Marked inhibition of Na+, K(+)- ATPase activity and the respiratory chain by phytanic acid in cerebellum from young rats: possible underlying mechanisms of cerebellar ataxia in Refsum disease.

Refsum disease is an autosomal recessive disorder of peroxisomal metabolism biochemically characterized by highly elevated concentrations of phytanic acid (Phyt) in a variety of tissues including the cerebellum. Reduction of plasma Phyt levels by dietary restriction intake ameliorates ataxia, a common clinical manifestation of this disorder, suggesting a neurotoxic role for this branched-chain fatty acid. Therefore, considering that the underlying mechanisms of cerebellum damage in Refsum disease are poorly known, in the present study we tested the effects of Phyt on important parameters of bioenergetics, such as the activities of the respiratory chain complexes I to IV, creatine kinase and Na(+), K(+)- ATPase in cerebellum preparations from young rats. The activities of complexes I, II, I-III and II-III and Na(+), K(+)- ATPase were markedly inhibited (65-85%) in a dose-dependent manner by Phyt. In contrast, creatine kinase and complex IV activities were not altered by this fatty acid. Therefore, it is presumed that impairment of the electron flow through the respiratory chain and inhibition of Na(+), K(+)- ATPase that is crucial for synaptic function may be involved in the pathophysiology of the cerebellar abnormalities manifested as ataxia in Refsum disease and in other peroxisomal disorders in which brain Phyt accumulates.

Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene.

Refsum disease is an autosomal-recessively inherited disorder characterized clinically by a tetrad of abnormalities: retinitis pigmentosa, peripheral neuropathy, cerebellar ataxia and elevated protein levels in the cerebrospinal fluid (CSF) without an increase in the number of cells in the CSF. All patients exhibit accumulation of an unusual branched-chain fatty acid, phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), in blood and tissues. Biochemically, the disease is caused by the deficiency of phytanoyl-CoA hydroxylase (PhyH), a peroxisomal protein catalyzing the first step in the alpha-oxidation of phytanic acid. We have purified PhyH from rat-liver peroxisomes and determined the N-terminal amino-acid sequence, as well as an additional internal amino-acid sequence obtained after Lys-C digestion of the purified protein. A search of the EST database with these partial amino-acid sequences led to the identification of the full-length human cDNA sequence encoding PhyH: the open reading frame encodes a 41.2-kD protein of 338 amino acids, which contains a cleavable peroxisomal targeting signal type 2 (PTS2). Sequence analysis of PHYH fibroblast cDNA from five patients with Refsum disease revealed distinct mutations, including a one-nucleotide deletion, a 111-nucleotide deletion and a point mutation. This analysis confirms our finding that Refsum disease is caused by a deficiency of PhyH.

Ataxia with loss of Purkinje cells in a mouse model for Refsum disease.

Refsum disease is caused by a deficiency of phytanoyl-CoA hydroxylase (PHYH), the first enzyme of the peroxisomal alpha-oxidation system, resulting in the accumulation of the branched-chain fatty acid phytanic acid. The main clinical symptoms are polyneuropathy, cerebellar ataxia, and retinitis pigmentosa. To study the pathogenesis of Refsum disease, we generated and characterized a Phyh knockout mouse. We studied the pathological effects of phytanic acid accumulation in Phyh(-/-) mice fed a diet supplemented with phytol, the precursor of phytanic acid. Phytanic acid accumulation caused a reduction in body weight, hepatic steatosis, and testicular atrophy with loss of spermatogonia. Phenotype assessment using the SHIRPA protocol and subsequent automated gait analysis using the CatWalk system revealed unsteady gait with strongly reduced paw print area for both fore- and hindpaws and reduced base of support for the hindpaws. Histochemical analyses in the CNS showed astrocytosis and up-regulation of calcium-binding proteins. In addition, a loss of Purkinje cells in the cerebellum was observed. No demyelination was present in the CNS. Motor nerve conduction velocity measurements revealed a peripheral neuropathy. Our results show that, in the mouse, high phytanic acid levels cause a peripheral neuropathy and ataxia with loss of Purkinje cells. These findings provide important insights in the pathophysiology of Refsum disease.

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.

Brain pyruvate and 2-oxoglutarate dehydrogenase complexes are mitochondrial targets of the CoA ester of the Refsum disease marker phytanic acid.

Pyruvate and 2-oxoglutarate dehydrogenase complexes are strongly inhibited by phytanoyl-CoA (IC(50) approximately 10(-6)-10(-7) M). Palmitoyl-CoA is 10-fold less potent. Phytanic or palmitic acids have no inhibitory effect up to 0.3 mM. At the substrate saturation, the acyl-CoA's affect the first and second enzymatic components of the 2-oxoglutarate dehydrogenase complex, while the third component is inhibited only at a low saturation with its substrate dihydrolipoamide. Thus, key regulatory branch points of mitochondrial metabolism are targets of a cellular derivative of phytanic acid. Decreased activity of the complexes might therefore contribute to neurological symptoms upon accumulation of phytanic acid in Refsum disease.

Phenotypic heterogeneity in cultured skin fibroblasts from patients with disorders of peroxisome biogenesis belonging to the same complementation group.

We have studied fibroblast cell lines derived from a control subject (cell line 85AD5035F) and three patients clinically described as having the Zellweger syndrome (cell line W78/515), the infantile form of Refsum disease (cell line BOV84AD) and hyperpipecolic acidaemia (cell line GM3605), respectively. The mutant cell lines belonged to the same complementation group. The fibroblasts were cultured under identical conditions and were harvested at different time intervals after reaching confluence. Several peroxisomal parameters were determined. In agreement with previous reports, a lowered enzymic activity of acyl-CoA: dihydroxyacetonephosphate acyltransferase and a decrease in latent catalase clearly distinguished the patient cell lines from the control cell line. However, the cell lines exhibited a phenotypic heterogeneity. This was most strikingly encountered when cells were processed for indirect immunofluorescence microscopy and stained with anti-(catalase). The control cells exhibited a punctate fluorescence, which is indicative of the presence of catalase in peroxisomes. In the mutant cell line W78/515 a diffuse fluorescence was observed, indicative of the presence of catalase in the cytosol. In the other two mutant cell lines a punctate fluorescence was observed in some of the cells. Moreover, clear differences in the extent of proteolytic processing of acyl-CoA oxidase were detected. The mutant cell line BOV84AD displayed a control-like pattern with all molecular forms of acyl-CoA oxidase (72, 52 and 20 kDa) present, whereas in the W78/515 cell line only the 72 kDa component could be visualised. The GM3605 cell line was intermediate in this respect.

Identification of genetic heterogeneity in Refsum's disease.

Refsum's disease (MIM 266500) is a recessive disorder characterised by defective peroxisomal alpha-oxidation of phytanic acid. A Refsum's disease gene, phytanoyl-CoA hydroxylase (PAHX), has been localised to chromosome 10p13 between the markers D10S226-D10S223. This study investigated whether all cases of Refsum's disease were linked with chromosome 10p13. Eight genetically informative families comprising 92 individuals including 17 living patients with a Refsum's disease phenotype and initial plasma phytanic acid > 200 micromol/L were recruited. Linkage to the 10pter-10p11.2 region was investigated using a panel of eight dinucleotide repeat markers. Linkage analysis of this phenotypically identical cohort suggested that Refsum's disease was genetically heterogeneous (Zmax = 5.28, alpha = 0.45). Two subgroups were identified. One group of four families with eight affected individuals had a maximum multipoint lod score for linkage of 3.89 in the region D10S547 to D10S191, whilst in another three families with nine affected individuals linkage to this region was definitely excluded. Our results show that Refsum's disease is genetically heterogeneous, with up to 55% of cases not being linked to the PAHX gene locus at D10S547 to D10S223. This suggests that Refsum's disease, in common with other peroxisomal 'diseases', may be more accurately described as a heterogeneous syndrome.

Restoration of phytanic acid oxidation in Refsum disease fibroblasts from patients with mutations in the phytanoyl-CoA hydroxylase gene.

Refsum disease (RD) is biochemically characterized by the excessive accumulation of phytanic acid in tissues and body fluids due to deficiency of phytanoyl-CoA hydroxylase (PAHX). In this study, we screened three RD patients and identified a novel deletion (88 amino acids), and a missense mutation (Arg275Trp) in the previously reported PAHX cDNA (Jansen et al., 1997; Mihalik et al., 1997). Moreover, transfection of skin fibroblasts from two RD patients with wild-type PAHX gene restored the activity for alpha-oxidation of phytanic acid. Southern analysis on a somatic cell hybrid panel detected the PAHX gene on chromosome 10, corroborating radiation hybrid and homozygosity mapping data (Mihalik et al., 1997; Nadal et al., 1995).

Hepatic peroxisomes are deficient in infantile refsum disease: a cytochemical study of 4 cases.

We examined liver biopsies from 4 patients with the infantile form of Refsum disease. No peroxisomes were visualized by light microscopy after cytochemical staining for catalase, a marker enzyme for this organelle. Absence of peroxisomes was confirmed by electron microscopy in 3 patients; in the 4th patient we observed organelles of peculiar size and structure and with minimal catalase activity. Light microscopy also showed birefringent macrophages containing P.A.S.-positive material; they were abundant in the 3 older children, and rare in the youngest (8 months). Peroxisomes and birefringent macrophages were absent in 2 patients with the cerebrohepatorenal syndrome of Zellweger. The simultaneous presence of these unique light microscopical characteristics may be of diagnostic value.

Identification of a brain specific protein that associates with a refsum disease gene product, phytanoyl-CoA alpha-hydroxylase.

Refsum disease is an autosomal recessive neurologic disorder of the lipid metabolism. Major diagnostic clinical findings include retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia, increased cerebrospinal fluid protein without pleocytosis, nerve deafness, and cardiac involvement. We have identified a novel protein (PAHX-AP #1) associated with phytanoyl-CoA alpha-hydroxylase (PAHX), a Refsum disease gene product, using the yeast-based two-hybrid assay. The middle portion (amino acids 83-264) of PAHX was used as a bait and a mouse brain cDNA library was searched. The ability of PAHX-AP #1 to interact with PAHX was confirmed using immunoprecipitation and Western blot studies in NIH3T3 cells which stably expressed both PAHX and PAHX-AP #1. Northern and Western blot analyses demonstrated a unique pattern of developmental PAHX-AP #1 expression which was targeted to the adult brain, but ubiquitous expressions of PAHX were observed in all examined tissues. In situ hybridization analyses of the brain showed specific localization of PAHX-AP #1 to the supragranular layer in the cerebral cortex, dentate gyrus, hippocampus, Purkinje cell layer, deep cerebellar nucleus, trigeminal nucleus, abducent nucleus, facial nucleus, cochlear and vestibular nucleus, ganglion cell and nuclear layer of the retina. These data indicate that localization of PAHX-AP #1 in the brain is correlated with central neurologic symptoms of Refsum disease such as retinitis pigmentosa, cerebellar ataxia, nerve deafness and suggest that PAHX-AP #1 may be involved in the development of the central neurologic deficits of Refsum disease.

Clinical and biochemical heterogeneity in conditions with phytanic acid accumulation.

Phytanic acid accumulation has for more than 20 years been used as a diagnostic criterion of Refsum's disease. Recently, however, phytanic acid has also been found in peroxisomal disorders (Zellweger's syndrome, neonatal adrenoleukodystrophy, infantile Refsum's syndrome, rhizomelic chondrodysplasia punctata). The 17 patients with Refsum's disease in the present study had serum phytanic acid values differing from 73 to less than 0.5 mg/dl (normal). alpha-Oxidation of phytanic acid in skin fibroblast cultures showed a defective capacity in all, with only small differences in residual activity. Phytanic acid determinations in serum from 3 of the 7 patients with peroxisomal disorders showed slightly elevated levels in 2. The alpha-oxidation capacity in the fibroblasts was defective in all, with a residual activity similar to that of Refsum's disease. An assay of the alpha-oxidation capacity may be useful in the diagnosis of both Refsum's disease and the peroxisomal disorders. The distinction between Refsum's disease and the peroxisomal disorders can easily be done on a clinical basis.

Peroxisomal functions in classical Refsum's disease: comparison with the infantile form of Refsum's disease.

The infantile and classical forms of Refsum's disease are generally considered to belong to the newly recognized group of peroxisomal disorders. In this study we carried out a detailed investigation into different peroxisomal functions in classical Refsum's disease by analyses of plasma (very long chain fatty acids, di- and trihydroxycoprostanoic acid and pipecolic acid) and cultured skin fibroblasts from the patients (de novo plasmalogen biosynthesis, very long chain fatty acid oxidation and amount of particle-bound catalase). The results obtained indicate that, except for a deficient phytanic acid oxidation, peroxisomal functions were found to be normal in classical Refsum's disease in contrast with the findings in infantile Refsum's disease, in which there is a general impairment of peroxisomal functions. Based on these results it is concluded that peroxisomal biogenesis is normal in classical (but not in infantile) Refsum's disease and that the classical and infantile form of Refsum's disease hence represent distinct entities. Since available evidence suggests that phytanic acid is oxidized in mitochondria rather than in peroxisomes, at least in rat liver, it remains to be established whether classical Refsum's disease is a peroxisomal disorder or not.

[Zellweger syndrome, neonatal adrenoleukodystrophy or infantile Refsum's disease in a case with generalized peroxisome defect?]. / Zellweger-Syndrom, neonatale Adrenoleukodystrophie oder infantile Refsumsche Erkrankung bei einem Fall mit generalisiertem Peroxisomendefekt?

An eleven month-old boy presented clinically with craniofacial dysmorphia, severe psychomotor retardation, neurological deterioration, no response to visual and acoustic stimuli, failure to thrive, hepatomegaly and adrenal insufficiency. Specific biochemical markers for a peroxisomal deficiency disorder (Zellweger's syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease) revealed pathological results for very long chain fatty acids, phytanic acid, pristanic acid, plasmalogen biosynthesis and catalase, thus confirming the clinical diagnosis. Comparison of clinical and biochemical findings in the patient with the characteristics of the three peroxisomal deficiency disorders showed overlapping with each of these disorders, which corresponds to the current view that these three peroxisomal disorders differ only with respect to onset and severity of the clinical manifestations, but not with regard to the biochemical defects.

Non-manifesting Refsum heterozygotes carrying the c.135-2A>G PAHX gene transition.

So far, subjects heterozygous for PAHX mutations are regarded as non-symptomatic. In the 24-year-old, HIV-negative daughter and the 26-year-old, HIV-negative son of a patient with Refsum disease due to the homozygous c.135-2A>G transition at the splice site before exon 3 of the PAHX gene, slight abnormalities suggestive of the disease became apparent. The daughter reported a single fever cramp in childhood, recurrent, short-lived amaurotic episodes after getting up from supine, short-sightedness, hypoacusis, and restless legs. The son complained about restless legs, hyperhidrosis, hypoacusis, and bulbar oscillations. Though both children carried the same mutation as their mother in the heterozygous form, clinical neurologic examination, nerve conduction studies and serum phytanic acid concentration were normal in both of them, implying that the described abnormalities were not causally related to the PAHX mutation. In the absence of elevated phytanic acid concentrations, clinical neurologic abnormalities in heterozygous relatives of Refsum patients are not attributable to heterozygosity for PAHX mutations.

CYP4 isoform specificity in the omega-hydroxylation of phytanic acid, a potential route to elimination of the causative agent of Refsum's disease.

The saturated C20 isoprenoid phytanic acid is physiologically derived from phytol released in the degradation of chlorophyll. The presence of a C-3 methyl group in this substrate blocks normal beta-oxidation, so phytanic acid degradation primarily occurs by initial peroxisomal alpha-oxidation to shift the register of the methyl group. However, individuals with Refsum's disease are genetically deficient in the required phytanoyl-CoA alpha-hydroxylase and suffer from neurological pathologies caused by the accumulation of phytanic acid. Recent work has shown that phytanic acid can also be catabolized by a pathway initiated by omega-hydroxylation of the hydrocarbon chain, followed by oxidation of the alcohol to the acid and conventional beta-oxidation. However, the enzymes responsible for the omega-hydroxylation of phytanic acid have not been identified. In this study, we have determined the activities of all of the rat and human CYP4A enzymes and two of the rat CYP4F enzymes, with respect to the omega-hydroxylation of phytanic acid. Furthermore, we have shown that the ability to omega-hydroxylate phytanic acid is elevated in microsomes from rats pretreated with clofibrate. The results support a possible role for CYP4 enzyme elevation in the elimination of phytanic acid in Refsum's disease patients.

Structure of human phytanoyl-CoA 2-hydroxylase identifies molecular mechanisms of Refsum disease.

Refsum disease (RD), a neurological syndrome characterized by adult onset retinitis pigmentosa, anosmia, sensory neuropathy, and phytanic acidaemia, is caused by elevated levels of phytanic acid. Many cases of RD are associated with mutations in phytanoyl-CoA 2-hydroxylase (PAHX), an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the initial alpha-oxidation step in the degradation of phytenic acid in peroxisomes. We describe the x-ray crystallographic structure of PAHX to 2.5 A resolution complexed with Fe(II) and 2OG and predict the molecular consequences of mutations causing RD. Like other 2OG oxygenases, PAHX possesses a double-stranded beta-helix core, which supports three iron binding ligands (His(175), Asp(177), and His(264)); the 2-oxoacid group of 2OG binds to the Fe(II) in a bidentate manner. The manner in which PAHX binds to Fe(II) and 2OG together with the presence of a cysteine residue (Cys(191)) 6.7 A from the Fe(II) and two further histidine residues (His(155) and His(281)) at its active site distinguishes it from that of the other human 2OG oxygenase for which structures are available, factor inhibiting hypoxia-inducible factor. Of the 15 PAHX residues observed to be mutated in RD patients, 11 cluster in two distinct groups around the Fe(II) (Pro(173), His(175), Gln(176), Asp(177), and His(220)) and 2OG binding sites (Trp(193), Glu(197), Ile(199), Gly(204), Asn(269), and Arg(275)). PAHX may be the first of a new subfamily of coenzyme A-binding 2OG oxygenases.