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RNAi-mediated silencing of ABCD3 gene expression in rat C6 glial cells: a model system to study PMP70 function
Rita Di Benedetto1, Michela Alessandra Denti, Serafina Salvati
1Department of Food Science, Nutrition and Health, Istituto Superiore di Sanità, Rome, Italy.
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 (C26:0) 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 C26:0 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.
Insights
Researchers created a cell model to study peroxisome membrane protein 70 (PMP70) by reducing ABCD3 gene expression. This knockdown impaired fatty acid oxidation and increased disease-specific lipid accumulation, suggesting PMP70
Area of Science:
- Cell Biology
- Biochemistry
- Genetics
Background:
- The function of peroxisome membrane protein 70 (PMP70), encoded by the ABCD3 gene, remains largely unknown.
- Previous studies suggested a potential clinical relevance of PMP70 in X-linked adrenoleukodystrophy (X-ALD) by observing partial correction of fatty acid oxidation defects upon PMP70 overexpression.
- Glia are considered a potential target tissue for X-ALD, necessitating a suitable model system to study PMP70's role in these cells.
Purpose of the Study:
- To establish a loss-of-function model system for studying PMP70 function.
- To investigate the impact of ABCD3 gene knockdown on fatty acid beta-oxidation and lipid metabolism in a rat glial cell line.
- To explore the potential role of PMP70 in X-linked adrenoleukodystrophy.
Main Methods:
- Utilized RNA interference (RNAi) to specifically target and reduce ABCD3 mRNA expression in rat C6 glial cells.
- Generated a stable knockdown cell line (abcd3kd) exhibiting a significant decrease (>70%) in PMP70 protein levels.
- Assessed beta-oxidation of lignoceric and palmitic acids, mitochondrial membrane potential, and lipid/fatty acid profiles (including hexacosenoic acid, C26:0).
Main Results:
- ABCD3 knockdown in abcd3kd cells led to a substantial reduction (approximately one-third) in both peroxisomal and mitochondrial beta-oxidation activities.
- The mitochondrial membrane potential was significantly affected in the abcd3kd cells.
- Lipid analysis revealed an accumulation of hexacosenoic acid (C26:0) in the cholesteryl-ester fraction of abcd3kd cells, a hallmark of X-ALD.
Conclusions:
- The abcd3kd cell line serves as a valuable tool for further investigating PMP70 function.
- The observed results suggest an overlapping function between PMP70 and ALDP (the protein implicated in X-ALD).
- The findings provide additional insights into the potential role of PMP70 in the pathogenesis of X-linked adrenoleukodystrophy, particularly concerning C26:0 accumulation.
