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. 2013 Jun;27(6):2468-75.
doi: 10.1096/fj.12-219055. Epub 2013 Feb 21.

The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system

Shao-Chiang Lai  1 , Yasumi Nakayama, Jeffrey M Sequeira, Bogdan J Wlodarczyk, Robert M Cabrera, Richard H Finnell, Teodoro Bottiglieri, Edward V Quadros
Affiliations

The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system

Shao-Chiang Lai et al. FASEB J. 2013 Jun.

Abstract

The membrane receptor (TCblR/CD320) for transcobalamin (TC)-bound cobalamin (Cbl) facilitates the cellular uptake of Cbl. A genetically modified mouse model involving ablation of the CD320 gene was generated to study the effects on cobalamin homeostasis. The nonlethal nature of this knockout and the lack of systemic cobalamin deficiency point to other mechanisms for cellular Cbl uptake in the mouse. However, severe cobalamin depletion in the central nervous system (CNS) after birth (P<0.01) indicates that TCblR is the only receptor responsible for Cbl uptake in the CNS. Metabolic Cbl deficiency in the brain was evident from the increased methylmalonic acid (P<0.01-0.04), homocysteine (P<0.01), cystathionine (P<0.01), and the decreased S-adenosylmethionine/S-adenosyl homocysteine ratio (P<0.01). The CNS pathology of Cbl deficiency seen in humans may not manifest in this mouse model; however, it does provide a model with which to evaluate metabolic pathways and genes affected.

Keywords: CD320; cellular uptake; homocysteine; methylmalonic acid; nullizygous.

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Figures

Figure 1.
Figure 1.
TCblR and megalin expression in WT (+/+) and KO (−/−) mice during embryonic development and in adult tissues, determined by qPCR. A) Relative mRNA levels in WT embryo were normalized to the expression in GD16 embryos. B) TCblR mRNA expression in KO embryos compared to WT. Values are means for 2 embryos analyzed in triplicate for each time point. C) Relative mRNA level in adult mouse tissues. D, E) Megalin mRNA in embryos, normalized to GD16 expression (D), and in adult mouse tissues, relative to the expression in the liver (E). Data represent means ± se, n = 4/genotype.
Figure 2.
Figure 2.
Total B12 and transcobalamin in mouse serum. Total B12 (A), B12-saturated Holo TC (B), and unsaturated apo TC (C) were measured at 3, 8, and 20 wk of age. For each genotype, serum from 6 mice was pooled for a single analysis. Number above each bar indicates percentage increase or decrease compared to WT.
Figure 3.
Figure 3.
Total homocysteine (HCY) and methylmalonic acid (MMA) levels in serum. HCY (A) and MMA (B) levels in mouse serum were measured at 20 and 52 wk. Serum samples of WT and KO mice were pooled from 3 mice. Data represent single analysis in an aliquot of the pooled serums.
Figure 4.
Figure 4.
Total B12 level in mouse tissues. B12 level of liver (A), kidney (B), and spleen (C) was measured at 3, 8, and 20 wk of age. Six mice from each WT (+/+) and KO (−/−) group were sacrificed to obtain tissues. Data represent means ± se. *P < 0.01.
Figure 5.
Figure 5.
Total B12 levels in mouse CNS tissues. A) B12 level in brain was measured at age of 3, 8, and 20 wk. Six mice from each genotype were sacrificed to obtain tissues. B) B12 level of spinal cord was measured at age of 10 mo from 4 mice of each WT (+/+) and KO (−/−) group. Data represent means ± se. *P < 0.01.
Figure 6.
Figure 6.
Analysis of metabolites in CNS tissues. Levels of MMA (A), SAM (B), SAH (C), SAM/SAH ratio (D), methionine (E), cystathionine (F), betaine (G), and choline (H) were obtained from 5 WT and 5 KO mice. The whole brain was removed and separated into cortex, cerebellum, medulla, and hippocampus by microdissection. Spinal cord was collected from the same animals. Data represent means ± se. *P < 0.01, **P < 0.04.
Figure 7.
Figure 7.
Regional distribution of homocysteine in mouse brain. Homocysteine in WT and KO mice was determined at 3 mo (A) and 6 mo (B). At both time periods, the regional distribution of HCY appears to be similar with significantly elevated levels in all regions of the KO mouse brain.
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