Many studies indicate a crucial role for the vitamin B12 and folate-dependent enzyme methionine synthase (MS) in brain development and function, but vitamin B12 status in the brain across the lifespan has not been previously investigated. Vitamin B12 (cobalamin, Cbl) exists in multiple forms, including methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), serving as cofactors for MS and methylmalonylCoA mutase, respectively. We measured levels of five Cbl species in postmortem human frontal cortex of 43 control subjects, from 19 weeks of fetal development through 80 years of age, and 12 autistic and 9 schizophrenic subjects. Total Cbl was significantly lower in older control subjects (> 60 yrs of age), primarily reflecting a >10-fold age-dependent decline in the level of MeCbl. Levels of inactive cyanocobalamin (CNCbl) were remarkably higher in fetal brain samples. In both autistic and schizophrenic subjects MeCbl and AdoCbl levels were more than 3-fold lower than age-matched controls. In autistic subjects lower MeCbl was associated with decreased MS activity and elevated levels of its substrate homocysteine (HCY). Low levels of the antioxidant glutathione (GSH) have been linked to both autism and schizophrenia, and both total Cbl and MeCbl levels were decreased in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice, which exhibit low GSH levels. Thus our findings reveal a previously unrecognized decrease in brain vitamin B12 status across the lifespan that may reflect an adaptation to increasing antioxidant demand, while accelerated deficits due to GSH deficiency may contribute to neurodevelopmental and neuropsychiatric disorders.
Citation: Zhang Y, Hodgson NW, Trivedi MS, Abdolmaleky HM, Fournier M, Cuenod M, et al. (2016) Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PLoS ONE 11(1): e0146797. https://doi.org/10.1371/journal.pone.0146797
Editor: Joseph Alan Bauer, Bauer Research Foundation, UNITED STATES
Received: June 4, 2015; Accepted: December 22, 2015; Published: January 22, 2016
Copyright: © 2016 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Data are available within the paper, its Supporting Information files and Dryad (doi:10.5061/dryad.28jn6).
Funding: This work was supported by the Autism Research Institute (http://www.autism.com) to RD and the National Center of Competence in Research (http://www.snf.ch) to KD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Metabolically active forms of vitamin B12, methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), serve as essential cofactors for two reactions: MeCbl for folate-dependent methylation of HCY to methionine by methionine synthase (MS) in the cytoplasm, and AdoCbl for conversion of methylmalonylCoA to succinylCoA by methylmalonyl CoA mutase in mitochondria (Fig 1) [1,2]. Since MS activity determines the ratio of the methyl donor S-adenosylmethionine (SAM) to the endogenous methylation inhibitor S-adenosylhomocysteine (SAH), MeCbl is poised to influence hundreds of SAM-dependent methylation reactions, affecting nearly every aspect of metabolism. Important among these reactions is methylation of DNA and histones, which combine to exert dynamic epigenetic control over gene expression . MeCbl is also required for dopamine-stimulated phospholipid methylation, a unique activity of D4 dopamine receptors , which depends upon MS activity  and has been proposed to play an important role in neuronal synchronization and attention . Genetic variants of the D4 receptor have been linked to attention-deficit hyperactivity disorder (ADHD) [7,8], schizophrenia risk [9,10], and drug addiction , as well as to human longevity .
Endocytosis brings TC-bound Cbl species to lysosomes where axial ligands are removed by MMACHC and MeCbl or AdoCbl are subsequently formed by SAM and ATP-dependent pathways, respectively. MeCbl is a required cofactor for methionine synthase, whose activity supports a large number of methylation reactions, including DNA methylation, as well as dopamine-stimulated phospholipid methylation, carried out by the D4 dopamine receptor (D4R). AdoCbl supports MMACoA mutase in mitochondria. Cysteine, which is rate-limiting for GSH synthesis, can be provided either by cellular uptake via the cysteine/glutamate transporter EAAT3 (excitatory amino acid transporter 3) or by transsulfuration of HCY via cystathionine. The latter pathway is restricted in human brain, increasing the importance of growth factor-dependent cysteine uptake by EAAT3.
Vitamin B12 is only synthesized by certain bacteria and humans obtain it from animal source foods such as meat, dairy, eggs, and fish.. A series of chaperones, transport proteins and their receptors (e.g. haptocorrin, intrinsic factor, cubilin, amnionless and megalin) protect vitamin B12 and facilitate its GI absorption and renal reabsorption for its retention. In the general circulation vitamin B12 primarily exists bound to transcobalamin (TC) . As illustrated in Fig 1, cell surface receptors (TC receptor and/or megalin) bring the Cbl·TC complex into lysosomes where Cbl is processed by MMACHC (methylmalonic aciduria type C and homocystinuria, also known as CblC). MMACHC carries out dealkylation of alkylCbls and decyanation of cyanocobalamin (CNCbl) in glutathione (GSH)-dependent and NADPH-dependent reactions, respectively . Formation of active cofactors MeCbl and AdoCbl is then carried out by MMACHC in conjunction with MMADHC (methylmalonic aciduria type D and homocystinuria, also known as CblD) in the cytoplasm and mitochondria, respectively.
The brain exists within a distinct compartment and levels of metabolic resources, including vitamin B12, are reflective of their transport into and out of cerebral spinal fluid (CSF) across the neuroepithelial barrier in the choroid plexus. While factors responsible for vitamin B12 entry into brain have not been fully elucidated, cubilin and megalin, which combine to participate in transport of vitamin B12 in other tissues, are expressed in the choroid plexus [14,15], and a role for amnionless has been postulated based upon disturbed vitamin B12 transport into the brain in a patient with a mutation causing Imerslund-Gräsbeck syndrome . While diet or genetic defects in transport/ processing can affect systemic vitamin B12 availability [17,18], there have been relatively few direct studies of vitamin B12 status in human brain [19,20] and none have provided a comprehensive analysis of different Cbl species.
Methylation of DNA and histone proteins complexly regulates gene expression and this form of epigenetic regulation is particularly important during development, including pre- and postnatal brain development . Neural tube defects, as well as Rett and Angelman/Prader-Willi neurodevelopmental syndromes are linked to defects in methylation-dependent epigenetic regulation [22–24]. Turnover of DNA methylation marks is very fast in prefrontal cortex during fetal development but is 2–3 orders lower during childhood and later life . We previously showed that the level of MS mRNA in human prefrontal cortex decreases several hundred-fold across the lifespan, indicating a dynamic role for vitamin B12-dependent MS activity in brain development and function, and MS mRNA levels were prematurely decreased in autistic subjects . Abnormal DNA methylation [27, 28] has been reported in postmortem brain of autistic subjects, in conjunction with low levels of the antioxidant GSH and elevated markers of oxidative stress [29,30]. Increased oxidative stress and impaired methylation have also been implicated in schizophrenia [31,32].
We utilized a novel HPLC/electrochemical detection-based assay to quantify individual Cbl species in postmortem human cerebral cortex of control subjects from fetal to 80 yrs of age, as well as autistic and schizophrenic subjects. Changes in Cbl species were compared with the status of methylation and antioxidant pathway metabolites and the influence of decreased GSH production on brain Cbl levels was evaluated in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice in which GSH synthesis was impaired, leading to a brain GSH level decrease of 60–70% . Our results reveal an unexpected decrease in cortical Cbl and MeCbl levels across the lifespan, as well as premature decreases in both autism and schizophrenia, which were replicated in GCLM-KO mice.
Materials and Methods
Tissue sample acquisition
Institutional approval for the use of postmortem brain samples was provided by the Northeastern University IRB (# 04-11-09). Postmortem samples of frontal cerebral cortex (Brodmann areas 9, 10, 44 or 45) were obtained from the Autism Tissue Program, now part of the Autism Brain Network (http://www.autismbrainnet.com), the Australian Brain Bank Network (http://www.austbrainbank.org.au) and the Harvard Brain Tissue Resource Center (http://www.brainbank.mclean.org). Samples included 43 control subjects of different age, from 19 weeks of gestation through 80 yrs (Table A in S1 File), as well as 12 autistic subjects (4–9 yrs) (Table B in S1 File) and 9 schizophrenic subjects (36–49 yrs) (Table C in S1 File). Placenta samples were commercially obtained from Advanced Tissue Services (Phoenix, AZ). All tissues samples were maintained in liquid nitrogen until their use and experiments were completed within four months of their receipt.
Vitamin B12 analysis
Cbl extraction and HPLC mobile phase selection were based on a previously published method . Extraction was performed under dim-red light due to Cbl light sensitivity. Brain tissues were thawed on ice and a 10% homogenate was prepared. 150 μL of ice-cold absolute ethanol was added to 100 μL of each sample homogenate and incubated for 10 min. Protein precipitates were removed by centrifugation at 10,600 RPM for 3 min at 20°C. The resulting supernatant was evaporated to dryness, re-suspended with 300 μL PBS and passed through a syringe-driven filter (0.22 μm). The Cbl extract was then transferred to a conical micro autosampler vial, blown with nitrogen, capped and kept at 4°C in the autosampler cooling tray, covered by aluminum foil to avoid Cbl degradation. 30 μL of sample was injected into an Agilent Eclipse XDB-C8 (3 x 150mm; 3.5 μm) and Agilent Eclipse XDB-C8 (4.6 x 12.5mm; 5 μm) guard column by the autosampler. Samples were eluted using the following step gradient: 0–2 min 0% B, 2–14 min 17% B, 14–19 min 30% B, 24–31 min 58% B, 31–32 min 100% B, then equilibrate column with 0% B for 2 min at a flow rate of 0.6 mL/min. Mobile phase A contained 0.1% acetic acid/acetate buffer titrated to pH 3.5 with NH4OH. Mobile phase B was acetonitrile containing 0.1% acetic acid. Cbls were measured using electrochemical detection with an ESA CoulArray with BDD analytical cell model 5040 electrochemical detector at an operating potential of 1000 mV. Examples of chromatograms for cobalamin standards and brain samples are provided (Figures A-E in S2 File). Peak area analysis, based on standard curves generated for each compound, was performed using CoulArray software (version 3.06 ESA analysis program package). Sample Cbl levels were normalized against protein content. Based upon spiked tissue samples, the extraction procedure resulted in recovery of 94.7 +/- 1.8% of tissue Cbl, and replication studies yielded a coefficient of variation of 6.3%.
Thiol metabolite analysis
Thiol and thioether metabolites were measured using HPLC with electrochemical detection. Brain samples were thawed on ice, and a 10% homogenate was prepared. 50 mL of a 0.4 N perchloric acid solution was added to 200 μL of the sample, and samples were gently blown with nitrogen gas before being centrifuged at 13,000 RPM for 60 min. 100 μL of sample was added to a microautosampler vial, blown with nitrogen gas, capped and loaded at 4°C in the autosampler cooling tray. 10 μL of sample was injected into the HPLC system and measured by electrochemical detection. HPLC columns and running conditions were as same as previously published .
Methionine synthase assay
A 5% homogenate of postmortem brain samples was prepared in lysis buffer at 4°C. The assay was conducted under anaerobic and dark conditions, as previously described . 385 μl of a 5% brain homogenate was mixed with 1 M K2HPO4, 10 mM HCY, 100 mM DTT, 3.8 mM SAM, adding either 10 μl water or 10 μl of 5 mM OHCbl, in a final volume of 500μl. The assay was initiated by addition of [14C-methyl] methyltetrahydrofolate, incubated for 60 min at 37°C and terminated by heating at 98°C for 2 min. MS activity was determined by measuring incorporation of 14C into methionine, which was separated by passing through a Dowex 1-X8 column.
GCLM knockout mice studies
GCLM-KO mice were generated from C57Bl/6J mice  and kindly provided by TP Dalton (Cincinnati University, Ohio). Experiments were performed in accordance with the guidelines of the Veterinary Office of the Canton de Vaud, Switzerland and approved by the Swiss Federal Food Safety and Veterinary Office (FSVO). Animals were maintained in a temperature-and humidity-controlled environment under a 12-h light–dark cycle with free access to food and water. Animal welfare was checked 3-times per week; mice displaying signs of dysfunction, wounds or important loss of weight were sacrificed. Heterozygous mice were bred and after genotyping, male littermates were decapitated at 40 and 90 days of age and sections of frontal cortex were dissected and frozen at—80°C until analysis of thiol and Cbl levels.
Statistical analyses were carried out using Graph Pad Prism® version 5.01. Results were expressed as mean ± SEM. A two-tailed Student’s t-test and one-way analysis of variance (ANOVA) with Tukey’s post hoc test were used to determine statistical significance, using p < 0.05 as a criterion. Correlations were evaluated by Pearson’s correlation coefficient.
Frontal cortex Cbl across the lifespan
Different species of vitamin B12 are distinguished by the ligand attached to the upper face of the corrin ring cobalt atom, and they include MeCbl, AdoCbl, CNCbl, hydroxocobalamin (OHCbl) and glutathionylcobalamin (GSCbl) (Fig 2A). These five Cbl species were detected in postmortem frontal cortex brain samples from subjects grouped by age and we observed that the combined Cbl total was 2.7-fold lower in 61–80 yr old vs. 0–20 yr old subjects (Fig 2B). Levels of individual Cbl species were similar in 0–20 and 21–40 yr old control subjects, but significant differences were observed in 41–60 and 61–80 yr old subjects (Fig 2C). Among individual Cbl species, the greatest difference was an age-dependent decline in MeCbl, such that its level in 61–80 yr old subjects was 12.4-fold lower than in 0–20 yr-olds and 6.7-fold lower than 41–60 yr-olds. At younger ages MeCbl was the predominant Cbl species, but its level decreased across the lifespan and was eclipsed by OHCbl, GSCbl and CNCbl in older subjects (61–80 yrs). This progressive age-dependent decrease in frontal cortex MeCbl levels was quasi-linear (r2 = 0.61) and MeCbl was negatively correlated with age across the lifespan (p < 0.0001) (Fig 2D). AdoCbl was significantly lower in 61–80 yr old vs. 0–20 yr old subjects (p < 0.001), but its level was not correlated with age across the lifespan. In contrast, the level of OHCbl was increased in 61–80 yr old subjects and was positively correlated with age (p = 0.01). This substantial age-dependent decrease in total vitamin B12, MeCbl and AdoCbl in frontal cortex contrasts with the comparatively stable level of serum vitamin B12 levels, as reported for samples derived from the National Health and Nutrition Examination Survey (NHANES)  (Fig 2D, inset). Thus frontal cortex levels of vitamin B12 appear to be more dynamically regulated across the lifespan than blood levels.
(a) The general structure of Cbl species in which “X” represents various ligands linked to the cobalt atom, giving rise to the five different Cbl species measured in postmortem frontal cortex. (b) Total Cbl levels in frontal cortex of control subjects divided into four age groups: 0–20 yrs (n = 12), 21–40 yrs (n = 5), 41–60 yrs (n = 10) and 61–80 yrs (n = 12). (c) Levels of five individual Cbl species of control subjects in four age groups. (d) Age-dependent decrease of MeCbl in human frontal cortex (n = 43). Inset: Age trends of serum Cbl, frontal cortex total Cbl and MeCbl. Serum Cbl data is from Ref. 30. (e) Total Cbl levels in placenta (n = 6), frontal cortex of fetal (n = 4) and control (0–20 yrs) subjects (n = 12). (f) Levels of five individual Cbl species in placenta (n = 6), frontal cortex of fetal (n = 4) and control (0–20 yrs) subjects (n = 12). * Indicates a significant difference from 0–20 yrs group (* p < 0.05, ** p < 0.01, *** p < 0.001).
To investigate possible differences between prenatal and postnatal vitamin B12 status, we compared Cbl levels in fetal frontal cortex with levels in young subjects (0–20 yrs). Strikingly, the level of CNCbl was almost 15-fold higher in fetal samples vs. young subjects, while the level of MeCbl was 65% higher (Fig 2E). We further examined placental vitamin B12 status as a potential source of higher CNCbl for fetal brain. However, total Cbl levels in placenta were 10-fold lower than fetal brain and CNCbl levels were 20-fold lower, making it an unlikely source (Fig 2F).
Frontal cortex Cbl in autism and schizophrenia
Cbl levels were analyzed in frontal cortex from young autistic subjects (<10 yrs) and compared to levels in young control subjects (<13 yrs). As illustrated in Fig 3A, the average total Cbl level was 3.1-fold lower in autistic subjects vs. controls (3.1 vs. 8.9 pmol/mg protein). MeCbl and AdoCbl were each more than 3-fold lower in autistic subjects vs. control levels, although the decrease in AdoCbl did not reach statistical significance (p = 0.07) (Fig 3B). Thus the level of frontal cortex Cbl in autistic subjects corresponds to the level of control subjects >50 yrs. Notably, the level of GSCbl was 6-fold lower in autistic subjects, while the level of OHCbl was >3-fold higher, consistent with impairment of GSH-dependent synthesis of MeCbl and AdoCbl.
(a) Total Cbl levels in frontal cortex of autistic subjects (n = 12) and aged-matched controls (n = 9). (b) Levels of five individual Cbl species in frontal cortex of autistic subjects (n = 12) and aged-matched controls (n = 9). (c) Total Cbl levels in frontal cortex of schizophrenic subjects (n = 9) and aged-matched controls (n = 9). (d) Levels of five individual Cbl species in frontal cortex of schizophrenic subjects (n = 9) and aged-matched controls (n = 9). * Indicates a significant difference from control group (* p < 0.05, ** p < 0.01, *** p < 0.001).
Cortical Cbl levels were also measured in schizophrenic subjects (ages 36–49 yrs) and compared to levels in control subjects (ages 36–50 yrs). As illustrated in Fig 3C, the average total Cbl level was 3.3-fold lower in schizophrenic subjects (7.3 vs. 2.2 pmol/mg protein). MeCbl and AdoCbl were >5-fold lower in schizophrenic subjects vs. control levels (Fig 3D). GSCbl was 6-fold lower in schizophrenic subjects, while the level of OHCbl was 3.5-fold higher. Thus both autistic and schizophrenic subjects show similar abnormal patterns of frontal cortex Cbl, including lower levels of both MeCbl and GSCbl. Moreover, the lower levels of total Cbl, MeCbl and AdoCbl are similar to the pattern observed in elderly subjects.
Methylation and redox metabolites
Through its role as cofactor for MS, the status of MeCbl can influence the level of methionine methylation cycle metabolites as well as metabolites in the intersecting pathways which provide for GSH synthesis, as illustrated in Fig 1. A comparison of frontal cortex metabolite levels in younger (0–20 yrs) vs. older (61 to 80 yrs) subjects revealed several significant differences (Fig 4A). The level of HCY was 2-fold higher in older subjects, while the level of methionine was lower, indicative of lower MS activity. The level of SAM, whose formation is MS-dependent, was also lower in older subjects, in association with a decrease in the SAM to SAH ratio (Fig 4B), indicating an impaired methylation potential. Cysteine, GSH and GSSG levels, as well as the GSH to GSSG ratio, were unaffected by age. Remarkably, the level of cystathionine was 10-fold lower in older subjects. Cystathionine is an intermediate in the transsulfuration of HCY to cysteine and its level is higher in human brain compared to other species, reflecting restricted transsulfuration activity [39,40]. Thus the age-dependent decrease in frontal cortex MeCbl appears to be associated with a loss of this restriction, allowing increased HCY diversion toward GSH synthesis.
(a) Redox and methylation pathway metabolites in control subjects of 0–20 yrs (n = 12) compared to subjects of 61–80 yrs (n = 10). (b) GSH/GSSG ratio (left) and SAM/SAH ratio (right) in aging. (c) Redox and methylation pathway metabolites in frontal cortex of autistic subjects (n = 9) compared to age-matched controls (n = 9). (d) GSH/GSSG ratio (left) and SAM/SAH ratio (right) in autism. * Indicates a significant difference from 0–20 yrs group (panels a and b) or control group (panels c and d) (* p < 0.05, ** p < 0.01, *** p < 0.001).
A comparison of methionine cycle metabolites in autistic vs. age-matched young control subjects revealed a pattern generally similar to older subjects (Fig 4C). Thus HCY was higher, methionine and SAM levels were lower, and GSH and oxidized glutathione (GSSG) levels, as well as the GSH to GSSG ratio, were not different from age-matched controls, while the level of cystathionine was 3.5-fold lower in autistic subjects (Fig 4D). Similar to the influence of aging, the SAM to SAH ratio was significantly lower in autism, indicative of impaired methylation potential in association with an increase in transsulfuration and GSH synthesis at a younger than normal age.
To assess methylation capacity in the context of a deficit in Cbl, we measured MS activity (i.e. conversion of HCY to methionine) in frontal cortex samples of control and autistic subjects with or without provision of exogenous OHCbl. As illustrated in Fig 5, MS activity was 3-fold lower in autistic vs. control subjects when only endogenous Cbl was available, and was 38% lower under OHCbl-supplemented conditions, confirming the functional importance of decreased frontal cortex Cbl levels in autism.
Methionine synthase activity in frontal cortex of autistic and age-matched control subjects measured either with only endogenous Cbl or with the addition of OHCbl. * Indicates a significant difference from control group (* p < 0.05, ** p < 0.01, *** p < 0.001).
Decreased GSH synthesis lowers brain B12 levels
Since previous studies have reported lower levels of GSH in autism [27,28] and schizophrenia [29,30], we investigated whether a decrease in GSH affects brain Cbl levels. GCL is the rate-limiting step in GSH synthesis and its modulatory subunit increases GCL activity . Accordingly, we examined thiol metabolite and Cbl levels in cortex of GCLM-KO mice at 40 and 90 days of age, as compared to C57Bl/6J wild-type mice. Consistent with prior studies , the level of GSH in frontal cortex of GCLM-KO mice was decreased by 85% and 89% at 40 and 90 days, respectively, in comparison to wild-type mice, along with significant (p < 0.05) decreases in HCY and cysteine (at 90 days), while GSSG was increased at 40 days (Fig 6A). Lower levels of GSH were associated with a significantly lower level of total Cbl (p < 0.001) in GCLM-KO cortex, amounting to 63% and 56% at 40 and 90 days, respectively (Fig 6B inset). The decrease affected all Cbl species, including decreases of 68% and 39% in MeCbl (Fig 6B). An age-dependent decline in the level of MeCbl was evident in cortex of control mice, amounting to a decrease of 38% between 40 and 90 days. Notably, the total Cbl level was approximately 3-fold higher in human (20–40 yrs) vs. murine frontal cortex, while MeCbl levels were more than 4-fold higher (cf. Figs 2C and 6B).
(a) Redox and methylation metabolite levels in frontal cortex of GCLM KO mice at P40 and P90 (n = 7). Results are expressed as a percentage of the WT level of each metabolite. (b) Levels of five individual Cbl species in frontal cortex of GLCM KO and WT mice at P40 and P90. Inset indicates total Cbl levels. * Indicates a significant difference from control group (* p < 0.05, ** p < 0.01, *** p < 0.001).
Here we report novel findings suggesting that levels of vitamin B12, especially its MeCbl form, decrease with age in frontal cortex of control human subjects. Since serum Cbl levels do not show a similar decrease with age, our results further suggest that vitamin B12 status in the brain compartment is distinctly regulated from the rest of the body and dynamic changes in brain MeCbl across the lifespan may play an important functional role in methylation-dependent processes, including epigenetic regulation of gene expression. Additionally, we observed abnormally lower total Cbl and MeCbl levels in subjects with autism and schizophrenia, as compared to age-matched controls. To our knowledge, this is the first report of pathologically reduced levels of active Cbl species in autistic and schizophrenic brain. Although the number of brain samples analyzed was limited, our findings highlight a possible role for vitamin B12-dependent methylation reactions in brain function and in the etiology of neurological disorders.
As a compartment distinct from the rest of the body, the metabolic environment of the brain depends upon the bi-directional transport of nutrients and micronutrients across the choroid plexus neuroepithelial barrier into the CSF. The aging-related decrease in total brain levels of vitamin B12 observed in our studies is likely to reflect changes in the activity of one or more of these transport processes across the lifespan, particularly since serum levels do not show a similar decrease38. Although the precise molecular basis of vitamin B12 transport into CFS is not fully understood, one or more active and selective transport systems are likely to be involved. Transport across the choroid plexus is an important determinant of CSF composition, and megalin, protein product of the LRP2 gene, which has been implicated in vitamin B12 transport by the distal ileum and the renal proximal tubule, is expressed in choroid plexus epithelial cells, along with amnionless and cubilin [14,43]. Binding of Cbl-loaded TC to megalin leading to Cbl uptake has been described . Megalin also facilitates endocytosis of the TCR  and TCR knock out mice show a deficit in brain B12 concentrations accompanied by DNA hypomethylation . A recent study showed that LRP2 is robustly expressed in the distal ileum during fetal and early postnatal development, but is not expressed in adult human ileum , indicating an age-dependent decline in its expression. Thus a decline in megalin and/or TCR activity could underlie the lower brain levels of vitamin B12 we observed in elderly subjects. Interestingly, megalin promotes removal of amyloid precursor protein-derived Aβ peptide by the choroid plexus and an age-related decline in megalin function has been proposed as a contributor to increased brain levels of Aβ in Alzheimer’s disease (AD) . Impaired MS activity and elevated HCY have been linked to AD and supplementation with vitamin B12, folate and vitamin B6 decreases progression of cognitive impairment [49–51]. In combination with our results, these observations suggest a coordinated normal decrease in choroid plexus-mediated transport of vitamin B12 into the brain and Aβ out of the brain with advancing age (>40 yrs), while environmental and genetic factors introduce increased risk of neurodegenerative disorders in vulnerable individuals.
We found CNCbl to be 15-fold higher in fetal samples, as compared to 0–20 yr old subjects, suggesting unique Cbl metabolism during fetal development. However, the underlying cause of this higher CNCbl level remains unclear, as does the biological origin CNCbl. Maternal folate and vitamin B12 supplementation is a common recommendation during pregnancy, which could be a source of the elevated CNCbl we observed, although Cbl levels in placenta were comparatively low (Fig 1F). Conversion of CNCbl to active cofactors MeCbl and AdoCbl requires NADPH- or GSH-dependent decyanation by MMACHC [52,53] and it is possible that the developing fetal brain has diminished decyanation capacity. The markedly higher level of inactive CNCbl could potentially have functional consequences by competing with MeCbl and AdoCbl, restricting their cofactor activity.
MeCbl is the most abundant Cbl species in the brain of younger subjects and its 12-fold decrease was the largest contributor to the 2.7-fold decrease in total Cbl between 0–20 and 61–80 yr old subjects (Fig 2C and 2D). As an essential cofactor for MS, MeCbl availability regulates methylation capacity, reflected as a decrease in the SAM to SAH ratio in elderly subjects (Fig 4B). Among hundreds of methylation reactions which are dependent upon [SAM]/[SAH], methylation of DNA and histones merit special attention for their contribution to epigenetic regulation of gene expression. Dynamic changes in DNA methylation status at specific loci in frontal cortex are closely correlated with chronological age , consistent with the age-dependent decrease in MeCbl we observed in frontal cortex. Moreover, changes in DNA methylation provide an epigenetic mechanism of memory formation  and the capacity of the brain for learning is therefore a reflection of its ability to modify patterns of DNA and histone methylation and to sustain these patterns over time. D4 dopamine receptor-mediated phospholipid methylation is completely dependent upon MS activity  and D4 receptor activation promotes gamma frequency synchronization of neural networks during attention [5,6]. Thus MeCbl-dependent MS activity is poised to play a critical role in both attention and learning.
Autism is a complex neurodevelopmental disorder and a number of studies have reported low plasma levels of GSH and a decrease in [SAM]/[SAH] [56–61], leading us to propose a “Redox/Methylation Hypothesis of Autism” whereby the sensitivity of MS to oxidative stress could lead to impairments in epigenetic regulation and D4 receptor-mediated attention . Our current finding that frontal cortex levels of MeCbl are 3.5-fold lower in autistic subjects vs. age-matched controls (Fig 2B) lends support to this hypothesis, linking decreased methylation capacity in the brain to the deficits in neurodevelopment and learning capacity which are hallmarks of autism. Serum and plasma levels of vitamin B12 are reported to be normal in autism [60,63], except under conditions of overt nutritional deficiency , suggesting that the lower brain levels we observed might result from a limitation in its transport into the brain compartment. While lower serum B12 levels have been reported for schizophrenia in several studies [65–67], others found no difference [68,69] or higher levels . In an earlier study we showed that MS mRNA levels in frontal cortex decreased dramatically across the lifespan and levels in autistic subjects were approximately one-half of age-matched controls, although protein levels were not decreased in autistic and elderly subjects . We found that MS enzyme activity is significantly reduced in autistic subjects when measured with endogenous Cbl, and this deficit can be largely, but not completely, reversed by addition of OHCbl (Fig 5). Thus deficits in MS transcription and availability of its vitamin B12 cofactor may both contribute to impaired methylation in autism. It remains unclear whether these deficits occur prenatally or postnatally, or if they reflect an acceleration of the normal age-dependent decline caused by one or more environmental factors.
Decreased GSH levels may contribute to impaired vitamin B12 transport into the brain, as indicated by lower total Cbl and MeCbl in GCLM-KO mice (Fig 6B). However, while total Cbl and MeCbl levels were lower in frontal cortex of autistic subjects (Fig 3A and 3B), GSH levels were not decreased (Fig 4C). GSH levels have been reported to be decreased in some brain regions (e.g. cerebellum and temporal cortex) in autism, but not decreased in other regions (e.g. frontal, parietal and occipital cortex) [29,30]. The metabolic basis for these regional differences remains obscure, but may relate to their different functional roles. For example, some brain regions may be metabolically keyed to maintain GSH/GSSG (stable redox status), while other regions may maintain SAM/SAH (stable methylation status). We speculate that the former would exhibit more dynamic methylation-dependent epigenetic responses and a higher level of neuroplasticity, while epigenetic stability in the latter would favor memory. We recently showed that vitamin B12 levels in cultured human neuronal cells is strongly linked to GSH levels and neurotrophic factor activation of the PI3 kinase signaling pathway augments GSH synthesis in parallel with increased MeCbl and AdoCbl levels . Taken together, our results suggest that the well-documented systemic deficit of GSH in autism, as measured in the blood [56–61], may be linked to decreased vitamin B12 transport into the brain. However, the GCLM-KO model, which genetically restricts GSH synthesis throughout the body, does not replicate our brain findings in autistic subjects. Further studies are needed to assess the impact of systemic GSH depletion on vitamin B12 transport into the brain.
A GSH deficit has been proposed to be a key factor in the etiology of schizophrenia [30–32] and GCLM-KO mice have been extensively characterized as an animal model showing many schizophrenia-related phenotypes [42,72–74]. GCLM-KO mice exhibit a significant decrease in γ-frequency synchronized oscillations, a shared feature of schizophrenia and autism , and D4 dopamine receptor activation in parvalbumin-expressing GABAergic interneurons is essential for synchronized γ oscillations . Since D4 receptor-mediated phospholipid methylation is absolutely dependent upon MS activity , lower levels of MeCbl may contribute to diminished γ-frequency synchronized oscillations in autism and schizophrenia. Indeed, autism was initially described as “childhood onset schizophrenia” and these two disorders share many risk genes and core psychiatric/neurological features .
The finding of decreased brain vitamin B12 in autism is analogous to cerebral folate deficiency (CFD) syndrome , and approximately 75% of autistic subjects exhibit autoantibodies capable of blocking folate receptor-mediated folate transport in the choroid plexus . Megalin-directed autoantibodies are relatively common in autoimmune diseases , decreasing its function could restrict transport of both folate and vitamin B12, combining to limit MS and methylation activity in the brain. Decreased levels of total vitamin B12 and MeCbl in autistic subjects (average age 7.5 yrs), were accompanied by a pattern of methylation and transsulfuration metabolites more typical of control subjects 50–60 yrs of age (cf. Figs 1C and 2B), similar to the premature decrease in MS expression we previously reported (26), which can be expected to have neurodevelopmental consequences.
Low levels of MeCbl may help explain long-standing observations of abnormal single-carbon metabolism in schizophrenia. For example, as reviewed by Cohen et al. , more than ten studies demonstrated that intake of 20 mg of L-methionine/day induces an acute psychotic reaction in 40% of schizophrenic individuals, but is without effect in normal subjects. We previously reported that phospholipid methylation is significantly lower in lymphocytes from schizophrenic subjects  and similarly abnormal epigenetic patterns are present in both lymphocytes and corticolimbic brain regions . Moreover, the age-dependent decline in MeCbl we observed in this study, in conjunction with the previously reported decline in MS transcription  may be responsible for the characteristic post-adolescence onset of schizophrenia, which is associated with elevated levels of HCY .
Individual risk for brain disorders associated with the age-dependent decrease in MeCbl may depend upon genetic factors affecting methylation capacity. In accord with this notion, single-nucleotide polymorphisms (SNPs) in genes for methionine synthase (MTR), methionine synthase reductase (MTRR), transcobalamin (TCN2) and 5,10-methylenetetrahydrofolate reductase (MTHFR), which limit their respective activities, are associated with increased risk of autism [57,84] and schizophrenia [83,85,86], as well as major depression and bipolar disorder [85,86], Parkinson’s disease  and Alzheimer’s disease [86,88]. The breadth of these disorders indicates a central role for methylation in maintaining normal brain function and suggests that vulnerability to brain disorders at different stages of life involves impaired methylation, in combination with other risk factors specific to a particular disorder. Indeed, restriction of methylation-dependent epigenetic regulation may enhance the risk of genetic variants which might otherwise be benign.
While provision of supplemental vitamin B12 may be helpful in treating the aforementioned brain disorders, several issues must be considered. The required dosage may significantly exceed the Recommended Dietary Allowance (RDA) of 2.4 μg/day. Adequate absorption from the GI tract is essential for oral dosage and transport across the choroid plexus is critical for raising brain levels. Nasal administration may provide an alternative route of administration. The supplemented form of vitamin B12 (commonly CNCbl) must be converted to the active MeCbl and AdoCbl species, which requires adequate levels of GSH and NADPH, and a functional vitamin B12 deficiency state may result when levels of these reducing factors are low, as in oxidative stress . Thus supplementation with supraphysiological levels of the active Cbl species (i.e. MeCbl and AdoCbl) may be required to address an oxidative stress-related functional deficiency .
Our findings are subject to several limitations, most important of which is the number of samples analyzed. A larger study is warranted, particularly with regard to autism and schizophrenia samples, although availability of the former is particularly limited. Despite this limitation, the differences we observed are robust. We were not able to measure serum and brain B12 levels of the same subjects, which would provide more definitive comparison of age-dependent changes. Such studies could be carried out in animals. We did not investigate gender differences, which would be particularly relevant for autism, since it is more prevalent in males. The changes we observed in frontal cortex may not occur in other brain regions, so our findings should not be generalized to the entire brain, subject to further studies. The absence of further demographic or nutritional data, including the use of vitamin supplements, did not allow us to evaluate their potential contribution to brain B12 levels.
In conclusion, vitamin B12 levels in human frontal cortex decrease with age, especially MeCbl, which plays a crucial role in regulating all methylation reactions, including those providing epigenetic regulation of gene expression. MeCbl deficits in autistic and schizophrenic subjects suggest that impaired methylation may be a critical pathological component of these brain disorders, as well as other neurological and neuropsychiatric conditions. Our findings provide a novel redox/methylation-based perspective on the metabolic systems which support normal brain function across the lifespan.
S1 File. Clinical details for control, autism and schizophrenia subjects.
Some tissue, biological specimens or data used in this research was obtained from Autism BrainNet that is sponsored by the Simons Foundation and Autism Speaks. The authors also acknowledge the Autism Tissue Program that was the predecessor to Autism BrainNet and the Australian Brain Bank Network (ABBN) for their assistance in providing samples.
Conceived and designed the experiments: YZ RD MC KD. Performed the experiments: YZ NH MT MF. Analyzed the data: YZ MT RD. Contributed reagents/materials/analysis tools: MC KD HMA. Wrote the paper: YZ MC KD RD.
- 1. Guéant JL, Caillerez-Fofou M, Battaglia-Hsu S, Alberto JM, Freund JN, Dulluc I et al. Molecular and cellular effects of vitamin B12 in brain, myocardium and liver through its role as co-factor of methionine synthase. Biochimie. 2013; 95:1033–1040. pmid:23415654
- 2. Gherasim C, Lofgren M, Banerjee R. Navigating the B(12) road: assimilation, delivery, and disorders of cobalamin. J Biol Chem. 2013;288: 13186–13193. pmid:23539619
- 3. Park LK, Friso S, Choi SW. Nutritional influences on epigenetics and age-related disease. Proc Nutr Soc. 2012; 71:75–83. pmid:22051144
- 4. Sharma A, Kramer ML, Wick PF, Liu D, Chari S, Shim S. et al. D4 dopamine receptor-mediated phospholipid methylation and its implications for mental illnesses such as schizophrenia. Mol Psychiatry. 1999; 4:235–246. pmid:10395213
- 5. Waly M, Olteanu H, Banerjee R, Choi SW, Mason JB, Parker BS, et al. Activation of methionine synthase by insulin-like growth factor-1 and dopamine: a target for neurodevelopmental toxins and thimerosal. Mol Psychiatry. 2004; 9:358–370. pmid:14745455
- 6. Kuznetsova AY, Deth RC. A model for modulation of neuronal synchronization by D4 dopamine receptor-mediated phospholipid methylation. J Comput Neurosci. 2008; 24:314–329. pmid:17929154
- 7. Swanson JM, Kinsbourne M, Nigg J, Lanphear B, Stefanatos GA, Volkow N et al. Etiologic subtypes of attention-deficit/hyperactivity disorder: brain imaging, molecular genetic and environmental factors and the dopamine hypothesis. Neuropsychol Rev. 2007;17: 39–59. pmid:17318414
- 8. Tarazi FI, Baldessarini RJ. Dopamine D4 receptors: significance for molecular psychiatry at the millennium. Mol Psychiatry 1999; 4:529–538. pmid:10578234
- 9. Lai JH, Zhu YS, Huo ZH, Sun RF, Yu B, Wang YP, et al. Association study of polymorphisms in the promoter region of DRD4 with schizophrenia, depression, and heroin addiction. Brain Res 2010; 1359:227–232. pmid:20801104
- 10. Cheng J, Wang Y, Zhou K, Wang L, Li J, Zhuang Q, et al. Male-specific association between dopamine receptor D4 gene methylation and schizophrenia. PLoS One. 2014; 9:e89128. pmid:24586542
- 11. Grady DL, Thanos PK, Corrada MM, Barnett JC Jr, Ciobanu V, Shustarovich D. et al. DRD4 genotype predicts longevity in mouse and human. J Neurosci. 2013; 33:286–291. pmid:23283341
- 12. Gille D, Schmid A. Vitamin B12 in meat and dairy products. Nutr Rev. 2015; 73:106–115. pmid:26024497
- 13. Kozyraki R, Cases O. Vitamin B12 absorption: mammalian physiology and acquired and inherited disorders. Biochimie. 2013; 95:1002–1007. pmid:23178706
- 14. Carro E, Spuch C, Trejo JL, Antequera D, Torres-Aleman I. Choroid plexus megalin is involved in neuroprotection by serum insulin-like growth factor I. J Neurosci. 2005; 25:10884–10893. pmid:16306401
- 15. Christensen EI, Birn H. Megalin and cubilin: multifunctional endocytic receptors. Nat Rev Mol Cell Biol 2002; 3:256–266. pmid:11994745
- 16. Luder AS, Tanner SM, de la Chapelle A, Walter JH. Amnionless (AMN) mutations in Imerslund-Gräsbeck syndrome may be associated with disturbed vitamin B(12) transport into the CNS. J Inherit Metab Dis. 2008; 31 Suppl 3:493–6. pmid:18181028
- 17. Stabler SP. Clinical practice. Vitamin B12 deficiency. N Engl J Med. 2013; 368:149–160. pmid:23301732
- 18. Kirsch SH, Herrmann W, Obeid R. Genetic defects in folate and cobalamin pathways affecting the brain. Clin Chem Lab Med. 2013; 51:139–155. pmid:23183749
- 19. Worm-Peterson J. Vitamin B12 haemoglobin and iron concentration in human brain tissue. Acta Neurol Scand. 1964; 40: 241–8. pmid:14192302
- 20. Baker H, Frank O, Chen T, Feingold S, DeAngelis B, Baker E. Vitamin content of some normal human brain segments. J Neurosci Res. 1984; 11: 419–35. pmid:6235380
- 21. Numata S, Ye T, Hyde TM, Guitart-Navarro X, Tao R, Wininger M. et al. DNA methylation signatures in development and aging of the human prefrontal cortex. Am J Hum Genet. 2012; 90:260–272. pmid:22305529
- 22. Gapp K, Woldemichael BT, Bohacek J, Mansuy IM. Epigenetic regulation in neurodevelopment and neurodegenerative diseases. Neuroscience. 2014; 264:99–111. pmid:23256926
- 23. Imbard A, Benoist JF, Blom HJ. Neural tube defects, folic acid and methylation. Int J Environ Res Public Health. 2013; 10:4352–43589. pmid:24048206
- 24. Guy J, Cheval H, Selfridge J, Bird A. The role of MeCP2 in the brain. Annu Rev Cell Dev Biol. 2011; 27:631–652. pmid:21721946
- 25. Numata S et al. DNA methylation signatures in development and aging of the human prefrontal cortex. Am J Hum Genet. 2012, 90: 260–272. Erratum in: Am J Hum Genet. 2012 Oct 5; 91: 765. pmid:22305529
- 26. Muratore CR et al. Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism. PloS One. 2013; 8: e56927. pmid:23437274
- 27. Ladd-Acosta C, Hansen KD, Briem E, Fallin MD, Kaufmann WE, Feinberg AP. Common DNA methylation alterations in multiple brain regions in autism. Mol Psychiatry. 2014; 19:862–871. pmid:23999529
- 28. James SJ, Shpyleva S, Melnyk S, Pavliv O, Pogribny IP. Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum. Transl Psychiatry. 2014; 4: e460. pmid:25290267
- 29. Rose S, Melnyk S, Pavliv O, Bai S, Nick TG, Frye RE. et al. Evidence of oxidative damage and inflammation associated with low glutathione redox status in the autism brain. Transl Psychiatry. 2012; 2:e134. pmid:22781167
- 30. Chauhan A, Audhya T, Chauhan V. Brain region-specific glutathione redox imbalance in autism. Neurochem Res. 2012; 37:1681–1689. pmid:22528835
- 31. Prabakaran S, Swatton JE, Ryan MM, Huffaker SJ, Huang JT, Griffin JL. et al. Mitochondrial dysfunction in schizophrenia: evidence for compromised brain metabolism and oxidative stress. Mol Psychiatry. 2004; 9:684–697. pmid:15098003
- 32. Do KQ, Cabungcal JH, Frank A, Steullet P, Cuenod M. Redox dysregulation, neurodevelopment and schizophrenia. Curr Opin Neurobiol. 2009; 19:220–230. pmid:19481443
- 33. Steullet P, Cabungcal JH, Kulak A, Kraftsik R, Chen Y, Dalton TP. et al. Chronic Redox Dysregulation Affects the Ventral But Not Dorsal Hippocampus: Impairment of Parvalbumin Neurons, Gamma Oscillations and Related Behaviors. J Neurosci. 2010; 30:2547–2558. pmid:20164340
- 34. Hannibal L, Axhemi A, Glushchenko AV, Moreira ES, Brasch NE, Jacobsen DW. Accurate assessment and identification of naturally occurring cellular cobalamins. Clin Chem Lab Med. 2008; 46:1739–1746. pmid:18973458
- 35. Hodgson N, Trivedi M, Muratore C, Li S, Deth R. Soluble oligomers of amyloid-β cause changes in redox state, DNA methylation, and gene transcription by inhibiting EAAT3 mediated cysteine uptake. J Alzheimers Dis. 2013; 36:197–209. pmid:23579332
- 36. Waly MI, Kharbanda KK, Deth RC. Ethanol lowers glutathione in rat liver and brain and inhibits methionine synthase in a cobalamin-dependent manner. Alcohol Clin Exp Res. 2011; 35:277–283. pmid:21121936
- 37. Yang Y, Dieter MZ, Chen Y, Shertzer HG, Nebert DW, Dalton TP. Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Novel model system for a severely compromised oxidative stress response. J Biol Chem. 2002; 277:49446–49452. pmid:12384496
- 38. Selhub J, Jacques PF, Rosenberg IH, Rogers G, Bowman BA, Gunter EW. et al. Serum total homocysteine concentrations in the third National Health and Nutrition Examination Survey (1991–1994): population reference ranges and contribution of vitamin status to high serum concentrations. Ann Intern Med. 1999; 131:331–339. pmid:10475885
- 39. Tallan HH, Moore S, Stein WH. L-cystathionine in human brain. J Biol Chem. 1958; 230:707–716. pmid:13525388
- 40. Sturman JA, Rassin DK, Gaull GE. Relation of three enzymes of transsulphuration to the concentration of cystathionine in various regions of monkey brain. J Neurochem. 1970; 17:1117–1119. pmid:4987664
- 41. Do KQ, Trabesinger AH, Kirsten-Krüger M, Lauer CJ, Dydak U, Hell D. et al. Schizophrenia: glutathione deficit in cerebrospinal fluid and frontal cortex in vivo. Eur J Neurosci. 2000; 12:3721–3728. pmid:11029642
- 42. das Neves Duarte JM, Kulak A, Gholam-Razaee MM, Cuenod M, Gruetter R, Do KQ. N-acetylcysteine normalizes neurochemical changes in the glutathione-deficient schizophrenia mouse model during development. Biol Psychiatry. 2012; 71:1006–1014. pmid:21945305
- 43. Spuch C, Navarro C. Transport Mechanisms at the Blood-Cerebrospinal-Fluid Barrier: Role of Megalin (LRP2). Recent Pat Endocr Metab Immune Drug Discov. 2010; 4:190–205.
- 44. Moestrup SK et al. Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis. Proc Natl Acad Sci U S A. 1996; 93: 8612–8617. pmid:8710919
- 45. Bose S, Kalra S, Yammani RR, Ahuja R, Seetharam B. Plasma membrane delivery, endocytosis and turnover of transcobalamin receptor in polarized human intestinal epithelial cells. J Physiol. 2007; 581:457–466. pmid:17347267
- 46. Fernàndez-Roig S, Lai S-C, Murphy MM, Fernandez-Ballart J, Quadros EV. Vitamin B12 deficiency in the brain leads to DNA hypomethylation in the TCblR/CD320 knockout mouse. Nutr Metab. 2012; 9:41.
- 47. Jensen LL, Andersen RK, Hager H, Madsen M. Lack of megalin expression in adult human terminal ileum suggests megalin-independent cubilin/amnionless activity during vitamin B12 absorption. Physiol Rep. 2014; 2:e12086. pmid:25052491
- 48. Pascale CL, Miller MC, Chiu C, Boylan M, Caralopoulos IN, Gonzalez L, et al. Amyloid-beta transporter expression at the blood-CSF barrier is age-dependent. Fluids Barriers CNS. 2011; 8:21. pmid:21740544
- 49. Bosco P. Guéant-Rodríguez RM, Anello G, Romano A, Namour B, Spada RS, et al. Association of IL-1 RN*2 allele and methionine synthase 2756 AA genotype with dementia severity of sporadic Alzheimer’s disease. J Neurol Neurosurg Psychiatry. 2004; 75:1036–1038. pmid:15201366
- 50. Smith AD, Smith SM, de Jager CA, Whitbread P, Johnston C, Agacinski G, et al. Homocysteine-lowering by B vitamins slows the rate of accelerated brain atrophy in mild cognitive impairment: a randomized controlled trial. PLoS One 2010; 5:e12244. pmid:20838622
- 51. Douaud G. Refsum H, de Jager CA, Jacoby R, Nichols TE, Smith SM, et al. Preventing Alzheimer’s disease-related gray matter atrophy by B-vitamin treatment. Proc Natl Acad Sci U S A. 2013; 110:9523–9528. pmid:23690582
- 52. Kim J, Gherasim C, Banerjee R. Decyanation of vitamin B12 by a trafficking chaperone. Proc Natl Acad Sci USA. 2008 Sep 23;105(38):14551–14554. pmid:18779575
- 53. Li Z, Gherasim C, Lesniak NA, Banerjee R. Glutathione-dependent one-electron transfer reactions catalyzed by a B₁₂ trafficking protein. J Biol Chem. 2014; 289:16487–16497. pmid:24742678
- 54. Horvath S. DNA methylation age of human tissues and cell types. Genome Biol. 2013; 14:R115. pmid:24138928
- 55. Zovkic IB, Guzman-Karlsson MC, Sweatt JD. Epigenetic regulation of memory formation and maintenance. Learn Mem. 2013; 20:61–74. pmid:23322554
- 56. James SJ, Cutler P, Melnyk S, Jernigan S, Janak L, Gaylor DW, et al. Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism. Am J Clin Nutr. 2004; 80:1611–1617. pmid:15585776
- 57. James SJ. Melnyk S, Jernigan S, Cleves MA, Halsted CH, Wong DH, et al. Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism. Am J Med Genet B Neuropsychiatr Genet. 2006; 141B: 947–956. pmid:16917939
- 58. Al-Gadani Y. El-Ansary A, Attas O, Al-Ayadhi L. Metabolic biomarkers related to oxidative stress and antioxidant status in Saudi autistic children. Clin Biochem. 2009; 42:1032–1040. pmid:19306862
- 59. Paşca SP, Dronca E, Kaucsár T, Craciun EC, Endreffy E, Ferencz BK, et al. One Carbon Metabolism Disturbances and the C667T MTHFR Gene Polymorphism in Children with Autism Spectrum Disorders. J Cell Mol Med. 2009; 13:4229–4238. pmid:19267885
- 60. Melnyk S. Fuchs GJ, Schulz E, Lopez M, Kahler SG, Fussell JJ, et al. Metabolic imbalance associated with methylation dysregulation and oxidative damage in children with autism. J Autism Dev Disord. 2012; 42:367–377. pmid:21519954
- 61. Frustaci A, Neri M, Cesario A, Adams JB, Domenici E, Dalla Bernardina B, et al. Oxidative stress-related biomarkers in autism: systematic review and meta-analyses. Free Radic Biol Med. 2012; 52:2128–2141. pmid:22542447
- 62. Deth R, Muratore C, Benzecry J, Power-Charnitsky V- A, Waly M. How environmental and genetic factors combine to cause autism: A redox/methylation hypothesis. Neurotoxicology. 2008; 29:190–201. pmid:18031821
- 63. Adams JB, Audhya T, McDonough-Means S, Rubin RA, Quig D, Geis E, et al. Nutritional and metabolic status of children with autism vs. neurotypical children, and the association with autism severity. Nutr Metab (Lond). 2011; 8:34.
- 64. Al-Farsi YM, Waly MI, Deth RC, Al-Sharbati MM, Al-Shafaee M, Al-Farsi O, et al. Low folate and vitamin B12 nourishment is common in Omani children with newly diagnosed autism. Nutrition. 2013; 29:537–541. pmid:23287069
- 65. Kemperman RF, Veurink M, van der Wal T, Knegtering H, Bruggeman R, Fokkema MR, et al. Low essential fatty acid and B-vitamin status in a subgroup of patients with schizophrenia and its response to dietary supplementation. Prostaglandins Leukot Essent Fatty Acids. 2006; 74:75–85. pmid:16384692
- 66. Kale A, Naphade N, Sapkale S, Kamaraju M, Pillai A, Joshi S, et al. Reduced folic acid, vitamin B12 and docosahexaenoic acid and increased homocysteine and cortisol in never-medicated schizophrenia patients: implications for altered one-carbon metabolism. Psychiatry Res 2010; 175: 47–53. pmid:19969375
- 67. Ssonko M, Ddungu H, Musisi S. Low serum vitamin B12 levels among psychiatric patients admitted in Butabika mental hospital in Uganda. BMC Res Notes. 2014; 7: 90. pmid:24533701
- 68. Haidemenos A, Kontis D, Gazi A, Kallai E, Allin M, Lucia B, et al. Plasma homocysteine, folate and B12 in chronic schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry. 2007; 31:1289–1296. pmid:17597277
- 69. Eren E, Yeğin A, Yilmaz N, Herken H. Serum total homocysteine, folate and vitamin B12 levels and their correlation with antipsychotic drug doses in adult male patients with chronic schizophrenia. Clin Lab. 2010; 56:513–518. pmid:21141434
- 70. Saedisomeolia A, Djalali M, Moghadam AM, Ramezankhani O, Najmi L. Folate and vitamin B12 status in schizophrenic patients. J Res Med Sci. 2011; 16:S437–441. pmid:22247731
- 71. Zhang Y, Hodgson N, Trivedi M, Deth R. Neuregulin-1 promotes glutathione-dependent neuronal cobalamin metabolism by stimulating cysteine uptake. Oxid Med and Cell Longevity. (In Press).
- 72. Kulak A, Cuenod M, Do KQ. Behavioral phenotyping of glutathione-deficient mice: relevance to schizophrenia and bipolar disorder. Behav Brain Res. 2012; 226:563–570. pmid:22033334
- 73. das Neves Duarte JM, Kulak A, Gholam-Razaee MM, Cuenod M, Gruetter R, Do KQ. N-acetylcysteine normalizes neurochemical changes in the glutathione-deficient schizophrenia mouse model during development. Biol Psychiatry. 2012; 71:1006–1014. pmid:21945305
- 74. Cabungcal JH, Steullet P, Morishita H, Kraftsik R, Cuenod M, Hensch TK, et al. Perineuronal nets protect fast-spiking interneurons against oxidative stress. Proc Natl Acad Sci USA. 2013; 110:9130–9135. pmid:23671099
- 75. Uhlhaas PJ, Pipa G, Lima B, Melloni L, Neuenschwander S, Nikolić D, et al. Neural synchrony in cortical networks: history, concept and current status. Front Integr Neurosci. 2009; 3:17. pmid:19668703
- 76. Andersson RH, Johnston A, Herman PA, Winzer-Serhan UH, Karavanova I, Vullhorst D et al. Neuregulin and dopamine modulation of hippocampal gamma oscillations is dependent on dopamine D4 receptors. Proc Natl Acad Sci USA. 2012; 109:13118–13123. pmid:22822214
- 77. Rapoport J, Chavez A, Greenstein D, Addington A, Gogtay N. Autism spectrum disorders and childhood-onset schizophrenia: clinical and biological contributions to a relation revisited. J Am Acad Child Adolesc Psychiatry. 2009; 48:10–8. pmid:19218893
- 78. Ramaekers VT, Blau N, Sequeira JM, Nassogne MC, Quadros EV. Folate receptor autoimmunity and cerebral folate deficiency in low-functioning autism with neurological deficits. Neuropediatrics. 2007; 38:276–281. pmid:18461502
- 79. Frye RE, Sequeira JM, Quadros EV, James SJ, Rossignol DA. Cerebral folate receptor autoantibodies in autism spectrum disorder. Mol Psychiatry. 2013; 18:369–381. pmid:22230883
- 80. Ooka S, Matsui T, Nishioka K, Kato T. Autoantibodies to low-density-lipoprotein-receptor-related protein 2 (LRP2) in systemic autoimmune diseases. Arthritis Res Ther. 2003; 5:R174–180. pmid:12723989
- 81. Cohen SM, Nichols A, Wyatt R, Pollin W. The administration of methionine to chronic schizophrenic patients: a review of ten studies. Psychiatry. 1974; 8:209–225.
- 82. Auta J, Smith RC, Dong E, Tueting P, Sershen H, Boules S, et al. DNA-methylation gene network dysregulation in peripheral blood lymphocytes of schizophrenia patients. Schizophr Res. 2013; 150:312–318. pmid:23938174
- 83. Muntjewerff JW, Kahn RS, Blom HJ, den Heijer M. Homocysteine, methylenetetrahydrofolate reductase and risk of schizophrenia: a meta-analysis. Mol Psychiatry. 2006; 11:143–149. pmid:16172608
- 84. Pu D, Shen Y, Wu J. Association between MTHFR gene polymorphisms and the risk of autism spectrum disorders: a meta-analysis. Autism Res. 2013; 6:384–392. pmid:23653228
- 85. Peerbooms OL, van Os J, Drukker M, Kenis G, Hoogveld L; MTHFR in Psychiatry Group, et al. Meta-analysis of MTHFR gene variants in schizophrenia, bipolar disorder and unipolar depressive disorder: evidence for a common genetic vulnerability? Brain Behav Immun. 2011; 25:1530–1543. pmid:21185933
- 86. Mitchell ES, Conus N, Kaput J. B vitamin polymorphisms and behavior: evidence of associations with neurodevelopment, depression, schizophrenia, bipolar disorder and cognitive decline. Neurosci Biobehav Rev. 2014; 47:307–320. pmid:25173634
- 87. Wu YL, Ding XX, Sun YH, Yang HY, Sun L. Methylenetetrahydrofolate reductase (MTHFR) C677T/A1298C polymorphisms and susceptibility to Parkinson's disease: a meta-analysis. J Neurol Sci. 2013; 335:14–21. pmid:24064257
- 88. Beyer K, Lao JI, Latorre P, Riutort N, Matute B, Fernández-Figueras MT, et al. Methionine synthase polymorphism is a risk factor for Alzheimer disease. Neuroreport. 2003; 14:1391–1394. pmid:12876480
- 89. Solomon LR. Functional cobalamin (vitamin B12) deficiency: role of advanced age and disorders associated with increased oxidative stress. Eur J Clin Nutr. 2015; 69:687–692. pmid:25563739
- 90. Froese DS, Gravel RA. Genetic disorders of vitamin B(1)(2) metabolism: eight complementation groups—eight genes. Expert Rev Mol Med 2010;12:e37 pmid:21114891