levels and LDL uptake. In murine oligodendrocytes, Ndrg1 silencing not only results in reduced LDL uptake but also in downregulation of the oligodendrocyte differentiation factor Olig2. Both phenotypes are rescued by co-silencing of Idol, suggesting that ligand uptake through LDLR family members controls oligodendrocyte differentiation. These findings identify NDRG1 as a novel regulator of multivesicular body formation and endosomal LDLR trafficking. The deficiency of functional NDRG1 in CMT4D might impair lipid processing and differentiation of myelinating cells.

Identification of PAHX, a Refsum disease gene.

Refsum disease is an autosomal recessive disorder characterized by retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia and increased cerebrospinal fluid protein. Biochemically, the disorder is defined by two related properties: pronounced accumulation of phytanic acid and selective loss of the peroxisomal dioxygenase required for alpha-hydroxylation of phytanoyl-CoA2. Decreased phytanic-acid oxidation is also observed in human cells lacking PEX7, the receptor for the type-2 peroxisomal targetting signal (PTS2; refs 3,4), suggesting that the enzyme defective in Refsum disease is targetted to peroxisomes by a PTS2. We initially identified the human PAHX and mouse Pahx genes as expressed sequence tags (ESTs) capable of encoding PTS2 proteins. Human PAHX is targetted to peroxisomes, requires the PTS2 receptor for peroxisomal localization, interacts with the PTS2 receptor in the yeast two-hybrid assay and has intrinsic phytanoyl-CoA alpha-hydroxylase activity that requires the dioxygenase cofactor iron and cosubstrate 2-oxoglutarate. Radiation hybrid data place PAHX on chromosome 10 between the markers D10S249 and D10S466, a region previously implicated in Refsum disease by homozygosity mapping. We find that both Refsum disease patients examined are homozygous for inactivating mutations in PAHX, demonstrating that mutations in PAHX can cause Refsum disease.

Evidence of increases of phytol and chlorophyllide by enzymatic dephytylation of chlorophylls in smoothie made from spinach leaves.

Phytol is a diterpene alcohol found abundantly in nature as the phytyl side chain of chlorophylls. Free form of phytol and its metabolites have been attracting attention because they have a potential to improve the lipid and glucose metabolism. On the other hand, phytol is unfavorable for those who suffering from Refsum's disease. However, there is little information on the phytol contents in leafy vegetables rich in chlorophylls. This study indicated that raw spinach leaves contain phytol of 0.4-1.5 mg/100 g fresh weight. Furthermore, crude enzyme extracted from the leaves showed the enzyme activities involved in dephytylation of chlorophyll derivatives and they were high at mild alkaline pH and around 45°C, and lowered at 55°C or above. Under the optimum pH and temperature for such enzymes determined in the model reaction using the crude enzyme, phytol content in the smoothie made from raw spinach leaves increased with an increase of chlorophyllide, another reaction product. Comparison between the increased amounts of phytol and chlorophyllide showed that the enzymatic dephytylation of chlorophylls was critically responsible for the increase of phytol in the smoothie. PRACTICAL APPLICATION: Phytol, which is released by the enzymes related to chlorophyll metabolism in plants, has been investigated because of its potential abilities to improve the lipid metabolism and blood glucose level. In contrast to such health benefits, they are known to be toxic for patients suffering from Refsum's disease. This research for the first time reports the phytol content in raw spinach leaves and that phytol can be increased in the smoothie made from spinach leaves by the action of endogenous enzymes on chlorophyll derivatives under a certain condition. These results help control phytol content in the smoothies.

Disruption of mitochondrial bioenergetics and calcium homeostasis by phytanic acid in the heart: Potential relevance for the cardiomyopathy in Refsum disease.

Refsum disease is an inherited peroxisomal disorder caused by severe deficiency of phytanoyl-CoA hydroxylase activity. Affected patients develop severe cardiomyopathy of poorly known pathogenesis that may lead to a fatal outcome. Since phytanic acid (Phyt) concentrations are highly increased in tissues of individuals with this disease, it is conceivable that this branched-chain fatty acid is cardiotoxic. The present study investigated whether Phyt (10-30 µM) could disturb important mitochondrial functions in rat heart mitochondria. We also determined the influence of Phyt (50-100 µM) on cell viability (MTT reduction) in cardiac cells (H9C2). Phyt markedly increased mitochondrial state 4 (resting) and decreased state 3 (ADP-stimulated) and uncoupled (CCCP-stimulated) respirations, besides reducing the respiratory control ratio, ATP synthesis and the activities of the respiratory chain complexes I-III, II, and II-III. This fatty acid also reduced mitochondrial membrane potential and induced swelling in mitochondria supplemented by exogenous Ca2+, which were prevented by cyclosporin A alone or combined with ADP, suggesting the involvement of the mitochondrial permeability transition (MPT) pore opening. Mitochondrial NAD(P)H content and Ca2+ retention capacity were also decreased by Phyt in the presence of Ca2+. Finally, Phyt significantly reduced cellular viability (MTT reduction) in cultured cardiomyocytes. The present data indicate that Phyt, at concentrations found in the plasma of patients with Refsum disease, disrupts by multiple mechanisms mitochondrial bioenergetics and Ca2+ homeostasis, which could presumably be involved in the cardiomyopathy of this disease.

Phytanic acid metabolism in health and disease.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid which cannot be beta-oxidized due to the presence of the first methyl group at the 3-position. Instead, phytanic acid undergoes alpha-oxidation to produce pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) plus CO(2). Pristanic acid is a 2-methyl branched-chain fatty acid which can undergo beta-oxidation via sequential cycles of beta-oxidation in peroxisomes and mitochondria. The mechanism of alpha-oxidation has been resolved in recent years as reviewed in this paper, although some of the individual enzymatic steps remain to be identified. Furthermore, much has been learned in recent years about the permeability properties of the peroxisomal membrane with important consequences for the alpha-oxidation process. Finally, we present new data on the omega-oxidation of phytanic acid making use of a recently generated mouse model for Refsum disease in which the gene encoding phytanoyl-CoA 2-hydroxylase has been disrupted.

Phytanic acid disturbs mitochondrial homeostasis in heart of young rats: a possible pathomechanism of cardiomyopathy in Refsum disease.

Phytanic acid (Phyt) accumulates in tissues and biological fluids of patients affected by Refsum disease. Although cardiomyopathy is an important clinical manifestation of this disorder, the mechanisms of heart damage are poorly known. In the present study, we investigated the in vitro effects of Phyt on important parameters of oxidative stress in heart of young rats. Phyt significantly increased thiobarbituric acid-reactive substances levels (P < 0.001) and carbonyl formation (P < 0.01), indicating that this fatty acid induces lipid and protein oxidative damage, respectively. In contrast, Phyt did not alter sulfhydryl oxidation. Phyt also decreased glutathione (GSH) concentrations (P < 0.05), an important non-enzymatic antioxidant defense. Moreover, Phyt increased 2',7'-dichlorofluorescin oxidation (DCFH) (P < 0.01), reflecting increased reactive species generation. We also found that the induced lipid and protein oxidative damage, as well as the decreased GSH levels and increased DCFH oxidation provoked by this fatty acid were prevented or attenuated by the reactive oxygen species scavengers melatonin, trolox, and GSH, but not by the nitric oxide inhibitor N: (ω)-nitro-L: -arginine methyl ester, suggesting that reactive oxygen species were involved in these effects. Next, we verified that Phyt strongly inhibited NADH-cytochrome c oxidoreductase (complex I-III) activity (P < 0.001) in heart supernatants, and decreased membrane potential and the NAD(P)H pool in heart mitochondria, indicating that Phyt acts as a metabolic inhibitor and as an uncoupler of the electron transport chain. Therefore, it can be presumed that disturbance of cellular energy and redox homeostasis induced by Phyt may possibly contribute to the cardiomyopathy found in patients affected by Refsum disease.

NDRG1 is induced by antigen-receptor signaling but dispensable for B and T cell self-tolerance.

Peripheral tolerance prevents the initiation of damaging immune responses by autoreactive lymphocytes. While tolerogenic mechanisms are tightly regulated by antigen-dependent and independent signals, downstream pathways are incompletely understood. N-myc downstream-regulated gene 1 (NDRG1), an anti-cancer therapeutic target, has previously been implicated as a CD4+ T cell clonal anergy factor. By RNA-sequencing, we identified Ndrg1 as the third most upregulated gene in anergic, compared to naïve follicular, B cells. Ndrg1 is upregulated by B cell receptor activation (signal one) and suppressed by co-stimulation (signal two), suggesting that NDRG1 may be important in B cell tolerance. However, though Ndrg1-/- mice have a neurological defect mimicking NDRG1-associated Charcot-Marie-Tooth (CMT4d) disease, primary and secondary immune responses were normal. We find that B cell tolerance is maintained, and NDRG1 does not play a role in downstream responses during re-stimulation of in vivo antigen-experienced CD4+ T cells, demonstrating that NDGR1 is functionally redundant for lymphocyte anergy.

Aberrant Neuregulin 1/ErbB Signaling in Charcot-Marie-Tooth Type 4D Disease.

Charcot-Marie-Tooth type 4D (CMT4D) is an autosomal recessive demyelinating form of CMT characterized by progressive motor and sensory neuropathy. N-myc downstream regulated gene 1 (NDRG1) is the causative gene for CMT4D. Although more CMT4D cases have been reported, the comprehensive molecular mechanism underlying CMT4D remains elusive. Here, we generated a novel knockout mouse model in which the fourth and fifth exons of the Ndrg1 gene were removed. Ndrg1-deficient mice develop early progressive demyelinating neuropathy and limb muscle weakness. The expression pattern of myelination-related transcriptional factors, including SOX10, OCT6, and EGR2, was abnormal in Ndrg1-deficient mice. We further investigated the activation of the ErbB2/3 receptor tyrosine kinases in Ndrg1-deficient sciatic nerves, as these proteins play essential roles in Schwann cell myelination. In the absence of NDRG1, although the total ErbB2/3 receptors expressed by Schwann cells were significantly increased, levels of the phosphorylated forms of ErbB2/3 and their downstream signaling cascades were decreased. This change was not associated with the level of the neuregulin 1 ligand, which was increased in Ndrg1-deficient mice. In addition, the integrin ß4 receptor, which interacts with ErbB2/3 and positively regulates neuregulin 1/ErbB signaling, was significantly reduced in the Ndrg1-deficient nerve. In conclusion, our data suggest that the demyelinating phenotype of CMT4D disease is at least in part a consequence of molecular defects in neuregulin 1/ErbB signaling.

Phytanic acid and pristanic acid, branched-chain fatty acids associated with Refsum disease and other inherited peroxisomal disorders, mediate intracellular Ca2+ signaling through activation of free fatty acid receptor GPR40.

The accumulation of the two branched-chain fatty acids phytanic acid and pristanic acid is known to play an important role in several diseases with peroxisomal impairment, like Refsum disease, Zellweger syndrome and α-methylacyl-CoA racemase deficiency. Recent studies elucidated that the toxic activity of phytanic acid and pristanic acid is mediated by multiple mitochondrial dysfunctions, generation of reactive oxygen species and Ca2+ deregulation via the InsP3-Ca2+ signaling pathway in glial cells. However, the exact signaling mechanism through which both fatty acids mediate toxicity is still under debate. Here, we studied the ability of phytanic acid and pristanic acid to activate the free fatty acid receptor GPR40, a G-protein-coupled receptor, which was described to be involved in the Ca2+ signaling of fatty acids. We treated HEK 293 cells expressing the GPR40 receptor with phytanic acid or pristanic acid. This resulted in a significant increase in the intracellular Ca2+ level, similar to the effect seen after treatment with the synthetic GPR40 agonist GW9508. Furthermore, we demonstrate that the GPR40 activation might be due to an interaction of the carboxylate moiety of fatty acids with the receptor. Our findings indicate that the phytanic acid- and pristanic acid-mediated Ca2+ deregulation can involve the activation of GPR40. Therefore, we suppose that activation of GPR40 might be part of the signaling cascade of the toxicity of phytanic and pristanic acids.

Ndrg1 in development and maintenance of the myelin sheath.

CMT4D disease is a severe autosomal recessive demyelinating neuropathy with extensive axonal loss leading to early disability, caused by mutations in the N-myc downstream regulated gene 1 (NDRG1). NDRG1 is expressed at particularly high levels in the Schwann cell (SC), but its physiological function(s) are unknown. To help with their understanding, we characterise the phenotype of a new mouse model, stretcher (str), with total Ndrg1 deficiency, in comparison with the hypomorphic Ndrg1 knock-out (KO) mouse. While both models display normal initial myelination and a transition to overt pathology between weeks 3 and 5, the markedly more severe str phenotype suggests that even low Ndrg1 expression results in significant phenotype rescue. Neither model replicates fully the features of CMT4D: although axon damage is present, regenerative capacity is unimpaired and the mice do not display the early severe axonal loss typical of the human disease. The widespread large fibre demyelination coincides precisely with the period of rapid growth of the animals and the dramatic (160-500-fold) increase in myelin volume and length in large fibres. This is followed by stabilisation after week 10, while small fibres remain unaffected. Gene expression profiling of str peripheral nerve reveals non-specific secondary changes at weeks 5 and 10 and preliminary data point to normal proteasomal function. Our findings do not support the proposed roles of NDRG1 in growth arrest, terminal differentiation, gene expression regulation and proteasomal degradation. Impaired SC trafficking failing to meet the considerable demands of nerve growth, emerges as the likely pathogenetic mechanism in NDRG1 deficiency.

Fibroblast-specific genome-scale modelling predicts an imbalance in amino acid metabolism in Refsum disease.

Refsum disease (RD) is an inborn error of metabolism that is characterised by a defect in peroxisomal α-oxidation of the branched-chain fatty acid phytanic acid. The disorder presents with late-onset progressive retinitis pigmentosa and polyneuropathy and can be diagnosed biochemically by elevated levels of phytanate in plasma and tissues of patients. To date, no cure exists for RD, but phytanate levels in patients can be reduced by plasmapheresis and a strict diet. In this study, we reconstructed a fibroblast-specific genome-scale model based on the recently published, FAD-curated model, based on Recon3D reconstruction. We used transcriptomics (available via GEO database with identifier GSE138379), metabolomics and proteomics (available via ProteomeXchange with identifier PXD015518) data, which we obtained from healthy controls and RD patient fibroblasts incubated with phytol, a precursor of phytanic acid. Our model correctly represents the metabolism of phytanate and displays fibroblast-specific metabolic functions. Using this model, we investigated the metabolic phenotype of RD at the genome scale, and we studied the effect of phytanate on cell metabolism. We identified 53 metabolites that were predicted to discriminate between healthy and RD patients, several of which with a link to amino acid metabolism. Ultimately, these insights in metabolic changes may provide leads for pathophysiology and therapy. DATABASES: Transcriptomics data are available via GEO database with identifier GSE138379, and proteomics data are available via ProteomeXchange with identifier PXD015518.

The influence of the branched-chain fatty acids pristanic acid and Refsum disease-associated phytanic acid on mitochondrial functions and calcium regulation of hippocampal neurons, astrocytes, and oligodendrocytes.

Pristanic acid and phytanic acid are branched-chain fatty acids, which play an important role in diseases with peroxisomal impairment, like Refsum disease (MIM 266500), Zellwegers syndrome and alpha-methylacyl-CoA racemase deficiency (MIM 604489). Several studies revealed that the toxic activity of phytanic acid is mediated by multiple mitochondrial dysfunctions. However, the action of pristanic acid on brain cells is still completely unknown. Here, we exposed astrocytes, oligodendrocytes and neurons in mixed culture to pristanic acid and phytanic acid to analyse cellular consequences. Pristanic acid exerts a strong cytotoxic activity on brain cells, displayed by dramatic Ca2+ deregulation, in situ mitochondrial depolarization and cell death. Interestingly, pristanic acid strongly induced generation of reactive oxygen species (ROS), whereas phytanic acid exerts weaker effects on ROS production. In conclusion, pristanic acid as well as phytanic acid induced a complex array of toxic activities with mitochondrial dysfunction and Ca2+ deregulation.

Refsum's disease: nature of the enzyme defect.

Two siblings with Refsum's disease, an inherited disorder of lipid metabolism, oxidized intravenously injected uniformly labeled phytanic acid-C(14) at rates less than 5 percent of those found in normal subjects. The defect in oxidation of phytanic acid persisted in cultures of fibroblasts from the patients' skin. The rate of oxidation of the phytanic acid-C(14) was less than 1 percent of that found in cultures of fibroblasts from normal skin. However, pristanic acid, previously shown to be the first product of phytanic acid degradation, was oxidized at a normal rate in the patients' cultures. These results indicate that the enzymatic defect in Refsum's disease is in the first step of the pathway for degradation of phytanic acid, that is, in the unusual alpha-oxidative process that leads to a shortening of phytanic acid by one carbon atom.

The import receptor Pex7p and the PTS2 targeting sequence.

This chapter concerns one branch of the peroxisome import pathway for newly-synthesized peroxisomal proteins, specifically the branch for matrix proteins that contain a peroxisome targeting sequence type 2 (PTS2). The structure and utilization of the PTS2 are discussed, as well as the properties of the receptor, Pex7p, which recognizes the PTS2 sequence and conveys these proteins to the common translocation machinery in the peroxisome membrane. We also describe the recent evidence that this receptor recycles into the peroxisome matrix and back out to the cytosol in the course of its function. Pex7p is assisted in its functioning by several species-specific auxiliary proteins that are described in the following chapter.

Refsum's disease: characterization of the enzyme defect in cell culture.

Refsum's disease (heredopathia atactica polyneuritiformis, HAP) is an inherited neurological disorder associated with storage of the branched-chain fatty acid, phytanic acid (3,7,11,15-tetramethylhexadecanoic acid). Cultured fibroblasts derived from skin biopsies of HAP patients did not contain elevated levels of phytanate, yet showed rates of phytanate-C-(14)C oxidation less than 3% of those seen in cells from control subjects. Cells of control subjects converted phytanate to alpha-hydroxyphytanate, to pristanate (the [n-1] homologue of phytanate) and to 4,8,12-trimethyltridecanoate, compounds previously identified as intermediates on the major pathway for phytanate metabolism in animals, providing the first direct evidence that this same oxidative pathway is operative in human cells. None of these breakdown products could be found after incubation of phytanate with HAP cells. Labeled alpha-hydroxyphytanate and labeled pristanate were oxidized at normal rates by HAP cells. Oxidation of the latter proceeded at normal rates both when added to the medium at very low tracer levels and at levels 100 times greater. Phytanate was incorporated into and released from lipid esters at normal rates by HAP cells. Elevated levels of free phytanate in the medium were no more toxic to HAP cells than to control cells over the 48- to 72-hr exposures involved in these studies, as evidenced by morphologic criteria and by ability to oxidize labeled palmitate. These findings are consistent with the hypothesis that the cells from HAP patients are deficient in a single enzyme involved in the alpha-hydroxylation of phytanate, while the enzymes involved in later steps are present at normal or near-normal levels.

Studies on the metabolic error in Refsum's disease.

Studies utilizing mevalonic acid-2-(14)C and D(2)O as precursors failed to provide evidence for an appreciable rate of endogenous biosynthesis of phytanic acid in a patient with Refsum's disease. Orally administered tracer doses of phytol-U-(14)C were well absorbed both by seven normal control subjects (61 to 94%) and by two patients with Refsum's disease (74 and 80%). The fraction of the absorbed dose converted to (14)CO(2) in 12 hours was 3.5 and 5.8% in Refsum's disease patients and averaged 20.9% in seven control subjects. Labeled phytanic acid was demonstrated in the plasma of both control subjects and patients given phytol-U-(14)C, establishing phytol in the diet as a potential precursor of phytanic acid. This labeled phytanic acid had disappeared almost completely from the plasma of the seven control subjects by 24 to 48 hours, whereas it persisted at high concentrations in the plasma of the two patients for many days. We conclude that the phytanic acid accumulating in Refsum's disease is primarily of exogenous origin and that patients with Refsum's disease have a relative block in the degradation of phytanic acid and possibly other similar branched-chain compounds. This may relate to a deficiency in mechanisms for release of phytanic acid from stored ester forms or, more probably, to reactions essential to oxidative degradation of the carbon skeleton.

Attenuated prostaglandin formation in peroxisomal-deficient human skin fibroblasts.

Peroxisomal-deficient skin fibroblasts from patients with Zellweger's syndrome or infantile Refsum's disease produced fewer prostaglandins than normal skin fibroblasts. Radioimmunoassay indicated a 45-55% decrease in prostaglandin E2 (PGE2) production when Zellweger's fibroblasts were incubated with arachidonic acid. This deficiency was not overcome by pretreatment of the Zellweger's fibroblasts with media containing arachidonic acid, and it was not due to channeling of arachidonic acid into other eicosanoid products. Modifications in the peroxide tone of the Zellweger's fibroblasts by addition of H2O2 or catalase failed to increase PGE2 production. Using Northern analysis, we were unable to detect an mRNA transcript for PGH synthase in unstimulated Zellweger fibroblasts but identified a 4.2-kb mRNA transcript after treatment with phorbol myristate acetate (PMA). Treatment for 6 h with 10 nM PMA raised PGE2 production in normal and Zellweger fibroblasts to equivalent levels. These increases were prevented by addition of H-7, staurosporine, cycloheximide, or actinomycin D. Our findings suggest that the reduced PGE2 production in peroxisomal deficient fibroblasts is due to a decrease in PGH synthase mRNA. The reduction in PGH synthase can be overcome by treatment of the cells with agents which enhance gene expression.

Presence of cytoplasmic factors functional in peroxisomal protein import implicates organelle-associated defects in several human peroxisomal disorders.

Cells from patients with peroxisome-deficient disorders contain membrane ghosts devoid of most matrix contents instead of normal peroxisomes indicating that the underlying molecular defects impair the import of matrix proteins into these peroxisome ghosts. Genetic heterogeneity for the molecular defects was inferred from the assignment of patients with peroxisome-deficient disorders into nine complementation groups. The aim of our studies was to analyze cell lines from six different complementation groups in a systematic manner for the presence of peroxisome ghosts, the ability to import Ser-Lys-Leu-containing proteins into peroxisome ghosts and for the presence of cytosolic factors required for peroxisomal protein import. We show that each of the cell lines analyzed contains peroxisome ghosts, but is unable to import matrix proteins as judged by a peroxisomal import assay using permeabilized cells. The addition of wild type cytosol did not restore the capacity to import matrix proteins but cytosol prepared from these cell lines was functional in stimulation of peroxisomal protein import in a heterologous system. These results implicate organelle-associated molecular defects in each of the six cell lines analyzed.

Localization of the oxidative defect in phytanic acid degradation in patients with Refsum's disease.

The rate of oxidation of phytanic acid-U-(14)C to (14)CO(2) in three patients with Refsum's disease was less than 5% of that found in normal volunteers. In contrast, the rate of oxidation of alpha-hydroxyphytanic acid-U-(14)C and of pristanic acid-U-(14)C to (14)CO(2), studied in two patients, while somewhat less than that in normal controls, was not grossly impaired. These studies support the conclusion that the defect in phytanic acid oxidation in Refsum's disease is located in the first step of phytanic acid degradation, that is, in the alpha oxidation step leading to formation of alpha-hydroxyphytanic acid. The initial rate of disappearance of plasma free fatty acid radioactivity after intravenous injection of phytanic acid-U-(14)C (t(1/2) = 5.9 min) was slower than that seen with pristanic acid-U-(14)C (t(1/2) = 2.7 min) or palmitic acid-1-(14)C (t(1/2) = 2.5 min). There were no differences between patients and normal controls in these initial rates of free fatty acid disappearance for any of the three substrates tested. There was no detectable lipid radioactivity found in the plasma 7 days after the injection of palmitic acid-1-(14)C or pristanic acid-U-(14)C in either patients or controls. After injection of phytanic acid-U-(14)C, however, the two patients showed only a very slow decline in plasma lipid radioactivity (estimated t(1/2) = 35 days), in contrast to the normals who had no detectable radioactivity after 2 days. Incorporation of radioactivity from phytanic acid-U-(14)C into the major lipid ester classes of plasma was studied in one of the patients; triglycerides accounted for by far the largest fraction of the total present between 1 and 4 hr.