RNAi-mediated silencing of ABCD3 gene expression in rat C6 glial cells: a model system to study PMP70 function.

ABSTRACT

The function of PMP70, one of the four ABC half-transporters of mammalian peroxisomes, encoded by ABCD3 gene, is still unclear. The finding that PMP70 over-expression partially corrected very long-chain fatty acid oxidation defects in fibroblasts of X-linked adrenoleukodystrophy patients, has unveiled its potential clinical relevance, prompting us to set up a model system to study PMP70 function. We used the RNA interference technique, a powerful approach to loss-of-function gene expression analysis, to knockdown the ABCD3 gene in the rat glial C6 cell line, since glia could represent the target tissue of X-linked adrenoleukodystrophy disease. Cells were transfected with a vector for RNA interference generating small interfering RNAs that specifically target the ABCD3 mRNA. By using a puromycin-selectable version of the plasmid, we generated a stable cell line (abcd3kd), in which we observed a stable decrease of PMP70 protein expression greater than 70%. We thus examined the effect of ABCD3 knockdown on lignoceric and palmitic acids beta-oxidation and we found that in abcd3kd cells the rate of peroxisomal and mitochondrial beta-oxidation activities were both reduced about one-third compared with control cells. The mitochondrial membrane potential, determined by cytofluorometric analysis, was also affected. Lipid and fatty acid analyses of abcd3kd cells showed an increase of hexacosenoic acid (C260) in the cholesteryl-ester fraction. These results add another clue about the overlapping function of PMP70 and ALDP, the peroxisomal protein involved in X-linked adrenoleukodystrophy, since C260 is the biochemical marker of the disease and in the brain lesions it is accumulated in the cholesteryl-ester fraction. Considered as a whole, our results indicate that the abcd3kd cell line is a valuable tool to further study the function of PMP70 and eventually its role in X-linked adrenoleukodystrophy.