Refsum disease is caused by a deficiency of phytanoyl-CoA hydroxylase (PHYH), the first enzyme of the peroxisomal ␣-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.fatty acid oxidation ͉ metabolic disorder ͉ peroxisomes

Phytanic acid and pristanic acid are branchedchain fatty acids, present at micromolar concentrations in the plasma of healthy individuals. Here we show that both phytanic acid and pristanic acid activate the peroxisome proliferator-activated receptor ␣ (PPAR ␣ ) in a concentrationdependent manner. Activation is observed via the ligandbinding domain of PPAR ␣ as well as via a PPAR response element (PPRE). Via the PPRE significant induction is found with both phytanic acid and pristanic acid at concentrations of 3 and 1 M, respectively. The trans -activation of PPAR ␦ and PPAR ␥ by these two ligands is negligible. Besides PPAR ␣ , phytanic acid also trans -activates all three retinoic X receptor subtypes in a concentration-dependent manner. In primary human fibroblasts, deficient in phytanic acid ␣ -oxidation, trans -activation through PPAR ␣ by phytanic acid is observed. This clearly demonstrates that phytanic acid itself, and not only its metabolite, pristanic acid, is a true physiological ligand for PPAR ␣ . Because induction of PPAR ␣ occurs at ligand concentrations comparable to the levels found for phytanic acid and pristanic acid in human plasma, these fatty acids should be seen as naturally occurring ligands for PPAR ␣ . These results demonstrate that both pristanic acid and phytanic acid are naturally occurring ligands for PPAR ␣ , which are present at physiological concentrations. -Zomer, A. W. M., B. van der Burg, G. A. Jansen, R. J. A. Wanders, B. T. Poll-The, and P. T. van der Saag. Pristanic acid and phytanic acid: naturally occurring ligands for the nuclear receptor peroxisome proliferatoractivated receptor ␣ .

CRH-binding protein (CRH-BP) regulates activation of the hypothalamic-pituitary-adrenal (HPA) axis by binding and inhibiting CRH. We investigated for the first time transcriptional regulation of the human CRH-BP promoter using transient transfections. Estrogen receptors (ERs) contributed to ligand-independent constitutive activation of the promoter, whereas in the presence of estradiol ERalpha induced and ERbeta repressed promoter activity in a dose-dependent manner. TNFalpha inhibited promoter induction by ERalpha in the absence and presence of estradiol. Three ERE half-sites in the CRH-BP promoter bound ERalpha and ERbeta in an EMSA, and disruption of ERE half-sites by site-directed mutagenesis abolished ligand-independent induction by ERalpha and ERbeta and promoter enhancement by estradiol-activated ERalpha. Repression by estradiol/ERbeta was unaffected by disruption of ERE half-sites, activating protein 1, cAMP response element, GATA, or nuclear factor kappaB sites, and reversed to promoter induction by estrogen antagonists, tamoxifen and ICI 182,780, suggesting corepressor involvement. In hypothalamic GT1-7 cells, Western blotting demonstrated rapid induction of endogenous CRH-BP expression by estradiol-bound ER, which was inhibited by TNFalpha. We propose a model in which ERs maintain basal CRH-BP expression in pituitary and neurosecretory cells, whereas in the presence of ERalpha estrogen enhances CRH-BP transcription, causing down-regulation of the HPA axis, and nuclear factor kappaB-activating cytokines activate the HPA axis by inhibiting ERalpha.

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Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid present in various dietary products such as milk, cheese and fish. In patients with Refsum disease, accumulation of phytanic acid occurs due to a deficiency of phytanoyl-CoA hydroxylase, a peroxisomal enzyme containing a peroxisomal targeting signal 2. Recently, phytanoyl-CoA hydroxylase cDNA has been isolated and functional mutations have been identified. As it has been shown that phytanic acid activates the nuclear hormone receptors peroxisome proliferator-activated receptor (PPAR)a and all three retinoid X receptors (RXRs), the intracellular concentration of this fatty acid should be tightly regulated.When various cell lines were grown in the presence of phytanic acid, the activity of phytanoyl-CoA hydroxylase increased up to four times, depending on the particular cell type. In one cell line, HepG2, no induction of phytanoyl-CoA hydroxylase activity was observed. After addition of phytanic acid to COS-1 cells, an increase in phytanoyl-CoA hydroxylase activity was observed within 2 h, indicating a quick cell response. No stimulation of phytanoyl-CoA hydroxylase was observed when COS-1 cells were grown in the presence of clofibric acid, 9-cis-retinoic acid or both ligands together. This indicates that the activation of phytanoyl-CoA hydroxylase is not regulated via PPARa or RXR. However, stimulation of PPARa and all RXRs by clofibric acid and 9-cis-retinoic acid was observed in transient transfection assays. These results suggest that the induction of phytanoyl-CoA hydroxylase by phytanic acid does not proceed via one of the nuclear hormone receptors, RXR or PPARa.