Fig 1.
Generation and characterization of Abcd1;CMV-ELOVL1 mice.
(A) Schematic presentation of the Rosa26CAG-STOPflox/flox-ELOVL1TG transgenic construct (black line). The grey line indicates the wild type Rosa26 gene. ELOVL1 transgene expression is prevented by the upstream floxed STOP element. The ELOVL1 transgenic construct consists of a loxP flanked STOP cassette positioned between the human ELOVL1 coding cDNA and the upstream ubiquitous CAG promoter. Crossing the transgenic mice with a Cre-driver strain activates ELOVL1 transgene expression. (B) ELOVL1 protein expression in total homogenates, organelle and cytosolic fractions and purified microsomes derived from livers from wild type mice (control) and wild type mice heterozygous (CMV-ELOVL1+/-) for the activated ELOVL1 transgene. (C) C16:0-CoA and C22:0-CoA elongation capacity in mouse liver microsomes (n = 5) derived from wild type mice (control) and wild types heterozygous (CMV-ELOVL1+/-) for the activated ELOVL1 transgene. (D) ELOVL1 protein expression in brain, adrenal gland and testis from wild type (WT), wild type heterozygous (CMV-ELOVL1+/-) or homozygous (CMV-ELOVL1+/+) for the activated ELOVL1 transgene, Abcd1y/- knockout and Abcd1y/- knockout heterozygous for the activated ELOVL1 transgene (Abcd1y/-;CMV-ELOVL1+/-) mice. (E) C26:0 levels (in nmol/mg protein) in tissues derived from wild type (n = 6), Abcd1y/- knockout (n = 6) and Abcd1y/-;CMV-ELOVL1+/- (n = 2) mice. Data are mean ± SD. ***p<0.001 by unpaired student’s t-test.
Table 1.
MRM, cone voltage and collision energy for each acylcarnitine and lysoPC.
Fig 2.
Possibly early lethal phenotype in Abcd1;CMV-ELOVL1 mice.
(A) Distribution of the actual F1 genotypes of 4 independent crosses between Abcd1 knockout females (Abcd1-/-) with males heterozygous for the activated ELOVL1 transgene (CMV-ELOVL1+/-). Males are in blue and females in pink. The dashed lines indicate the expected percentages. (B) The actual male to female ratio in the F1 generation of 4 independent crosses between Abcd1 knockout females (Abcd1-/-) with males homozygous for the activated ELOVL1 transgene (CMV-ELOVL1+/+). (C) Untargeted metabolomics using bloodspots derived from Swiss and C57BL6/J wild type and Abcd1 knockout mice reveals strong differences in the metabolome between both background strains. The color in the heat map reflects the global metabolite abundance level according to the z-score. The 50 most significant different metabolites are shown (P < 0.05) as determined by a Welch’s t-test.
Fig 3.
Generation and characterization of Abcd1;Cnp-ELOVL1 mice.
(A) ELOVL1 protein expression in brain and spinal cord from wild type, Abcd1y/- knockout and Abcd1y/-;Cnp-ELOVL1+/-. (B) C26:0 levels in brain and spinal cord from wild type (n = 6), Abcd1y/- knockout (n = 6) and Abcd1y/-;Cnp-ELOVL1+/- (n = 6) mice. Data are mean ± SD. ***p<0.001 by unpaired student’s t-test.
Fig 4.
C26:0-lysoPC is highly elevated in central nervous tissue.
C22:0-lysoPC, C24:0-lysoPC and C26:0-lysoPC levels in brain and spinal cord from wild type (n = 6), Abcd1y/- knockout (n = 6) and Abcd1y/-;Cnp-ELOVL1+/- (n = 6) mice. Data are mean ± SD. *p<0.05, **p<0.01, ***p<0.001 by unpaired student’s t-test.
Fig 5.
C26:0-carnitine is highly elevated in central nervous tissue.
C22:0-carnitine, C24:0-carnitine and C26:0-carnitine levels in brain and spinal cord from wild type (n = 6), Abcd1y/- knockout (n = 6) and Abcd1y/-;Cnp-ELOVL1+/- (n = 6) mice. Data are mean ± SD. **p<0.01, ***p<0.001 by unpaired student’s t-test.
Fig 6.
C26:0-carnitine is highly elevated in mouse and human bloodspots.
(A) plasma total C26:0 in wild type (n = 8), Abcd1y/- knockout (n = 10) and Abcd1y/-;Cnp-ELOVL1+/- (n = 6) mice. (B) bloodspot C26:0-lysoPC in wild type (n = 8), Abcd1y/- knockout (n = 10) and Abcd1y/-;Cnp-ELOVL1+/- (n = 6) mice. (C) bloodspot C26:0-carnitine in wild type (n = 8), Abcd1y/- knockout (n = 10) and Abcd1y/-;Cnp-ELOVL1+/- (n = 6) mice. (D) plasma total C26:0 in controls (n = 23) and ALD patients (n = 10). (E) bloodspot C26:0-lysoPC in controls (n = 23) and ALD patients (n = 10). (F) bloodspot C26:0-carnitine in controls (n = 23) and ALD patients (n = 10). Data are mean ± SD. ****p<0.0001 by unpaired student’s t-test.