Knockout of Slc25a19 causes mitochondrial thiamine pyrophosphate depletion, embryonic lethality, CNS malformations, and anemia

Abstract

SLC25A19 mutations cause Amish lethal microcephaly (MCPHA), which markedly retards brain development and leads to alpha-ketoglutaric aciduria. Previous data suggested that SLC25A19, also called DNC, is a mitochondrial deoxyribonucleotide transporter. We generated a knockout mouse model of Slc25a19. These animals had 100% prenatal lethality by embryonic day 12. Affected embryos at embryonic day 10.5 have a neural-tube closure defect with ruffling of the neural fold ridges, a yolk sac erythropoietic failure, and elevated alpha-ketoglutarate in the amniotic fluid. We found that these animals have normal mitochondrial ribo- and deoxyribonucleoside triphosphate levels, suggesting that transport of these molecules is not the primary role of SLC25A19. We identified thiamine pyrophosphate (ThPP) transport as a candidate function of SLC25A19 through homology searching and confirmed it by using transport assays of the recombinant reconstituted protein. The mitochondria of Slc25a19(-/-) and MCPHA cells have undetectable and markedly reduced ThPP content, respectively. The reduction of ThPP levels causes dysfunction of the alpha-ketoglutarate dehydrogenase complex, which explains the high levels of this organic acid in MCPHA and suggests that mitochondrial ThPP transport is important for CNS development.

Fig. 1.

Diagram of the Slc25a19 knockout strategy. A 2.5-kb EcoRI (E)/BamHI (B) fragment (5′) and a 3.9-kb SpeI (S)/EcoRI (3′) fragment were cloned into pPNT-far. The construct is missing exons 4–6. Transfected ES cell clones were digested with either BamHI (B) (3′ and neo probes) or NheI (N) (5′ probe) to test for homologous recombination. Thick lines represent mouse sequence, thin lines represent vector sequence, and the arrow represents the Neo^R cassette. The stippled boxes represent the Southern blot analysis probes. Black numbered boxes are exons (the translation start site is in exon 2).

Fig. 2.

Examples of unaffected and mutant embryos at E10.5. (A) Unaffected Slc25a19^+/− embryo. (B) Slc25a19^−/− embryo taken at the same magnification as in A. (C) Higher magnification of embryo in B. Note the ruffling along the open neural tube (yellow arrowheads), point of fusion of neural tube (green arrowhead), and lack of color in heart and blood vessels. (D) Example of an amorphous embryo arrested early in development.

Fig. 3.

Coronal sections of unaffected (Left) and mutant (Right) E10.5 embryos. These images are higher magnifications of the last of the serial sections shown in Fig. 10 (154 and 130, respectively). Yellow arrowheads mark the erythrocytes in the heart and vessels in the section from the unaffected embryo. The rostral neural tube is not fused (green arrows) but is fused caudally (red arrow) in the mutant embryo.

Fig. 4.

AKG levels in amniotic fluid from 25 embryos at E10.5. AKG was measured by using tandem MS and an internal standard. Five embryos were Slc25a19^+/+ (triangles), 13 embryos were Slc25a19^+/− (squares), and 7 embryos were Slc25a19^−/− (circles). The samples represented by each color were measured during the same experiment. The mean values were 6.17 ± 0.72 μmol/liter for the unaffected and 30.8 ± 9.91 μmol/liter for the mutants (P = 0.0155 Mann–Whitney). The data also were dichotomized by using the mean of the entire data set (13.06) and tested with Fisher's exact test (P = 0.0004). The t test could not be used because the data were not normally distributed.

Fig. 5.

Mitochondrial dNTP levels in wild-type and mutant MEFs and human lymphoblasts. For the MEFs, mitochondria were isolated from a wild-type (blue bars) or mutant (red bars) culture on two occasions. dNTP levels were measured in duplicate and averaged (±SD). Lymphoblast nucleotide levels were determined from mitochondria isolated from two control (yellow bars) or patient (green bars) cultures and averaged (±SD) according to affection status.

Fig. 6.

Transport assays of wild-type and mutant SLC25A19. Proteoliposomes reconstituted with recombinant wild-type (gray bars) and G177A mutant (white bars) SLC25A19 were preloaded internally with 200 μM each dATP, ThPP, ThMP, or thiamine. Transport was started with 20 μM α-³⁵S-dATP and terminated at 2 min. The values are means ±SD of at least three experiments.

Fig. 7.

KGDH and PDH complex assays. The KGDH (A and B) and PDH (C and D) complex activities were assayed by measuring NADH formed in the presence or absence of ThPP. (A and C) Wild-type (white bar) and knockout (gray bar) MEFs. (B and D) Human lymphoblasts from a normal individual (white bar) and a MCPHA patient (cross-hatched bar). The data represent the mean ± SEM of three independent experiments in duplicate. ANOVA analysis (two-way) was applied to compare the two groups, with and without ThPP, and ∗∗ denotes significance compared with control (P < 0.05).