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Our understanding of brain biology and function is one of the least characterized and therefore, there are no effective treatments for most of neurological disorders. The influence of vitamins, and particularly vitamin B

SummaryFTY720 (fingolimod) is a sphingosine 1-phosphate (S1P) receptor modulator and sphingosine analogue approved for multiple sclerosis (MS) therapy, which can functionally antagonize the S1P receptor, S1P1. Vitamin B12 (B12) deficiency produces neurological manifestations resembling MS. Here, we report a new mechanism where FTY720 suppresses neuroinflammation by regulating B12 metabolic pathways. Nuclear RNA-seq of c-Fos-activated astrocytes (called ieAstrocytes) from experimental autoimmune encephalomyelitis (EAE) spinal cords identified up-regulation of CD320, a transcobalamin 2 (TCN2)-B12 receptor, by S1P1 inhibition. CD320 was reduced in MS plaques. Deficiency of CD320 or dietary B12 worsened EAE and eliminated FTY720’s efficacy, while concomitantly down-regulating type I interferon signaling. TCN2 functioned as a chaperone for FTY720 and sphingosine, which induced astrocytic CD320 internalization. An accompanying paper identified a requirement for astrocyte sphingosine kinases in FTY720 efficacy and its altered expression in MS brains, molecularly linking MS and B12 deficiency that can be accessed by sphingolipid/fingolimod metabolic pathways.

International audience; Autism spectrum disorders (ASD) are influenced by interacting maternal and environmental risk factors. High-dose folinic acid has shown improvement in verbal communication in ASD children. The EFFET randomized placebo-controlled trial (NCT02551380) aimed to evaluate the efficacy of folinic acid (FOLINORAL®) at a lower dose of 5 mg twice daily.Nineteen children were included in the EFFET trial. The primary efficacy outcome was improvement of Autism Diagnostic Observation Schedule (ADOS) score. The secondary outcomes were the improvement in ADOS sub scores communication, social interactions, Social Responsiveness Score (SRS) and treatment safety.The global ADOS score and social interaction and communication sub scores were significantly improved at week 12 compared to baseline in the folinic acid group (P = 0.003, P = 0.004 and P = 0.022, respectively), but not in the placebo group (P = 0.574, P = 0.780, P = 0.269, respectively). We observed a greater change of ADOS global score (−2.78 vs. −0.4 points) and (−1.78 vs. 0.20 points) in the folinic acid group, compared to the placebo group. No serious adverse events were observed.This pilot study showed significant efficacy of folinic acid with an oral formulation that is readily available. It opens a perspective of therapeutic intervention with folinic acid but needs to be confirmed by a multi-center trial on a larger number of children.

Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR (or CD320), encoded by the CD320 gene(1). Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells such as cancer cells(2–4), this uptake route is a potential target for cancer therapy(5). We developed and characterized four camelid nanobodies that bind TC or the interface of the TC:TCblR complex with nanomolar affinities. We determined X-ray crystal structures of all four nanobodies in complex with TC:TCblR, which enabled us to map their binding sites. When conjugated to a toxin, three of these nanobodies are capable of inhibiting the growth of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TCNB4) using fluorescent light microscopy. The co-crystal structures of TC:TCblR with another nanobody (TCNB34) revealed novel features of the interface of TC and the LDLR-A1 domain of TCblR. Our findings rationalize the structural basis for a decrease in affinity of TC-B12 binding caused by the TCblR-Glu88 deletion mutant.

Folate deficiency and folate receptor autoimmune disorder are major contributors to infertility, pregnancy related complications and abnormal fetal development including structural and functional abnormalities of the brain. Food fortification and prenatal folic acid supplementation has reduced the incidence of neural tube defect (NTD) pregnancies but is unlikely to prevent pregnancy-related complications in the presence of folate receptor autoantibodies (FRAb). In pregnancy, these autoantibodies can block folate transport to the fetus and in young children, folate transport to the brain. These antibodies are prevalent in neural tube defect pregnancies and in developmental disorders such as cerebral folate deficiency (CFD) syndrome and autism spectrum disorder (ASD). In the latter conditions, folinic acid treatment has shown clinical improvement in some of the core ASD deficits. Early testing for folate receptor autoantibodies and intervention is likely to result in a positive outcome. This review discusses the first identification of FRAb in women with a history of neural tube defect pregnancy and FRAb’s association with sub-fertility and preterm birth. Autoantibodies against folate receptor alpha (FRα) are present in about 70% of the children with a diagnosis of ASD, and a significant number of these children respond to oral folinic acid with overall improvements in speech, language and social interaction. The diagnosis of folate receptor autoimmune disorder by measuring autoantibodies against FRα in the serum provides a marker with the potential for treatment and perhaps preventing the pathologic consequences of folate receptor autoimmune disorder.

In humans, vitamin B12 deficiency causes peripheral and CNS manifestations. Loss of myelin in the peripheral nerves and the spinal cord (SC) contributes to peripheral neuropathy and motor deficits. The metabolic basis for the demyelination and brain disorder is unknown. The transcobalamin receptor-knockout mouse ( Cd320-/-) develops cobalamin (Cbl) deficiency in the nervous system, with mild anemia. A decreased S-adenosylmethionine: S-adenosylhomocysteine ratio and increased methionine were seen in the brain with no significant changes in neurotransmitter metabolites. The structural pathology in the SC presented as loss of myelin in the axonal tracts with inflammation. The sciatic nerve (SN) showed increased nonuniform, internodal segments suggesting demyelination, and remyelination in progress. Consistent with these changes, the Cd320-/- mouse showed an increased latency to thermal nociception. Further, lower amplitude of compound action potential in the SN suggested that the functional capacity of the heavily myelinated axons were preferentially compromised, leading to loss of peripheral sensation. Although the metabolic basis for the demyelination and the structural and functional alterations of the nervous system in Cbl deficiency remain unresolved, the Cd320-/- mouse provides a unique model to investigate the pathologic consequences of vitamin B12 deficiency. -Arora, K., Sequeira, J. M., Alarcon, J. M., Wasek, B., Arning, E., Bottiglieri, T., Quadros, E. V. Neuropathology of vitamin B12 deficiency in the Cd320-/- mouse.

Cobalamin (Cbl, vitamin B12) deficiency or inborn errors of Cbl metabolism can produce neurologic disorders resistant to therapies, including cognitive dysfunction, mild mental retardation, memory impairment, and confusion. We used Cd320 KO mouse as a model for studying the pathological mechanisms of these disorders. Cd320 encodes the receptor (TCblR) needed for the cellular uptake of Cbl in the brain. The Cd320-/- mouse model presented an impaired learning memory that could be alleviated by a moderate stress, which produced also a greater increase of plasma corticosterone, compared to wild type animals. The present study investigated such a putative rescue mechanism in Cbl-deficient mice. At the molecular level in the brain of Cd320-/- mouse, the decreased methylation status led to a downregulation of glucocorticoid nuclear receptor (GR)/PPAR-gamma co-activator-1 alpha (PGC-1α) pathway. This was evidenced by the decreased expression of GR, decreased methylation of GR and PGC1α, and decreased dimerization and interaction of GR with PGC1α. This led to altered synaptic activity evidenced by decreased interaction between the NMDA glutamatergic receptor and the PSD95 post-synaptic protein and a lower expression of Egr-1 and synapsin 1, in Cd320-/- mice compared to the wild type animals. Intraperitoneal injection of hydrocortisone rescued these molecular changes and normalized the learning memory tests. Our study suggests adaptive influences of moderate stress on loss of memory and cognition due to brain Cbl deficiency. The GR pathway could be a potential target for innovative therapy of cognitive manifestations in patients with poor response to conventional Cbl treatment.

Cellular uptake of vitamin B

Cobalamin (Cbl, vitamin B

Background. Biomarkers such as oxidative stress, folate receptor alpha (FRα) autoimmunity, and abnormal brain serotonin turnover are common in autism. Methods. Oxidative stress biomarkers with pro- and antioxidants were measured in the severe form of infantile autism (n = 38) and controls (n = 24). Children and parents had repeated testing for serum FR autoantibodies, spinal fluid dopamine and serotonin metabolites, pterins, and N5-methyltetrahydrofolate (MTHF). Statistical analysis assessed correlations between variables. Genetic analysis included the SLC6A4 and SLC29A4 genes encoding synaptic serotonin reuptake proteins. Results. Compared to controls, the autism group showed a significant increase in oxidative DNA damage in lymphocytes, plasma ceruloplasmin and copper levels with a high copper/zinc ratio, thiol proteins, and superoxide dismutase (SOD) activity. Vitamin C levels were significantly diminished. In most autistic patients, the vitamin A (64%) and D (70%) levels were low. Serum FR autoantibodies fluctuating over 5–7 week periods presented in 68% of all autistic children, 41% of parents vs. 3.3% of control children and their parents. CSF showed lowered serotonin 5-hydroxyindole acetic acid (5HIAA) metabolites in 13 (34%), a low 5HIAA to HVA (dopamine metabolite) ratio in 5 (13%), low 5HIAA and MTHF in 2 (5%), and low MTHF in 8 patients (21%). A known SLC6A4 mutation was identified only in 1 autistic child with low CSF 5HIAA and a novel SLC29A4 mutation was identified in identical twins. Low CSF MTHF levels among only 26% of subjects can be explained by the fluctuating FR antibody titers. Two or more aberrant pro-oxidant and/or antioxidant factors predisposed to low CSF serotonin metabolites. Three autistic children having low CSF 5HIAA and elevated oxidative stress received antioxidative supplements followed by CSF 5HIAA normalisation. Conclusion. In autism, we found diverse combinations for FR autoimmunity and/or oxidative stress, both amenable to treatment. Parental and postnatal FR autoantibodies tend to block folate passage to the brain affecting folate-dependent pathways restored by folinic acid treatment, while an abnormal redox status tends to induce reduced serotonin turnover, corrected by antioxidant therapy. Trial Registration. The case-controlled study was approved in 2008 by the IRB at Liège University (Belgian Number: B70720083916). Lay Summary. Children with severe infantile autism frequently have serum folate receptor autoantibodies that block the transport of the essential vitamin folate across the blood-brain barrier to the brain. Parents are often asymptomatic carriers of these serum folate receptor autoantibodies, which in mothers can block folate passage across the placenta to their unborn child. This folate deficiency during the child’s intrauterine development may predispose to neural tube defects and autism. Oxidative stress represents a condition with the presence of elevated toxic oxygen derivatives attributed to an imbalance between the formation and protection against these toxic reactive oxygen derivatives. Oxidative stress was found to be present in autistic children where these reactive oxygen derivatives can cause damage to DNA, which changes DNA function and regulation of gene expression. In addition, excessive amounts of these toxic oxygen derivatives are likely to damage the enzyme producing the neuromessenger serotonin in the brain, diminished in about 1/3 of the autistic children. Testing children with autism for oxidative stress and its origin, as well as testing for serum folate receptor autoantibodies, could open new approaches towards more effective treatments.

The molecular mechanisms that underlie the neurological manifestations of patients with inherited diseases of vitamin B12 (cobalamin) metabolism remain to date obscure. We observed transcriptomic changes of genes involved in RNA metabolism and endoplasmic reticulum stress in a neuronal cell model with impaired cobalamin metabolism. These changes were related to the subcellular mislocalization of several RNA binding proteins, including the ELAVL1/HuR protein implicated in neuronal stress, in this cell model and in patient fibroblasts with inborn errors of cobalamin metabolism and Cd320 knockout mice. The decreased interaction of ELAVL1/HuR with the CRM1/exportin protein of the nuclear pore complex and its subsequent mislocalization resulted from hypomethylation at R-217 produced by decreased S-adenosylmethionine and protein methyl transferase CARM1 and dephosphorylation at S221 by increased protein phosphatase PP2A. The mislocalization of ELAVL1/HuR triggered the decreased expression of SIRT1 deacetylase and genes involved in brain development, neuroplasticity, myelin formation, and brain aging. The mislocalization was reversible upon treatment with siPpp2ca, cobalamin, S-adenosylmethionine, or PP2A inhibitor okadaic acid. In conclusion, our data highlight the key role of the disruption of ELAVL1/HuR nuclear export, with genomic changes consistent with the effects of inborn errors of Cbl metabolisms on brain development, neuroplasticity and myelin formation.

Folate deficiency can affect fetal and neonatal brain development Considering the reported association of Folate receptor alpha (FRα) autoantibodies (Abs) with autism and developmental disorders, we sought to confirm this in families of 82 children with ASD, 53 unaffected siblings, 65 fathers, and 70 mothers, along with 52 unrelated normal controls. Overall, 76% of the affected children, 75% of the unaffected siblings, 69% of fathers and 59% of mothers were positive for either blocking or binding Ab, whereas the prevalence of this Ab in the normal controls was 29%. The Ab was highly prevalent in affected families including unaffected siblings. The appearance of these antibodies may have a familial origin but the risk of developing ASD is likely influenced by other mitigating factors since some siblings who had the antibodies were not affected. The antibody response appears heritable with the blocking autoantibody in the parents and affected child increasing the risk of ASD. Autism Res 2018, 11: 707-712. © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY Folate is an essential nutrient during fetal and infant development. Autoantibodies against the folate receptor alpha can block folate transport from the mother to the fetus and to the brain in infants. Children diagnosed with autism and their immediate family members were evaluated for the prevalence of folate receptor autoantibodies. The autoantibody was highly prevalent in affected families with similar distribution in parents, normal siblings and affected children. The presence of these antibodies appears to have a familial origin and may contribute to developmental deficits when combined with other factors.

Autism spectrum disorder (ASD) has been linked to abnormalities in folate metabolism. Polymorphisms in folate genes may act in complex polygenic ways to increase the risk of developing ASD. Autoantibodies that block folate transport into the brain have been associated with ASD and children with ASD and these autoantibodies respond to high doses of a reduced form of folate known as folinic acid (leucovorin calcium). Some of the same abnormalities are also found in mothers of children with ASD and supplementing folate during preconception and gestational periods reduces the risk to the offspring from developing ASD. These data suggest that folate pathway abnormalities may be a major metabolic disturbance underlying ASD that can be leveraged as biomarkers to improve symptoms and prevent ASD.

Vitamin B12 deficiency causes megaloblastic anemia and neurologic disorder in humans. Gene defects of transcobalamin (TC) and the transcobalamin receptor (TCblR), needed for cellular uptake of the TC-bound B12, do not confer embryonic lethality. TC deficiency can produce the hematologic and neurologic complications after birth, whereas TCblR/CD320 gene defects appear to produce mild metabolic changes. Alternate maternofetal transport mechanisms appear to provide adequate B12 to the fetus. To understand this mechanism, we evaluated the role of TC, TCblR/CD320, and megalin in maternofetal transport of B12 in a TCblR/CD320-knockout (KO) mouse. Our results showed high expression of TCblR/CD320 in the labyrinth of the placenta, embryonic brain, and spinal column in wild-type (WT) mice. Megalin expression was about the same in both WT and KO mouse visceral yolk sac, brain, and spinal column. Megalin mRNA was down-regulated in the KO embryonic spinal cord (SC) and kidneys. Megalin expression remained unaltered in adult WT and KO mouse brain, SC, and kidneys. Injected dsRed-TC-B12 and TC-57CoB12 accumulated in the visceral yolk sac of KO mice where megalin is expressed and provides an alternate mechanism for the maternofetal transport of Cbl during fetal development.-Arora, K., Sequeira, J. M., Quadros, E. V. Maternofetal transport of vitamin B12: role of TCblR/CD320 and megalin.

Background. In contrast to multiple rare monogenetic abnormalities, a common biomarker among children with infantile autism and their parents is the discovery of serum autoantibodies directed to the folate receptor alpha (FRα) localized at blood-brain and placental barriers, impairing physiologic folate transfer to the brain and fetus. Since outcome after behavioral intervention remains poor, a trial was designed to treat folate receptor alpha (FRα) autoimmunity combined with correction of deficient nutrients due to abnormal feeding habits. Methods. All participants with nonsyndromic infantile autism underwent a routine protocol measuring CBC, iron, vitamins, coenzyme Q10, metals, and trace elements. Serum FRα autoantibodies were assessed in patients, their parents, and healthy controls. A self-controlled therapeutic trial treated nutritional derangements with addition of high-dose folinic acid if FRα autoantibodies tested positive. The Childhood Autism Rating Scale (CARS) monitored at baseline and following 2 years of treatment was compared to the CARS of untreated autistic children serving as a reference. Results. In this self-controlled trial (82 children; mean age ± SD: 4.4 ± 2.3 years; male:female ratio: 4.8:1), FRα autoantibodies were found in 75.6 % of the children, 34.1 % of mothers, and 29.4 % of fathers versus 3.3 % in healthy controls. Compared to untreated patients with autism (n=84) whose CARS score remained unchanged, a 2-year treatment decreased the initial CARS score from severe (mean ± SD: 41.34 ± 6.47) to moderate or mild autism (mean ± SD: 34.35 ± 6.25; paired t-test pα autoantibody titers, positive maternal FRα autoantibodies, or FRα antibodies in both parents. Conclusions. Correction of nutritional deficiencies combined with high-dose folinic acid improved outcome for autism, although the trend of a poor prognosis due to maternal FRα antibodies or FRα antibodies in both parents may warrant folinic acid intervention before conception and during pregnancy.

Cobalamin [Cbl (or B(12))] deficiency causes megaloblastic anemia and a variety of neuropathies. However, homeostatic mechanisms of cyanocobalamin (CNCbl) and other Cbls by vascular endothelial cells are poorly understood. Herein, we describe our investigation into whether cultured bovine aortic endothelial cells (BAECs) perform transcytosis of B(12), namely, the complex formed between serum transcobalamin and B(12), designated as holo-transcobalamin (holo-TC). We show that cultured BAECs endocytose [(57)Co]-CNCbl-TC (source material) via the CD320 receptor. The bound Cbl is transported across the cell both via exocytosis in its free form, [(57)Co]-CNCbl, and via transcytosis as [(57)Co]-CNCbl-TC. Transcellular mobilization of Cbl occurred in a bidirectional manner. A portion of the endocytosed [(57)Co]-CNCbl was enzymatically processed by methylmalonic aciduria combined with homocystinuria type C (cblC) with subsequent formation of hydroxocobalamin, methylcobalamin, and adenosylcobalamin, which were also transported across the cell in a bidirectional manner. This demonstrates that transport mechanisms for Cbl in vascular endothelial cells do not discriminate between various β-axial ligands of the vitamin. Competition studies with apoprotein- and holo-TC and holo-intrinsic factor showed that only holo-TC was effective at inhibiting transcellular transport of Cbl. Incubation of BAECs with a blocking antibody against the extracellular domain of the CD320 receptor inhibited uptake and transcytosis by ∼40%. This study reveals that endothelial cells recycle uncommitted intracellular Cbl for downstream usage by other cell types and suggests that the endothelium is self-sufficient for the specific acquisition and subsequent distribution of circulating B(12) via the CD320 receptor. We posit that the endothelial lining of the vasculature is an essential component for the maintenance of serum-tissue homeostasis of B(12).—Hannibal, L., Bolisetty, K., Axhemi, A., DiBello, P. M., Quadros, E. V., Fedosov, S., Jacobsen, D. W. Transcellular transport of cobalamin in aortic endothelial cells.

Cellular uptake of vitamin B12 (cobalamin, Cbl) is mediated by a cell surface receptor (TCblR/CD320) that binds transcobalamin (TC) saturated with Cbl. TC is secreted by the vascular endothelium, has a relatively short half-life, binds Cbl with high affinity and presents the vitamin to the receptor for cellular uptake. Here we show binding and internalization of the TC-Cbl complex along with its’ receptor (TCblR) in several human cell lines. The expression of TCblR is linked to the cell cycle with highest expression in actively proliferating cells. Upon binding TC-Cbl, the receptors appear to segregate on the plasma membrane and are internalized over the course of 30–60 min. Subsequently, the receptors appear to be destroyed along with the TC, which results in the release of free Cbl in the lysosome. The appearance of TCblR on the cell surface is limited to newly synthesized protein without contribution from recycling of the receptor. Therefore, Cbl uptake into cells is fully dependent on the expression of newly synthesized TCblR that is up-regulated in actively proliferating cells. The cell cycle-associated up-regulation of TCblR in cancers provides a route for targeted drug delivery.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder that currently has no approved medical therapy to address core symptoms or underling pathophysiological processes. Several compounds are under development that address both underlying pathophysiological abnormalities and core ASD symptoms. This article reviews one of these treatments, d,l-leucovorin calcium (also known as folinic acid) for treatment of folate pathway abnormalities in children with ASD. Folate is a water-soluble B vitamin that is essential for normal neurodevelopment and abnormalities in the folate and related pathways have been identified in children with ASD. One of these abnormalities involves a partial blockage in the ability of folate to be transported into the brain utilizing the primary transport mechanism, the folate receptor alpha. Autoantibodies which interfere with the function of the folate receptor alpha called folate receptor alpha autoantibodies have been identified in 58%-76% of children with ASD and independent studies have demonstrated that blood titers of these autoantibodies correlate with folate levels in the cerebrospinal fluid. Most significantly, case-series, open-label, and single and double-blind placebo-controlled studies suggest that d,l-leucovorin, a reduced folate that can bypass the blockage at the folate receptor alpha by using the reduced folate carrier, an alternate pathway, can substantially improve particular symptoms in children with ASD, especially those positive for folate receptor alpha autoantibodies. This article reviews the current evidence for treating core and associated symptoms and underlying pathophysiological mechanisms in children with ASD with d,l-leucovorin.

Multiple factors such as genetic and extraneous causes (drugs, toxins, adverse psychological events) contribute to neuro-psychiatric conditions. In a subgroup of these disorders, systemic folate deficiency has been associated with macrocytic anemia and neuropsychiatric phenotypes. In some of these, despite normal systemic levels, folate transport to the brain is impaired in the so-called cerebral folate deficiency (CFD) syndromes presenting as developmental and psychiatric disorders. These include infantile-onset CFD syndrome, infantile autism with or without neurologic deficits, a spastic-ataxic syndrome and intractable epilepsy in young children expanding to refractory schizophrenia in adolescents, and finally treatment-resistant major depression in adults. Folate receptor alpha (FRα) autoimmunity with low CSF N(5)-methyl-tetrahydrofolate (MTHF) underlies most CFD syndromes, whereas FRα gene abnormalities and mitochondrial gene defects are rarely found. The age at which FRα antibodies of the blocking type emerge, determines the clinical phenotype. Infantile CFD syndrome and autism with neurological deficits tend to be characterized by elevated FRα antibody titers and low CSF MTHF. In contrast, in infantile autism and intractable schizophrenia, abnormal behavioral signs and symptoms may wax and wane with fluctuating FRα antibody titers over time accompanied by cycling changes in CSF folate, tetrahydrobiopterin (BH4) and neurotransmitter metabolites ranging between low and normal levels. We propose a hypothetical model explaining the pathogenesis of schizophrenia. Based on findings from clinical, genetic, spinal fluid and MRI spectroscopic studies, we discuss the neurochemical changes associated with these disorders, metabolic and regulatory pathways, synthesis and catabolism of neurotransmitters, and the impact of oxidative stress on the pathogenesis of these conditions. A diagnostic algorithm and therapeutic regimens using high dose folinic acid, corticosteroids and milk-free diet is presented which has proven to be beneficial in providing adequate folate to the brain and decreasing the FRα autoantibody titer in those positive for the antibody.

Objective Cellular uptake of folate is mediated by folate receptor (FR)α. Prior studies indicate that a FRα autoantibody (FRAb) is implicated in poor pregnancy outcomes. The aims of this study were to determine the prevalence of FRAbs in women with preterm and term pregnancies, and to investigate the role of maternal FRAbs in preterm birth. Methods This prospective observational study included 23 mothers and 25 preterm infants (two twin births) born at gestational age (GA) ≤32 wk and/or birth weight ≤1500 g (group 1) and 25 mother–term infant pairs (infants born at GA ≥37 wk, group 2). Blocking and binding FRAbs in maternal and in cord blood were determined. The association between maternal FRAbs and pregnancy outcome was measured using multiple logistic regression, adjusted for maternal age and previous preterm birth. Results The prevalence of FRAbs was 65.2% in women with preterm birth, which was twofold higher than in those with term pregnancy (28%; relative risk [RR], 2.3; 95% confidence interval [CI], 1.2–4.7). The prevalence of FRAbs in preterm infants (64%) was significantly higher than in term infants (24%; RR, 2.7; 95% CI, 1.3–5.7). Pregnant women with positive FRAbs had 4.9 times higher odds of having preterm birth (odds ratio, 4.9; 95% CI, 1.4–17.7), adjusted for maternal age and previous preterm birth. Conclusions These findings suggest that the presence of FRAbs might be a contributing factor to preterm birth, which could be prevented with appropriate testing and therapeutic interventions. Further studies are warranted to investigate the possible mechanisms of fetal sensitization resulting in FRAb production in utero and its possible clinical correlates.

International audience; The pathomechanisms that associate a deficit in folate and/or vitamin B12 and the subsequent hyperhomocysteinemia with pathological brain ageing are unclear. We investigated the homocysteinylation of microtubule-associated proteins (MAPs) in brains of patients with Alzheimer's disease or vascular dementia, and in rats depleted in folate and vitamin B12, Cd320 KO mice with selective B12 brain deficiency and H19-7 neuroprogenitors lacking folate. Compared with controls, N-homocysteinylated tau and MAP1 were increased and accumulated in protein aggregates and tangles in the cortex, hippocampus and cerebellum of patients and animals. N-homocysteinylation dissociated tau and MAPs from β-tubulin, and MS analysis showed that it targets lysine residues critical for their binding to β-tubulin. N-homocysteinylation increased in rats exposed to vitamin B12 and folate deficit during gestation and lactation and remained significantly higher when they became 450 days-old, despite returning to normal diet at weaning, compared with controls. It was correlated with plasma homocysteine (Hcy) and brain expression of methionine tRNAsynthetase (MARS), the enzyme required for the synthesis of Hcy-thiolactone, the substrate of N-homocysteinylation. Experimental inactivation of MARS prevented the N-homocysteinylation of tau and MAP1, and the dissociation of tau and MAP1 from β-tubulin and PSD95 in cultured neuroprogenitors. In conclusion, increased N-homocysteinylation of tau and MAP1 is a mechanism of brain ageing that depends on Hcy concentration and expression of MARS enzyme. Its irreversibility and cumulative occurrence throughout life may explain why B12 and folate supplementation of the elderly has limited effects, if any, to prevent pathological brain ageing and cognitive decline. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

International audience; INTRODUCTION: Cerebral folate deficiency (CFD) syndromes are defined as neuro-psychiatric conditions with low CSF folate and attributed to different causes such as autoantibodies against the folate receptor-alpha (FR) protein that can block folate transport across the choroid plexus, FOLR1 gene mutations or mitochondrial disorders. High-dose folinic acid treatment restores many neurologic deficits. STUDY AIMS AND METHODS: Among 36 patients from 33 families the infantile-onset CFD syndrome was diagnosed based on typical clinical features and low CSF folate. All parents were healthy. Three families had 2 affected siblings, while parents from 4 families were first cousins. We analysed serum FR autoantibodies and the FOLR1 and FOLR2 genes. Among three consanguineous families homozygosity mapping attempted to identify a monogenetic cause. Whole exome sequencing (WES) was performed in the fourth consanguineous family, where two siblings also suffered from polyneuropathy as an atypical finding. RESULTS: Boys (72%) outnumbered girls (28%). Most patients (89%) had serum FR autoantibodies fluctuating over 5-6 weeks. Two children had a genetic FOLR1 variant without pathological significance. Homozygosity mapping failed to detect a single autosomal recessive gene. WES revealed an autosomal recessive polynucleotide kinase 3\textasciiacutephosphatase (PNKP) gene abnormality in the siblings with polyneuropathy. DISCUSSION: Infantile-onset CFD was characterized by serum FR autoantibodies as its predominant pathology whereas pathogenic FOLR1 gene mutations were absent. Homozygosity mapping excluded autosomal recessive inheritance of any single responsible gene. WES in one consanguineous family identified a PNKP gene abnormality that explained the polyneuropathy and also its contribution to the infantile CFD syndrome because the PNKP gene plays a dual role in both neurodevelopment and immune-regulatory function. Further research for candidate genes predisposing to FRα-autoimmunity is suggested to include X-chromosomal and non-coding DNA regions.

BACKGROUND: Auto-antibodies against folate receptor alpha (FRα) at the choroid plexus that block N(5)-methyltetrahydrofolate (MTHF) transfer to the brain were identified in catatonic schizophrenia. Acoustic hallucinations disappeared following folinic acid treatment. Folate transport to the CNS prevents homocysteine accumulation and delivers one-carbon units for methyl-transfer reactions and synthesis of purines. The guanosine derivative tetrahydrobiopterin acts as common co-factor for the enzymes producing dopamine, serotonin and nitric oxide. METHODS: Our study selected patients with schizophrenia unresponsive to conventional treatment. Serum from these patients with normal plasma homocysteine, folate and vitamin B12 was tested for FR autoantibodies of the blocking type on serial samples each week. Spinal fluid was analyzed for MTHF and the metabolites of pterins, dopamine and serotonin. The clinical response to folinic acid treatment was evaluated. RESULTS: Fifteen of 18 patients (83.3%) had positive serum FR auto-antibodies compared to only 1 in 30 controls (3.3%) (χ(2)=21.6; p

Folate deficiency can affect fetal and neonatal brain development Considering the reported association of Folate receptor alpha (FRα) autoantibodies (Abs) with autism and developmental disorders, we sought to confirm this in families of 82 children with ASD, 53 unaffected siblings, 65 fathers, and 70 mothers, along with 52 unrelated normal controls. Overall, 76% of the affected children, 75% of the unaffected siblings, 69% of fathers and 59% of mothers were positive for either blocking or binding Ab, whereas the prevalence of this Ab in the normal controls was 29%. The Ab was highly prevalent in affected families including unaffected siblings. The appearance of these antibodies may have a familial origin but the risk of developing ASD is likely influenced by other mitigating factors since some siblings who had the antibodies were not affected. The antibody response appears heritable with the blocking autoantibody in the parents and affected child increasing the risk of ASD. Autism Res 2018, 11: 707-712. © 2018 International Society for Autism Research, Wiley Periodicals, Inc.Folate is an essential nutrient during fetal and infant development. Autoantibodies against the folate receptor alpha can block folate transport from the mother to the fetus and to the brain in infants. Children diagnosed with autism and their immediate family members were evaluated for the prevalence of folate receptor autoantibodies. The autoantibody was highly prevalent in affected families with similar distribution in parents, normal siblings and affected children. The presence of these antibodies appears to have a familial origin and may contribute to developmental deficits when combined with other factors.

Vitamin B12 deficiency causes megaloblastic anemia and neurologic disorder in humans. Gene defects of transcobalamin (TC) and the transcobalamin receptor (TCblR), needed for cellular uptake of the TC-bound B12, do not confer embryonic lethality. TC deficiency can produce the hematologic and neurologic complications after birth, whereas TCblR/CD320 gene defects appear to produce mild metabolic changes. Alternate maternofetal transport mechanisms appear to provide adequate B12 to the fetus. To understand this mechanism, we evaluated the role of TC, TCblR/CD320, and megalin in maternofetal transport of B12 in a TCblR/CD320-knockout (KO) mouse. Our results showed high expression of TCblR/CD320 in the labyrinth of the placenta, embryonic brain, and spinal column in wild-type (WT) mice. Megalin expression was about the same in both WT and KO mouse visceral yolk sac, brain, and spinal column. Megalin mRNA was down-regulated in the KO embryonic spinal cord (SC) and kidneys. Megalin expression remained unaltered in adult WT and KO mouse brain, SC, and kidneys. Injected dsRed-TC-B12 and TC-57CoB12 accumulated in the visceral yolk sac of KO mice where megalin is expressed and provides an alternate mechanism for the maternofetal transport of Cbl during fetal development.—Arora, K., Sequeira, J. M., Quadros, E. V. Maternofetal transport of vitamin B12: role of TCblR/CD320 and megalin.

Cellular uptake of cobalamin is facilitated by a receptor mediated endocytosis process involving transcobalamin, a plasma protein that binds cobalamin and a cell surface receptor that specifically binds transcobalamin saturated with cobalamin. Intracellular Cbl concentration is maintained by modulating the expression of the receptor, which is cell cycle associated with highest expression in actively proliferating cells and an efflux system that shunts the excess cobalamin out of the cells for mobilization to other tissues where it is most needed. This review describes the process, proteins involved and genes encoding these proteins.

The membrane receptor TCblR/CD320 binds transcobalamin (TC) saturated with vitamin B12 [cobalamin (Cbl)] and mediates cellular uptake of the vitamin. The specificity of TC for Cbl and of the receptor for TC-Cbl ensures efficient uptake of Cbl into cells. The high-affinity interaction of TCblR with TC-Cbl (Ka=10 nM−1) was investigated using deletions and mutations of amino acid sequences in TCblR. Only the extracellular region (aa 32–229) is needed for TC-Cbl binding, but the N-glycosylation sites (N126, N195, and N213) are of no importance for this function. Deleting the cysteine-rich region (aa 95–141) that separates the two low-density lipoprotein receptor type A (LDLR-A) domains does not affect TC-Cbl binding (Ka = 19–24 nM−1). The two LDLR-A domains (aa 54–89 and 132–167) with the negatively charged acidic residues involved in Ca2+ binding are critical determinants of ligand binding. The cytoplasmic tail is apparently crucial for internalization of the ligand. Within this region, the RPLGLL motif and the PDZ binding motifs (QERL/KESL) appear to be involved in initiating and completing the process of ligand internalization. Mutations and deletions of these regions involved in binding and internalization of TC-Cbl are likely to produce the biochemical and clinical phenotype of Cbl deficiency.—Jiang, W., Nakayama, Y., Sequeira, J. M., Quadros, E. V. Mapping the functional domains of TCblR/CD320, the receptor for cellular uptake of transcobalamin-bound cobalamin.

Cobalamin uptake into cells is mediated by the CD320 receptor for transcobalamin-bound cobalamin. Optimum receptor expression is associated with proliferating cells and therefore, in many cancers this receptor expression is up regulated. Delivering drugs or toxins via this receptor provides increased targeting to cancer cells while minimizing toxicity to the normal tissues. Saporin conjugated monoclonal antibodies to the extracellular domain of TCblR were effectively internalized to deliver a toxic dose of Saporin to some cancer cell lines propagating in culture. Antibody concentration of 2.5 nM was effective in producing optimum inhibition of cell proliferation. The cytotoxic effect of mAb-Saporin appears to be dictated primarily by the level of receptor expression and therefore normal primary cells expressing low levels of CD320 were spared while tumor cell lines with higher CD320 expression were destroyed. Targeting the pathway for cellular uptake of vitamin B12 via the CD320 receptor with toxin-antibody conjugates appears to be a viable treatment strategy for certain cancers that over expresses this receptor.

We sought to determine whether high-dose folinic acid improves verbal communication in children with non-syndromic autism spectrum disorder (ASD) and language impairment in a double-blind placebo control setting. Forty-eight children (mean age 7 years 4 months; 82% male) with ASD and language impairment were randomized to receive 12 weeks of high-dose folinic acid (2 mg kg−1 per day, maximum 50 mg per day; n=23) or placebo (n=25). Children were subtyped by glutathione and folate receptor-α autoantibody (FRAA) status. Improvement in verbal communication, as measured by a ability-appropriate standardized instrument, was significantly greater in participants receiving folinic acid as compared with those receiving placebo, resulting in an effect of 5.7 (1.0,10.4) standardized points with a medium-to-large effect size (Cohen’s d=0.70). FRAA status was predictive of response to treatment. For FRAA-positive participants, improvement in verbal communication was significantly greater in those receiving folinic acid as compared with those receiving placebo, resulting in an effect of 7.3 (1.4,13.2) standardized points with a large effect size (Cohen’s d=0.91), indicating that folinic acid treatment may be more efficacious in children with ASD who are FRAA positive. Improvements in subscales of the Vineland Adaptive Behavior Scale, the Aberrant Behavior Checklist, the Autism Symptom Questionnaire and the Behavioral Assessment System for Children were significantly greater in the folinic acid group as compared with the placebo group. There was no significant difference in adverse effects between treatment groups. Thus, in this small trial of children with non-syndromic ASD and language impairment, treatment with high-dose folinic acid for 12 weeks resulted in improvement in verbal communication as compared with placebo, particularly in those participants who were positive for FRAAs.

Folate receptor alpha (FRα) autoantibodies have been associated with fetal abnormalities and cerebral folate deficiency-related developmental disorders. Over 70% of the children with autism spectrum disorders (ASD) are positive for these autoantibodies and high-dose folinic acid is beneficial in treating these children. Here we show that antibodies (Abs) to the rat FRα administered during gestation produce communication, learning and cognitive deficits in a rat model that can be prevented by folinic acid and dexamethasone. FRα Ab can trigger inflammation as well as block folate transport to the fetus and to the developing brain to produce the functional deficits. In humans, exposure to FRα autoantibodies during fetal development and infancy could contribute to brain dysfunction such as that seen in ASD and other developmental disorders. Identifying women positive for the autoantibody and treating them with high-dose folinic acid along with other interventions to lower the autoantibody titer are effective strategies that may be considered to reduce the risk of having a child with developmental deficits.

Folate receptor α (FRα) autoantibodies (FRAAs) are prevalent in autism spectrum disorder (ASD). They disrupt the transportation of folate across the blood-brain barrier by binding to the FRα. Children with ASD with FRAAs have been reported to respond well to treatment with a form of folate known as folinic acid, suggesting that they may be an important ASD subgroup to identify and treat. There has been no investigation of whether they manifest unique behavioral and physiological characteristics. Thus, in this study we measured both blocking and binding FRAAs, physiological measurements including indices of redox and methylation metabolism and inflammation as well as serum folate and B12 concentrations and measurements of development and behavior in 94 children with ASD. Children positive for the binding FRAA were found to have higher serum B12 levels as compared to those negative for binding FRAAs while children positive for the blocking FRAA were found to have relatively better redox metabolism and inflammation markers as compared to those negative for blocking FRAAs. In addition, ASD children positive for the blocking FRAA demonstrated better communication on the Vineland Adaptive Behavior Scale, stereotyped behavior on the Aberrant Behavioral Checklist and Mannerisms on the Social Responsiveness Scale. This study suggests that FRAAs are associated with specific physiological and behavioral characteristics in children with ASD and provides support for the notion that these biomarkers may be useful for subgrouping children with ASD, especially with respect to targeted treatments.

Cerebral folate deficiency (CFD) syndrome is a neurodevelopmental disorder typically caused by folate receptor autoantibodies (FRAs) that interfere with folate transport across the blood-brain barrier. Autism spectrum disorders (ASDs) and improvements in ASD symptoms with leucovorin (folinic acid) treatment have been reported in some children with CFD. In children with ASD, the prevalence of FRAs and the response to leucovorin in FRA-positive children has not been systematically investigated. In this study, serum FRA concentrations were measured in 93 children with ASD and a high prevalence (75.3%) of FRAs was found. In 16 children, the concentration of blocking FRA significantly correlated with cerebrospinal fluid 5-methyltetrahydrofolate concentrations, which were below the normative mean in every case. Children with FRAs were treated with oral leucovorin calcium (2 mg kg(-1) per day; maximum 50 mg per day). Treatment response was measured and compared with a wait-list control group. Compared with controls, significantly higher improvement ratings were observed in treated children over a mean period of 4 months in verbal communication, receptive and expressive language, attention and stereotypical behavior. Approximately one-third of treated children demonstrated moderate to much improvement. The incidence of adverse effects was low. This study suggests that FRAs may be important in ASD and that FRA-positive children with ASD may benefit from leucovorin calcium treatment. Given these results, empirical treatment with leucovorin calcium may be a reasonable and non-invasive approach in FRA-positive children with ASD. Additional studies of folate receptor autoimmunity and leucovorin calcium treatment in children with ASD are warranted.

Folate receptor α (FRα) autoantibodies (FRAAs) are prevalent in autism spectrum disorder (ASD). FRAAs disrupt folate transport across the blood-brain barrier by binding to the FRα. Thyroid dysfunction is frequently found in children with ASD. We measured blocking and binding FRAAs and thyroid-stimulating hormone (TSH), free thyroxine (T4) (FT4), total triiodothyronine (T3) (TT3), reverse T3 (rT3), thyroid-releasing hormone (TRH) and other metabolites in 87 children with ASD, 84 of whom also underwent behaviour and cognition testing and in 42 of whom FRAAs, TSH and FT4 were measured at two time points. To better understand the significance of the FRα in relation to thyroid development, we examined FRα expression on prenatal and postnatal thyroid. TSH, TT3 and rT3 were above the normal range in 7%, 33% and 51% of the participants and TRH was below the normal range in 13% of the participants. FT4 was rarely outside the normal range. TSH concentration was positively and the FT4/TSH, TT3/TSH and rT3/TSH ratios were inversely related to blocking FRAA titres. On repeated measurements, changes in TSH and FT4/TSH ratio were found to correspond to changes in blocking FRAA titres. TSH and the FT4/TSH, TT3/TSH and rT3/TSH ratios were related to irritability on the Aberrant Behavior Checklist and several scales of the Social Responsiveness Scale (SRS), whereas TT3 was associated with SRS subscales and TRH was related to Vineland Adaptive Behavior Scale subscales. The thyroid showed significant FRα expression during the early prenatal period, although expression decreased significantly in later gestation and postnatal thyroid tissue. The results of the present study suggest that thyroid dysfunction in ASD may be related to blocking FRAA. The high expression of FRα in the early foetal thyroid suggests that foetal and neonatal exposure to maternal FRAAs could affect the development of the thyroid and may contribute to the pathology in ASD.

Folates are essential in the intermediary metabolism of amino acids, synthesis of nucleotides and for maintaining methylation reactions. They are also linked to the production of neurotransmitters through GTP needed for the synthesis of tetrahydrobiopterin. During pregnancy, folate is needed for fetal development. Folate deficiency during this period has been linked to increased risk of neural tube defects. Disturbances of folate metabolism due to genetic abnormalities or the presence of autoantibodies to folate receptor alpha (FRα) can impair physiologic processes dependent on folate, resulting in a variety of developmental disorders including cerebral folate deficiency syndrome and autism spectrum disorders. Overall, adequate folate status has proven to be important during pregnancy as well as neurological development and functioning in neonates and children. Treatment with pharmacologic doses of folinic acid has led to reversal of some symptoms in many children diagnosed with cerebral folate deficiency syndrome and autism, especially in those positive for autoantibodies to FRα. Thus, as the brain continues to develop throughout fetal and infant life, it can be affected and become dysfunctional due to a defective folate transport contributing to folate deficiency. Treatment and prevention of these disorders can be achieved by identification of those at risk and supplementation with folinic acid.

The clinical phenotype of cobalamin (Cbl) deficiency is dictated by the essential role of this vitamin in two key enzymatic reactions. Multiple proteins and receptors participate in the absorption, transport and delivery of this vitamin to tissue cells. Cellular uptake of Cbl is mediated by transcobalamin (TC), a plasma protein and a transmembrane receptor (TCblR) with high affinity for TC saturated with Cbl. Knockdown of TCblR with siRNA results in decreased TC-Cbl uptake. The ensuing Cbl deficiency leads to an increase in doubling time and decreased proliferation of these cells. The study confirms the seminal role of this receptor in the cellular uptake of Cbl and its down-regulation as a potential strategy to inhibit proliferation of cancer cells.

Cellular uptake of cobalamin (Cbl) is mediated by the transcobalamin receptor (TCblR) that binds and internalizes transcobalamin (TC) saturated with Cbl. These receptors are expressed in actively proliferating cells and are down-regulated in quiescent cells. The 5’ region of TCblR gene was analyzed for promoter activity to determine transcriptional regulation of TCblR expression. The region −668 to −455 appears to regulate TCblR expression. We have identified transcription factors MZF-1 (myeloid zinc finger 1)/RREB-1 (Ras-responsive element binding protein 1), C/EBP (CCAAT/enhancer binding protein)/HNF-3β (hepatocyte nuclear factor 3) and AP-1(activator protein 1) as proteins likely to be involved in this regulation with the former region primarily involved in up regulation and the latter two regions involved in suppression of TCblR expression. These transcription factors are involved in cell proliferation and differentiation. Thus the cell cycle associated expression of TCblR appears to be tightly regulated in synchrony with the proliferative phase of the cell cycle.

The transcobalamin (TC, TCII) receptor (TCblR) on the plasma membrane binds TC- cobalamin (Cbl) and internalizes the complex by endocytosis. This receptor was purified from human placental membranes by affinity chromatography. Tryptic digest of the protein extracted from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subjected to liquid chromatography/mass spectrometry identified 4 peptides that matched with a membrane protein in the data bank. TCblR belongs to the low-density lipoprotein receptor family, with 2 low-density lipoprotein receptor type A domains separated by a complement-like cysteine-rich region. The 282-amino acid sequence includes a signal peptide of 31 residues, extracellular domain of 198 residues, a transmembrane region of 21 residues, and a cytoplasmic domain of 32 residues. The binding of TC-Cbl does not require the cytoplasmic domain or its orientation in the plasma membrane because the recombinant extracellular domain binds TC-Cbl with high affinity and specificity. The protein is heavily glycosylated and accounts for the 58-kDa size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The human gene first identified as 8D6A and more recently as CD 320 encoding TCblR is located at p13.2 on the short arm of chromosome 19, spans a length of 6.224 kb, and is composed of 5 exons and 4 introns.