Robert M. Cabrera

Genetic or nutritional disorders in homocysteine or folate metabolism increase protein N-homocysteinylation in mice.

Genetic disorders of homocysteine (Hcy) or folate metabolism or high‐methionine diets elevate plasma Hcy and its atherogenic metabolite Hcy‐thiolac‐tone. In humans, severe hyperhomocysteinemia due to genetic alterations in cystathionine ς‐synthase (Cbs) or methylenetetrahydrofolate reductase (Mthfr) results in neurological abnormalities and premature death from vascular complications. In mouse models, dietary or genetic hyperhomocysteinemia results in liver or brain pathological changes and accelerates atherosclerosis. Hcy‐thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates modified proteins (A‐Hcy‐protein) with autoimmunogenic and prothrombotic properties. Our aim was to determine how A–Hcy‐protein levels are affected by genetic or nutritional disorders in Hcy or folate metabolism in mice. We found that plasma A‐Hcy‐protein was elevated 10‐fold in mice fed a high‐methionine diet compared with the animals fed a normal commercial diet. We also found that inactivation of Cbs, Mthfr, or the proton‐coupled folate transporter (Pcft) gene resulted in a 10‐to 30‐fold increase in plasma or serum AHcy‐protein levels. Liver AHcy‐protein was elevated 3.4‐fold in severely and 11‐fold in extremely hyperhomocysteinemic Cbs‐deficient mice, 3.6‐fold in severely hyperhomocysteinemic Pcft mice, but was not elevated in mildly hyperhomocysteinemic Mthfr‐deficient animals, suggesting that mice have a capacity to prevent accumulation of A‐Hcy‐protein in their organs. These findings provide evidence that A‐Hcy‐protein is an important metabolite associated with Hcy pathophysiology in the mouse.—Jakubowski, H.,Peria‐Kajan, J., Finnell, R.H., Cabrera, R.M., Wang, H., Gupta, S., Kruger, W.D., Kraus, J.P., Shih, D.M. Genetic or nutritional disorders in homocysteine or folate metabolism increase protein A‐homocysteinylation in mice. FASEB J. 23, 1721–1727 (2009)

Autoantibodies to folate receptor during pregnancy and neural tube defect risk

Periconceptional folic acid can reduce the occurrence of neural tube defects (NTDs) by up to 70%, and autoantibodies for folate receptors (FRs) have been observed in serum from women with a pregnancy complicated by an NTD. This population-based cohort study has examined serum from pregnant mothers for autoantibodies to FRs, antibodies to bovine folate binding protein (FBP), and inhibition of folic acid binding to FR and FBP in association with NTD risk. The mid-gestational maternal serum specimens used for this study were collected during the 15-18th week of pregnancy. Samples were obtained from the California Birth Defects Monitoring Program; 29 mothers had a pregnancy complicated by spina bifida and 76 mothers had unaffected children. The presence of IgG and IgM antibodies to human FR, bovine FBP, and inhibition of folic acid binding to FR and FBP was determined. Higher activity of IgM to FBP in cases verses controls was observed (P=0.04). Higher activity of IgM and IgG autoantibodies to FR was observed (P<0.001 and P=0.04, respectively). Risk estimates at two standard deviations above average control antibody concentrations were OR=2.07 (CI=1.02, 4.06) for anti-FBP IgM, OR=2.15 (CI=1.02, 4.69) for anti-FR IgG and OR=3.19 (CI=1.47, 6.92) for anti-FR IgM. These data support the hypothesis that high titers of antibodies and blocking of folic acid binding to FRs by maternal serum should be regarded as risk factors for NTDs.

A mouse model of hereditary folate malabsorption: Deletion of the PCFT gene leads to systemic folate deficiency

The human proton coupled folate transporter (PCFT) is involved in low pH-dependent intestinal folate transport. In this report, we describe a new murine model of the hereditary folate malabsorption syndrome that we developed through targeted disruption of the first 3 coding exons of the murine homolog of the PCFT gene. By 4 weeks of age, PCFT-deficient (PCFT(-/-)) mice developed severe macrocytic normochromic anemia and pancytopenia. Immature erythroblasts accumulated in the bone marrow and spleen of PCFT(-/-) mice and failed to differentiate further, showing an increased rate of apoptosis in intermediate erythroblasts and reduced release of reticulocytes. In response to the inefficient hematologic development, the serum of the PCFT(-/-) animals contained elevated concentrations of erythropoietin, soluble transferrin receptor (sCD71), and thrombopoietin. In vivo folate uptake experiments demonstrated a systemic folate deficiency caused by disruption of PCFT-mediated intestinal folate uptake, thus confirming in vivo a critical and nonredundant role of the PCFT protein in intestinal folate transport and erythropoiesis. The PCFT-deficient mouse serves as a model for the hereditary folate malabsorption syndrome and is the most accurate animal model of folate deficiency anemia described to date that closely captures the spectrum of pathology typical of this disease.

LRP2 mediates folate uptake in the developing neural tube.

ABSTRACT The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2) is a multifunctional cell-surface receptor expressed in the embryonic neuroepithelium. Loss of LRP2 in the developing murine central nervous system (CNS) causes impaired closure of the rostral neural tube at embryonic stage (E) 9.0. Similar neural tube defects (NTDs) have previously been attributed to impaired folate metabolism in mice. We therefore asked whether LRP2 might be required for the delivery of folate to neuroepithelial cells during neurulation. Uptake assays in whole-embryo cultures showed that LRP2-deficient neuroepithelial cells are unable to mediate the uptake of folate bound to soluble folate receptor 1 (sFOLR1). Consequently, folate concentrations are significantly reduced in Lrp2−/− embryos compared with control littermates. Moreover, the folic-acid-dependent gene Alx3 is significantly downregulated in Lrp2 mutants. In conclusion, we show that LRP2 is essential for cellular folate uptake in the developing neural tube, a crucial step for proper neural tube closure.

Differentially expressed genes in embryonic cardiac tissues of mice lacking Folr1 gene activity.

BackgroundHeart anomalies are the most frequently observed among all human congenital defects. As with the situation for neural tube defects (NTDs), it has been demonstrated that women who use multivitamins containing folic acid peri-conceptionally have a reduced risk for delivering offspring with conotruncal heart defects [1–3]. Cellular folate transport is mediated by a receptor or binding protein and by an anionic transporter protein system. Defective function of the Folr1 (also known as Folbp1; homologue of human FRα) gene in mice results in inadequate transport, accumulation, or metabolism of folate during cardiovascular morphogenesis.ResultsWe have observed cardiovascular abnormalities including outflow tract and aortic arch arterial defects in genetically compromised Folr1 knockout mice. In order to investigate the molecular mechanisms underlying the failure to complete development of outflow tract and aortic arch arteries in the Folr1 knockout mouse model, we examined tissue-specific gene expression difference between Folr1 nullizygous embryos and morphologically normal heterozygous embryos during early cardiac development (14-somite stage), heart tube looping (28-somite stage), and outflow track septation (38-somite stage). Microarray analysis was performed as a primary screening, followed by investigation using quantitative real-time PCR assays. Gene ontology analysis highlighted the following ontology groups: cell migration, cell motility and localization of cells, structural constituent of cytoskeleton, cell-cell adhesion, oxidoreductase, protein folding and mRNA processing. This study provided preliminary data and suggested potential candidate genes for further description and investigation.ConclusionThe results suggested that Folr1 gene ablation and abnormal folate homeostasis altered gene expression in developing heart and conotruncal tissues. These changes affected normal cytoskeleton structures, cell migration and motility as well as cellular redox status, which may contribute to cardiovascular abnormalities in mouse embryos lacking Folr1 gene activity.

Folate-regulated changes in gene expression in the anterior neural tube of folate binding protein-1 (Folbp1)-deficient murine embryos.

Inactivation of the murine folate binding protein-1 (Folbp1) has been shown to play a vital role in embryonic development. Nullizygous embryos (Folbp1−/−) have significant malformations of the neural tube, craniofacies, and conotruncus, and invariably die in utero by gestational day(E) 10. Administration of 25 mg·kg−1·day−1 folinic acid to dams prior to and throughout gestation rescues the majority of embryos from premature death; however, a portion of surviving embryos develops neural tube defects. Using antisense RNA amplification and cDNA microarrays, we examined the expression of approximately 5700 genes in the anterior neural tube of gestational day 9 Folbp1−/− embryos that were supplemented with folinic acid. Genes that appear to be folate regulated include transcription factors, G-proteins, growth factors, methyltransferases, and those that are related to cell proliferation. The potential impact of such changes during neural tube closure is considered in light of the phenotype of Folbp1−/− embryos.

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

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.—Lai, S.‐C., Nakayama, Y., Sequeira, J. M., Wlodarczyk, B. J., Cabrera, R. M., Finnell, R. H., Bottiglieri, T., Quadros, E. V. The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system. FASEB J. 27, 2468–2475 (2013). www.fasebj.org

Gene Expression Profiling Within the Developing Neural Tube

The developing mammalian nervous system is subject to devastating congenital malformations with clinical significance that extends into the billions of health care dollars annually worldwide. Neural tube defects (NTDs) are among the most common of all human congenital defects, yet their etiology remains poorly understood. This is largely due to the complexity of the genetic factors regulating the intricate events involved in neurulation. Using mouse model systems and the application of modern molecular biological technologies, we have recently gained a greater appreciation for the factors that not only regulate normal neural tube closure (NTC), but those genetic factors that predispose an embryo to significant birth defects such as anencephaly or spina bifida. We have selected prominent murine mutants, both spontaneous and genetically modified, as well as the use of teratogenic agents, to examine the impact of altering the normal pattern of gene expression in the developing neural tube.

Autism-Like Behavior and Epigenetic Changes Associated with Autism as Consequences of In Utero Exposure to Environmental Pollutants in a Mouse Model

We tested the hypothesis that in utero exposure to heavy metals increases autism-like behavioral phenotypes in adult animals and induces epigenetic changes in genes that have roles in the etiology of autism. Mouse dams were treated with cadmium, lead, arsenate, manganese, and mercury via drinking water from gestational days (E) 1–10. Valproic acid (VPA) injected intraperitoneally once on (E) 8.5 served as a positive control. Young male offspring were tested for behavioral deficits using four standardized behavioral assays. In this study, in utero exposure to heavy metals resulted in multiple behavioral abnormalities that persisted into adulthood. VPA and manganese induced changes in perseverative/impulsive behavior and social dominance behavior, arsenic caused changes only in perseverative/impulsive behavior, and lead induced abnormalities in social interaction in comparison to the control animals. Brain samples from Mn, Pb, and VPA treated and control animals were evaluated for changes in CpG island methylation in promoter regions and associated changes in gene expression. The Chd7 gene, essential for neural crest cell migration and patterning, was found to be hypomethylated in each experimental animal tested compared to water-treated controls. Furthermore, distinct patterns of CpG island methylation yielded novel candidate genes for further investigation.

Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice

Folate deficiency has been associated with many adverse clinical manifestations. The blood‐brain barrier (BBB), formed by brain capillary endothelial cells, protects the brain from exposure to neurotoxicants. The function of BBB is modulated by multiple ABC transporters, particularly P‐glycoprotein. A proton‐coupled folate transporter (PCFT)‐deficient mouse has been previously described as a model for systemic folate deficiency. Herein, we demonstrate that exposing mouse brain capillaries to the antiepileptic drug, valproic acid (VPA; 5 μM), significantly increased P‐glycoprotein transport function in the wild‐type animals. A ligand to the aryl hydrocarbon receptor, 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), produced a similar induction of P‐glycoprotein, which tightened the BBB, thereby increasing the neuroprotection. However, VPA‐ or TCDD‐induced P‐glycoprotein transport was blocked in the PCFT‐nullizygous mice, indicating that multiple neuroprotective mechanisms are compromised under folate‐deficient conditions. Brain capillaries from S‐folinic acid (SFA; 40 mg/kg)‐treated PCFT‐nullizygous mice exhibited increased P‐glycoprotein transport following VPA exposure. This suggests that SFA supplementation restored the normal BBB function. In addition, we show that tight‐junction proteins are disintegrated in the PCFT mutant mice. Taken together, these findings strongly suggest that folate deficiency disrupts the BBB function by targeting the transporter and tight junctions, which may contribute to the development of neurological disorders.—Wang, X., Cabrera, R. M., Li, Y., Miller, D. S., Finnell, R. H. Functional regulation of P‐glycoprotein at the blood‐brain barrier in proton‐coupled folate transporter (PCFT) mutant mice. FASEB J. 27, 1167–1175 (2013). www.fasebj.org