Cysteine is considered a nonessential amino acid in mammals as it is synthesized from methionine via trans-sulfuration. However, premature infants or patients with hepatic failure may require dietary cysteine due to a lack of cystathionine ␥-lyase (CTH), a key trans-sulfuration enzyme. Here, we generated CTH-deficient (Cth ؊/؊ ) mice as an animal model of cystathioninemia/cystathioninuria. Cth ؊/؊ mice developed normally in general but displayed hypercystathioninemia/hyperhomocysteinemia though not hypermethioninemia. When fed a low cyst(e)ine diet, Cth ؊/؊ mice showed acute skeletal muscle atrophy (myopathy) accompanied by enhanced gene expression of asparagine synthetase and reduced contents of glutathione in livers and skeletal muscles, and intracellular accumulation of LC3 and p62 in skeletal myofibers; they finally died of severe paralysis of the extremities. Cth ؊/؊ hepatocytes required cystine in a culture medium and showed greater sensitivity to oxidative stress. Cth ؊/؊ mice exhibited systemic vulnerability to oxidative injury, which became more prominent when they were fed the low cyst(e)ine diet. These results reveal novel roles of trans-sulfuration previously unrecognized in mice lacking another trans-sulfuration enzyme cystathionine ␤-synthase (Cbs ؊/؊ ). Because Cbs ؊/؊ mice display hyperhomocysteinemia and hypermethioninemia, our results raise questions against the homocysteine-based etiology of CBS deficiency and the current newborn screening for homocysteinemia using Guthrie's method, which detects hypermethioninemia.

Cystathionine gamma-lyase (CSE) is the last key enzyme in the trans-sulphuration pathway for biosynthesis of cysteine from methionine. Cysteine could be provided through diet; however, CSE has been shown to be important for the adequate supply of cysteine to synthesize glutathione, a major intracellular antioxidant. With a view to determining physiological roles of CSE in mice, we report the sequence of a complete mouse CSE cDNA along with its associated genomic structure, generation of specific polyclonal antibodies, and the tissue distribution and developmental expression patterns of CSE in mice. A 1.8 kb full-length cDNA containing an open reading frame of 1197 bp, which encodes a 43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb mouse genomic fragment was obtained by lambda genomic library screening. It contained promoter regions, 12 exons, ranging in size from 53 to 579 bp, spanning over 30 kb, and exon/intron boundaries that were conserved with rat and human CSE. The GC-rich core promoter contained canonical TATA and CAAT motifs, and several transcription factor-binding consensus sequences. The CSE transcript, protein and enzymic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. In developing mouse liver and kidney, the expression levels of CSE protein and activity gradually increased with age until reaching their peak value at 3 weeks of age, following which the expression levels in liver remained constant, whereas those in kidney decreased significantly. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli. These results suggest important physiological roles for CSE in mice.

al cooperation between LPA4 and LPA6 is essential for embryonic development. Since Lpa4;Lpa5-DKO and Lpa5;Lpa6-DKO mice were born at the expected Mendelian ratios and had no obvious abnormalities (Supplemental Tables 4 and 5), LPA5 is unlikely to be involved in embryonic development. To examine the roles of LPA4 and LPA6 in embryonic development, yolk sac and embryo proper were observed at various stages of gestation. At E8.5, both Lpa4;Lpa6-DKO yolk sac and embryo proper appeared normal (Supplemental Figure 1, A and B). However, at E9.5-10.5, almost all of Lpa4;Lpa6-DKO embryos proper had various morphological abnormalities, such as severe pericardial effusion (Figure 1A and Supplemental Figure 1, B-D), axial turning abnormality (Supplemental Figure 1, B, E, and F), and developmental delay (Figure 1, A and B, and Supplemental Figure 1, B, G, and H). DKO yolk sacs lacked large blood vessels at E9.5-10.5, whereas WT yolk sacs had well-developed blood vessels (Figure 1B and Supplemental Figure 1, A, I, and J). We observed that all DKO embryos died by E11.5 (Supplemental Figure 1, B, K, and L, and Supplemental Table 6). Histological analysis showed that the endoderm and mesoderm of the yolk sacs were more widely separated in the DKO tissue (Figure 1C). Despite this vascular defect, erythrocytes were present in Lpa4;Lpa6-DKO yolk sacs (Figure 1C). Blood vessel-stained whole-mount embryos at E10.5 revealed that DKO embryos had enlarged dorsal aortae and poor vascular networks in the head and intersomitic regions, compared with WT embryos (Figure 1D). Furthermore, blood vessel-stained cross sections of embryos at E9.5 also revealed that DKO embryos had enlarged dorsal aortae and thinned neural tubes, compared with WT embryos (Figure 1E). These results strongly suggest that both LPA4 and LPA6 are indispensable for embryonic angiogenesis. Endothelial LPA4 and LPA6 are involved in retinal angiogenesis. Previously, we detected mRNA expression of Lpa4 and LPA6 in vascular ECs (8, 16). To investigate the functional roles of endothelial LPA4 and LPA6, we generated a tamoxifen-inducible and EC-specific Lpa4/Lpa6 deletion mouse line by crossing Lpa4;Lpa6 double-floxed mutants with transgenic mice carrying Cdh5-CreER T2 (ref. 37, Figure 2A, and Supplemental Figure 2A). The resulting Lpa4 fl/fl(Y) Lpa6 fl/fl Cdh5-CreER T2 mice (termed hereafter Lpa4; Lpa6 iΔEC) were treated with tamoxifen from P1 to P3. Allele-specific PCR confirmed recombination of both floxed alleles in the tails of Lpa4;Lpa6 iΔEC mice at P5 (Supplemental Figure 2B). At P5, we found that the radial expansion, EC area, and retinal blood vessel branching were significantly reduced in Lpa4;Lpa6 iΔEC mice compared with those in control CreER T2-negative Lpa4 fl/fl(Y) ;Lpa6 fl/fl littermates (Figure 2, C and F-H). At the angiogenic front, sprouts of retinal vessels were significantly reduced in Lpa4;Lpa6 iΔEC retina (Figure 2, D and I). Additionally, both the number and length of sprouting filopodia were significantly reduced in Lpa4;Lpa6 iΔEC retina (Figure 2, E, J...

Cystathionine beta-synthase-deficient mice (Cbs(-/-)) exhibit several pathophysiological features similar to hyperhomocysteinemic patients, including endothelial dysfunction and hepatic steatosis. Heterozygous mutants (Cbs(+/-)) on the C57BL/6J background are extensively analyzed in laboratories worldwide; however, detailed analyses of Cbs(-/-) have been hampered by the fact that they rarely survive past the weaning age probably due to severe hepatic dysfunction. We backcrossed the mutants with four inbred strains (C57BL/6J(Jcl), BALB/cA, C3H/HeJ and DBA/2J) for seven generations, and compared Cbs(-/-) phenotypes among the different genetic backgrounds. Although Cbs(-/-) on all backgrounds were hyperhomocysteinemic/hypermethioninemic and suffered from lipidosis/hepatic steatosis at 2 weeks of age, >30% of C3H/HeJ-Cbs(-/-) survived over 8 weeks whereas none of DBA/2J-Cbs(-/-) survived beyond 5 weeks. At 2 weeks, serum levels of total homocysteine and triglyceride were lowest in C3H/HeJ-Cbs(-/-). Adult C3H/HeJ-Cbs(-/-) survivors showed hyperhomocysteinemia but escaped hypermethioninemia, lipidosis and hepatic steatosis. They appeared normal in general behavioral tests but showed cerebellar malformation and impaired learning ability in the passive avoidance step-through test, and required sufficient dietary supplementation of cyst(e)ine for survival, demonstrating the essential roles of cystathionine beta-synthase in the central nervous system function and cysteine biosynthesis. Our C3H/HeJ-Cbs(-/-) mice could be useful tools for investigating clinical symptoms such as mental retardation and thromboembolism that are found in homocysteinemic patients.