ABCD2 AND ABCD3 EXPRESSION IN CONTROL AND X-ALD FIBROBLASTS by Real-Time PCR

1. FENOFIBRATE CLA UNTREATED control cells ALD cells Control cells ALD cells 45 45 25 25 40 40 PPAR-EXPRESSION IN UNTREATED AND X-ALD FIBROBLASTS FENOFIBRATE 35 35 20 20 CLA 30 30 UNTREATED 25 25 15 15 mRNA expression mRNA expression * 20 20 mRNA expression mRNA expression 10 10 15 15 10 10 5 5 5 5 0 0 0 0 18 24 48 18 24 48 18 24 48 18 24 48 hours hours * hours hours * * P<0.01 considered statistically significant versus untreated, according to Tukey’s test. INTRODUCTION X-linked adrenoleukodystrophy (X-ALD) is a rare, inherited, metabolic disorder characterized by progressive demyelination. Due to defective peroxisomal beta-oxidation, Very Long Chain Fatty Acids (VLCFA) accumulate in different tissues including plasma, fibroblasts, nervous system and adrenal cortex and are thus regarded as the main biochemical markers of the disease. X-ALD is due to mutations in the ABCD1 (Xq28) gene, which normally encodes a peroxisomal ATP-binding cassette transporter (ALDP). It has been shown that overexpression of ABCD1 closest homologue, ABCD2, and of other related genes such as ABCD3 and ABCD4, can partially compensate for ALDP deficiency. Various ALDP agonists can induce the expression of the above mentioned genes, partially compensating the pathological abnormalities of the disease, such as enhancing VLCFA beta-oxidation and we have previously demonstrated (Petroni et al., 2003) that the androgen dihydrotestosterone (DHT) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) are among such compounds. OBJECTIVES “Lorenzo’s oil therapy” is the only available and partially effective treatment for the patients, It consists of a 4:1 mixture of glycerol trioleate and glycerol trierucate. Lorenzo’s oil does not directly affect peroxisomal beta oxidation but just slows down the production of VLCFAs. Peroxisomal Beta-oxidation defect in the liver of ABCD1-deficient mice could be restored by stimulation of ABCD2 and ABCD4 gene expression through treatment with fenofibrate. Our goal is to investigate, as a possible candidate in ABCD2 induction , conjugated linoleic acid (CLA), which is a naturally ocurring fatty acid (FA) and a good ligand for PPAR (regulator of peroxisomal beta-oxidation key enzymes.) Moreover CLA passes the blood-brain barrier and is partially metabolized by the peroxisomes. METHODS X-ALD fibroblasts were obtained and cultured as previously described (Petroni et al., 2000) from two affected brothers and normal subjects. The two brothers presented the same mutation in exon 9, but different clinical manifestations of the disease, ALD1 being severely affected, while ALD2 being asymptomatic. CLA isomers and fenofibrate were supplemented separately to the cells for different times. Gene expression was evaluated by real-time PCR . CLA metabolites were measured by HPLC-DAD-MS. ABC half-transporter relative gene expression and metabolism of conjugated linoleic acid (CLA) in X- linked AdrenoleukodystrophyPetroni A1.1, Carissimi R.1, Iannone A.2, Della Casa L.2, Giordano E.3, Cordeddu L.3, Banni S.31University of Milan, 2University of Modena and Reggio Emilia, 3University of Cagliari,ITALY ABCD2 AND ABCD3 EXPRESSION IN CONTROL AND X-ALD FIBROBLASTS by Real-Time PCR MELTING CURVES OF ABCD2 AND ABCD3 ABCD2 Total RNA was extracted with Trizol method ABCD2 The use of the SYBR Green dye in all the experiments also provided us with melting curves for each gene. Melting curves for ABCD2, ABCD3 and the housekeeping gene 18S confirmed the generation of a specific amplicone. Further proof that the amplification was specific for our target genes was obtained by electrophoresis on a 3% agarose gel. 135 pb The amplification of the indicated genes and of the housekeeping gene 18S in three cell lines (control, ALD1, and ALD2 fibroblasts) either in untreated conditions or after incubation were evaluated by Real-Time PCR. Threshold cycle (CT) was determined as the point at which amplification resulted in significant fluorescence above background and later used to calculate fold change. ABCD3 ABCD3 Fluorescence Fluorescence 77 pb 18S 18S 170 pb Cycle Temperature, Celsius ABCD2 EXPRESSION IN UNTREATED AND X-ALD FIBROBLASTS * * * * * * * * * The fibroblasts were incubated at different time (16,24,48 H) with 50 μM of CLA (isomer c9,t11) or 100 μM Fenofibrate (PPAR-alpha’s agonist). Primers for Real-Time PCR were designed by human cDNA ABCD2 (GenBank Accession Number NM_005164), PPAR-alpha (GenBank Accession Number BC000052) and 18SrRNA (GenBank Accession NumberNM_X03205) using the programs: Primer3, Operon Toolkit and NCBI. * P<0.01 considered statistically significant versus untreated, according to Tukey’s test. RESULTS In untreated conditions ABCD2 was upregulated in X-ALD fibroblasts towards the controls. Fenofibrate, used as positive control, increased both PPAR-  and ABCD2 mRNA levels after 18 h. CLA differently induced PPAR-  and ABCD2 mRNA, the latter was particularly enhanced after 24 and 48h in X-ALD fibroblasts. DISCUSSION In untreated conditions ABCD2 was upregulated in X-ALD fibroblasts towards the controls, indicating a compensation for ALDP deficiency. CLA differently induced PPAR-  and ABCD2 mRNA, the latter was particularly enhanced after 24 and 48h in X-ALD fibroblasts. The higher induction of PPAR-  and ABCD2 mRNA by CLA was associated to its conjugated diene metabolites , derived from peroxisomal beta-oxidation (data not shown). CONCLUSIONS Inflammation could contribute to the pathogenesis of X-ALD, being involved in demyelinating processes (Moser et al.1997), as well as to other neurodegenerative disorders such as Alzheimer’s disease. CLA passes the blood-brain barrier, it is incorporated and metabolized in the brain and isolated astrocytes (Fa et al. 2005). The effect of CLA on ABCD2 and PPAR, here reported,and its possible antinflammatory effect , due to its conjugated diene metabolites, can indicate CLA as a promising therapeutic approach for X-ALD. REFERENCES Moser HW. Adrenoleukodystrophy: phenotype, genetics, pathogenesis and therapy. Brain. 1997 Aug;120 ( Pt 8):1485-508. Petroni A, Blasevich M, Uziel G. Effects of the testosterone metabolite dihydrotestosterone and 5-alpha-androstan-3 alpha,17 beta-diol on very long chain fatty acid metabolism in X-adrenoleukodystrophic fibroblasts. Life Sci. 2003 Aug 8;73(12):1567-75 Petroni A, Papini N, Blasevich M, Magnaghi V, Cavarretta I, Galli C, Melcangi RC. Testosterone metabolites in patients reduce the levels of very long chain fatty acids accumulated in X-adrenoleukodystrophic fibroblasts. Neurosci Lett. 2000 Aug 4;289(2):139-42 Fa M, Diana A, Carta G, et al. Incorporation and metabolism of c9,t11 and t10,c12 conjugated linoleic acid (CLA) isomers in rat brain. Biochimica et Biophysica Acta 2005;1736:61-6. Acknowledgement This research was funded by the BioMedicine and Nutrition Association (www.biomedicinanutrizione.org).