Identification and characterization of novel molecular mechanisms involved in initiation and progression of myelination

Schwann cells synthesize a large amount of membrane that form a specialized structure called myelin that surrounds axons and facilitate the transmission of electrical signal along neurons in peripheral nervous system (PNS). Previous studies demonstrated that both Schwann cell differentiation and de-differentiation (in the situation of a nerve injury or demyelinating disease) are regulated by cell-intrinsic regulators including several transcription factors. In particular, the de-differentiation of mature Schwann cells is driven by the activation of multiple negative regulators of myelination including Sox2, c-Jun, Notch and Pax3, all usually expressed in immature Schwann cells and suppressed at the onset of myelination. In order to identify new regulators of myelination involved in the development of the PNS, we analyzed the gene-expression profiling data from developing PNS and from three models of demyelinating neuropathies. This analysis led to the identification of Sox4, a member of the Sox family of transcription factors, as a potential candidate. To characterize the molecular function of Sox4 in PNS, we generated two transgenic lines of mice, which overexpress Sox4 specifically in Schwann cells. Detailed analysis of these mice showed that the overexpression of Sox4 in Schwann cells causes a delay in progression of myelination between post-natal day 2 (P2) and P5. Our in vitro analysis suggested that Sox4 cDNA can be overexpressed while the protein translation is tightly regulated. Interestingly, we observed that Sox4 protein is stabilized in nerves of the CMT4C mouse, a model of the human neuropathy. We therefore crossed Sox4 transgenic mice with CMT4C mice and we observed that Sox4 overexpression exacerbated the neuropathy phenotype in these mice. While recognized as being crucial for the normal function of both neurons and myelinating glial cells, the processes that regulate the beginning of myelination and the nature of the neuro-glial cross-talk remains mostly unknown. In order to gain insight into the molecular pathways involved in the interactions between neurons and associated glial cells, we developed a neuron-glia co-culture system based on microfluidic chambers and successfully induced myelination in this system by ascorbic acid. Importantly, we observed that in addition to acting on Schwann cells, ascorbic acid also modulate neuronal/axonal NRG1/ErbB2-B3 signalling. The experimental setting used in our study thus allowed us to discover a novel phenomena of propagation for myelination in vitro. The further characterization of this event brought us to identify other compounds able to induce myelination: ADAMs secretases inhibitor GM6001 and cyclic-AMP. The results generated during my thesis project are therefore not only important for the advancement of our understanding of how the PNS works, but may also potentially help to develop new therapies aiming at improvement of PNS myelination under disease conditions. - Les cellules de Schwann synthétisent une grande quantité de membrane formant une structure spécialisée appelée myéline qui entoure les axones et facilite la transmission du signal électrique le long des neurones du système nerveux périphérique (SNP). Des études antérieures ont démontré que la différenciation et la dédifférenciation des cellules de Schwann (dans la situation d'une lésion nerveuse ou d'une maladie démyélinisante) sont régulées par des régulateurs cellulaires intrinsèques, incluant plusieurs facteurs de transcription. En particulier, la dédifférenciation des cellules de Schwann matures est contrôlée par l'activation de plusieurs régulateurs négatifs de la myélinisation dont Sox2, c-Jun, Notch et Pax3, tous habituellement exprimés dans des cellules de Schwann immatures et supprimés au début de la myélinisation. Afin d'identifier de nouveaux régulateurs de myélinisation impliqués dans le développement du SNP, nous avons analysé le profil d'expression génique durant le développement du SNP ainsi que dans trois modèles de neuropathies démyélinisantes. Cette analyse a mené à l'identification de Sox4, un membre de la famille des facteurs de transcription Sox, comme étant un candidat potentiel. Dans le but de caractériser la fonction moléculaire de Sox4 dans le SNP, nous avons généré deux lignées transgéniques de souris qui surexpriment Sox4 spécifiquement dans les cellules de Schwann. L'analyse détaillée de ces souris a montré que la surexpression de Sox4 dans les cellules de Schwann provoque un retard dans la progression de la myélinisation entre le jour postnatal 2 (P2) et P5. Notre analyse in vitro a suggéré que l'ADNc de Sox4 peut être surexprimé alors que la traduction des protéines est quand à elle étroitement régulée. De façon intéressante, nous avons observé que la protéine Sox4 est stabilisée dans les nerfs des souris CMT4C, un modèle de neuropathie humaine. Nous avons donc croisé les souris transgéniques Sox4 avec des souris CMT4C et avons observé que la surexpression de Sox4 exacerbe le phénotype de neuropathie chez ces souris. Bien que reconnus comme étant cruciaux pour le fonctionnement normal des neurones et des cellules gliales myélinisantes, les processus qui régulent le début de la myélinisation ainsi que la nature des interactions neurone-glie restent largement méconnus. Afin de mieux comprendre les mécanismes moléculaires impliqués dans les interactions entre les neurones et les cellules gliales leur étant associés, nous avons développé un système de co-culture neurone-glie basé sur des chambres microfluidiques et y avons induit avec succès la myélinisation avec de l'acide ascorbique. Étonnamment, nous avons remarqué que, en plus d'agir sur les cellules de Schwann, l'acide ascorbique module également la voie de signalisation neuronale/axonale NRG1/ErbB2-B3. Le protocole expérimental utilisé dans notre étude a ainsi permis de découvrir un nouveau phénomène de propagation de la myélinisation in vitro. La caractérisation plus poussée de ce phénomène nous a menés à identifier d'autres composés capables d'induire la myélinisation: L'inhibiteur de sécrétases ADAMs GM6001 et l'AMP cyclique. Les résultats obtenus au cours de mon projet de thèse ne sont donc pas seulement importants pour l'avancement de notre compréhension sur la façon dont le SNP fonctionne, mais peuvent aussi potentiellement aider à développer de nouvelles thérapies visant à l'amélioration de la myélinisation du SNP dans des conditions pathologiques.

Comparison of three acellular tests for assessing the oxidation potential of nanomaterials

Great effort is put into developing reliable, predictive, high-throughput, and low-cost screening approaches for the toxicity evaluation of ambient and manufactured nanoparticles (NP). These tests often consider oxidative reactivity, as oxidative stress is a well-documented pathway in particle toxicology. Based on a panel of six carbonaceous and five metal/metal oxide (Me/MeOx) nanoparticles, we: (i) compared the specifications (linearity, detection limits, repeatability) of three acellular reactivity tests using either dithiothreitol (DTT assay), dichlorofluorescein (DCFH assay), or ascorbic acid (AA-assay) as the reducing agent; and (ii) evaluated which physicochemical properties were important for explaining the observed reactivity. The selected AA assay was found to be neither sensitive nor robust enough to be retained. For the other tests, the surface properties of carbonaceous NP were of utmost importance for explaining their reactivity. In particular, the presence of "strongly reducing" surface functions explained most of its DCFH reactivity and a large part of its DTT reactivity. For the selected Me/MeOx, a different picture emerged. Whereas all particles were able to oxidize DCFH, dissolution and complexation processes could additionally influence the measured reactivity, as observed using the DTT assay. This study suggests that a combination of the DTT and DCFH assays provides complementary information relative to the quantification of the oxidative capacity of NP.

Vitamin C requirements in parenteral nutrition.

Some biochemical functions of vitamin C make it an essential component of parenteral nutrition (PN) and an important therapeutic supplement in other acute conditions. Ascorbic acid is a strong aqueous antioxidant and is a cofactor for several enzymes. The average body pool of vitamin C is 1.5 g, of which 3%-4% (40-60 mg) is used daily. Steady state is maintained with 60 mg/d in nonsmokers and 140 mg/d in smokers. Shocked surgical, trauma, and septic patients have a drastic reduction of circulating plasma ascorbate concentrations. These low concentrations require 3-g doses/d to restore normal plasma ascorbate concentrations, questioning the recommended PN dose of 100 mg/d. Determination of intravenous requirements is usually based on plasma concentrations, which are altered during the inflammatory response. There is no clear indicator of deficiency: serum or plasma ascorbate concentrations <0.3 mg/dL (20 micromol/L) indicates inadequate vitamin C status. On the basis of available pharmacokinetic data the 100 mg/d dose for patients receiving home PN and 200 mg/d for stable adult patients receiving PN are adequate, but requirements have been shown to be higher in perioperative, trauma, burn, and critically ill patients, paralleling oxidative stress. One recommendation cannot fit all categories of patients. Large vitamin C supplements may be considered in severe critical illness, major trauma, and burns because of increased requirements resulting from oxidative stress and wound healing. Future research should distinguish therapeutic use of high-dose ascorbic acid antioxidant therapy from nutritional PN requirements.

Transitory glutathione deficit during brain development induces cognitive impairment in juvenile and adult rats: relevance to schizophrenia.

Glutathione (GSH) metabolism dysfunction is one risk factor in schizophrenia. A transitory brain GSH deficit was induced in Wistar (WIS) and mutant (ODS; lacking ascorbic acid synthesis) rats using BSO (l-buthionine-(S,R)-sulfoximine) from post-natal days 5-16. When GSH was re-established to physiological levels, juvenile BSO-ODS rats were impaired in the water maze task. Long after treatment cessation, adult BSO-WIS/-ODS rats showed impaired place discrimination in the homing board with distributed visual or olfactory cues. Their accuracy was restored when a single cue marked the trained position. Similarly, more working memory errors were made by adult BSO-WIS in the radial maze when several olfactory cues were present. These results reveal that BSO rats did not suffer simple sensory impairment. They were selectively impaired in spatial memory when the task required the integration of multimodal or olfactory cues. These results, in part, resemble some of the reported olfactory discrimination and cognitive impairment in schizophrenia.

Increased vulnerability of neurones and glial cells to low concentrations of methylmercury in a prooxidant situation.

Using reaggregating rat brain cell cultures at two different stages of differentiation, we examined the biochemical effects of a 10-day treatment with nanomolar concentrations of methylmercuric chloride (monomethylmercury), in the presence or absence of promoters of hydroxyl radical formation (10 microM copper sulphate plus 100 microM ascorbate). A decrease in total protein content accounted for the general cytotoxicity of these compounds, whereas selective effects were assessed by determining the activities of cell type-specific enzymes. Methylmercury, up to 100 nM, as well as the copper ascorbate mixture, when applied separately, induced no general cytotoxicity, and only slight effects on neuronal parameters. However, when applying 100 nM methylmercury and the copper-ascorbate mixture together, a drastic decrease in neuronal and glial parameters was found. Under these conditions, the content of reactive oxygen species, assessed by 2',7'-dichlorofluorescin oxidation, increased greatly, while the activities of antioxidant enzymes decreased. In the presence of copper and ascorbate, differentiated cultures appeared more resistant than immature ones to low methylmercury concentrations (1-10 mM), but did undergo similar changes in both cell type-specific and antioxidant enzyme activities at 100 nM methylmercury. These results suggest that in prooxidant conditions low doses of mercury can become much more deleterious for the central nervous system.

Effect of an 8-weeks aerobic training program in elderly on oxidative stress and HSP72 expression in leukocytes during antioxidant supplementation.

OBJECTIVE: To investigate the effect of aerobic training in the context of antioxidant supplementation on systemic oxidative stress and leukocytes heat shock protein (Hsp)72 expression in the elderly. DESIGN: Sixteen septuagenarians (8 males and 8 females, mean age 74.6) were supplemented with Vitamin C and E (respectively 500 and 100mg per day) and randomly assigned either to sedentary (AS) or individualized aerobically trained (AT) group for 8 weeks. METHODS: Plasma Vitamin C and E concentrations and aerobic fitness, as well as resting and post graded exercise (GXT) Hsp72 expression in leukocytes, plasma levels of thiobarbituric acid reactive substances (TBARS) and advanced oxidation protein product (AOPP) were measured pre and post training / supplementation. RESULTS: At the end of the intervention, the two groups showed a significant increase in resting plasma vitamin C and E (approximately 50 and 20% increase respectively) and a significant decrease in both resting and post GXT plasma TBARS and AOPP (approximately 25 and 20% decrease respectively). These changes were of similar magnitude in the two groups. The reduced oxidative stress was concomitant with a 15% decreased expression of Hsp72 in monocytes and granulocytes in both groups. CONCLUSION: This study provides evidence that in elderly, increased concentration of antioxidant vitamins C and E is associated with a reduction in oxidative stress and leukocytes Hsp72. In this context, 8 weeks of aerobic training has no impact on oxidative stress or leukocytes Hsp72 expression in elderly people.