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.

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.

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.

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.

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.

Further studies on the substrate spectrum of phytanoyl-CoA hydroxylase: implications for Refsum disease?

Refsum disease is a peroxisomal disorder characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy, ataxia, and an accumulation of phytanic acid in plasma and tissues. Approximately 45% of cases are caused by mutations in phytanoyl-CoA hydroxylase (PAHX), the enzyme catalyzing the second step in the peroxisomal alpha-oxidation of 3-methyl-branched fatty acids. To study the substrate specificity of human PAHX, different 3-alkyl-branched substrates were synthesized and incubated with a recombinant polyhistidine-tagged protein. The enzyme showed activity not only toward racemic phytanoyl-CoA and the isomers of 3-methylhexadecanoyl-CoA, but also toward a variety of other mono-branched 3-methylacyl-CoA esters with a chain length down to seven carbon atoms. Furthermore, PAHX hydroxylated a 3-ethylacyl-CoA quite well, whereas a 3-propylacyl-CoA was a poor substrate. Hydroxylation of neither 2- or 4-methyl-branched acyl-CoA esters, nor long or very long straight-chain acyl-CoA esters could be detected. The results presented in this paper show that the substrate specificity of PAHX, with regard to the length of both the acyl-chain and the branch at position 3, is broader than expected. Hence, Refsum disease might be characterized by an accumulation of not only phytanic acid but also other 3-alkyl-branched fatty acids.

Stereochemistry of the peroxisomal branched-chain fatty acid alpha- and beta-oxidation systems in patients suffering from different peroxisomal disorders.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid derived from dietary sources and broken down in the peroxisome to pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) via alpha-oxidation. Pristanic acid then undergoes beta-oxidation in peroxisomes. Phytanic acid naturally occurs as a mixture of (3S,7R,11R)- and (3R,7R,11R)-diastereomers. In contrast to the alpha-oxidation system, peroxisomal beta-oxidation is stereospecific and only accepts (2S)-isomers. Therefore, a racemase called alpha-methylacyl-CoA racemase is required to convert (2R)-pristanic acid into its (2S)-isomer. To further investigate the stereochemistry of the peroxisomal oxidation systems and their substrates, we have developed a method using gas-liquid chromatography-mass spectrometry to analyze the isomers of phytanic, pristanic, and trimethylundecanoic acid in plasma from patients with various peroxisomal fatty acid oxidation defects. In this study, we show that in plasma of patients with a peroxisomal beta-oxidation deficiency, the relative amounts of the two diastereomers of pristanic acid are almost equal, whereas in patients with a defect of alpha-methylacyl-CoA racemase, (2R)-pristanic acid is the predominant isomer. Furthermore, we show that in alpha-methylacyl-CoA racemase deficiency, not only pristanic acid accumulates, but also one of the metabolites of pristanic acid, 2610-trimethylundecanoic acid, providing direct in vivo evidence for the requirement of this racemase for the complete degradation of pristanic acid.

Structure-function analysis of phytanoyl-CoA 2-hydroxylase mutations causing Refsum's disease.

Refsum's disease is a neurological syndrome characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy and phytanic acidaemia. Many cases are caused by mutations in peroxisomal oxygenase phytanoyl-CoA 2-hydroxylase (PAHX) which catalyses the initial alpha-oxidation step in the degradation of phytanic acid. Both pro and mature forms of recombinant PAHX were produced in Escherichia coli, highly purified, and shown to have a requirement for iron(II) as a co-factor and 2-oxoglutarate as a co-substrate. Sequence analysis in the light of crystallographic data for other members of the 2-oxoglutarate-dependent oxygenase super-family led to secondary structural predictions for PAHX, which were tested by site-directed mutagenesis. The H175A and D177A mutants did not catalyse hydroxylation of phytanoyl-CoA, consistent with their assigned role as iron(II) binding ligands. The clinically observed P29S, Q176K, G204S, N269H, R275Q and R275W mutants were assayed for both 2-oxoglutarate and phytanoyl-CoA oxidation. The P29S mutant was fully active, implying that the mutation resulted in defective targeting of the protein to peroxisomes. Mutation of Arg-275 resulted in impaired 2-oxoglutarate binding. The Q176K, G204S and N269H mutations caused partial uncoupling of 2-oxoglutarate conversion from phytanoyl-CoA oxidation. The results demonstrate that the diagnosis of Refsum's disease should not solely rely upon PAHX assays for 2-oxoglutarate or phytanoyl-CoA oxidation.

Identification of pristanal dehydrogenase activity in peroxisomes: conclusive evidence that the complete phytanic acid alpha-oxidation pathway is localized in peroxisomes.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid which, due to the methyl-group at the 3-position, can not undergo beta-oxidation unless the terminal carboxyl-group is removed by alpha-oxidation. The structure of the phytanic acid alpha-oxidation machinery in terms of the reactions involved, has been resolved in recent years and includes a series of four reactions: (1) activation of phytanic acid to phytanoyl-CoA, (2) hydroxylation of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, (3) cleavage of 2-hydroxyphytanoyl-CoA to pristanal and formyl-CoA, and (4) oxidation of pristanal to pristanic acid. The subcellular localization of the enzymes involved has remained enigmatic, with the exception of phytanoyl-CoA hydroxylase and 2-hydroxyphytanoyl-CoA lyase which are both localized in peroxisomes. The oxidation of pristanal to pristanic acid has been claimed to be catalysed by the microsomal aldehyde dehydrogenase FALDH encoded by the ALDH10-gene. Making use of mutant fibroblasts deficient in FALDH activity, we show that phytanic acid alpha-oxidation is completely normal in these cells. Furthermore, we show that pristanal dehydrogenase activity is not fully deficient in FALDH-deficient cells, implying the existence of one or more additional aldehyde dehydrogenases reacting with pristanal. Using subcellular localization studies, we now show that peroxisomes contain pristanal dehydrogenase activity which leads us to conclude that the complete phytanic acid alpha-oxidation pathway is localized in peroxisomes.

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.

Human phytanoyl-CoA hydroxylase: resolution of the gene structure and the molecular basis of Refsum's disease.

Refsum's disease (RD) is an inherited neurological syndrome biochemically characterized by the accumulation of phytanic acid in plasma and tissues. Patients with RD are unable to degrade phytanic acid due to a deficient activity of phytanoyl-CoA hydroxyl-ase (PhyH), a peroxisomal enzyme catalysing the first step of phytanic acid alpha-oxidation. To enable mutation analysis of RD at the genome level, we have elucidated the genomic organization of the PHYH gene. The gene is approximately 21 kb and contains nine exons and eight introns. Mutation analysis of PHYH cDNA from 22 patients with RD revealed 14 different missense mutations, a 3 bp insertion, and a 1 bp deletion, which were all confirmed at the genome level. A 111 bp deletion identified in the PHYH cDNA of several patients with RD was due to either one of two different mutations in the same splice acceptor site, which result in skipping of exon 3. Six mutations, including a large in-frame deletion and five missense mutations, were expressed in the yeast Saccharomyces cerevisiae to study their effect on PhyH activity. The results showed that all these mutations lead to an enzymatically inactive PhyH protein.

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.

Atypical refsum disease with pipecolic acidemia and abnormal catalase distribution.

We describe an 18-year-old patient with psychomotor retardation and abnormally short metatarsi and metacarpals but no other signs of classic Refsum disease. Molecular analysis of the phytanoyl-coenzyme A hydroxylase gene revealed a homozygous deletion causing a frameshift. Surprisingly, L-pipecolic acid was elevated in plasma, and microscopy of the liver showed a reduced number of peroxisomes per cell and a larger average peroxisome size. These abnormal peroxisomes lacked catalase as did peroxisomes in fibroblasts of this patient. Such generalized peroxisomal abnormalities are not present in classic Refsum disease.

Immunophilins, Refsum disease, and lupus nephritis: the peroxisomal enzyme phytanoyl-COA alpha-hydroxylase is a new FKBP-associated protein.

FKBP52 (FKBP59, FKBP4) is a "macro" immunophilin that, although sharing high structural and functional homologies in its amino-terminal domain with FKBP12 (FKBP1), does not have immunosuppressant activity when complexed with FK506, unlike FKBP12. To investigate the physiological function of FKBP52, we used the yeast two-hybrid system as an approach to find its potential protein partners and, from that, its cellular role. This methodology, which already has allowed us to find the FK506-binding protein (FKBP)-associated protein FAP48, also led to the detection of another FKBP-associated protein. Determination of the sequence of this protein permitted its identification as phytanoyl-CoA alpha-hydroxylase (PAHX), a peroxisomal enzyme that so far was unknown as an FKBP-associated protein. Inactivation of this enzyme is responsible for Refsum disease in humans. The protein also corresponds to the mouse protein LN1, which could be involved in the progress of lupus nephritis. We show here that PAHX has the physical capacity to interact with the FKBP12-like domain of FKBP52, but not with FKBP12, suggesting that it is a particular and specific target of FKBP52. Whereas the binding of calcineurin to FKBP12 is potentiated by FK506, the specific association of PAHX and FKBP52 is maintained in the presence of FK506. This observation suggests that PAHX is a serious candidate for studying the cellular signaling pathway(s) involving FKBP52 in the presence of immunosuppressant drugs.

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).

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.