The role of ELOVL1 in very long-chain fatty acid homeostasis and X-linked adrenoleukodystrophy

Abstract

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). X-ALD is characterized by the accumulation of very long-chain fatty acids (VLCFA; > or =C24) in plasma and tissues. In this manuscript we provide insight into the pathway underlying the elevated levels of C26:0 in X-ALD. ALDP transports VLCFacyl-CoA across the peroxisomal membrane. A deficiency in ALDP impairs peroxisomal beta-oxidation of VLCFA but also raises cytosolic levels of VLCFacyl-CoA which are substrate for further elongation. We identify ELOVL1 (elongation of very-long-chain-fatty acids) as the single elongase catalysing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1). ELOVL1 expression is not increased in X-ALD fibroblasts suggesting that increased levels of C26:0 result from increased substrate availability due to the primary deficiency in ALDP. Importantly, ELOVL1 knockdown reduces elongation of C22:0 to C26:0 and lowers C26:0 levels in X-ALD fibroblasts. Given the likely pathogenic effects of high C26:0 levels, our findings highlight the potential of modulating ELOVL1 activity in the treatment of X-ALD.

Figure 1. D₃-VLCFA levels in control and X-ALD fibroblasts

1. D₃-C22:0 levels in control versus X-ALD cell lines.

2. D₃-C26:0 levels in control versus X-ALD cell lines.

3. D₃-C26:0/D₃-C22:0 ratio in control versus X-ALD cell lines.

   Eight control and 25 X-ALD cell lines were cultured for 72 h with 50 µM D₃-C16:0. Fatty acids are in nmol/mg protein. Data are mean ± SD. **p < 0.01, ***p < 0.001 by unpaired student's t-test.

Figure 2. VLCFacyl-CoA levels are increased in X-ALD fibroblasts

1. Pristanoyl-CoA levels in control versus X-ALD cell lines.

2. C24:0-CoA levels in control versus X-ALD cell lines.

3. C26:0-CoA levels in control versus X-ALD cell lines.

4. Fatty acyl-CoAs are in pmol/mg protein. Data are mean ± SD in 3 control and 6 X-ALD cell lines. ***p < 0.001 by unpaired student's t-test.

Figure 3. ELOVL1 expression is not increased in X-ALD fibroblasts

1. ELOVL1 mRNA expression in 6 control and 9 X-ALD cell lines. Data are mean ± SD.

2. ELOVL1 protein expression in control and X-ALD cell lines. (+) positive control: CHO cells expressing ELOVL1.

Figure 4. ELOVL1 knockdown reduces C26:0 levels in X-ALD fibroblasts

1. ELOVL1 expression in X-ALD fibroblasts cultured for 3, 4, 5 or 6 days in medium devoid of (−) or supplemented with (+) ELOVL1 siRNA.

2. ELOVL1 expression in X-ALD fibroblasts which were untransfected (−) or transfected (+) with siRNA targeting either cyclophilin-B (control) or ELOVL1.

3. Effect of control siRNA (white bars) or ELOVL1 siRNA (grey bars) on cell proliferation in comparison with untransfected cells (black bars).

4. D₃-C26:0 levels in ELOVL1 siRNA transfected (grey bars) versus untransfected (black bars) X-ALD fibroblasts.

5. C26:0 levels in control and X-ALD fibroblasts which were untransfected (black bars) and in X-ALD fibroblasts which were transfected with either control siRNA (white bars) or ELOVL1 siRNA (grey bars). Fatty acids are in nmol/mg protein. Data are mean ± SD. ***p < 0.001 by unpaired student's t-test.