Folate and Cobalamin Deficiencies during Pregnancy Disrupt the Glucocorticoid Response in Hypothalamus through N-Homocysteinilation of the Glucocorticoid Receptor.

Vitamin B9 (folate)/B12 (cobalamin) deficiency is known to induce brain structural and/or functional retardations. In many countries, folate supplementation, targeting the most severe outcomes such as neural tube defects, is discontinued after the first trimester. However, adverse effects may occur after birth because of some mild misregulations. Various hormonal receptors were shown to be deregulated in brain tissue under these conditions. The glucocorticoid receptor (GR) is particularly sensitive to epigenetic regulation and post-translational modifications. In a mother-offspring rat model of vitamin B9/B12 deficiency, we investigated whether a prolonged folate supplementation could restore the GR signaling in the hypothalamus. Our data showed that a deficiency of folate and vitamin B12 during the in-utero and early postnatal periods was associated with reduced GR expression in the hypothalamus. We also described for the first time a novel post-translational modification of GR that impaired ligand binding and GR activation, leading to decrease expression of one of the GR targets in the hypothalamus, AgRP. Moreover, this brain-impaired GR signaling pathway was associated with behavioral perturbations during offspring growth. Importantly, perinatal and postnatal supplementation with folic acid helped restore GR mRNA levels and activity in hypothalamus cells and improved behavioral deficits.

Cognitive Impairment Is Associated with AMPAR Glutamatergic Dysfunction in a Mouse Model of Neuronal Methionine Synthase Deficiency.

Impairment of one-carbon metabolism during pregnancy, either due to nutritional deficiencies in B9 or B12 vitamins or caused by specific genetic defects, is often associated with neurological defects, including cognitive dysfunction that persists even after vitamin supplementation. Animal nutritional models do not allow for conclusions regarding the specific brain mechanisms that may be modulated by systemic compensations. Using the Cre-lox system associated to the neuronal promoter Thy1.2, a knock-out model for the methionine synthase specifically in the brain was generated. Our results on the neurobehavioral development of offspring show that the absence of methionine synthase did not lead to growth retardation, despite an effective reduction of both its expression and the methylation status in brain tissues. Behaviors were differently affected according to their functional outcome. Only temporary retardations were recorded in the acquisition of vegetative functions during the suckling period, compared to a dramatic reduction in cognitive performance after weaning. Investigation of the glutamatergic synapses in cognitive areas showed a reduction of AMPA receptors phosphorylation and clustering, indicating an epigenomic effect of the neuronal deficiency of methionine synthase on the reduction of glutamatergic synapses excitability. Altogether, our data indicate that cognitive impairment associated with methionine synthase deficiency may not only result from neurodevelopmental abnormalities, but may also be the consequence of alterations in functional plasticity of the brain.

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.

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.

The Stimulation of Neurogenesis Improves the Cognitive Status of Aging Rats Subjected to Gestational and Perinatal Deficiency of B9-12 Vitamins.

A deficiency in B-vitamins is known to lead to persistent developmental defects in various organs during early life. The nervous system is particularly affected with functional retardation in infants and young adults. In addition, even if in some cases no damage appears evident in the beginning of life, correlations have been shown between B-vitamin metabolism and neurodegenerative diseases. However, despite the usual treatment based on B-vitamin injections, the neurological outcomes remain poorly rescued in the majority of cases, compared with physiological functions. In this study, we explored whether a neonatal stimulation of neurogenesis could compensate atrophy of specific brain areas such as the hippocampus, in the case of B-vitamin deficiency. Using a physiological mild transient hypoxia within the first 24 h after birth, rat-pups, submitted or not to neonatal B-vitamin deficiency, were followed until 330-days-of-age for their cognitive capacities and their hippocampus status. Our results showed a gender effect since females were more affected than males by the deficiency, showing a persistent low body weight and poor cognitive performance to exit a maze. Nevertheless, the neonatal stimulation of neurogenesis with hypoxia rescued the maze performance during adulthood without modifying physiological markers, such as body weight and circulating homocysteine. Our findings were reinforced by an increase of several markers at 330-days-of-age in hypoxic animals, such as Ammon's Horn 1hippocampus (CA1) thickness and the expression of key actors of synaptic dynamic, such as the NMDA-receptor-1 (NMDAR1) and the post-synaptic-density-95 (PSD-95). We have not focused our conclusion on the neonatal hypoxia as a putative treatment, but we have discussed that, in the case of neurologic retardation associated with a reduced B-vitamin status, stimulation of the latent neurogenesis in infants could ameliorate their quality of life during their lifespan.

The Fate of Transplanted Olfactory Progenitors Is Conditioned by the Cell Phenotypes of the Receiver Brain Tissue in Cocultures.

Among the numerous candidates for cell therapy of the central nervous system (CNS), olfactory progenitors (OPs) represent an interesting alternative because they are free of ethical concerns, are easy to collect, and allow autologous transplantation. In the present study, we focused on the optimization of neuron production and maturation. It is known that plated OPs respond to various trophic factors, and we also showed that the use of Nerve Growth Factor (NGF) allowed switching from a 60/40 neuron/glia ratio to an 80/20 one. Nevertheless, in order to focus on the integration of OPs in mature neural circuits, we cocultured OPs in primary cultures obtained from the cortex and hippocampus of newborn mice. When dissociated OPs were plated, they differentiated into both glial and neuronal phenotypes, but we obtained a 1.5-fold higher viability in cortex/OP cocultures than in hippocampus/OP ones. The fate of OPs in cocultures was characterized with different markers such as BrdU, Map-2, and Synapsin, indicating a healthy integration. These results suggest that the integration of transplanted OPs might by affected by trophic factors and the environmental conditions/cell phenotypes of the host tissue. Thus, a model of coculture could provide useful information on key cell events for the use of progenitors in cell therapy.

SIRT1 activation rescues the mislocalization of RNA-binding proteins and cognitive defects induced by inherited cobalamin disorders.

BACKGROUND: The molecular consequences of inborn errors of vitamin B12 or cobalamin metabolism are far from being understood. Moreover, innovative therapeutic strategies are needed for the treatment of neurological outcomes that are usually resistant to conventional treatments. Our previous findings suggest a link between SIRT1, cellular stress and RNA binding proteins (RBP) mislocalization in the pathological mechanisms triggered by impaired vitamin B12 metabolism. OBJECTIVES AND METHODS: The goal of this study was to investigate the effects of the pharmacological activation of SIRT1 using SRT1720 on the molecular mechanisms triggered by impaired methionine synthase activity. Experiments were performed in vitro with fibroblasts from patients with the cblG and cblC inherited defects of vitamin B12 metabolism and in vivo with an original transgenic mouse model of methionine synthase deficiency specific to neuronal cells. Subcellular localization of the RBPs HuR, HnRNPA1, RBM10, SRSF1 and Y14 was investigated by immunostaining and confocal microscopy in patient fibroblasts. RBPs methylation and phosphorylation were studied by co-immunoprecipitation and proximity ligation assay. Cognitive performance of the transgenic mice treated with SRT1720 was measured with an aquatic maze. RESULTS: Patient fibroblasts with cblC and cblG defects of vitamin B12 metabolism presented with endoplasmic reticulum stress, altered methylation, phosphorylation and subcellular localization of HuR, HnRNPA1 and RBM10, global mRNA mislocalization and increased HnRNPA1-dependent skipping of IRF3 exons. Incubation of fibroblasts with cobalamin, S-adenosyl methionine and okadaic acid rescued the localization of the RBPs and mRNA. The SIRT1 activating compound SRT1720 inhibited ER stress and rescued RBP and mRNA mislocalization and IRF3 splicing. Treatment with this SIRT1 agonist prevented all these hallmarks in patient fibroblasts but it also improved the deficient hippocampo-dependent learning ability of methionine synthase conditional knock-out mice. CONCLUSIONS: By unraveling the molecular mechanisms triggered by inborn errors of cbl metabolism associating ER stress, RBP mislocalization and mRNA trafficking, our study opens novel therapeutic perspectives for the treatment of inborn errors of vitamin B12 metabolism.

Brain Susceptibility to Methyl Donor Deficiency: From Fetal Programming to Aging Outcome in Rats.

Deficiencies in methyl donors, folate, and vitamin B12 are known to lead to brain function defects. Fetal development is the most studied but data are also available for such an impact in elderly rats. To compare the functional consequences of nutritional deficiency in young versus adult rats, we monitored behavioral outcomes of cerebellum and hippocampus circuits in the offspring of deficient mother rats and in adult rats fed a deficient diet from 2 to 8 months-of-age. We present data showing that the main deleterious consequences are found in young ages compared to adult ones, in terms of movement coordination and learning abilities. Moreover, we obtained sex and age differences in the deleterious effects on these functions and on neuronal layer integrity in growing young rats, while deficient adults presented only slight functional alterations without tissue damage. Actually, the cerebellum and the hippocampus develop and maturate according to different time lap windows and we demonstrate that a switch to a normal diet can only rescue circuits that present a long permissive window of time, such as the cerebellum, whereas the hippocampus does not. Thus, we argue, as others have, for supplements or fortifications given over a longer time than the developmental period.

Methyl Donor Deficiency during Gestation and Lactation in the Rat Affects the Expression of Neuropeptides and Related Receptors in the Hypothalamus.

The micronutrients vitamins B9 and B12 act as methyl donors in the one-carbon metabolism involved in transmethylation reactions which critically influence epigenetic mechanisms and gene expression. Both vitamins are essential for proper development, and their deficiency during pregnancy has been associated with a wide range of disorders, including persisting growth retardation. Energy homeostasis and feeding are centrally regulated by the hypothalamus which integrates peripheral signals and acts through several orexigenic and anorexigenic mediators. We studied this regulating system in a rat model of methyl donor deficiency during gestation and lactation. At weaning, a predominance of the anorexigenic pathway was observed in deficient pups, with increased plasma peptide YY and increased hypothalamic pro-opiomelanocortin (POMC) mRNA, in line with abnormal leptin, ghrelin, and insulin secretion and/or signaling during critical periods of fetal and/or postnatal development of the hypothalamus. These results suggest that early methyl donor deficiency can affect the development and function of energy balance circuits, resulting in growth and weight deficits. Maternal administration of folic acid (3 mg/kg/day) during the perinatal period tended to rectify peripheral metabolic signaling and central neuropeptide and receptor expression, leading to reduced growth retardation.

Developmental Impairments in a Rat Model of Methyl Donor Deficiency: Effects of a Late Maternal Supplementation with Folic Acid.

Vitamins B9 (folate) and B12 act as methyl donors in the one-carbon metabolism which influences epigenetic mechanisms. We previously showed that an embryofetal deficiency of vitamins B9 and B12 in the rat increased brain expression of let-7a and miR-34a microRNAs involved in the developmental control of gene expression. This was reversed by the maternal supply with folic acid (3 mg/kg/day) during the last third of gestation, resulting in a significant reduction of associated birth defects. Since the postnatal brain is subject to intensive developmental processes, we tested whether further folate supplementation during lactation could bring additional benefits. Vitamin deficiency resulted in weaned pups (21 days) in growth retardation, delayed ossification, brain atrophy and cognitive deficits, along with unchanged brain level of let-7a and decreased expression of miR-34a and miR-23a. Whereas maternal folic acid supplementation helped restore the levels of affected microRNAs, it led to a reduction of structural and functional defects taking place during the perinatal/postnatal periods, such as learning/memory capacities. Our data suggest that a gestational B-vitamin deficiency could affect the temporal control of the microRNA regulation required for normal development. Moreover, they also point out that the continuation of folate supplementation after birth may help to ameliorate neurological symptoms commonly associated with developmental deficiencies in folate and B12.

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.

Wnt Signaling Pathways Are Dysregulated in Rat Female Cerebellum Following Early Methyl Donor Deficiency.

Gestational methyl donor (especially B9 and B12 vitamins) deficiency is involved in birth defects and brain development retardation. The underlying molecular mechanisms that are dysregulated still remain poorly understood, in particular in the cerebellum. As evidenced from previous data, females are more affected than males. In this study, we therefore took advantage of a validated rat nutritional model and performed a microarray analysis on female progeny cerebellum, in order to identify which genes and molecular pathways were disrupted in response to methyl donor deficiency. We found that cerebellum development is altered in female pups, with a decrease of the granular cell layer thickness at postnatal day 21. Furthermore, we investigated the involvement of the Wnt signaling pathway, a major molecular pathway involved in neuronal development and later on in synaptic assembly and neurotransmission processes. We found that Wnt canonical pathway was disrupted following early methyl donor deficiency and that neuronal targets were selectively enriched in the downregulated genes. These results could explain the structural brain defects previously observed and highlighted new genes and a new molecular pathway affected by nutritional methyl donor deprivation.

Late Maternal Folate Supplementation Rescues from Methyl Donor Deficiency-Associated Brain Defects by Restoring Let-7 and miR-34 Pathways.

The micronutrients folate and vitamin B12 are essential for the proper development of the central nervous system, and their deficiency during pregnancy has been associated with a wide range of disorders. They act as methyl donors in the one-carbon metabolism which critically influences epigenetic mechanisms. In order to depict further underlying mechanisms, we investigated the role of let-7 and miR-34, two microRNAs regulated by methylation, on a rat model of maternal deficiency. In several countries, public health policies recommend periconceptional supplementation with folic acid. However, the question about the duration and periodicity of supplementation remains. We therefore tested maternal supply (3 mg/kg/day) during the last third of gestation from embryonic days (E) 13 to 20. Methyl donor deficiency-related developmental disorders at E20, including cerebellar and interhemispheric suture defects and atrophy of selective cerebral layers, were associated with increased brain expression (by 2.5-fold) of let-7a and miR-34a, with subsequent downregulation of their regulatory targets such as Trim71 and Notch signaling partners, respectively. These processes could be reversed by siRNA strategy in differentiating neuroprogenitors lacking folate, with improvement of their morphological characteristics. While folic acid supplementation helped restoring the levels of let-7a and miR-34a and their downstream targets, it led to a reduction of structural and functional defects taking place during the perinatal period. Our data outline the potential role of let-7 and miR-34 and their related signaling pathways in the developmental defects following gestational methyl donor deficiency and support the likely usefulness of late folate supplementation in at risk women.

Foetal programming by methyl donor deficiency produces steato-hepatitis in rats exposed to high fat diet.

Non-alcoholic steatohepatitis (NASH) is a manifestation of metabolic syndrome, which emerges as a major public health problem. Deficiency in methyl donors (folate and vitamin B12) during gestation and lactation is frequent in humans and produces foetal programming effects of metabolic syndrome, with small birth weight and liver steatosis at day 21 (d21), in rat pups. We investigated the effects of fetal programming on liver of rats born from deficient mothers (iMDD) and subsequently subjected to normal diet after d21 and high fat diet (HF) after d50. We observed increased abdominal fat, ASAT/ALAT ratio and angiotensin blood level, but no histological liver abnormality in d50 iMDD rats. In contrast, d185 iMDD/HF animals had hallmarks of steato-hepatitis, with increased markers of inflammation and fibrosis (caspase1, cleaved IL-1ß, α1(I) and α2(I) collagens and α-SMA), insulin resistance (HOMA-IR and Glut 2) and expression of genes involved in stellate cell stimulation and remodelling and key genes triggering NASH pathomechanisms (transforming growth factor beta super family, angiotensin and angiotensin receptor type 1). Our data showed a foetal programming effect of MDD on liver inflammation and fibrosis, which suggests investigating whether MDD during pregnancy is a risk factor of NASH in populations subsequently exposed to HF diet.

Folate- and vitamin B12-deficient diet during gestation and lactation alters cerebellar synapsin expression via impaired influence of estrogen nuclear receptor α.

Deficiency in the methyl donors vitamin B12 and folate during pregnancy and postnatal life impairs proper brain development. We studied the consequences of this combined deficiency on cerebellum plasticity in offspring from rat mothers subjected to deficient diet during gestation and lactation and in rat neuroprogenitor cells expressing cerebellum markers. The major proteomic change in cerebellum of 21-d-old deprived females was a 2.2-fold lower expression of synapsins, which was confirmed in neuroprogenitors cultivated in the deficient condition. A pathway analysis suggested that these proteomic changes were related to estrogen receptor α (ER-α)/Src tyrosine kinase. The influence of impaired ER-α pathway was confirmed by abnormal negative geotaxis test at d 19-20 and decreased phsophorylation of synapsins in deprived females treated by ER-α antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP). This effect was consistent with 2-fold decreased expression and methylation of ER-α and subsequent decreased ER-α/PPAR-γ coactivator 1 α (PGC-1α) interaction in deficiency condition. The impaired ER-α pathway led to decreased expression of synapsins through 2-fold decreased EGR-1/Zif-268 transcription factor and to 1.7-fold reduced Src-dependent phosphorylation of synapsins. The treatment of neuroprogenitors with either MPP or PP1 (4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline, 6,7-dimethoxy-N-(4-phenoxyphenyl)-4-quinazolinamine, SKI-1, Src-l1) Src inhibitor produced similar effects. In conclusion, the deficiency during pregnancy and lactation impairs the expression of synapsins through a deregulation of ER-α pathway.

Gestational methyl donor deficiency alters key proteins involved in neurosteroidogenesis in the olfactory bulbs of newborn female rats and is associated with impaired olfactory performance.

Gestational methyl donor deficiency (MDD) leads to growth retardation as well as to cognitive and motor disorders in 21-d-old rat pups. These disorders are related to impaired neurogenesis in the cerebral neurogenic areas. Olfactory bulbs (OB), the main target of neuronal progenitors originating from the subventricular zone, play a critical role during the postnatal period by allowing the pups to identify maternal odour. We hypothesised that growth retardation could result from impaired suckling due to impaired olfactory discrimination through imbalanced apoptosis/neurogenesis in the OB. Since neurosteroidogenesis modulates neurogenesis in OB, in the present study, we investigated whether altered neurosteroidogenesis could explain some these effects. Pups born to dams fed a normal diet (n 24) and a MDD diet (n 27) were subjected to olfactory tests during the lactation and weaning periods (n 24 and 20, respectively). We studied the markers of apoptosis/neurogenesis and the expression levels of the key neurosteroidogenic enzyme aromatase, the cholesterol-transfer protein StAR (steroidogenic acute regulatory protein) and the ERα oestrogen receptor and the content of oestradiol in OB. The 21-d-old MDD female pups displayed lower body weight and impaired olfactory discrimination when compared with the control pups. MDD led to greater homocysteine accumulation and more pronounced apoptosis, along with impaired cell proliferation in the OB of female pups. The expression levels of aromatase, StAR and ERα as well as the content of oestradiol were lower in the OB of the MDD female pups than in those of the control female pups. In conclusion, gestational MDD may alter olfactory discrimination performances by affecting neurogenesis, apoptosis and neurosteroidogenesis in OB in a sex-dependent manner. It may be involved in growth retardation through impaired suckling.

Ghrelin, neuropeptide Y, and other feeding-regulatory peptides active in the hippocampus: role in learning and memory.

The hippocampus is a brain region of primary importance for neurogenesis, which occurs during early developmental states as well as during adulthood. Increases in neuronal proliferation and in neuronal death with age have been associated with drastic changes in memory and learning. Numerous neurotransmitters are involved in these processes, and some neuropeptides that mediate neurogenesis also modulate feeding behavior. Concomitantly, feeding peptides, which act primarily in the hypothalamus, are also present in the hippocampus. This review aims to ascertain the role of several important feeding peptides in cognitive functions, either through their local synthesis in the hippocampus or through their actions via specific receptors in the hippocampus. A link between neurogenesis and the orexigenic or anorexigenic properties of feeding peptides is discussed.

Early methyl donor deficiency produces severe gastritis in mothers and offspring through N-homocysteinylation of cytoskeleton proteins, cellular stress, and inflammation.

We examined the gastric mucosa structure and inflammatory status in control well-nourished Wistar dams and in Wistar dams deprived of choline, folate, and vitamin B12 during gestation and suckling periods, and in their offspring just before birth and at weaning. In this model of methyl donor deficiency (MDD), structural protein (E-cadherin and actin) N-homocysteinylation was measured through immunoprecipitation and proximity ligation assays. Cellular stress, inflammation, and apoptosis were estimated by the analysis of the NF-κB pathway, and the expression of superoxide dismutase, cyclooxygenase-2, tumor necrosis factor α, caspases 3 and 9, and TUNEL assay. Aberrant gastric mucosa formation and signs of surface layer erosion were detected in MDD fetuses and weanlings. E-cadherin and actin were N-homocysteinylated (+215 and +249% vs. controls, respectively; P<0.001). Expression of ß-catenin staining drastically decreased (-98%; P<0.01). NF-κB pathway was activated (+124%; P<0.01). Expressions of all inflammatory factors (+70%; P<0.01), superoxide dismutase (+55%; P<0.01), and caspases (+104%; P<0.01) were markedly increased. These changes were also observed in dams, to a lesser extent. Early MDD induced gastric mucosa injury similar to atrophic gastritis through structural protein N-homocysteinylation, marked inflammation, and apoptosis, despite activation of repair machinery.

Non-injurious neonatal hypoxia confers resistance to brain senescence in aged male rats.

Whereas brief acute or intermittent episodes of hypoxia have been shown to exert a protective role in the central nervous system and to stimulate neurogenesis, other studies suggest that early hypoxia may constitute a risk factor that influences the future development of mental disorders. We therefore investigated the effects of a neonatal "conditioning-like" hypoxia (100% N2, 5 min) on the brain and the cognitive outcomes of rats until 720 days of age (physiologic senescence). We confirmed that such a short hypoxia led to brain neurogenesis within the ensuing weeks, along with reduced apoptosis in the hippocampus involving activation of Erk1/2 and repression of p38 and death-associated protein (DAP) kinase. At 21 days of age, increased thicknesses and cell densities were recorded in various subregions, with strong synapsin activation. During aging, previous exposure to neonatal hypoxia was associated with enhanced memory retrieval scores specifically in males, better preservation of their brain integrity than controls, reduced age-related apoptosis, larger hippocampal cell layers, and higher expression of glutamatergic and GABAergic markers. These changes were accompanied with a marked expression of synapsin proteins, mainly of their phosphorylated active forms which constitute major players of synapse function and plasticity, and with increases of their key regulators, i.e. Erk1/2, the transcription factor EGR-1/Zif-268 and Src kinase. Moreover, the significantly higher interactions between PSD-95 scaffolding protein and NMDA receptors measured in the hippocampus of 720-day-old male animals strengthen the conclusion of increased synaptic functional activity and plasticity associated with neonatal hypoxia. Thus, early non-injurious hypoxia may trigger beneficial long term effects conferring higher resistance to senescence in aged male rats, with a better preservation of cognitive functions.

Homocysteinylation of neuronal proteins contributes to folate deficiency-associated alterations of differentiation, vesicular transport, and plasticity in hippocampal neuronal cells.

Despite the key role in neuronal development of a deficit in the methyl donor folate, little is known on the underlying mechanisms. We therefore studied the consequences of folate deficiency on proliferation, differentiation, and plasticity of the rat H19-7 hippocampal cell line. Folate deficit reduced proliferation (17%) and sensitized cells to differentiation-associated apoptosis (+16%). Decreased production (-58%) of S-adenosylmethionine (the universal substrate for transmethylation reactions) and increased expression of histone deacetylases (HDAC4,6,7) would lead to epigenomic changes that may impair the differentiation process. Cell polarity, vesicular transport, and synaptic plasticity were dramatically affected, with poor neurite outgrowth (-57%). Cell treatment by an HDAC inhibitor (SAHA) led to a noticeable improvement of cell polarity and morphology, with longer processes. Increased homocysteine levels (+55%) consecutive to folate shortage produced homocysteinylation, evidenced by coimmunoprecipitations and mass spectrometry, and aggregation of motor proteins dynein and kinesin, along with functional alterations, as reflected by reduced interactions with partner proteins. Prominent homocysteinylation of key neuronal proteins and subsequent aggregation certainly constitute major adverse effects of folate deficiency, affecting normal development with possible long-lasting consequences.