Sébastien Blaise

Neonatal Hypoxia Triggers Transient Apoptosis Followed by Neurogenesis in the Rat CA1 Hippocampus

Continuous generation of new neurons has been demonstrated in the adult mammalian brain, and this process was shown to be stimulated by various pathologic conditions, including cerebral ischemia. Because brain oxygen deprivation is particularly frequent in neonates and represents the primary event of asphyxia, we analyzed long-term consequences of transient hypoxia in the newborn rat. Within 24 h after birth, animals were exposed to 100% N2 for 20 min at 36°C, and temporal changes in the vulnerable CA1 hippocampus were monitored. Cell density measurements revealed delayed cell death in the pyramidal cell layer reflecting apoptosis, as shown by characteristic nuclear morphology and expression levels of Bcl-2, Bax, and caspase-3. Neuronal loss was confirmed by reduced density of neuron-specific enolase (NSE)–labeled cells, and peaked by 1 wk post insult, to reach 27% of total cells. A gradual recovery then occurred, and no significant difference in cell density could be detected between controls and hypoxic rats at postnatal d 21. Repeated injections of bromodeoxyuridine (50 mg/kg) showed that newly divided cells expressing neuronal markers increased by 225% in the germinative subventricular zone, and they tended to migrate along the posterior periventricle toward the hippocampus. Therefore, transient hypoxia in the newborn rat triggered apoptosis in the CA1 hippocampus followed by increased neurogenesis and apparent anatomical recovery, suggesting that the developing brain may have a high capacity for self-repair.

Methyl donor deficiency impairs fatty acid oxidation through PGC-1α hypomethylation and decreased ER-α, ERR-α, and HNF-4α in the rat liver.

BACKGROUND & AIMSnFolate 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.nnnMETHODSnWe evaluated the consequences of methyl donor deficient diet in liver of pups from dams subjected to deficiency during gestation and lactation.nnnRESULTSnThe 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.nnnCONCLUSIONSnThe 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.

Mild, non-lesioning transient hypoxia in the newborn rat induces delayed brain neurogenesis associated with improved memory scores

Although neonatal hypoxia can lead to brain damage, mild hypoxic episodes may be beneficial, as illustrated by tolerance induction by preconditioning, a process that might involve neurogenesis. To examine if brief hypoxia in newborn rats could stimulate the generation of neurons, pups were exposed for 5 min to 100% N2. Cell density and apoptosis were monitored in various brain regions and cell proliferation was studied by the incorporation of bromodeoxyuridine. Hypoxia did not result in detectable cell death but promoted cell proliferation in the ensuing three weeks in the subventricular zone and hippocampal dentate gyrus, with increased cell density in hippocampus CA1 pyramidal cells and granular layer of the dentate gyrus. Newly generated cells expressed neuronal markers (NeuroD or neuronal nuclear antigen) and were able to migrate from germinative zones to specific sites, in particular from the subventricular zone to the CA1 layer along the posterior periventricle. Neurogenesis was associated with an early activation of the extracellular regulated kinase 1/2 pathway, and pre-hypoxic administration of U0126, an inhibitor of mitogen-activated protein kinase kinase, impaired hypoxia effect on cell proliferation. Neurobehavioral capacities of hypoxic rats paralleled those of controls, but early exposure to hypoxia was associated with significantly improved memory retrieval scores at 40 days. In conclusion, brief neonatal hypoxia may trigger delayed generation of potentially functional neurons without concomitant cell death. This may constitute an interesting model for studying cell key events involved in the induction of neurogenesis.

Mild neonatal hypoxia exacerbates the effects of vitamin-deficient diet on homocysteine metabolism in rats.

Elevated plasma homocysteine has been linked to pregnancy complications and developmental diseases. Whereas hyperhomocysteinemia is frequently observed in populations at risk of malnutrition, hypoxia may alter the remethylation of homocysteine in hepatocytes. We aimed to investigate the combined influences of early deficiency in nutritional determinants of hyperhomocysteinemia and of neonatal hypoxia on homocysteine metabolic pathways in developing rats. Dams were fed a standard diet or a diet deficient in vitamins B12, B2, folate, month, and choline from 1 mo before pregnancy until weaning of the offspring. The pups were divided into four treatment groups corresponding to “no hypoxia/standard diet,” “hypoxia (100% N2 for 5 min at postnatal d 1)/standard diet,” “no hypoxia/deficiency,” and “hypoxia/deficiency,” and homocysteine metabolism was analyzed in their liver at postnatal d 21. Hypoxia increased plasma homocysteine in deficient pups (21.2 ± 1.6 versus 13.3 ± 1.2 μM, p < 0.05). Whereas mRNA levels of cystathionine β-synthase remained unaltered, deficiency reduced the enzyme activity (48.7 ± 2.9 versus 83.6 ± 6.3 nmol/h/mg, p < 0.01), an effect potentiated by hypoxia (29.4 ± 4.7 nmol/h/mg, p < 0.05). The decrease in methylene-tetrahydrofolate reductase activity measured in deficient pups was attenuated by hypoxia (p < 0.05), and methionine-adenosyltransferase activity was slightly reduced only in the “hypoxia/deficiency” group (p < 0.05). Finally, hypoxia enhanced the deficiency-induced drop of the S-adenosylmethionine/S-adenosylhomocysteine ratio, which is known to influence DNA methylation and gene expression. In conclusion, neonatal hypoxia may increase homocysteinemia mainly by decreasing homocysteine transsulfuration in developing rats under methyl-deficient regimen. It could therefore potentiate the well-known adverse effects of hyperhomocysteinemia.

Methyl Donor Deficiency Affects Fetal Programming of Gastric Ghrelin Cell Organization and Function in the Rat

Methyl donor deficiency (MDD) during pregnancy influences intrauterine development. Ghrelin is expressed in the stomach of fetuses and influences fetal growth, but MDD influence on gastric ghrelin is unknown. We examined the gastric ghrelin system in MDD-induced intrauterine growth retardation. By using specific markers and approaches (such as periodic acid-Schiff, bromodeoxyuridine, homocysteine, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunostaining, reverse transcription-polymerase chain reaction), we studied the gastric oxyntic mucosa cellular organization and ghrelin gene expression in the mucosa in 20-day-old fetuses and weanling pups, and plasma ghrelin concentration in weanling rat pups of dams either normally fed or deprived of choline, folate, vitamin B6, and vitamin B12 during gestation and suckling periods. MDD fetuses weighed less than controls; the weight deficit reached 57% at weaning (P < 0.001). Both at the end of gestation and at weaning, they presented with an aberrant gastric oxyntic mucosa formation with loss of cell polarity, anarchic cell migration, abnormal progenitor differentiation, apoptosis, and signs of surface layer erosion. Ghrelin cells were abnormally located in the pit region of oxyntic glands. At weaning, plasma ghrelin levels were decreased (-28%; P < 0.001) despite unchanged mRNA expression in the stomach. This decrease was associated with lower body weight. Taken together, these data indicate that one mechanism through which MDD influences fetal programming is the remodeling of gastric cellular organization, leading to dysfunction of the ghrelin system and dramatic effects on growth.

In Vivo Evidence That TRAF4 Is Required for Central Nervous System Myelin Homeostasis

Tumor Necrosis Factor Receptor-Associated Factors (TRAFs) are major signal transducers for the TNF and interleukin-1/Toll-like receptor superfamilies. However, TRAF4 does not fit the paradigm of TRAF function in immune and inflammatory responses. Its physiological and molecular functions remain poorly understood. Behavorial analyses show that TRAF4-deficient mice (TRAF4-KO) exhibit altered locomotion coordination typical of ataxia. TRAF4-KO central nervous system (CNS) ultrastructure shows strong myelin perturbation including disorganized layers and disturbances in paranode organization. TRAF4 was previously reported to be expressed by CNS neurons. Using primary cell culture, we now show that TRAF4 is also expressed by oligodendrocytes, at all stages of their differentiation. Moreover, histology and electron microscopy show degeneration of a high number of Purkinje cells in TRAF4-KO mice, that was confirmed by increased expression of the Bax pro-apoptotic marker (immunofluorescence), TUNEL analysis, and caspase-3 activation and PARP1 cleavage (western blotting). Consistent with this phenotype, MAG and NogoA, two myelin-induced neurite outgrowth inhibitors, and their neuron partners, NgR and p75NTR were overexpressed (Q-RT-PCR and western blotting). The strong increased phosphorylation of Rock2, a RhoA downstream target, indicated that the NgR/p75NTR/RhoA signaling pathway, known to induce actin cytoskeleton rearrangement that favors axon regeneration inhibition and neuron apoptosis, is activated in the absence of TRAF4 (western blotting). Altogether, these results provide conclusive evidence for the pivotal contribution of TRAF4 to myelination and to cerebellar homeostasis, and link the loss of TRAF4 function to demyelinating or neurodegenerative diseases.

Vitamin B deficiency causes neural cell loss and cognitive impairment in the developing rat

The article by Troen et al. (1) adds interesting evidence that vitamin-B deficiency is associated with selective brain damage and cognitive decline. It reports a rarefaction of hippocampal microvasculature without neurodegeneration or gliosis in male C57BL6/J mice fed a B-vitamin-deficient diet. Cognitive impairment was shown by using a Morris water maze. According to the authors, this may result from the microvascular rarefaction. In a recent article (2) we showed that rat pups from dams fed …

Short hypoxia could attenuate the adverse effects of hyperhomocysteinemia on the developing rat brain by inducing neurogenesis.

Gestational deficiency in methyl donors such as folate and vitamin B12 impairs homocysteine metabolism and can alter brain development in the progeny. Since short hypoxia has been shown to be neuroprotective in preconditioning studies, we aimed to investigate the effects of brief, non-lesioning neonatal hypoxia (100% N2 for 5 min) on the developing brain of rats born to dams fed either a standard diet or a diet lacking vitamins B12, B2, folate and choline until offsprings weaning. While having no influence on brain accumulation of homocysteine and concomitant apoptosis in 21-day-old deficient pups, exposure to hypoxia reduced morphological injury of the hippocampal CA1 layer. It also markedly stimulated the incorporation of bromodeoxyuridine (BrdU) in permissive areas such as the subventricular zone and the hippocampus followed by the migration of new neurons. Scores in a locomotor coordination test (days 19-21) and learning and memory behavior in the eight-arm maze (days 80-84) were found to be significantly improved in rats exposed to hypoxia in addition to the deficient diet. Therefore, by stimulating neurogenesis in rat pups, brief neonatal hypoxia appeared to attenuate the long-term effects of early exposure to a deficiency in nutritional determinants of hyperhomocysteinemia.

New Insights into Molecular Organization of Human Neuraminidase-1: Transmembrane Topology and Dimerization Ability

Neuraminidase 1 (NEU1) is a lysosomal sialidase catalyzing the removal of terminal sialic acids from sialyloconjugates. A plasma membrane-bound NEU1 modulating a plethora of receptors by desialylation, has been consistently documented from the last ten years. Despite a growing interest of the scientific community to NEU1, its membrane organization is not understood and current structural and biochemical data cannot account for such membrane localization. By combining molecular biology and biochemical analyses with structural biophysics and computational approaches, we identified here two regions in human NEU1 - segments 139–159 (TM1) and 316–333 (TM2) - as potential transmembrane (TM) domains. In membrane mimicking environments, the corresponding peptides form stable α-helices and TM2 is suited for self-association. This was confirmed with full-size NEU1 by co-immunoprecipitations from membrane preparations and split-ubiquitin yeast two hybrids. The TM2 region was shown to be critical for dimerization since introduction of point mutations within TM2 leads to disruption of NEU1 dimerization and decrease of sialidase activity in membrane. In conclusion, these results bring new insights in the molecular organization of membrane-bound NEU1 and demonstrate, for the first time, the presence of two potential TM domains that may anchor NEU1 in the membrane, control its dimerization and sialidase activity.

Impact of sialic acids on the molecular dynamic of bi-antennary and tri-antennary glycans

Sialic acids (SA) are monosaccharides that can be located at the terminal position of glycan chains on a wide range of proteins. The post-translational modifications, such as N-glycan chains, are fundamental to protein functions. Indeed, the hydrolysis of SA by specific enzymes such as neuraminidases can lead to drastic modifications of protein behavior. However, the relationship between desialylation of N-glycan chains and possible alterations of receptor function remains unexplored. Thus, the aim of the present study is to establish the impact of SA removal from N-glycan chains on their conformational behavior. We therefore undertook an in silico investigation using molecular dynamics to predict the structure of an isolated glycan chain. We performed, for the first time, 3 independent 500u2009ns simulations on bi-antennary and tri-antennary glycan chains displaying or lacking SA. We show that desialylation alters both the preferential conformation and the flexibility of the glycan chain. This study suggests that the behavior of glycan chains induced by presence or absence of SA may explain the changes in the protein function.