Abstract

Cystathionine β-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 β-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.

INTRODUCTION

Homocysteinemia, a clinical condition with an elevated plasma level of homocyst(e)ine, is caused by a deficiency of vitamins (vitamins B6 and B12, and folic acid) and/or the enzymes such as cystathionine β-synthase (CBS; EC 4.2.1.22) and methylenetetrahydrofolate reductase ( 1 , 2 ). Homocysteine is emerging as an independent risk factor for atherosclerotic cardiovascular diseases, stroke, peripheral arterial occlusive diseases and venous thrombosis ( 1–3 ). Hyperhomocysteinemia is caused by several genetic defects, including deficiency of CBS: MIM 236200. CBS-deficient patients exhibit various clinical manifestations including thromboembolism, osteoporosis, hepatic steatosis (fatty liver), ectopia lentis, skeletal abnormalities and mental retardation. The molecular mechanisms by which accumulated homocysteine promotes such diseases are currently under extensive investigations, and endothelial dysfunction appears to play a key role in cardiovascular diseases ( 4–6 ). While mental retardation was one of the first clinical symptoms described for homocysteinemia ( 7 ), some clues to the mechanisms underlying such central nervous system (CNS) defects are just emerging ( 8–10 ).

CBS-deficient ( Cbs−/− ) mice were generated and analyzed as an animal model for hyperhomocysteinemia on the 129/Ola/C57BL/6J mixed background ( 11 ). They exhibit some pathophysiological features similar to CBS-deficient patients including hyperhomocysteinemia (∼200 µM in plasma) and hepatic steatosis, and the majority of them die within 4 weeks after birth probably due to severe hepatic dysfunction ( 11 ). Heterozygous ( Cbs+/− ) mice on the C57BL/6J background are currently maintained in and supplied from the Jackson laboratory, and researchers worldwide have confirmed that Cbs−/− suffer from hepatic dysfunction and rarely survive past the weaning age ( 12–14 ). Because total homocysteine levels in serum vary among inbred mouse strains ( 15 ), we tried to obtain greater numbers of and more mature Cbs−/− that are suitable for analyses of the CNS function and general behaviors by altering their genetic backgrounds. The Cbs+/− were backcrossed with four different inbred strains for seven generations, and the produced Cbs+/− progenies were bred to obtain Cbs−/− and their wild-type littermates ( Cbs+/+ ) for comparative analyses. In the present study, we found that Cbs−/− mice on the C3H/HeJ background (C3H/HeJ- Cbs−/− ) have higher survival rates than Cbs−/− on other backgrounds, and that C3H/HeJ- Cbs−/− survivors escape from lipidosis/hepatic steatosis but show some CNS abnormalities.

RESULTS

Survival and growth of Cbs−/− mice on different genetic backgrounds

The Cbs+/− mice were backcrossed onto four inbred strains (C57BL/6J(Jcl), BALB/cA, C3H/HeJ and DBA/2J) for seven generations (backcross generation number 7; N7) to achieve >99.2% genetic homogeneity for each, which took over 2 years. The produced N7 Cbs+/− males and females were bred to obtain Cbs+/+ , Cbs+/− and Cbs−/− littermates for comparative analyses. Kaplan–Meier survival plots indicate that the majority of C57BL/6J- Cbs−/− died between 2 and 4 postnatal weeks, and only 7.7% (2/26) survived beyond 8 weeks (Fig.  1 A); this result is generally consistent with previous studies using Cbs−/− on the 129/Ola/C57BL/6J mixed background ( 11 ). The survival rates at 8 weeks of age were greater in BALB/cA- Cbs−/− and C3H/HeJ- Cbs−/− (20.0% (5/25) and 30.8% (8/26), respectively) than in C57BL/6J- Cbs−/− . In contrast, none of 21 DBA/2J- Cbs−/− survived beyond 5 weeks of age. While most C57BL/6J- Cbs−/− and BALB/cA- Cbs−/− died within 6 months, all C3H/HeJ- Cbs−/− that survived beyond 8 weeks of age lived over 12 months (data not shown). Both C57BL/6J- Cbs−/− and BALB/cA- Cbs−/− were severely retarded in juvenile growth whereas C3H/HeJ- Cbs−/− developed progressively to match their Cbs+/+ and Cbs+/− littermates (Fig.  1 B). The 8-week-old Cbs−/− survivors on the three backgrounds showed similar skinny faces with pointed snouts and sparse fur (Fig.  1 C).

Figure 1.

Genetic background modifies survival and growth in Cbs−/− . ( A ) Kaplan–Meier survival analysis over 8 weeks. The cohorts of 36 Cbs+/+ (blue circles), 76 Cbs+/− (green triangles) and 26 Cbs−/− (red diamonds) mice including both sexes (36 Cbs+/+ : 76 Cbs+/− : 26 Cbs−/− ) were monitored on C57BL/6J, (29 Cbs+/+ : 52 Cbs+/− : 25 Cbs−/− ) on BALB/cA, (22 Cbs+/+ : 50 Cbs+/− : 26 Cbs−/− ) on C3H/HeJ and (24 Cbs+/+ : 54 Cbs+/− : 21 Cbs−/− ) on DBA/2J backgrounds. Significant differences in survival were observed between Cbs+/+ (or Cbs+/− ) and Cbs−/− on each of the four backgrounds ( P < 0.0001), as well as between C3H/HeJ- Cbs−/− and DBA/2J- Cbs−/− ( P < 0.02). ( B ) Body weight change in females after weaning at 3 weeks of age. Weight data were collected from mice that survived over 8 weeks, and thus there is no data for DBA/2J- Cbs−/− . The means ± SD of 5–25 samples for each genotype are shown. Significant differences were observed between Cbs−/− and aged-matched Cbs+/+ with Student t -test (* P < 0.05 and ** P < 0.01). ( C ) Typical appearance of 8-week-old Cbs−/− and littermate Cbs+/+ (or Cbs+/− ) on the three genetic backgrounds.

Figure 1.

Genetic background modifies survival and growth in Cbs−/− . ( A ) Kaplan–Meier survival analysis over 8 weeks. The cohorts of 36 Cbs+/+ (blue circles), 76 Cbs+/− (green triangles) and 26 Cbs−/− (red diamonds) mice including both sexes (36 Cbs+/+ : 76 Cbs+/− : 26 Cbs−/− ) were monitored on C57BL/6J, (29 Cbs+/+ : 52 Cbs+/− : 25 Cbs−/− ) on BALB/cA, (22 Cbs+/+ : 50 Cbs+/− : 26 Cbs−/− ) on C3H/HeJ and (24 Cbs+/+ : 54 Cbs+/− : 21 Cbs−/− ) on DBA/2J backgrounds. Significant differences in survival were observed between Cbs+/+ (or Cbs+/− ) and Cbs−/− on each of the four backgrounds ( P < 0.0001), as well as between C3H/HeJ- Cbs−/− and DBA/2J- Cbs−/− ( P < 0.02). ( B ) Body weight change in females after weaning at 3 weeks of age. Weight data were collected from mice that survived over 8 weeks, and thus there is no data for DBA/2J- Cbs−/− . The means ± SD of 5–25 samples for each genotype are shown. Significant differences were observed between Cbs−/− and aged-matched Cbs+/+ with Student t -test (* P < 0.05 and ** P < 0.01). ( C ) Typical appearance of 8-week-old Cbs−/− and littermate Cbs+/+ (or Cbs+/− ) on the three genetic backgrounds.

Lipid components and lecithin-cholesterol acyltransferase activity in 2-week-old Cbs−/− serum

We previously reported that 2–3-week-old Cbs−/− (on a mixed background) suffer from severe lipidosis ( 12 ) which may account for their retarded growth (Fig.  1 B). Cbs−/− on all four backgrounds showed hepatic steatosis, which was histochemically characterized by multivesicular lipid droplets at 2 weeks of age (Fig.  2 B and F for C57BL/6J- Cbs−/− and C3H/HeJ- Cbs−/− , respectively; data not shown for BALB/cA- Cbs−/− and DBA/2J- Cbs−/− ). Serum lipid components such as total cholesterol (T-Cho), free cholesterol (F-Cho), triglycerides (TG), non-esterified fatty acids (NEFA) and phospholipids (PL) were determined at 2 weeks of age (Table  1 ), just before most Cbs−/− mice start to die (Fig.  1 A). On all four backgrounds, body sizes were smaller and serum F-Cho levels were higher in Cbs−/− than in Cbs+/+ significantly (Table  1 ). Although the impact of Cbs deletion on serum T-Cho levels varied among the backgrounds, the major fractions of serum cholesterol in Cbs−/− were consistently non-esterified forms (F-Cho/T-Cho = 66.5–84.7%) while those in Cbs+/+ were esterified forms (F-Cho/T-Cho = 31.0–37.1%). Serum activity of lecithin-cholesterol acyltransferase (LCAT), the enzyme that esterifies cholesterol to form cholesterol ester and is mainly found in high-density lipoprotein (HDL) particles, was significantly lower in Cbs−/− than in the respective Cbs+/+ on all four backgrounds (Table  1 ). Serum TG, NEFA and PL levels were significantly greater in Cbs−/− than in the respective Cbs+/+ on two or three of the four backgrounds (Table  1 ). Serum TG levels in C3H/HeJ- Cbs−/− (135 mg/dl) were much lower than those in Cbs−/− on other backgrounds (186–226 mg/dl) (Table  1 ). High-performance liquid chromatography (HPLC) was performed to determine cholesterol and TG contents in each of the lipoprotein fractions: chylomicron (CM), very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and HDL. On all four backgrounds, serum cholesterol was mainly retained in the HDL and LDL fractions in Cbs+/+ (and Cbs+/− ; data not shown) while it was mainly found in the LDL and VLDL fractions in Cbs−/− (Fig.  3 ). On all four backgrounds, serum TG was mainly retained in the CM and LDL fractions in Cbs+/+ (Fig.  4 ) and Cbs+/− (data not shown). Serum TG levels in CM/VLDL fractions of C57BL/6J- Cbs−/− and BALB/cA- Cbs−/− were much higher than those in the respective Cbs+/+ or Cbs+/− samples (Fig.  4 ). No such remarkable increase in serum TG levels in the CM/VLDL fractions was observed in C3H/HeJ- Cbs−/− or DBA/2J- Cbs−/− (Fig.  4 ).

Figure 2.

Formation of lipid droplets and enlarged/multinucleated hepatocytes, and their disappearance in Cbs−/− . Liver sections from 2- and 10-week-old male mice ( Cbs+/+ and Cbs+/+ ) were stained with hematoxylin and oil red O ( AH ), or hematoxylin and eosin ( IP ). Multivesicular lipid droplets were observed in 2-week-old Cbs−/− liver (B and F) but not in 10-week-old Cbs−/− liver (D and H) on both C57BL/6J and C3H/HeJ backgrounds. In addition, enlarged/multinucleated pleomorphic hepatocytes were observed in 2-week-old Cbs−/− liver (J and N), but such abnormalities became much less apparent in 10-week-old Cbs−/− liver (L and P) on both backgrounds, especially on the C3H/HeJ background (P). Scale bars are 10 µm.

Figure 2.

Formation of lipid droplets and enlarged/multinucleated hepatocytes, and their disappearance in Cbs−/− . Liver sections from 2- and 10-week-old male mice ( Cbs+/+ and Cbs+/+ ) were stained with hematoxylin and oil red O ( AH ), or hematoxylin and eosin ( IP ). Multivesicular lipid droplets were observed in 2-week-old Cbs−/− liver (B and F) but not in 10-week-old Cbs−/− liver (D and H) on both C57BL/6J and C3H/HeJ backgrounds. In addition, enlarged/multinucleated pleomorphic hepatocytes were observed in 2-week-old Cbs−/− liver (J and N), but such abnormalities became much less apparent in 10-week-old Cbs−/− liver (L and P) on both backgrounds, especially on the C3H/HeJ background (P). Scale bars are 10 µm.

Figure 3.

HPLC analysis of cholesterol distribution in serum lipoprotein fractions. Serum samples from 2-week-old mice of each background and each genotype were analyzed for lipoprotein profiles by HPLC, and cholesterol content was measured. Representative data from 4 to 6 independent samples are shown. The CM, VLDL, LDL and HDL fractions are labeled.

Figure 3.

HPLC analysis of cholesterol distribution in serum lipoprotein fractions. Serum samples from 2-week-old mice of each background and each genotype were analyzed for lipoprotein profiles by HPLC, and cholesterol content was measured. Representative data from 4 to 6 independent samples are shown. The CM, VLDL, LDL and HDL fractions are labeled.

Figure 4.

HPLC analysis of TG distribution in serum lipoprotein fractions. Serum samples from 2-week-old mice of each background and each genotype were analyzed for lipoprotein profiles by HPLC, and TG content was measured. Representative data from 4 to 6 independent samples are shown. The CM, VLDL, LDL, HDL and FG (free glycerol) fractions are labeled.

Figure 4.

HPLC analysis of TG distribution in serum lipoprotein fractions. Serum samples from 2-week-old mice of each background and each genotype were analyzed for lipoprotein profiles by HPLC, and TG content was measured. Representative data from 4 to 6 independent samples are shown. The CM, VLDL, LDL, HDL and FG (free glycerol) fractions are labeled.

Table 1.

Body weights and serum levels of T-Cho, F-Cho, TG, NEFA, PL and LCAT activity in 2-week-old Cbs+/+ and Cbs−/− mice on the four genetic backgrounds

Background Genotype Body weights (g) T-Cho (mg/dl) F-Cho (mg/dl) TG (mg/dl) NEFA (µEq/l) PL (mg/dl) LCAT (nmol/ml/h) 
C57BL/6J +/+  6.59 ± 0.69 ( 24 )   115 ± 8 ( 22 )   42.7 ± 3.4 ( 22 )   91.9 ± 27.1 ( 22 )   488 ± 108 ( 22 )   213 ± 18 ( 22 )   965 ± 109 ( 5 )  
 −/−  5.42 ± 0.83 ( 17 )*   177 ± 56 ( 12 )*   150 ± 55 ( 12 )*   226 ± 92 ( 12 )*   1076 ± 344 ( 12 )*   362 ± 103 ( 12 )*   231 ± 288 ( 5 )**  
BALB/cA +/+  7.27 ± 1.66 ( 13 )   165 ± 17 ( 9 )   51.2 ± 4.3 ( 9 )   74.7 ± 43.4 ( 13 )   478 ± 130 ( 13 )   281 ± 25 ( 13 )   1236 ± 153 ( 5 )  
 −/−  6.16 ± 1.88 ( 17 )*   118 ± 44 ( 12 )**   86.8 ± 50.5 ( 12 )***   199 ± 120 ( 17 )*   797 ± 271 ( 17 )*   266 ± 70 ( 17 )   502 ± 167 ( 5 )*  
C3H/HeJ +/+  7.47 ± 1.21 ( 11 )   165 ± 31 ( 7 )   56.1 ± 8.0 ( 7 )   86.4 ± 20.0 ( 11 )   419 ± 92 ( 11 )   273 ± 25 ( 11 )   894 ± 217 ( 5 )  
 −/−  6.18 ± 0.86 ( 14 )*   178 ± 52 ( 8 )   142 ± 45 ( 8 )*   135 ± 46 ( 15 )*   450 ± 145 ( 15 )   420 ± 117 ( 15 )*   161 ± 125 ( 5 )*  
DBA/2J +/+  5.27 ± 0.86 ( 20 )   150 ± 8 ( 9 )   49.2 ± 3.1 ( 9 )   122 ± 89 ( 20 )   616 ± 169 ( 20 )   251 ± 26 ( 20 )   919 ± 194 ( 5 )  
 −/−  4.92 ± 1.04 ( 17 )*   167 ± 57 ( 15 )   111 ± 70 ( 16 )**   186 ± 102 ( 17 )   710 ± 230 ( 17 )   316 ± 78 ( 17 )**   273 ± 191 ( 5 )*  
Background Genotype Body weights (g) T-Cho (mg/dl) F-Cho (mg/dl) TG (mg/dl) NEFA (µEq/l) PL (mg/dl) LCAT (nmol/ml/h) 
C57BL/6J +/+  6.59 ± 0.69 ( 24 )   115 ± 8 ( 22 )   42.7 ± 3.4 ( 22 )   91.9 ± 27.1 ( 22 )   488 ± 108 ( 22 )   213 ± 18 ( 22 )   965 ± 109 ( 5 )  
 −/−  5.42 ± 0.83 ( 17 )*   177 ± 56 ( 12 )*   150 ± 55 ( 12 )*   226 ± 92 ( 12 )*   1076 ± 344 ( 12 )*   362 ± 103 ( 12 )*   231 ± 288 ( 5 )**  
BALB/cA +/+  7.27 ± 1.66 ( 13 )   165 ± 17 ( 9 )   51.2 ± 4.3 ( 9 )   74.7 ± 43.4 ( 13 )   478 ± 130 ( 13 )   281 ± 25 ( 13 )   1236 ± 153 ( 5 )  
 −/−  6.16 ± 1.88 ( 17 )*   118 ± 44 ( 12 )**   86.8 ± 50.5 ( 12 )***   199 ± 120 ( 17 )*   797 ± 271 ( 17 )*   266 ± 70 ( 17 )   502 ± 167 ( 5 )*  
C3H/HeJ +/+  7.47 ± 1.21 ( 11 )   165 ± 31 ( 7 )   56.1 ± 8.0 ( 7 )   86.4 ± 20.0 ( 11 )   419 ± 92 ( 11 )   273 ± 25 ( 11 )   894 ± 217 ( 5 )  
 −/−  6.18 ± 0.86 ( 14 )*   178 ± 52 ( 8 )   142 ± 45 ( 8 )*   135 ± 46 ( 15 )*   450 ± 145 ( 15 )   420 ± 117 ( 15 )*   161 ± 125 ( 5 )*  
DBA/2J +/+  5.27 ± 0.86 ( 20 )   150 ± 8 ( 9 )   49.2 ± 3.1 ( 9 )   122 ± 89 ( 20 )   616 ± 169 ( 20 )   251 ± 26 ( 20 )   919 ± 194 ( 5 )  
 −/−  4.92 ± 1.04 ( 17 )*   167 ± 57 ( 15 )   111 ± 70 ( 16 )**   186 ± 102 ( 17 )   710 ± 230 ( 17 )   316 ± 78 ( 17 )**   273 ± 191 ( 5 )*  

Means ± SD from independent samples (sample numbers in parentheses) are presented. T-Cho, total cholesterol; F-Cho, free cholesterol; TG, triglycerides; NEFA, non-esterified fatty acids; PL, phospholipids; LCAT, lecithin-cholesterol acyltransferase.

* P < 0.001 versus +/+ samples in t -test.

** P < 0.01 versus +/+ samples in t -test.

*** P < 0.05 versus +/+ samples in t -test.

Altered amino acid levels in 2-week-old Cbs−/− serum

Serum amino acid levels in 2-week-old mice were measured (Table  2 ). Total homocysteine concentrations in serum were measured after reducing all forms of disulfide-bound homocysteine; those in Cbs−/− were 13.9-, 14.9-, 16.7- and 17.5-fold greater than those in Cbs+/+ on the C57BL/6J, BALB/cA, C3H/HeJ and DBA/2J backgrounds, respectively (Table  2 ). Total homocysteine levels in both Cbs+/+ and Cbs−/− serums were lowest on the C3H/HeJ background (15.4 and 257 µM, versus 20.9–25.4 and 353–365 µM on other backgrounds, respectively). Amino acid contents were remarkably altered in Cbs−/− serum on all four backgrounds (Table  2 ); taurine levels were markedly down-regulated whereas homocystine (which was not detectable in Cbs+/+ ), glycine, histidine, methionine, proline, serine and threonine levels were all up-regulated. It should be noted that serum methionine accumulated to over 1 m m in Cbs−/− on all four backgrounds while 89.4–144 µM in Cbs+/+ . Genetic background-specific alterations were also observed: arginine and lysine levels were significantly decreased in DBA/2J- Cbs−/− and BALB/cA- Cbs−/− , respectively (Table  2 ). Both cysteine and cystine were under detectable levels in all serum samples.

Table 2.

Serum amino acid concentrations of 2-week-old Cbs+/+ and Cbs−/− mice on the four genetic backgrounds

Cbs genotype  C57BL/6J BALB/cA C3H/HeJ DBA/2J 
 +/+ −/− +/+ −/− +/+ −/− +/+ −/− 
Total homocysteine (µM) 25.4 ± 4.8 353 ± 81* 24.4 ± 5.8 363 ± 152* 15.4 ± 4.2 257 ± 74* 20.9 ± 8.1 365 ± 81* 
Free amino acids (µM)         
 Homocystine N.D. 59.2 ± 13.3 N.D. 75.5 ± 27.9 N.D. 52.0 ± 7.1 N.D. 52.8 ± 19.2 
 Taurine 646 ± 259 145 ± 36** 554 ± 164 144 ± 18* 795 ± 209 148 ± 33* 539 ± 188 114 ± 30** 
 Ala 448 ± 73 507 ± 115 276 ± 71 252 ± 115 291 ± 72 421 ± 91 435 ± 34 536 ± 102 
 Arg 232 ± 29 150 ± 99 234 ± 52 179 ± 74 304 ± 99 293 ± 58 280 ± 9 98.1 ± 92.3** 
 Asp and Asn 18.5 ± 5.2 23.5 ± 13.9 13.7 ± 2.6 11.6 ± 1.3 23.1 ± 9.4 14.1 ± 2.0 26.8 ± 15.1 19.0 ± 2.7 
 Glu and Gln 43.4 ± 8.5 53.1 ± 20.8 37.4 ± 6.5 31.9 ± 5.9 51.7 ± 10.7 37.1 ± 7.1 60.3 ± 13.9 48.7 ± 7.7 
 Gly 398 ± 37 710 ± 40* 367 ± 38 679 ± 109* 224 ± 23 513 ± 66* 304 ± 28 674 ± 134* 
 His 87.2 ± 4 145 ± 33** 74.1 ± 15.6 123 ± 25** 73.9 ± 11.5 112 ± 11* 81.3 ± 13.8 99.3 ± 14.4 
 Ile 104 ± 15 105 ± 31 72.7 ± 17.7 58.2 ± 21.2 104 ± 16 87.9 ± 25.8 115 ± 14 102 ± 20 
 Leu 164 ± 33 164 ± 25 116 ± 36 89 ± 43 161 ± 37 139 ± 40 175 ± 36 158 ± 43 
 Lys 701 ± 93 495 ± 133 504 ± 95 308 ± 87** 479 ± 74 452 ± 85 588 ± 61 475 ± 105 
 Met 144 ± 12 2545 ± 241* 89.4 ± 16.0 1619 ± 108* 109 ± 27 1808 ± 273* 112 ± 16 1514 ± 154* 
 Phe 174 ± 22 225 ± 41 166 ± 27 154 ± 32 167 ± 29 236 ± 55 179 ± 17 240 ± 42 
 Pro 300 ± 39 587 ± 99* 209 ± 38 335 ± 92 221 ± 43 452 ± 43* 272 ± 32 457 ± 45* 
 Ser 317 ± 35 724 ± 141* 253 ± 49 556 ± 112* 255 ± 46 808 ± 142* 297 ± 22 739 ± 138* 
 Thr 310 ± 25 585 ± 73* 221 ± 50 380 ± 83** 311 ± 49 666 ± 100* 293 ± 22 553 ± 57* 
 Tyr 273 ± 54 203 ± 90 197 ± 51 108 ± 42 174 ± 46 200 ± 35 212 ± 31 148 ± 19** 
 Val 275 ± 33 299 ± 65 187 ± 43 174 ± 70 314 ± 100 277 ± 51 279 ± 37 277 ± 42 
Cbs genotype  C57BL/6J BALB/cA C3H/HeJ DBA/2J 
 +/+ −/− +/+ −/− +/+ −/− +/+ −/− 
Total homocysteine (µM) 25.4 ± 4.8 353 ± 81* 24.4 ± 5.8 363 ± 152* 15.4 ± 4.2 257 ± 74* 20.9 ± 8.1 365 ± 81* 
Free amino acids (µM)         
 Homocystine N.D. 59.2 ± 13.3 N.D. 75.5 ± 27.9 N.D. 52.0 ± 7.1 N.D. 52.8 ± 19.2 
 Taurine 646 ± 259 145 ± 36** 554 ± 164 144 ± 18* 795 ± 209 148 ± 33* 539 ± 188 114 ± 30** 
 Ala 448 ± 73 507 ± 115 276 ± 71 252 ± 115 291 ± 72 421 ± 91 435 ± 34 536 ± 102 
 Arg 232 ± 29 150 ± 99 234 ± 52 179 ± 74 304 ± 99 293 ± 58 280 ± 9 98.1 ± 92.3** 
 Asp and Asn 18.5 ± 5.2 23.5 ± 13.9 13.7 ± 2.6 11.6 ± 1.3 23.1 ± 9.4 14.1 ± 2.0 26.8 ± 15.1 19.0 ± 2.7 
 Glu and Gln 43.4 ± 8.5 53.1 ± 20.8 37.4 ± 6.5 31.9 ± 5.9 51.7 ± 10.7 37.1 ± 7.1 60.3 ± 13.9 48.7 ± 7.7 
 Gly 398 ± 37 710 ± 40* 367 ± 38 679 ± 109* 224 ± 23 513 ± 66* 304 ± 28 674 ± 134* 
 His 87.2 ± 4 145 ± 33** 74.1 ± 15.6 123 ± 25** 73.9 ± 11.5 112 ± 11* 81.3 ± 13.8 99.3 ± 14.4 
 Ile 104 ± 15 105 ± 31 72.7 ± 17.7 58.2 ± 21.2 104 ± 16 87.9 ± 25.8 115 ± 14 102 ± 20 
 Leu 164 ± 33 164 ± 25 116 ± 36 89 ± 43 161 ± 37 139 ± 40 175 ± 36 158 ± 43 
 Lys 701 ± 93 495 ± 133 504 ± 95 308 ± 87** 479 ± 74 452 ± 85 588 ± 61 475 ± 105 
 Met 144 ± 12 2545 ± 241* 89.4 ± 16.0 1619 ± 108* 109 ± 27 1808 ± 273* 112 ± 16 1514 ± 154* 
 Phe 174 ± 22 225 ± 41 166 ± 27 154 ± 32 167 ± 29 236 ± 55 179 ± 17 240 ± 42 
 Pro 300 ± 39 587 ± 99* 209 ± 38 335 ± 92 221 ± 43 452 ± 43* 272 ± 32 457 ± 45* 
 Ser 317 ± 35 724 ± 141* 253 ± 49 556 ± 112* 255 ± 46 808 ± 142* 297 ± 22 739 ± 138* 
 Thr 310 ± 25 585 ± 73* 221 ± 50 380 ± 83** 311 ± 49 666 ± 100* 293 ± 22 553 ± 57* 
 Tyr 273 ± 54 203 ± 90 197 ± 51 108 ± 42 174 ± 46 200 ± 35 212 ± 31 148 ± 19** 
 Val 275 ± 33 299 ± 65 187 ± 43 174 ± 70 314 ± 100 277 ± 51 279 ± 37 277 ± 42 

Means ± SD from independent samples are presented ( n = 18 and 5 for total homocysteine and free amino acids, respectively). N.D., not detected.

*P < 0.001 versus +/+ samples in t -test.

**P < 0.01 versus +/+ samples in t -test.

Lipid components, LCAT activity and amino acid levels in 10-week-old Cbs−/− serum

The 10-week-old Cbs−/− survivors on the two backgrounds (C57BL6/J and C3H/HeJ) became free of hepatic steatosis (Fig.  2 D and H, respectively). Enlarged and multinucleated pleomorphic hepatocytes were observed in both 2-week-old C57BL6/J- Cbs−/− and C3H/HeJ- Cbs−/− (Fig.  2 J and N, respectively) ( 11 ), but such abnormalities became much less apparent at 10 weeks of age (Fig.  2 L and P, respectively), especially in C3H/HeJ- Cbs−/− (Fig.  2 P). When their serum samples of both backgrounds were analyzed, body weights, T-Cho and F-Cho levels were lower in Cbs−/− than those in the respective Cbs+/+ but the F-Cho/T-Cho ratios were comparable between Cbs+/+ and Cbs−/− (39.0 and 37.4% on C57BL6/J, and 29.8 and 31.1% on C3H/HeJ, respectively; Table  3 ). Serum levels of TG and PL (but not NEFA) were significantly lower in Cbs−/− than those in the respective Cbs+/+ while serum LCAT activity did not differ between the genotypes (Table  3 ). Lipoprotein profiling using HPLC revealed that the altered distributions of cholesterol and TG in each lipoprotein fraction observed in 2-week-old Cbs−/− (Figs  3 and 4 ) were dramatically recovered at 10 weeks of age, especially on the C3H/HeJ background ( Supplementary Material, Fig. S1 ). In contrast to the remarkable differences in serum amino acid contents between 2-week-old Cbs+/+ and Cbs−/− (Table  2 ), only slight changes were observed in those between 10-week-old Cbs+/+ and Cbs−/− except that Cbs−/− on both backgrounds became even more hyperhomocysteinemic ( Supplementary Material, Table S1 ). The excessive accumulation of methionine in 2-week-old Cbs−/− serum was no longer observed on either background. Taurine, glutamate/glutamine and glycine levels were significantly lower in C57BL/6J- Cbs−/− but not in C3H/HeJ- Cbs−/− when compared with their respective Cbs+/+ ( Supplementary Material, Table S1 ). These results indicate that the Cbs−/− abnormalities in lipid/amino acid metabolism and hepatocytic morphology at 2 weeks of age were mostly ameliorated at 10 weeks of age, particularly on the C3H/HeJ background.

Table 3.

Body weights and serum levels of T-Cho, F-Cho, TG, NEFA, PL and LCAT activity in 10-week-old Cbs+/+ and Cbs−/− male mice on the C57BL/6J or C3H/HeJ background

Background Genotype Body weights (g) T-Cho (mg/dl) F-Cho (mg/dl) TG (mg/dl) NEFA (µEq/l) PL (mg/dl) LCAT (nmol/ml/h) 
C57BL/6J +/+ 25.2 ± 0.8 54.3 ± 14.6 21.2 ± 4.6 97.6 ± 15.6 451 ± 95 188 ± 29 1177 ± 325 
 −/− 18.2 ± 1.5* 45.2 ± 4.1 16.9 ± 1.8 47.8 ± 11.9* 327 ± 90 134 ± 7* 1306 ± 112 
C3H/HeJ +/+ 23.3 ± 1.6 95.2 ± 8.1 28.4 ± 3.1 167 ± 52 215 ± 25 245 ± 13 1445 ± 288 
 −/− 20.1 ± 1.3** 60.2 ± 10.5* 18.7 ± 4.2* 75.2 ± 41.7** 169 ± 66 174 ± 18* 1448 ± 102 
Background Genotype Body weights (g) T-Cho (mg/dl) F-Cho (mg/dl) TG (mg/dl) NEFA (µEq/l) PL (mg/dl) LCAT (nmol/ml/h) 
C57BL/6J +/+ 25.2 ± 0.8 54.3 ± 14.6 21.2 ± 4.6 97.6 ± 15.6 451 ± 95 188 ± 29 1177 ± 325 
 −/− 18.2 ± 1.5* 45.2 ± 4.1 16.9 ± 1.8 47.8 ± 11.9* 327 ± 90 134 ± 7* 1306 ± 112 
C3H/HeJ +/+ 23.3 ± 1.6 95.2 ± 8.1 28.4 ± 3.1 167 ± 52 215 ± 25 245 ± 13 1445 ± 288 
 −/− 20.1 ± 1.3** 60.2 ± 10.5* 18.7 ± 4.2* 75.2 ± 41.7** 169 ± 66 174 ± 18* 1448 ± 102 

Means ± SD from 7 to 8 independent samples are presented.

*P < 0.001 versus +/+ samples in t -test.

**P < 0.01 versus +/+ samples in t -test.

Cerebellar malformation in Cbs−/−

Recently, Enokido et al . ( 16 ) reported impaired Purkinje cell differentiation and cerebellar malformation (impaired foliation/sulcus formation) in 3-week-old Cbs−/− on a mixed background. We observed similar cerebellar malformation characterized by small lobules and small intercrural fissures between the lobules in both 2-week-old and 10-week-old C57BL/6J- Cbs−/− (Fig.  5 B and D, respectively) while gross appearances (except sizes) of the cerebrum, hippocampus and olfactory bulb were normal ( Supplementary Material, Fig. S2 ). It is difficult to determine if such malformation is caused by the accumulated teratogenic homocysteine ( 17 ) or general developmental impediments in C57BL/6J- Cbs−/− because they were severely retarded in body weight growth (Fig.  1 B) and had much smaller brain sizes ( Supplementary Material, Fig. S2 ). However, C3H/HeJ- Cbs−/− that were comparable to their littermate C3H/HeJ- Cbs+/+ in body and brain sizes (Fig.  1 B and Supplementary Material, Fig. S2 , respectively) also showed small intercrural fissures between the lobules but not small lobules at both 2 and 10 weeks of age (Fig.  5 F and H, respectively), demonstrating that such cerebellar morphological abnormalities may not be solely caused by general developmental impediments. The gross appearances of the cerebrum, hippocampus and olfactory bulb were normal in C3H/HeJ- Cbs−/− ( Supplementary Material, Fig. S2 ).

Figure 5.

Cerebellar malformation in Cbs−/− . Whole brain sections were stained with hematoxylin and eosin, and cerebellar images are shown. The number of lobule is indicated by roman numerals in ( A ), ( C ), ( E ) and ( G ). Smaller lobules (arrow heads) and smaller intercrural fissures (arrows) were observed in Cbs−/− cerebelli on the C57BL/6J background, but only the latter was observed on the C3H/HeJ background, when compared with the respective Cbs+/+ . See Supplementary Material, Fig. S2 for larger views of whole brain sections. Scale bars are 100 µm.

Figure 5.

Cerebellar malformation in Cbs−/− . Whole brain sections were stained with hematoxylin and eosin, and cerebellar images are shown. The number of lobule is indicated by roman numerals in ( A ), ( C ), ( E ) and ( G ). Smaller lobules (arrow heads) and smaller intercrural fissures (arrows) were observed in Cbs−/− cerebelli on the C57BL/6J background, but only the latter was observed on the C3H/HeJ background, when compared with the respective Cbs+/+ . See Supplementary Material, Fig. S2 for larger views of whole brain sections. Scale bars are 100 µm.

Behavioral analysis of 10-week-old C3H/HeJ- Cbs−/−

The relatively high survival rates (Fig.  1 A) and body sizes that almost match their respective Cbs+/+ (Fig.  1 B) in C3H/HeJ- Cbs−/− permitted general comparative behavioral assessments, which could not be pursued previously. In the 5-min open-field test, there were no significant differences between C3H/HeJ- Cbs+/+ and C3H/HeJ- Cbs−/− in periods of movements, distances moved and periods spent in each area (Fig.  6 A–C, respectively). No significant differences were observed in the numbers of rearing behaviors and duration of grooming events (data now shown). In both the hole-board and elevated plus-maze tests, no differences were observed among the genotypes (Fig.  6 D and E, respectively), suggesting no specific changes in locomotion and exploratory (anti-anxiety) activity. To examine learning and memory performances, a passive avoidance step-through test was employed (Fig.  6 F). During the training trial at day 1, all eight C3H/HeJ- Cbs−/− entered into the dark compartment just as quickly (∼30 s) as all 23 C3H/HeJ- Cbs+/+ . In the testing trial at day 2, most (18/23) of the C3H/HeJ- Cbs+/+ avoided entering the dark compartment during the 400 s observation period but all eight C3H/HeJ- Cbs−/− entered after a short-lag period. Five, six and seven of the eight C3H/HeJ- Cbs−/− avoided entering the dark compartment during the testing trial at day 3, 4 and 5, respectively (Fig.  6 F).

Figure 6.

Behavioral assessments. Behavioral analyses were done on 10-week-old C3H/HeJ- Cbs+/+ and C3H/HeJ- Cbs−/− males ( n = 7 and 5, respectively, in AE ). (A–C) Open-field test. Each mouse was allowed to roam the field for 5 min. (A) Periods of movement (s); (B) distance moved (cm) and (C) retention time (seconds) in the center, side, and corner areas. (D) Hole-board test. Each mouse was placed in the hole-board and the number of head dips was counted during the 5-min test. (E) Elevated plus-maze test. Each mouse was placed in the center zone of the apparatus facing an open arm and the amount of time spent in the open arms was counted. ( F ) Step-through test. At day 1, each mouse was placed in the bright compartment and received an electric foot shock upon entering the dark compartment. At ≥ day 2, the mouse was placed in the bright compartment for a maximum of 400 s. The latency to enter the dark compartment (s) was recorded. The difference between C3H/HeJ- Cbs+/+ ( n = 23) and C3H/HeJ- Cbs−/− ( n = 8) was significant at day 2 (* P < 0.001).

Figure 6.

Behavioral assessments. Behavioral analyses were done on 10-week-old C3H/HeJ- Cbs+/+ and C3H/HeJ- Cbs−/− males ( n = 7 and 5, respectively, in AE ). (A–C) Open-field test. Each mouse was allowed to roam the field for 5 min. (A) Periods of movement (s); (B) distance moved (cm) and (C) retention time (seconds) in the center, side, and corner areas. (D) Hole-board test. Each mouse was placed in the hole-board and the number of head dips was counted during the 5-min test. (E) Elevated plus-maze test. Each mouse was placed in the center zone of the apparatus facing an open arm and the amount of time spent in the open arms was counted. ( F ) Step-through test. At day 1, each mouse was placed in the bright compartment and received an electric foot shock upon entering the dark compartment. At ≥ day 2, the mouse was placed in the bright compartment for a maximum of 400 s. The latency to enter the dark compartment (s) was recorded. The difference between C3H/HeJ- Cbs+/+ ( n = 23) and C3H/HeJ- Cbs−/− ( n = 8) was significant at day 2 (* P < 0.001).

Cyst(e)ine is an essential amino acid in Cbs−/−

Cysteine is considered to be a non-essential amino acid in mammals because it is synthesized from methionine via transsulfuration and, to our knowledge, only CBS can catalyze the first part of the two-step reaction ( 18 ). Biosynthesized cysteine is utilized as a component of polypeptides and proteins as well as a precursor of glutathione and taurine, two major anti-oxidants ( 18 ). We examined if the dietary supplementation of cyst(e)ine is essential for normal development of C3H/HeJ- Cbs−/− . The C3H/HeJ- Cbs−/− males that survived with the standard CE-2 diet (containing 0.37% cystine; Supplementary Material, Table S2 ) for 6 postnatal weeks were fed a cystine-supplemented KR diet (KR+Cys containing 0.37% cystine; Supplementary Material, Table S2 ) during 6–8 postnatal weeks. At 8 weeks of age, the diet was changed to a cyst(e)ine-restricted KR diet (0.16% cystine; ∼43% of CE-2). After the first change of diet, C3H/HeJ- Cbs−/− gained weight for 2 weeks just like the C3H/HeJ- Cbs+/+ did (Fig.  7 A). However, after the second change, C3H/HeJ- Cbs−/− but not C3H/HeJ- Cbs+/+ steadily lost weight until they died ∼4 weeks later (Fig.  7 A). When mice were fed CE-2 diet between 3 and 5 weeks of age and then a cyst(e)ine-free AA (amino acid) diet, C3H/HeJ- Cbs+/+ developed while C3H/HeJ- Cbs−/− died within a couple of days (Fig.  7 B). Because the supplementation of cystine (up to final 0.37%) to the cyst(e)ine-free AA diet did not rescue C3H/HeJ- Cbs−/− (data not shown), the deaths may be due to their severely impaired hepatic function (incapability of synthesizing proteins sufficiently) rather than the dietary deprivation of cyst(e)ine. Taken together, these results indicate that dietary cyst(e)ine is dispensable for the survival/development of the wild-type mice while 0.16% cystine in the diet could be insufficient for Cbs−/− .

Figure 7.

Body weight changes caused by special diets. ( A ) After weaning at 3 weeks of age, the C3H/HeJ- Cbs+/+ and C3H/HeJ- Cbs−/− males ( n = 4 each) were fed the standard CE-2 diet. At 6 and 8 weeks of age, the diet was changed to the KR+Cys and KR diet, respectively. Cystine supplementation to the KR diet is essential for the Cbs−/− survival. ( B ) After weaning at 3 weeks of age, the C3H/HeJ- Cbs+/+ and C3H/HeJ- Cbs−/− males ( n = 7 and 4, respectively) were fed the standard diet. At 5 weeks of age, the diet was changed to the cyst(e)ine-free AA diet. Cyst(e)ine is dispensable for Cbs+/+ survival/growth but indispensable for Cbs−/− . Data are the means ± SD for 4–6 males.

Figure 7.

Body weight changes caused by special diets. ( A ) After weaning at 3 weeks of age, the C3H/HeJ- Cbs+/+ and C3H/HeJ- Cbs−/− males ( n = 4 each) were fed the standard CE-2 diet. At 6 and 8 weeks of age, the diet was changed to the KR+Cys and KR diet, respectively. Cystine supplementation to the KR diet is essential for the Cbs−/− survival. ( B ) After weaning at 3 weeks of age, the C3H/HeJ- Cbs+/+ and C3H/HeJ- Cbs−/− males ( n = 7 and 4, respectively) were fed the standard diet. At 5 weeks of age, the diet was changed to the cyst(e)ine-free AA diet. Cyst(e)ine is dispensable for Cbs+/+ survival/growth but indispensable for Cbs−/− . Data are the means ± SD for 4–6 males.

DISCUSSION

Previous studies reported that the majority of Cbs−/− mice die between 2 and 4 weeks of age, and only a few survive to adulthood ( 11–13 , 19 ). All such analyses were performed on the C57BL/6J or related mixed genetic backgrounds because the Jackson Laboratory has been the main distributor of the mice. Some tips exist for obtaining adult Cbs−/− on those backgrounds. Dietary supplementation of choline chloride or folic acid has been shown to improve the survivability of Cbs−/− ( 13 , 19 , 20 ). Heterozygous Cbs+/− fed high-methionine diets become hyperhomocysteinemic and thereby may mimic Cbs−/− phenotypes ( 21 ). However, such dietetics could modify transsulfuration or other native metabolic pathways ( 22 ). For example, C57BL/6J- Cbs−/− fed high-choline diets did not show hepatic steatosis by 8 weeks of age, but did thereafter ( 13 ). By altering genetic backgrounds, we obtained C3H/HeJ- Cbs−/− mice with improved survivability and juvenile growth that can be used for general analyses of behaviors and the CNS functions, and examined if they could serve as a model for CBS-deficient patients with brain atrophy, mental retardation or neurodegenerative diseases ( 8 , 22 , 23 ).

We first examined the biochemical basis for less severe phenotypes in C3H/HeJ- Cbs−/− by comparing lipid/amino acid metabolism in serum. Although Cbs−/− on all four backgrounds are hyperhomocysteinemic (Table  2 ) and suffer from fatty liver (Fig.  2 B and F, and data not shown) and severe lipidosis (decreased levels of esterified and HDL cholesterols, and increased levels of F-Cho, TG, NEFA and PL) (Table  1 , Figs  3 and 4 ) at 2 weeks of age, serum levels of total homocysteine and TG were lowest in C3H/HeJ- Cbs−/− (Table  2 ). The reasons for the differences in degree of severity are currently unknown, but one possibility is that genes involved in homocysteine homeostasis may be polymorphic among those inbred strains and the functions of their products (e.g. enzymes) may be different. Epidemiological studies have shown that even mild elevations of plasma homocyst(e)ine are associated with an increased risk of cardiovascular diseases ( 3 , 24 ). Therefore, the much lower total homocysteine levels in C3H/HeJ- Cbs−/− may contribute to less severe phenotypes, although the relationship between serum homocysteine levels and hepatic dysfunction (or semi-lethality) remains to be clarified. To our knowledge, this is the first study to determine serum amino acid (except for homocysteine) concentrations in Cbs−/− mice, and remarkable alterations were observed in 2-week-old Cbs−/− compared with their respective Cbs+/+ on all four backgrounds (Table  2 ). Serum levels of methionine were markedly increased in Cbs−/− (Table  2 ), and this is in accordance with CBS-deficient patients who also suffer from hypermethioninemia ( 2 ). On all four backgrounds, serum levels of serine, glycine and threonine were higher in Cbs−/− , probably because serine was not consumed by the CBS-catalyzing condensation reaction with homocysteine and the increase in serine could lead to an accumulation of glycine and threonine, two precursor amino acids for serine biosynthesis ( 25 ). The reason for histidine up-regulation in serum is unknown. Such abnormal amino acid metabolism was observed to a greater or lesser extent in Cbs−/− on all four backgrounds at 2 weeks of age (Table  2 ).

Serum levels of taurine, the most abundant free amino acid in mammals as well as a major anti-oxidant ( 26 ), were markedly decreased in Cbs−/− mice. Taurine is synthesized from cysteine ( 27 ) and, therefore, the deficiency of biosynthesized cysteine caused by the CBS deletion may lead to decreased serum levels of taurine (Table  2 ). Taurine (not glycine) and cholesterol are essential components of bile salt in mice ( 28 ). Dietary supplementation of taurine induces bile flow as well as bile acid excretion ( 29 ) and decreases serum VLDL/LDL cholesterols ( 30 ). Therefore, it is possible that a shortage of taurine led to decreased secretion of bile salt into the duodenum and impaired emulsification in the intestine, which in turn induced the malnutrition/retarded growth in Cbs−/− . Moreover, the shortage of taurine/bile salt may lead to increased hepatic secretion of excessive cholesterol into VLDL/LDL (Fig.  3 ). Abnormal (F-Cho-rich) VLDL in Cbs−/− may not be a good substrate of lipoprotein lipase ( 12 ), which could lead to the accumulation of TG in VLDL of Cbs−/− (Fig.  4 ).

At 10 weeks of age, both C57BL/6J- Cbs−/− and C3H/HeJ- Cbs−/− survivors became even more hyperhomocysteinemic, but showed rather normal serum lipid components except for slightly low TG and PL levels (Table  3 ) and became free of hepatic steatosis (Fig.  2 D and H, respectively) and malformation (enlarged/multinucleated pleomorphic hepatocytes) (Fig.  2 L and P, respectively). The serum LCAT activity was comparable between 10-week-old Cbs+/+ and Cbs−/− , and surprisingly, abnormal amino acid contents including high methionine were no longer observed ( Supplementary Material, Table S1 ). These results suggest that Cbs−/− survivors adapt to hyperhomocysteinemia by decreasing sensitivities to homocysteine rather than by metabolizing (or excreting) it to lower its serum level. The LCAT activity was severely decreased in Cbs−/− at 2 weeks of age on all four backgrounds (Table  1 ) as we previously reported in Cbs−/− on a mixed background ( 12 ), which led to elevated F-Cho/T-Cho in Cbs−/− serum (Table  1 ). We have proposed that homocysteine could covalently crosslink with two cystine residues near the active site of LCAT, thereby leading to the enzyme inhibition ( 12 ). However, this may not be the case because LCAT activities and F-Cho/T-Cho ratios in hyperhomocysteinemic 10-week-old C57BL/6J- Cbs−/− and C3H/HeJ- Cbs−/− mice were within normal ranges (Table  3 ), although some phenotypes including hypertriglyceridemia in LCAT-deficient mice ( 31 ) resemble with those in Cbs−/− .

In humans, a relationship between plasma homocysteine levels and brain (white matter) atrophy has been reported ( 23 ). Cerebellar but not cerebral malformation was observed in both 2- and 10-week-old C3H/HeJ- Cbs−/− (Fig.  5 and Supplementary Material, Fig. S2 ). Homocysteine has been shown to cause teratogenesis of the heart and neural tubes in chick embryo models ( 17 ) but not in a mouse model ( 32 ). However, mice lacking methylenetetrahydrofolate reductase, a model for homocystinuria (MIM 236250) with moderately elevated levels of plasma homocysteine, also exhibit cerebellar malformation ( 33 ). Therefore, homocysteine (or its active metabolites such as homocysteic acid) that is present in the brain ( 9 , 34 ) may influence cerebellar neuronal progenitors as hippocampal progenitors in vitro ( 35 ).

By using C3H/HeJ- Cbs−/− survivors, we were able to explore possible behavioral changes, without comparing wild-type mice with their half-size littermate Cbs−/− on the C57BL/6J or related backgrounds. Although general behavioral analyses did not reveal any specific alterations, the passive avoidance step-through test that is often used for examining cholinergic system dysfunction in the cortex and hippocampus ( 36 ) demonstrated a learning deficit in C3H/HeJ- Cbs−/− ; the deficit was apparent on day 2, but not thereafter (Fig.  6 F). This is in part consistent with previous studies that showed impaired learning and memory in amyloid precursor protein-overexpressing transgenic mice chronically exposed to homocysteine ( 37 ) or methionine-fed mildly homocysteinemic rats ( 38 ). The Morris water-maze learning test was not employed because C3H/HeJ mice were not good at swimming and performed poorly in it (data not shown), probably due to their retinal degeneration ( 39 ). Homocysteine is postulated to have neurotoxic properties such as activation of N -methyl- d -aspartate receptor subtype and oxidative injuries leading to neuronal cell death ( 8 , 22 ). In cultured cortical neurons, homocysteic acid induces amyloid β42 accumulation ( 9 ), and moreover, it induces α-synuclein expression in the presence of high concentrations of methionine ( 40 ). Whether or not the observed cerebellar malformation and learning deficit are related to homocysteine (or homocysteic acid in combination with methionine)-induced teratogenicity/neurotoxicity is currently under investigation. By using C3H/HeJ- Cbs−/− survivors, we were also able to add further support to the extensive evidence for the fact that cyst(e)ine is an essential amino acid in the absence of CBS/transsulfuration in mammals. The cyst(e)ine supplementation in diets was dispensable for C3H/HeJ- Cbs+/+ when >0.16% cystine in diets was required for C3H/HeJ- Cbs−/− survival (Fig.  7 A and B).

A variety of working hypotheses have been proposed for homocysteine-induced diseases, among which homocysteine-mediated protein modification has attracted attention in recent years ( 41 ). In normal human plasma, ∼75% of total homocysteine exists in the protein-bound form through disulfide bonds with cysteine residues in proteins (primarily albumin in blood) ( 42 ). Alternatively, homocysteine may be converted to homocysteine thiolactone (HTL) by methionyl-tRNA synthetase in editing reactions to avoid misincorporation of homocysteine into proteins ( 43 ), thereby mediating homocysteinylation of lysine residues in functional proteins ( 44–46 ). Several in vitro studies reported that homocysteinylation of LDL causes oxidative injury (e.g. lipid peroxidation and peroxynitrite production) in endothelial cells ( 47–49 ). Decreased gene expression of homocysteine thiolactonase [paraoxonase 1; ( 43 )] in Cbs−/− liver ( 50 ) that associates with HDL and converts HTL into homocysteine ( 44 ), may suggest the accumulation of HTL and hyper-homocysteinylation in Cbs−/− ( 46 ). We recently found that serum activity of homocysteine thiolactonase was markedly decreased in 2-week-old C57BL/6J- Cbs−/− (I Ishii, unpublished observation), which may be partly due to abnormal HDL maturation [Fig.  3 and ( 12 )]. Homocysteine (or HTL)-based protein modifications may impair LDL functions and cause the altered cholesterol/TG distribution in LDL in Cbs−/− (Figs  3 and 4 , respectively). Such modifications could occur in a protein sequence (genetic polymorphism)-dependent fashion, which may account for the observed phenotypic differences in Cbs−/− between the inbred strains.

Finally, C3H/HeJ- Cbs−/− will be powerful tools for defining the unknown molecular mechanisms for thromboembolism, osteoporosis and the CNS abnormalities including mental retardation that are found in CBS-deficient patients, all of which may require a long-term progression of the diseases. Moreover, the C3H/HeJ- Cbs−/− survivors may gradually adapt to hyperhomocysteinemia and ameliorate hepatic dysfunction, which could provide a clue to alternatives for current homocysteine-lowing therapies against CBS-deficient patients such as persistent diet or supplementation of folate, betaine or vitamin B6/B12.

MATERIALS AND METHODS

Mice and diets

Heterozygous Cbs+/− on the C57BL/6J background (B6.129P2- Cbstm1Unc /J) were obtained from the Jackson Laboratory (Bar Harbar, ME, USA). They were backcrossed for seven generations with four different inbred strains (C57BL/6J(Jcl: Japan Clea), BALB/cA, C3H/HeJ and DBA/2J) available from a domestic distributor, Clea Japan (Tokyo, Japan). The N7 Cbs+/− were bred and age-matched progenies were comparatively analyzed. Tail biopsies were done at 2 weeks of age and genotyping for the targeted Cbs allele was performed by polymerase chain reaction ( 11 , 12 ). All animal protocols have been approved by the Animal Care and Experimentation Committee in Gunma University. Mice were fed ad libitum usually with a standard CE-2 diet (Clea Japan), and in some experiments, with the KR (Kojin Rayon ® ), KR+Cys or cyst(e)ine-free AA diets (for formula and composition, see Supplementary Material, Table S2 ). The KR diet, which mainly consists of denucleated torula yeast (Oriental Yeast, Tokyo, Japan) and sucrose, was originally formulated as a selenium-deficient diet ( 51 ). But in this study, it was used as a cyst(e)ine-deficient (selenium-sufficient) diet that contains 0.16% cystine (versus 0.37% in CE-2 or KR+Cys) and 0.164 ppm Na 2 SeO 4 (as a component of the AIN-93G mineral mixture). The original KR diet is also deficient in glutathione and taurine, the active metabolites of cysteine (and the intracellular storage form of cysteine as for glutathione), and therefore they were supplemented in the KR and KR+Cys diets so as to match their contents in CE-2 (0.007 and 0.0074%, respectively). The cyst(e)ine-free AA diet is formulated as a protein-free diet; it contains analytical grade amino acids that quantitatively match with protein-constituent amino acids in CE-2 ( Supplementary Material, Table S2 ). The composition of the diets was analyzed in Japan Food Research Laboratories (Tokyo, Japan).

Measurement of lipids, lipoproteins and LACT activity in serum

Serum levels of T-Cho, F-Cho, TG, NEFA and PL were measured by using commercial enzymatic assay kits from Wako (Osaka, Japan) ( 12 ). The serum lipoprotein profiling was performed with the HPLC system as described previously ( 12 , 52 ). The serum LCAT activity was determined using a commercial assay kit from Daiichi Pure Chemicals (Tokyo, Japan) ( 12 ).

Measurement of total homocysteine and free amino acids in serum

Total homocysteine [homocysteine and all its derivatives that will give rise to the thiol homocysteine after reductive cleavage of disulfide bonds ( 42 )] levels were measured using a Homocysteine Microtiter Plate Assay kit (Diazyme Laboratories, San Diego, CA, USA) as described previously ( 53 ). Free amino acids in serum were measured using HPLC systems. Amino acids were derivatized with 4-fluoro-7-nitrobenzofurazan (NBD-F; Dojindo, Kumamoto, Japan) under acidic conditions according to the manufacturer’s instruction, and samples were injected onto a reversed-phase C18 column (CAPCELL PAK C 18 MGII S5; Shiseido, Tokyo, Japan) for separation of the fluorescent-derivatized amino acids by acetonitrile gradient elution. All gradient steps were controlled by a CLASS-VP chromatography workstation (Shimadzu, Kyoto, Japan). The peaks of fluorescence (excitation 480 nm; emission 530 nm) were measured with a RF-5300PC fluorescence detector (Shimadzu, Japan). The amino acids were identified based on their retention times, and their concentrations were calculated by comparison with calibrated amino acid standard solutions (Wako, Japan). Tryptophan is not detectable because it is not derivatized with NBD-F.

Histochemistry

Liver and whole brain sections (each genotype, 2- and 10-week-old, C57BL/6J and C3H/HeJ backgrounds) were analyzed. Anesthetized male mice were perfused through the heart with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS. The brains were dissected out, post-fixed overnight in 4% paraformaldehyde in PBS and then cryoprotected in 30% sucrose in PBS ( 54 ). After sinking, they were embedded, frozen, sectioned with a cryostat at 10 µm and stained with Mayer’s hematoxylin and oil red O (or eosin Y) solutions (Muto Pure Chemicals, Tokyo, Japan).

Behavioral assessment

Locomotion and exploratory activities were assessed using the open-field test. Each mouse was placed in the center of an ‘open-field’ (a round-shaped arena, 75 cm in diameter surrounded by a 50 cm high wall made of gray plastic board), and allowed to roam the field for 5 min. Activities of mice in the field were automatically recorded by a CompACT VAS Ver.3.0x video tracking system (Muromachi Kikai, Tokyo, Japan). Periods of movement and distances traveled in the field were measured, and retention times in the center (0–7.5 cm radius), side (7.5–22.5 cm radius) and corner (22.5–37.5 cm radius) areas (1/9, 3/9 and 5/9 of the total area, respectively) were recorded. The numbers of rearing behaviors and duration of grooming events were also recorded. Exploratory behavior was also assessed using the hole-board test ( 55 ). The apparatus consisted of a square gray plastic plate, 48 × 48 cm, 5 mm thick, with 16 holes (3 cm diameter) regularly spaced on the surface, at 4.5 cm from the edges. The apparatus was elevated to a height of 10 cm, and mice were placed in the center of the plate. The number of head dips into the holes was manually counted during the 5-min test. The elevated plus-maze consisted of four arms (50 × 10 cm), two enclosed by walls 40 cm high and two exposed, elevated 50 cm off the ground. Mice were placed in the center zone facing the same open arm, and their behavior was recorded and analyzed by the CompAct. The amount of time spent in the open arms (and the number of open arm entries; data not shown) during the 5-min test were determined as measures of anxiety ( 56 ). Passive avoidance learning was tested by the step-through test, which utilizes the natural preference of mice for a dark environment ( 56 , 57 ). The apparatus consisted of bright and dark compartments (O’hara Ika-Sangyo, Tokyo, Japan). During the learning trial, mice were placed in the bright compartment and received an electric foot shock (75 V) upon entering the dark compartment. During the test period, starting on day 2, mice were placed in the bright compartment again for a maximum of 400 s at the same time each day for 4 days. The step-through latency, which indicates the time elapsed before the mouse enters the dark compartment, was recorded in the learning and testing trials as the performance of each mouse. Mice that did not enter the dark compartment were assigned a cutoff of 400 s and were scored as 400 s.

Statistics

Statistical analysis was performed using Student’s t -test. Results are represented as means ± SD from independent mouse samples. Kaplan–Meier survival analysis was done using Prism 4 software (GraphPad, San Diego, CA, USA).

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online .

FUNDING

This work was supported by Grant-in-Aid for Scientific Research 18590047 from the MEXT of Japan (I.I.); the Nakatomi Foundation (I.I.) and the Research Foundation for Pharmaceutical Sciences (I.I.). Data collected by metabolome analysis were supported by Leading Project for Biosimulation (M.S.) and in part by Grant-in-Aid for Creative Science Research 17GS0419 (M.S.) from the MEXT of Japan.

ACKNOWLEDGEMENTS

We thank animal care staffs in the Institute of Experimental Animal Research, Gunma University Graduate School of Medicine. M.S. is the leader of Global COE Program for Human Metabolomic Systems Biology from the MEXT of Japan.

Conflict of Interest statement . None declared.

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