Grégory Pourié

Homocysteine and methylenetetrahydrofolate reductase polymorphism in Alzheimer's disease.

Homocysteine metabolism is influenced by genetic polymorphisms of the methylenetetrahydrofolate reductase (MTHFR 677 C→T and 1298 A→C) and transcobalamin genes (TCN1 776 C→G ). We evaluated the association of homocysteine with Alzheimers disease (AD) and the influence of related polymorphisms and APOE, in 180 cases and 181 controls from southern Italy. Homocysteine (upper tercile) was associated with AD risk, with an odds ratio of 2.8 (95% confidence interval (CI) 1.54–5.22, p=0.0008), which was increased 2.2- and 2.0-fold by MTHFR 677 T (odds ratio 6.28, 95% CI 2.88–16.20, p < 0.0001) and APOE &egr;4 (odds ratio: 5.60, 95% CI 1.12–28.05, p=0.0361), respectively. In conclusion, association of homocysteine with AD was aggravated by MTHFR 677 T and APOE &egr;4 alleles.

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.

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.

Enhancement of spatial learning by predator odor in mice: Involvement of amygdala and hippocampus

Olfaction has particular links with learning and memory compared with other sensory cues, due to the interrelations between their neural circuitry. The present study deals with the effects of a putative stressor (i.e. a predator odor) on visuo-spatial learning in mice. Firstly, the results show that a predator odor spread during the Morris water maze task led to learning enhancement. In addition, a stereotaxic approach was used to investigate the involvement of the amygdala in this hippocampus-dependent type of learning. Thus, the performance of mice in visuo-spatial learning under predator odor conditions was dramatically reduced by an ibotenate bilateral amygdala lesion. The involvement of the amygdala was confirmed by a reduced expression of c-fos in the CA1 hippocampus of amygdala-lesioned mice at the end of the learning procedure. Mild exposure to a predator odor during hippocampus-dependent learning therefore leads to an enhancement of performance through the co-activation of the amygdala, probably by a stress mediated mechanism.

Inhalation exposure to acetone induces selective damage on olfactory neuroepithelium in mice

Due to their specific position in the nasal cavity, the cells of olfactory neuroepithelium can be damaged by exposure to environmental airborne chemicals. However, few studies have been focused on selective damage, i.e. olfactory sensory neurons, basal cells, supporting and duct cells. As solvents are known to induce critical effects on olfactory neuroepithelium (OE), this study was designed to characterize histological and immunohistological effects induced by acetone exposure on OE in mice. Behavioral tests were conducted to evaluate olfactory sensitivity. Moreover, olfactory neuroepithelium was examined to evaluate the thickness and the total number of cells. Finally, different markers, olfactory marker protein (OMP) and proliferating cell nuclear antigen (PCNA), were used to characterize respectively olfactory sensory neurons and basal cells, and secondly to evaluate the dynamic of the tissue turnover. Results showed structural modifications, since the thickness and the number of cells in the OE were modified according to the time course of the exposure. Additionally, no changes for OMP-positive cells were observed whereas significant differences appeared for the density of PCNA-positive cells in relation to their location (main-body or basal layer of OE). These findings indicate that acetone exposure induces selective damage in olfactory neuroepithelium.

Differentiation and neural integration of hippocampal neuronal progenitors: signaling pathways sequentially involved.

In the context of their potential implication in regenerative strategies, we characterized cell mechanisms underlying the fate of embryonic rat hippocampal H19–7 progenitors in culture upon induction of their differentiation, and tested their capacities to integrate into a neuronal network in vitro. Without addition of growth factors, nearly 100% of cells expressed various neuronal markers, with a progressive rise of the expression of Synapsin I and II, suggesting that cells developed as mature neurons with synaptogenic capacities. Fully differentiated neurons were identified as glutamatergic and expressed the receptor‐associated protein PSD‐95. Quantification of ATP showed that 60% of cells died within 24 h after differentiation. Cell death was shown to imply Erk1/2‐dependent intrinsic mitochondrial apoptosis signaling pathway, with activation of caspase‐9 and ‐3, finally leading to single‐strand DNA. Surviving neurons displayed high levels of Akt, phospho‐Akt, and antiapoptotic proteins such as Bcl‐2 and Bcl‐XL, with decreased caspase activation. In the absence of trophic support, the proapoptotic death‐associated protein (DAP) kinase was dramatically stimulated by 24 h postdifferentiation, along with increased levels of p38 and phospho‐p38, and caspase reactivation. These findings show that different signaling pathways are sequentially triggered by differentiation, and highlight that ultimate cell death would involve p38 and DAP kinase activation. This was supported by the improvement of cell survival at 24‐h postdifferentiation when cells were treated by PD169316, a specific inhibitor of p38. Finally, when seeded on rat hippocampal primary cultured neurons, a significant number of differentiated H19–7 cells were able to survive and to develop cell–cell communication.

Postnatal exposure to synthetic predator odor (TMT) induces quantitative modification in fear-related behaviors during adulthood without change in corticosterone levels

Environmental stimuli and adverse experiences in early life may result in behavioral and physiological changes in adulthood. In several animal species, the odors cues are crucial in the setting of adaptive behaviors, especially towards predators. However, little is known about the effects of postnatal exposure to predator odor on the later physiological and behavioral responses to this natural stressor. Thus, the aim of this study was to investigate the effects of a postnatal exposure to synthetic predator odor (TMT) in mice pups on later adult fear-related behaviors and corticosterone levels in response to this specific stimulus. Pups postnatally exposed to only water showed later in adult life behavioral responses when exposed to TMT that were statistically different from mice that were exposed as neonates to TMT. In addition, mice exposed as neonates to TMT showed a decrease of fear-related behaviors while no differences occurred in the corticosterone levels between both groups.

Carbon dioxide effects on olfactory functioning: behavioral, histological and immunohistochemical measurements.

Most studies on toxic inhalation focus on solvent effects and few have dealt with gases on olfactory functioning. Among gases, the effects of carbon dioxide on general physiology have been well investigated contrary to the impact on olfactory neuroepithelium. Thus, this work was designed to evaluate in mice the possible effects of 3% CO(2) in two exposure periods: a 5h/day and a 12h/day conditions. Behavioral, histological and immunohistochemical observations were conducted every 2 weeks, i.e. before (W0), during (W2, W4) and after exposure (W6, W8). Firstly, behavioral evaluations of odor sensitivity showed differences in relation to the odor tested, i.e. no effect with congener urine odor and a reinforcement of 2,4,5-trimethythiazoline (TMT) (predator odor) repulsion. Secondly, histological evaluations showed a similar evolution of the epithelium thickness, i.e. a decrease along the exposure as well as during the post-exposure period and an increase of cell number (whatever the phenotype) although the kinetic appeared different in both experimental conditions. Thirdly, immunohistochemical quantification of olfactory marker protein (OMP)- and proliferating cell nuclear antigen (PCNA)-positive cells revealed that the number of mature olfactory neurons increased at the early beginning of exposure period in both conditions. While a decrease was observed in the following weeks (W4-W8) for the 12h/day condition, a stable amount of OMP-positive cells was maintained in the 5h/day condition. In contrast, the number of PCNA-positive cells followed a similar evolution, i.e. a constant decrease along the experiment. These findings indicate that the effects of CO(2) inhalation exposure are selectively dose-dependent.

Effects of pyridine inhalation exposure on olfactory epithelium in mice

Olfactory neurons in the nasal mucosa have the capacity to regenerate continuously along the lifespan by neurogenesis processes starting with progenitor cells close to the basal lamina. The cellular turnover into olfactory neuroepithelium may be modified by environmental stimuli insofar as nasal mucosa is directly in contact with airborne chemicals. However, few studies have been focused on selective changes, especially those concerning mature olfactory neurons and basal cells during specific inhalation exposure. Among chemicals, solvents are known to induce changes in smell abilities and concomitant histological and cellular modifications related to the type of molecule, concentration and time of exposure. This study was designed to characterize smell sensitivity (using behavioral tests) and immunohistochemical effects on olfactory neuroepithelium induced by pyridine exposure in mice. Olfactory marker protein (OMP) and proliferating cell nuclear antigen (PCNA) were used to characterize respectively mature olfactory neurons and basal cells. Results showed that inhalation exposure to pyridine had no impact on smell sensitivity whatever the concentration used and the time of exposure. These findings were in agreement with immunohistochemical measurements showing the same cellular kinetic whatever the condition of exposition to pyridine. Indeed, OMP-positive cells increased and PCNA-positive cells decreased as early as the beginning of exposure and cell amounts remained stable at this level until the end of exposure. These findings suggest that pyridine could have the property to rapidly activate a cellular turnover from basal cell progenitors. Rather than toxic effects, the present findings suggest that the metabolites of pyridine might have cell cycle activation properties.