Binding Folate Receptor Alpha Autoantibody Is a Biomarker for Leucovorin Treatment Response in Autism Spectrum Disorder.

Autism spectrum disorder (ASD) affects up to 1 in 36 children in the United States. It is a heterogeneous neurodevelopmental disorder with life-long consequences. Patients with ASD and folate pathway abnormalities have demonstrated improved symptoms after treatment with leucovorin (folinic acid), a reduced form of folate. However, biomarkers for treatment response have not been well investigated and clinical trials are lacking. In this retrospective analysis, a cohort of prospectively collected data from 110 consecutive ASD clinic patients [mean (SD) age: 10.5 (6.2) years; 74% male] was examined. These patients all underwent testing for folate receptor alpha autoantibodies (FRAAs) and soluble folate binding proteins (sFBPs) biomarkers and were treated with leucovorin, if appropriate. Analyses examined whether these biomarkers could predict response to leucovorin treatment as well as the severity of ASD characteristics at baseline. The social responsiveness scale (SRS), a measure of core ASD symptoms, and the aberrant behavior checklist (ABC), a measure of disruptive behavior, were collected at each clinic visit. Those positive for sFBPs had more severe ASD symptoms, and higher binding FRAA titers were associated with greater ABC irritability. Treatment with leucovorin improved most SRS subscales with higher binding FRAA titers associated with greater response. Leucovorin treatment also improved ABC irritability. These results confirm and expand on previous studies, underscore the need for biomarkers to guide treatment of folate pathways in ASD, and suggest that leucovorin may be effective for children with ASD.

FTY720 requires vitamin B12-TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis.

Vitamin B12 (B12) deficiency causes neurological manifestations resembling multiple sclerosis (MS); however, a molecular explanation for the similarity is unknown. FTY720 (fingolimod) is a sphingosine 1-phosphate (S1P) receptor modulator and sphingosine analog approved for MS therapy that can functionally antagonize S1P1. Here, we report that FTY720 suppresses neuroinflammation by functionally and physically regulating the B12 pathways. Genetic and pharmacological S1P1 inhibition upregulates a transcobalamin 2 (TCN2)-B12 receptor, CD320, in immediate-early astrocytes (ieAstrocytes; a c-Fos-activated astrocyte subset that tracks with experimental autoimmune encephalomyelitis [EAE] severity). CD320 is also reduced in MS plaques. Deficiency of CD320 or dietary B12 restriction worsens EAE and eliminates FTY720's efficacy while concomitantly downregulating type I interferon signaling. TCN2 functions as a chaperone for FTY720 and sphingosine, whose complex induces astrocytic CD320 internalization, suggesting a delivery mechanism of FTY720/sphingosine via the TCN2-CD320 pathway. Taken together, the B12-TCN2-CD320 pathway is essential for the mechanism of action of FTY720.

Folate and Other B Vitamins in Brain Health and Disease.

B vitamins as a group play essential roles in a multitude of metabolic reactions involved in cellular replication, energy production, the synthesis of intermediary compounds, and neurotransmitters [...].

Transcobalamin receptor gene polymorphisms and mutation in an elderly population.

BACKGROUND & AIMS: Cellular uptake of the essential nutrient vitamin B12 (cobalamin) occurs via the transcobalamin receptor (TCblR/CD320), a ubiquitous membrane receptor. Polymorphisms in the receptor exist, though the effect of such variants across patient populations is unknown. METHODS: We determined CD320 genotype in 377 randomly selected elderly individuals. RESULTS: Three polymorphisms and a codon deletion were identified in the exon 2 region. Haplotype variants had significantly higher holotranscobalamin (holo-TC) values and a higher holo-TC/total cobalamin ratio. TCblR haplotype explained 46% of the variability in holo-TC values. CONCLUSIONS: This has significant implications for the clinical utility of the 'combined indicator' of B12 status since it is based on a standard rate of intracellular flux via the TC-Cbl receptor. Modification of the model may be required to account for CD320 haplotype.

Brain Uptake of Folate Forms in the Presence of Folate Receptor Alpha Antibodies in Young Rats: Folate and Antibody Distribution.

In a rat model, following exposure to rat folate receptor alpha antibodies (FRαAb) during gestation, FRαAb accumulates in the placenta and the fetus and blocks folate transport to the fetal brain and produces behavioral deficits in the offspring. These deficits could be prevented with folinic acid. Therefore, we sought to evaluate folate transport to the brain in young rat pups and determine what effect FRαAb has on this process, to better understand the folate receptor autoimmune disorder associated with cerebral folate deficiency (CFD) in autism spectrum disorders (ASD). When injected intraperitoneally (IP), FRαAb localizes to the choroid plexus and blood vessels including the capillaries throughout the brain parenchyma. Biotin-tagged folic acid shows distribution in the white matter tracts in the cerebrum and cerebellum. Since these antibodies can block folate transport to the brain, we orally administered various folate forms to identify the form that is better-absorbed and transported to the brain and is most effective in restoring cerebral folate status in the presence of FRαAb. The three forms of folate, namely folic acid, D,L-folinic acid and levofolinate, are converted to methylfolate while L-methylfolate is absorbed as such and all are efficiently distributed to the brain. However, significantly higher folate concentration is seen in the cerebrum and cerebellum with levofolinate in the presence or absence of FRαAb. Our results in the rat model support testing levofolinate to treat CFD in children with ASD.

The Soluble Folate Receptor in Autism Spectrum Disorder: Relation to Autism Severity and Leucovorin Treatment.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder with life-long consequences that affects up to 1 in 44 children. Treatment with leucovorin (folinic acid), a reduced form of folate, has been shown to improve symptoms in those with ASD and folate pathway abnormalities in controlled clinical trials. Although soluble folate binding proteins (sFBPs) have been observed in the serum of some patients with ASD, the significance of this finding has not been studied. Here, we present a cohort of ASD patients with sFBPs. These patients had severe ASD and were medically complex. Using baseline controlled open-label methodology and standardized assessments, these patients were found to improve in both core and associated ASD symptoms with leucovorin treatment. No adverse effects were related to leucovorin treatment. This is the first report of the sFBPs in ASD. This study complements ongoing controlled clinical trials and suggests that leucovorin may be effective for children with ASD who are positive for sFBPs. Further, sFBPs might be important biomarkers for treatment response to leucovorin in children with ASD. This study paves the way for further controlled studies for patients with sFBPs.

Cerebral Folate Deficiency Syndrome: Early Diagnosis, Intervention and Treatment Strategies.

Cerebral folate deficiency syndrome (CFDS) is defined as any neuropsychiatric or developmental disorder characterized by decreased CSF folate levels in the presence of normal folate status outside the nervous system. The specific clinical profile appears to be largely determined by the presence or absence of intrauterine folate deficiency as well as postnatal age at which cerebral folate deficiency occurs. The primary cause of CFDS is identified as the presence of serum folate receptor-alpha (FRα) autoantibodies impairing folate transport across the choroid plexus to the brain whereas, in a minority of cases, mitochondrial disorders, inborn errors of metabolism and loss of function mutations of the FRα (FOLR1) gene are identified. Early recognition and diagnosis of CFDS and prompt intervention is important to improve prognosis with successful outcomes. In this article we focus on FRα autoimmunity and its different age-dependent clinical syndromes, the diagnostic criteria, and treatments to be considered, including prevention strategies in this at-risk population.

Absorption and Tissue Distribution of Folate Forms in Rats: Indications for Specific Folate Form Supplementation during Pregnancy.

Food fortification and folic acid supplementation during pregnancy have been implemented as strategies to prevent fetal malformations during pregnancy. However, with the emergence of conditions where folate metabolism and transport are disrupted, such as folate receptor alpha autoantibody (FRαAb)-induced folate deficiency, it is critical to find a folate form that is effective and safe for pharmacologic dosing for prolonged periods. Therefore, in this study, we explored the absorption and tissue distribution of folic acid (PGA), 5-methyl-tetrahydrofolate (MTHF), l-folinic acid (levofolinate), and d,l-folinic acid (Leucovorin) in adult rats. During absorption, all forms are converted to MTHF while some unconverted folate form is transported into the blood, especially PGA. The study confirms the rapid distribution of absorbed folate to the placenta and fetus. FRαAb administered, also accumulates rapidly in the placenta and blocks folate transport to the fetus and high folate concentrations are needed to circumvent or overcome the blocking of FRα. In the presence of FRαAb, both Leucovorin and levofolinate are absorbed and distributed to tissues better than the other forms. However, only 50% of the leucovorin is metabolically active whereas levofolinate is fully active and generates higher tetrahydrofolate (THF). Because levofolinate can readily incorporate into the folate cycle without needing methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) in the first pass and is relatively stable, it should be the folate form of choice during pregnancy, other disorders where large daily doses of folate are needed, and food fortification.

Behavioral profile of vitamin B12 deficiency: A reflection of impaired brain development, neuronal stress and altered neuroplasticity.

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 B12, in neurodegenerative disease is demonstrated but largely unresolved. Behaviors are often quantified to attest brain dysfunction alone or in parallel with neuro-imaging to identify regions involved. Nevertheless, attention should be paid to extending observations made in animal models to humans, since, first, behavioral tests have to be adjusted in each model to address the initial question and second, because brain analysis should not be conducted for a whole organ but rather to specific sub-structures to better define function. Indeed, cognitive functions such as psychiatric disorders and learning and memory are often cited as the most impacted by a vitamin B12 deficiency. In addition, differential dysfunctions and mechanisms could be defined according sub-populations and ages. Vitamin B12 enters the cell bound to Transcobalamin, through the Transcobalamin Receptor and serves in two cell compartments, the lipid metabolism in the mitochondrion and the one-carbon metabolism involved in methylation reactions. Dysfunctions in these mechanisms can lead to two majors outcomes; axons demyelinisation and upregulation of cellular stress involving mislocalization of RNA binding proteins such as the ELAVL1/HuR or the dysregulation of pro- or anti-oxidant NUDT15, TXNRD1, VPO1 and ROC genes. Finally, it appears that apart from developmental problems that have to be identified and treated as early as possible, other therapeutic approaches for behavioral dysfunctions should investigate cellular methylation, oxidative and endoplasmic reticulum stress and mitochondrial function.

Generation of nanobodies targeting the human, transcobalamin-mediated vitamin B12 uptake route.

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, encoded by the CD320 gene. Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo-TC (TC in complex with B12 ) or the interface of the human holo-TC:TCblR complex with nanomolar affinities. We determined X-ray crystal structures of these nanobodies bound to holo-TC:TCblR, which enabled us to map their binding epitopes. When conjugated to the model toxin saporin, three of our nanobodies caused growth inhibition 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 (TC-Nb4) using fluorescent light microscopy. The co-crystal structure of holo-TC:TCblR with another nanobody (TC-Nb34) revealed novel features of the interface of TC and the LDLR-A1 domain of TCblR, rationalizing the decrease in the affinity of TC-B12 binding caused by the Δ88 mutation in CD320.

Folate Receptor Alpha Autoantibodies in Autism Spectrum Disorders: Diagnosis, Treatment and Prevention.

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.

Glucocorticoid Receptor Activation Restores Learning Memory by Modulating Hippocampal Plasticity in a Mouse Model of Brain Vitamin B12 Deficiency.

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.

Oxidative Stress, Folate Receptor Autoimmunity, and CSF Findings in Severe Infantile Autism.

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.

Treatment of Folate Metabolism Abnormalities in Autism Spectrum Disorder.

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.

Cellular uptake of vitamin B12: Role and fate of TCblR/CD320, the transcobalamin receptor.

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.

Folinic acid improves the score of Autism in the EFFET placebo-controlled randomized trial.

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.

Improving Outcome in Infantile Autism with Folate Receptor Autoimmunity and Nutritional Derangements: A Self-Controlled Trial.

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 p<0.0001), achieving complete recovery in 17/82 children (20.7 %). Prognosis became less favorable with the finding of higher FRα 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.

N-homocysteinylation of tau and MAP1 is increased in autopsy specimens of Alzheimer's disease and vascular dementia.

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 ß-tubulin, and MS analysis showed that it targets lysine residues critical for their binding to ß-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 ß-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.

Neuropathology of vitamin B12 deficiency in the Cd320-/- mouse.

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.

Inherited disorders of cobalamin metabolism disrupt nucleocytoplasmic transport of mRNA through impaired methylation/phosphorylation of ELAVL1/HuR.

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.