RESUMO
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.
Assuntos
Ácido Fólico , Deficiência de Vitamina B 12 , Gravidez , Feminino , Animais , Ratos , Ácido Fólico/farmacologia , Receptores de Glucocorticoides/genética , Glucocorticoides , Epigênese Genética , Suplementos Nutricionais , Vitamina B 12/farmacologia , HipotálamoRESUMO
The molecular mechanisms that underlie the neurological manifestations of patients with inherited diseases of vitamin B12 (cobalamin) metabolism remain to date obscure. We observed transcriptomic changes of genes involved in RNA metabolism and endoplasmic reticulum stress in a neuronal cell model with impaired cobalamin metabolism. These changes were related to the subcellular mislocalization of several RNA binding proteins, including the ELAVL1/HuR protein implicated in neuronal stress, in this cell model and in patient fibroblasts with inborn errors of cobalamin metabolism and Cd320 knockout mice. The decreased interaction of ELAVL1/HuR with the CRM1/exportin protein of the nuclear pore complex and its subsequent mislocalization resulted from hypomethylation at R-217 produced by decreased S-adenosylmethionine and protein methyl transferase CARM1 and dephosphorylation at S221 by increased protein phosphatase PP2A. The mislocalization of ELAVL1/HuR triggered the decreased expression of SIRT1 deacetylase and genes involved in brain development, neuroplasticity, myelin formation, and brain aging. The mislocalization was reversible upon treatment with siPpp2ca, cobalamin, S-adenosylmethionine, or PP2A inhibitor okadaic acid. In conclusion, our data highlight the key role of the disruption of ELAVL1/HuR nuclear export, with genomic changes consistent with the effects of inborn errors of Cbl metabolisms on brain development, neuroplasticity and myelin formation.
Assuntos
Transporte Biológico/genética , Proteína Semelhante a ELAV 1/metabolismo , Carioferinas/metabolismo , Doenças Metabólicas/genética , Proteínas de Ligação a RNA/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Vitamina B 12/metabolismo , Animais , Encéfalo/patologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Estresse do Retículo Endoplasmático/genética , Humanos , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácido Okadáico/farmacologia , Fosforilação , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/farmacologia , RNA Mensageiro/metabolismo , S-Adenosilmetionina/farmacologia , Sirtuína 1/biossíntese , Proteína Exportina 1RESUMO
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.
Assuntos
Envelhecimento/patologia , Comportamento Animal , Disfunção Cognitiva/prevenção & controle , Ácido Fólico/metabolismo , Neurogênese , Deficiência de Vitamina B 12/complicações , Animais , Animais Recém-Nascidos , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/patologia , Feminino , Masculino , Aprendizagem em Labirinto , Gravidez , Ratos , Ratos Wistar , Vitamina B 12/metabolismo , Vitaminas/metabolismoRESUMO
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.
Assuntos
Encéfalo/metabolismo , Encéfalo/fisiopatologia , Deficiências Nutricionais/complicações , Deficiências Nutricionais/metabolismo , Desenvolvimento Fetal , Transtornos do Neurodesenvolvimento/etiologia , Transtornos do Neurodesenvolvimento/metabolismo , Animais , Cognição , Deficiências Nutricionais/etiologia , Dieta , Modelos Animais de Doenças , Feminino , Deficiência de Ácido Fólico/complicações , Deficiência de Ácido Fólico/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Masculino , Aprendizagem em Labirinto , RatosRESUMO
Deficiency in methyl donor (folate and vitamin B12) and in vitamin D is independently associated with altered bone development. Previously, methyl donor deficiency (MDD) was shown to weaken the activity of nuclear receptor coactivator, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), for nuclear signaling in rat pups, including estrogen receptor-α and estrogen-related receptor-α; its effect on vitamin D receptor (VDR) signaling, however, is unknown. We studied bone development under MDD in rat pups and used human MG-63 preosteoblast cells to better understand the associated molecular mechanism. In young rats, MDD decreased total body bone mineral density, reduced tibia length, and impaired growth plate maturation, and in preosteoblasts, MDD slowed cellular proliferation. Mechanistic studies revealed decreased expression of VDR, estrogen receptor-α, PGC1α, arginine methyltransferase 1, and sirtuin 1 in both rat proximal diaphysis of femur and in MG-63, as well as decreased nuclear VDR-PGC1α interaction in MG-63 cells. The weaker VDR-PGC1α interaction could be attributed to the reduced protein expression, imbalanced PGC1α methylation/acetylation, and nuclear VDR sequestration by heat shock protein 90 (HSP90). These together compromised bone development, which is reflected by lowered bone alkaline phosphatase and increased proadipogenic peroxisome proliferator-activated receptor-γ, adiponectin, and estrogen-related receptor-α expression. Of interest, under MDD, the bone development effects of 1,25-dihydroxyvitamin D3 were ineffectual and these could be rescued by the addition of S-adenosylmethionine, which restored expression of arginine methyltransferase 1, PGC1α, adiponectin, and HSP90. In conclusion, MDD inactivates vitamin D signaling via both disruption of VDR-PGC1α interaction and sequestration of nuclear VDR attributable to HSP90 overexpression. These data suggest that vitamin D treatment may be ineffective under MDD.-Feigerlova, E., Demarquet, L., Melhem, H., Ghemrawi, R., Battaglia-Hsu, S.-F., Ewu, E., Alberto, J.-M., Helle, D., Weryha, G., Guéant, J.-L. Methyl donor deficiency impairs bone development via peroxisome proliferator-activated receptor-γ coactivator-1α-dependent vitamin D receptor pathway.
Assuntos
Desenvolvimento Ósseo/fisiologia , PPAR gama/metabolismo , Receptores de Calcitriol/metabolismo , Animais , Calcitriol/metabolismo , Linhagem Celular Tumoral , Feminino , Proteínas de Choque Térmico/metabolismo , Humanos , Ratos , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
BACKGROUND: Methyl donor deficiency (MDD) aggravates experimental colitis in rats and increases endoplasmic reticulum (ER) stress through decreased sirtuin 1 (SIRT1) in neuronal cells and myocardium. ER stress plays a key role in IBD pathogenesis. AIM: We investigated whether the influence of MDD on colitis resulted from an ER stress response triggered by decreased SIRT1 expression. DESIGN: The unfolded protein response (UPR), chaperones proteins, heat shock factor protein 1 (HSF1) and SIRT1 were examined in rats with MDD and dextran sulfate sodium (DSS)-induced colitis in a Caco-2 cell model with stable expression of transcobalamin-oleosin (TO) chimera, which impairs cellular availability of vitamin B12, and in IBD. The effects of SIRT1 activation were studied both in vitro and in vivo. RESULTS: MDD aggravated DSS-induced colitis clinically, endoscopically and histologically. MDD activated ER stress pathways, with increased phosphorylate-PKR-like ER kinase, P-eiF-2α, P-IRE-1α, activating transcription factor (ATF)6, XBP1-S protein and ATF4 mRNA expression levels in rats. This was accompanied by reduced SIRT1 expression level and greater acetylation of HSF1, in relation with a dramatic decrease of chaperones (binding immunoglobulin protein (BIP), heat shock protein (HSP)27 and HSP90). Adding either vitamin B12, S-adenosylmethionine or an SIRT1 activator (SRT1720) reduced the UPR in vitro. In rats, SIRT1 activation by SRT1720 prevented colitis by reducing HSF1 acetylation and increasing expression of BIP, HSP27 and HSP90. Immunohistochemistry showed impaired expression of SIRT1 in the colonic epithelium of patients with IBD. CONCLUSIONS: SIRT1 is a master regulator of ER stress and severity of experimental colitis in case of MDD. It could deserve further interest as a therapeutic target of IBD.
Assuntos
Biópsia , Colite/induzido quimicamente , Dieta , Estresse do Retículo Endoplasmático , Sirtuína 1/metabolismo , Animais , Western Blotting , Células CACO-2 , Células Cultivadas , Deficiência de Colina , Proteínas de Ligação a DNA , Sulfato de Dextrana/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Deficiência de Ácido Fólico , Humanos , Técnicas Imunoenzimáticas , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição , Transfecção , Resposta a Proteínas não Dobradas , Deficiência de Vitamina B 12 , eIF-2 QuinaseRESUMO
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.
Assuntos
Encéfalo/metabolismo , Receptor alfa de Estrogênio/metabolismo , Deficiência de Ácido Fólico , Regulação da Expressão Gênica no Desenvolvimento , Lactação , Sinapsinas/biossíntese , Deficiência de Vitamina B 12 , Animais , Encéfalo/embriologia , Encéfalo/patologia , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/antagonistas & inibidores , Feminino , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , PPAR gama/metabolismo , Gravidez , RatosRESUMO
The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as 'cblG-variant'. In four 'cblG-variant' cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesis.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Anemia Megaloblástica/genética , Proteínas de Transporte/metabolismo , Isoformas de Proteínas/metabolismo , Deficiência de Vitamina B 12/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/química , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Sítios de Ligação/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Hidroxocobalamina/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Oxirredutases , Ligação Proteica/genética , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína , Vitamina B 12/análogos & derivados , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/genéticaRESUMO
Early deficiency of the methyl donors folate and vitamin B12 produces hyperhomocysteinemia and cognitive and motor disorders in 21-day-old rat pups from dams fed a diet deficient in methyl donors during gestation and lactation. These disorders are associated with impaired neurogenesis and altered synaptic plasticity in cerebellum. We aimed to investigate whether these disorders could be related to impaired expression of neurosteroidogenesis-associated proteins, key regulator receptors, and some steroid content in the cerebellum. The methyl donor deficiency produced a decreased concentration of folate and vitamin B12, along with accumulation of homocysteine in Purkinje cells in both sexes, whereas the S-adenosylmethionine/S-adenosylhomocysteine ratio was reduced only in females. The transcription level and protein expression of StAR, aromatase, ERα, ERß, and LH receptors were decreased only in females, with a marked effect in Purkinje cells, as shown by immunohistochemistry. Consistently, reduced levels of estradiol and pregnenolone were measured in cerebellar extracts of females only. The decreased expression levels of the transcriptional factors CREB, phospho-CREB, and SF-1, the lesser increase of cAMP concentration, and the lower level of phospho-PKC in the cerebellum of deficient females suggest that the activation of neurosteroidogenesis via cAMP-mediated signaling pathways associated with LHR activation would be altered. In conclusion, a gestational methyl donor deficiency impairs neurosteroidogenesis in cerebellum in a sex-dependent manner.
Assuntos
Cerebelo/metabolismo , AMP Cíclico/fisiologia , Deficiência de Ácido Fólico/metabolismo , Neurotransmissores/biossíntese , Transdução de Sinais/fisiologia , Deficiência de Vitamina B 12/metabolismo , Animais , Estradiol/metabolismo , Feminino , Microssomos/metabolismo , Mitocôndrias/metabolismo , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Pregnenolona/metabolismo , Ratos , Ratos Wistar , Transcrição Gênica/genética , Transcrição Gênica/fisiologiaRESUMO
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.
Assuntos
Animais Recém-Nascidos/metabolismo , Metilação de DNA/fisiologia , Neurotransmissores/biossíntese , Transtornos do Olfato/etiologia , Bulbo Olfatório/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Animais , Apoptose , Aromatase/análise , Aromatase/genética , Dieta , Receptor alfa de Estrogênio/análise , Receptor alfa de Estrogênio/genética , Feminino , Expressão Gênica , Homocisteína/metabolismo , Lactação , Masculino , Metilação , Neurogênese , Fosfoproteínas/análise , Fosfoproteínas/genética , Gravidez , Ratos , Ratos Wistar , DesmameRESUMO
BACKGROUND: The high variability in clinical and metabolic presentations of inborn errors of cobalamin (cbl) metabolism (IECM), such as the cblC/epicblC types with combined deficits in methylmalonyl-coA mutase (MUT) and methionine synthase (MS), are not well understood. They could be explained by the impaired expression/activity of enzymes from other metabolic pathways. METHODS: We performed metabolomic, genomic, proteomic, and post-translational modification (PTM) analyses in fibroblasts from three cblC cases and one epi-cblC case compared with three cblG cases with specific MS deficits and control fibroblasts. FINDINGS: CblC patients had metabolic profilings consistent with altered urea cycle, glycine, and energy mitochondrial metabolism. Metabolomic analysis showed partial disruption and increased glutamate/ketoglutarate anaplerotic pathway of the tricarboxylic acid cycle (TCA), in patient fibroblasts. RNA-seq analysis showed decreased expression of MT-TT (mitochondrial tRNA threonine), MT-TP (mitochondrial tRNA proline), OXCT1 (succinyl CoA:3-oxoacid CoA transferase deficiency), and MT-CO1 (cytochrome C oxidase subunit 1). Proteomic changes were observed for key mitochondrial enzymes, including NADH:ubiquinone oxidoreductase subunit A8 (NDUFA8), carnitine palmitoyltransferase 2 (CPT2), and ubiquinol-cytochrome C reductase, complex III subunit X (UQCR10). Propionaldehyde addition in ornithine aminotransferase was the predominant PTM in cblC cells and could be related with the dramatic cellular increase in propionate and methylglyoxalate. It is consistent with the decreased concentration of ornithine reported in 3 cblC cases. Whether the changes detected after multi-omic analyses underlies clinical features in cblC and cblG types of IECM, such as peripheral and central neuropathy, cardiomyopathy, pulmonary hypertension, development delay, remains to be investigated. INTERPRETATION: The omics-related effects of IECM on other enzymes and metabolic pathways are consistent with the diversity and variability of their age-related metabolic and clinical manifestations. PTMs are expected to produce cumulative effects, which could explain the influence of age on neurological manifestations. FUNDING: French Agence Nationale de la Recherche (Projects PREDICTS and EpiGONE) and Inserm.
Assuntos
Multiômica , Vitamina B 12 , Humanos , Vitamina B 12/metabolismo , Proteômica , Oxirredutases/metabolismo , Fibroblastos/metabolismo , RNA de Transferência/metabolismoRESUMO
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.
Assuntos
Deficiência de Ácido Fólico/metabolismo , Ácido Fólico/farmacologia , Hipocampo/citologia , Homocisteína/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Hep G2 , Humanos , Imuno-Histoquímica , Neurônios/metabolismo , Ligação Proteica , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Vitamina B 12/farmacologiaRESUMO
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.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Disfunção Cognitiva , Camundongos , Gravidez , Animais , Feminino , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Vitamina B 12RESUMO
BACKGROUND: MTR gene encodes the cytoplasmic enzyme methionine synthase, which plays a pivotal role in the methionine cycle of one-carbon metabolism. This cycle holds a significant importance in generating S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), the respective universal methyl donor and end-product of epigenetic transmethylation reactions. cblG type of inherited disorders of vitamin B12 metabolism due to mutations in MTR gene exhibits a wide spectrum of symptoms, including a retinopathy unresponsive to conventional therapies. METHODS: To unveil the underlying epigenetic pathological mechanisms, we conducted a comprehensive study of epigenomic-wide alterations of DNA methylation by NGS of bisulfited retinal DNA in an original murine model with conditional Mtr deletion in retinal tissue. Our focus was on postnatal day 21, a critical developmental juncture for ocular structure refinement and functional maturation. RESULTS: We observed delayed eye opening and impaired visual acuity and alterations in the one-carbon metabolomic profile, with a notable dramatic decline in SAM/SAH ratio predicted to impair DNA methylation. This metabolic disruption led to epigenome-wide changes in genes involved in eye development, synaptic plasticity, and retinoid metabolism, including promoter hypermethylation of Rarα, a regulator of Lrat expression. Consistently, we observed a decline in cone photoreceptor cells and reduced expression of Lrat, Rpe65, and Rdh5, three pivotal genes of eye retinoid metabolism. CONCLUSION: We introduced an original in vivo model for studying cblG retinopathy, which highlighted the pivotal role of altered DNA methylation in eye development, cone differentiation, and retinoid metabolism. This model can be used for preclinical studies of novel therapeutic targets.
Assuntos
Células Fotorreceptoras Retinianas Cones , Doenças Retinianas , Camundongos , Animais , Células Fotorreceptoras Retinianas Cones/metabolismo , Camundongos Transgênicos , Epigenoma , Metilação de DNA , S-Adenosilmetionina/metabolismo , Doenças Retinianas/metabolismo , Carbono/metabolismo , Retinoides/metabolismoRESUMO
BACKGROUND & AIMS: Folate and cobalamin are methyl donors needed for the synthesis of methionine, which is the precursor of S-adenosylmethionine, the substrate of methylation in epigenetic, and epigenomic pathways. Methyl donor deficiency produces liver steatosis and predisposes to metabolic syndrome. Whether impaired fatty acid oxidation contributes to this steatosis remains unknown. METHODS: We evaluated the consequences of methyl donor deficient diet in liver of pups from dams subjected to deficiency during gestation and lactation. RESULTS: The deprived rats had microvesicular steatosis, with increased triglycerides, decreased methionine synthase activity, S-adenosylmethionine, and S-adenosylmethionine/S-adenosylhomocysteine ratio. We observed no change in apoptosis markers, oxidant and reticulum stresses, and carnityl-palmitoyl transferase 1 activity, and a decreased expression of SREBP-1c. Impaired beta-oxidation of fatty acids and carnitine deficit were the predominant changes, with decreased free and total carnitines, increased C14:1/C16 acylcarnitine ratio, decrease of oxidation rate of palmitoyl-CoA and palmitoyl-L-carnitine and decrease of expression of novel organic cation transporter 1, acylCoA-dehydrogenase and trifunctional enzyme subunit alpha and decreased activity of complexes I and II. These changes were related to lower protein expression of ER-α, ERR-α and HNF-4α, and hypomethylation of PGC-1α co-activator that reduced its binding with PPAR-α, ERR-α, and HNF-4α. CONCLUSIONS: The liver steatosis resulted predominantly from hypomethylation of PGC1-α, decreased binding with its partners and subsequent impaired mitochondrial fatty acid oxidation. This link between methyl donor deficiency and epigenomic deregulations of energy metabolism opens new insights into the pathogenesis of fatty liver disease, in particular, in relation to the fetal programming hypothesis.
Assuntos
Receptor alfa de Estrogênio/fisiologia , Ácidos Graxos/metabolismo , Fator 4 Nuclear de Hepatócito/fisiologia , Fígado/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Estrogênio/fisiologia , Fatores de Transcrição/metabolismo , Animais , Transporte de Elétrons , Estresse do Retículo Endoplasmático , Metabolismo Energético , Receptor alfa de Estrogênio/análise , Fígado Gorduroso/etiologia , Ácido Fólico/sangue , Fator 4 Nuclear de Hepatócito/análise , Metilação , Oxirredução , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Ratos , Ratos Wistar , Receptores de Estrogênio/análise , Vitamina B 12/sangue , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Cardiomyopathies occur by mechanisms that involve inherited and acquired metabolic disorders. Both folate and vitamin B12 deficiencies are associated with left ventricular dysfunction, but mechanisms that underlie these associations are not known. However, folate and vitamin B12 are methyl donors needed for the synthesis of S-adenosylmethionine, the substrate required for the activation by methylation of regulators of energy metabolism. We investigated the consequences of a diet lacking methyl donors in the myocardium of weaning rats from dams subjected to deficiency during gestation and lactation. Positron emission tomography (PET), microscope and metabolic examinations evidenced a myocardium hypertrophy, with cardiomyocyte enlargement, disturbed mitochondrial alignment, lipid droplets, decreased respiratory activity of complexes I and II and decreased S-adenosylmethionine:S-adenosylhomocysteine ratio. The increased concentrations of triglycerides and acylcarnitines were consistent with a deficit in fatty acid oxidation. These changes were explained by imbalanced acetylation/methylation of PGC-1α, through decreased expression of SIRT1 and PRMT1 and decreased S-adenosylmethionine:S-adenosylhomocysteine ratio, and by decreased expression of PPARα and ERRα. The main changes of the myocardium proteomic study were observed for proteins regulated by PGC-1α, PPARs and ERRα. These proteins, namely trifunctional enzyme subunit α-complex, short chain acylCoA dehydrogenase, acylCoA thioesterase 2, fatty acid binding protein-3, NADH dehydrogenase (ubiquinone) flavoprotein 2, NADH dehydrogenase (ubiquinone) 1α-subunit 10 and Hspd1 protein, are involved in fatty acid oxidation and mitochondrial respiration. In conclusion, the methyl donor deficiency produces detrimental effects on fatty acid oxidation and energy metabolism of myocardium through imbalanced methylation/acetylation of PGC-1α and decreased expression of PPARα and ERRα. These data are of pathogenetic relevance to perinatal cardiomyopathies.
Assuntos
Cardiomiopatias/etiologia , Proteína-Arginina N-Metiltransferases/fisiologia , Proteínas de Ligação a RNA/metabolismo , Sirtuína 1/fisiologia , Fatores de Transcrição/metabolismo , Deficiência de Vitaminas do Complexo B/complicações , Acetilação , Animais , Apoptose/fisiologia , Cardiomiopatias/diagnóstico por imagem , Cardiomiopatias/metabolismo , Respiração Celular/fisiologia , Metabolismo Energético/fisiologia , Ácidos Graxos/metabolismo , Feminino , Ácido Fólico/sangue , Homocisteína/metabolismo , Metilação , Mitocôndrias Cardíacas/metabolismo , Oxirredução , PPAR alfa/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Tomografia por Emissão de Pósitrons/métodos , Proteômica/métodos , Ratos , Ratos Wistar , Receptores de Estrogênio/metabolismo , Estresse Fisiológico/fisiologia , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Vitamin B12 (cobalamin, Cbl) is indispensable for proper brain development and functioning, suggesting that it has neurotrophic effects beside its well-known importance in metabolism. The molecular basis of these effects remains hypothetical, one of the reasons being that no efficient cell model has been made available for investigating the consequences of B12 cellular deficiency in neuronal cells. Here, we designed an approach by stable transfection of NIE115 neuroblastoma cells to impose the anchorage of a chimeric B12-binding protein, transcobalamin-oleosin (TO) to the intracellular membrane. This model produced an intracellular sequestration of B12 evidenced by decreased methyl-Cbl and S-adenosylmethionine and increased homocysteine and methylmalonic acid concentrations. B12 deficiency affected the proliferation of NIE115 cells through an overall increase in catalytic protein phosphatase 2A (PP2A), despite its demethylation. It promoted cellular differentiation by improving initial outgrowth of neurites and, at the molecular level, by augmenting the levels of proNGF and p75(NTR). The up-regulation of PP2A and pro-nerve growth factor (NGF) triggered changes in ERK1/2 and Akt, two signaling pathways that influence the balance between proliferation and neurite outgrowth. Compared with control cells, a 2-fold increase of p75(NTR)-regulated intramembraneous proteolysis (RIP) was observed in proliferating TO cells (P < 0.0001) that was associated with an increased expression of two tumor necrosis factor (TNF)-alpha converting enzyme (TACE) secretase enzymes, Adam 10 and Adam 17. In conclusion, our data show that B12 cellular deficiency produces a slower proliferation and a speedier differentiation of neuroblastoma cells through interacting signaling pathways that are related with increased expression of PP2A, proNGF, and TACE.
Assuntos
Proteínas ADAM/metabolismo , Diferenciação Celular , Proliferação de Células , Fator de Crescimento Neural/metabolismo , Neuroblastoma/patologia , Proteína Fosfatase 2/metabolismo , Precursores de Proteínas/metabolismo , Regulação para Cima , Deficiência de Vitamina B 12/patologia , Proteína ADAM17 , Linhagem Celular Tumoral , Humanos , Neuroblastoma/metabolismo , Plasmídeos , Deficiência de Vitamina B 12/metabolismoRESUMO
Previously, the in vitro growth of cancer stem cells in the form of tumor spheres from five different brain cancer cell lines was found to be methionine-dependent. As this earlier work indicated that ALDH1L2, a folate-dependent mitochondria aldehyde dehydrogenase gene, is upregulated in glioblastoma stem cells, we invalidated this gene using CRISPR-cas 9 technique in this present work. We reported here that this invalidation was effective in U251 glioblastoma cells, and no cas9 off target site could be detected by genome sequencing of the two independent knockout targeting either exon I or exon III. The knockout of ALDH1L2 gene in U251 cells rendered the growth of the cancer stem cells of U251 methionine independent. In addition, a much higher ROS (reactive oxygen radicals) level can be detected in the knockout cells compared to the wild type cells. Our evidence here linked the excessive ROS level of the knockout cells to reduced total cellular NADPH. Our evidence suggested also that the cause of the slower growth of the knockout turmor sphere may be related to its partial differentiation.
Assuntos
Glioblastoma , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Humanos , Metionina/metabolismo , Células-Tronco Neoplásicas/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Inflammatory bowel diseases (IBD) result from complex interactions between environmental and genetic factors. Low blood levels of vitamin B12 and folate and genetic variants of related target enzymes are associated with IBD risk, in population studies. To investigate the underlying mechanisms, we evaluated the effects of a methyl-deficient diet (MDD, folate, vitamin B12 and choline) in an experimental model of colitis induced by dextran sodium sulphate (DSS), in rat pups from dams subjected to the MDD during gestation and lactation. Four groups were considered (n = 12-16 per group): C DSS(-) (control/DSS(-)), D DSS(-) (deficient/DSS(-)), C DSS(+) (control/DSS(+)) and D DSS(+) (deficient/DSS(+)). Changes in apoptosis, oxidant stress and pro-inflammatory pathways were studied within colonic mucosa. In rat pups, the MDD produced a decreased plasma concentration of vitamin B12 and folate and an increased homocysteine (7.8 ± 0.9 versus 22.6 ± 1.2 µmol/l, P < 0.001). The DSS-induced colitis was dramatically more severe in the D DSS(+) group compared with each other group, with no change in superoxide dismutase and glutathione peroxidase activity, but decreased expression of caspase-3 and Bax, and increased Bcl-2 levels. The mRNA levels of tumour necrosis factor (TNF)-α and protein levels of p38, cytosolic phospolipase A2 and cyclooxygenase 2 were significantly increased in the D DSS(+) pups and were accompanied by a decrease in the protein level of tissue inhibitor of metalloproteinases (TIMP)3, a negative regulator of TNF-α. MDD may cause an overexpression of pro-inflammatory pathways, indicating an aggravating effect of folate and/or vitamin B12 deficiency in experimental IBD. These findings suggest paying attention to vitamin B12 and folate deficits, frequently reported in IBD patients.
Assuntos
Deficiência de Colina , Colite Ulcerativa , Deficiência de Ácido Fólico , Deficiência de Vitamina B 12 , Animais , Apoptose/efeitos dos fármacos , Caspase 3/biossíntese , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Colo/efeitos dos fármacos , Ciclo-Oxigenase 2/biossíntese , Sulfato de Dextrana/farmacologia , Dieta , Ácido Fólico/sangue , Glutationa Peroxidase/metabolismo , Homocisteína/sangue , Estresse Oxidativo/efeitos dos fármacos , Fosfolipases A2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Vitamina B 12/sangue , Proteína X Associada a bcl-2/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/biossínteseRESUMO
SCOPE: Vitamin B12 and folate (methyl donors) deficiency is frequent during pregnancy. Experimental rat models with methyl donor deficit during pregnancy and lactation (Initial methyl donor deficit (iMDD)) produce impaired myocardium fatty acid oxidation and mitochondrial energy metabolism at weaning. METHODS AND RESULTS: The consequences of iMDD on heart of rat pups under normal diet after weaning and high fat diet (HF) between day (D) 50 and D185 are investigated. iMDD/HF induces increased histological fibrosis and increased B-type natriuretic peptide blood level. Inflammation is evidenced by increased protein expression of NFkB, Caspase1, and IL1ß and fibrosis by increased expression of αSMA, col1a1, and col1a2 in females, but not in males. Fibrosis is related to increased angiotensin at D50 and D185 and increased protein expression of TGFB1 and AT1 angiotensin receptors at D185. The limited fibrosis in males is consistent with increased expression of AT2, the antagonist receptor of AT1. The increased expression of GLUT4 and decreased expression of PGC1α and PPARα reflect a shift from fatty acid oxidation to glycolysis. CONCLUSION: Developmental programming by iMDD produces cardiomyopathy in female offspring exposed to HF. The cardiomyopathy is linked to inflammation and fibrosis through angiotensin-AT2 and TGFB1 pathways and alteration of energy metabolism.