Autoantibodies against homocysteinylated protein in a mouse model of folate deficiency-induced neural tube defects

Abstract

Background: Periconceptional supplementation with folic acid results in a significant reduction in the incidence of neural tube defects (NTDs). Nonetheless, NTDs remain a leading cause of perinatal morbidity and mortality worldwide, and the mechanism(s) by which folate exerts its protective effects are unknown. Homocysteine is an amino acid that accumulates under conditions of folate-deficiency, and is suggested as a risk factor for NTDs. One proposed mechanism of homocysteine toxicity is its accumulation into proteins in a process termed homocysteinylation.

Methods & results: Herein, we used a folate-deficient diet in pregnant mice to demonstrate that there is: (i) a significant inverse correlation between maternal serum folate levels and serum homocysteine; (ii) a significant positive correlation between serum homocysteine levels and titers of autoantibodies against homocysteinylated protein; and (iii) a significant increase in congenital malformations and NTDs in mice deficient in serum folate. Furthermore, in mice administered the folate-deplete diet before conception, supplementation with folic acid during the gestational period completely rescued the embryos from congenital defects, and resulted in homocysteinylated protein titers at term that are comparable to that of mice administered a folate-replete diet throughout both the pre- and postconception period. These results demonstrate that a low-folate diet that induces NTDs also increases protein homocysteinylation and the subsequent generation of autoantibodies against homocysteinylated proteins.

Conclusion: These data support the hypotheses that homocysteinylation results in neo-self antigen formation under conditions of maternal folate deficiency, and that this process is reversible with folic acid supplementation.

Keywords: autoantibodies; folate; folate-deficient diet; homocysteine; neural tube defects; pregnancy.

Figure 1

Maternal serum 5-MTHF levels at term in mice administered a folate-replete diet (FR), folate-deficient diet (FD), folate-deficient diet containing 1% succinylsulfathiazole (FD+SS), and a FD or FD+SS diet for six weeks prior to mating, followed by a FR “rescue” diet during gestation (FD/FR and FD+SS/FR, respectively). One-way ANOVA, followed by a Dunnett’s multiple comparisons test; ** p < 0.01; *** p < 0.001; ns, not significant.

Figure 2

Congenital abnormalities observed in FD+SS E18.5 fetuses included (A) exencephaly, (B) omphalocele, and (C) anophthalmia. (D) Quantification of malformation incidence in the different diet groups. (n) number of fetuses. Data were analysed by Fisher’s exact test. * p < 0.05.

Figure 3

(A) Maternal homocysteine levels at term, following a range of dietary regimens. One-way ANOVA, followed by a Dunnett’s multiple comparisons test; ** p<0.01; ns, not significant. (B) Maternal serum homocysteine levels with respect to 5-MTHF levels. Simple linear regression yielded r² = 0.18. Inset: data plotted on log-log axes. Non-linear regression analysis yielded r² = 0.49, p = 0.00002.

Figure 4

(A) Maternal levels of antibodies directed against homocysteinylated albumin (Anti-Hcy-albumin) at term, following a range of dietary regimens. One-way ANOVA, followed by a Dunnett’s multiple comparisons test; ** p<0.01; ns, not significant. (B) Maternal Anti-Hcy-albumin levels are positively correlated with homocysteine levels in maternal serum. Pearson’s correlation r = 0.46, p = 0.0033.