PATOLOGIE NEURODEGENERATIVE PEROSSISOMIALI. MODULAZIONE DI AGONISTI E FATTORI NUTRIZIONALI PER LO SVILUPPO DI NUOVE STRATEGIE TERAPEUTICHE

X-linked adrenoleukodystrophy (X-ALD) is an inherited demyelinating disorder that affects mainly the nervous system and the adrenal cortex. The disease is characterized by abnormal accumulation of very long chain fatty acids (VLCFA) in plasma, fibroblasts and tissues, owing to a defect in VLCFA peroxisomal Beta-oxidation. In particular, the uptake of VLCFA by peroxisomal VLC acyl-CoA synthetase, is impaired (Moser et al. 1995) In 1981 the ALD gene was mapped to Xq28 . Subsequently Higgins (Higgins et al. 1992) demonstrated that ALD protein (ALDP) was a peroxisomal membrane protein but it had no homology to VLC acyl-CoA synthetase and its role in lipid metabolism has not yet been elucidated. Moreover no correlation has been established between X-ALD phenotype and the mutation. Mild phenotype are associated with large deletions with consequent absence of ALDP, whereas severe phenotypes present abundant ALDP. The range of phenotypic expression in X-ALD is wide, but most patients have impaired adrenocortical function. The adrenal cortex is the main site for synthesis of steroids which cannot be normally synthetized since cholesterol is entrapped, as cholesterol ester, with VLCFA. Hypogonadism, which affects X-ALD prepubescent boys, might be a consequence of steroid metabolic alterations. “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 (the triglyceride forms of oleic and erucic acid). The oil was formulated by Augusto and Michaela Odone after their son Lorenzo was diagnosed with the disease in 1984, at the age of five. This mixture of fatty acids reduces the levels of very long chain fatty acids (VLCFA) known to cause ALD. It does so by decreasing the dietary intake of saturated fatty acids, it is in fact widely accepted that in humans synthesis of long chain saturated fatty acids is very low and thereby having as a main dietary source of fat Lorenzo’s oil with none SFA, in addition, erucic acid competitively inhibits the elongase that forms VLCFAs. Therefore, 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 (Netik et al.1999).These results implicate that a therapy of adrenoleukodystrophy might be possible by drug-induced overexpression or ectopic expression of ABCD genes. Furthermore, erucic acid barely passes the blood-brain barrier leaving open the question whether is effective in the central nervous system. One objective of this project was to study the hormonal involvement in X-ALD, as the ubiquitous accumulation of VLCFA can not explain the specific alterations in the nervous system, adrenal cortex and testes. These changes indicate a possible deficit in steroidogenesis. Had already demonstrated the involvement of androgens in the X-ALD, as incubating the fibroblasts of patients with dihydroxy-testosterone (DHT) or its metabolite 5alpha-androstane-3 alpha, 17 beta-diol (3alpha-diol), the VLCFA levels were significantly reduced. Conversely, fibroblasts incubated with testosterone is not had no effect Given the involvement of androgens in the pathology and based on the observation of structural homology proteins belonging to the subfamily ABCD in a first set of experiments in vitro was evaluated the effect of androgens on gene expression ABCD2 (Genbank Nm-005164) and ABCD3 (Genbank Nm-002858) in fibroblasts from patients with X-ALD by comparison with the expression of two genes in fibroblasts derived from clinically healthy subjects subjects in an attempt to find agents that could be used as new therapeutic approaches. Our hypothesis is that sostituitre fatty acid which may enhance peroxisomal beta oxidation and passes the blood-brain barrier. One of the possible candidate is conjugated linoleic acid (CLA). The term conjugated linoleic acid (CLA) refers to a collection of positional and geometric isomers of octadecadienoic acid with conjugated double bonds. In experimental models, CLA has a number of beneficial effects including protection against cancer (Belury et al.1996; Visonneau et al.1997; Cesano et al. 1998; Banni et al.1999), and atherosclerosis (Kritchevsk, et al. 2000), stimulation of certain immune functions, reduction in body fat (reviewed in Pariza et al.2001) and normalization of impaired glucose tolerance in diabetes. Several reports have also indicated that CLA is a high affinity ligand of the peroxisome proliferator activated receptor (PPAR) (Belury et al.1997; Houseknecht et al 1998; Moya-Camarena et al.1999, Belury et al.2002), a family of nuclear receptors that act as transcription factors for the genes involved in peroxisomal beta oxidation. Furthermore, it has found that CLA is promptly incorporated in rat’s brain and metabolised in the peroxisomes (FA et al. 2005). The objective of the study is to test CLA that, by inducing peroxisomal beta oxidation, should compensate for its decrease in X-ALD patients due to the genetic deficiency. In previous papers (Petroni et al.2007) we have shown that in untreated conditions the patients we showed an upregulation of ABCD2 versus the controls: the basal ABCD2 upregulation, or in general the functionality of ABC half -transporters, in patient might have been adequate and in relation to his normal metabolism. We have evaluated the effect of CLA as a promising therapeutic approach, on the expression of the ABC half-transporters encoded by ABCD2, in fibroblasts drawn from controls and from two affected brothers. To evaluate the gene’s expression and the transcription factors involved . the fibroblasts were incubated at different time (16,24 H) with oleic acid , the CLA isomers and Fenofibrate that’s PPAR-alpha’s agonist. Our in vitro studies have focused on the effect of CLA on gene expression of genes ABCD2 / 3 and PPAR-alpha in fibroblasts from patients with X-ALD. Tested the different isomers in vitro, later it was possible to conduct clinical trials based on the association between Lorenzo's Oil (LO) and the CLA, to assess the possible synergistic effects of CLA and extent of spread through the blood brain barrier. This study recruited patients, women heterozygous for X-ALD. In this study, were also taken into account the motor evoked potentials (MEP) of patients over the analysis of biochemical markers of disease, the VCLFA.