Distribution of monocarboxylate transporters in the peripheral nervous system suggests putative roles in lactate shuttling and myelination.

Lactate, a product of glycolysis, has been shown to play a key role in the metabolic support of neurons/axons in the CNS by both astrocytes and oligodendrocytes through monocarboxylate transporters (MCTs). Despite such importance in the CNS, little is known about MCT expression and lactate function in the PNS. Here we show that mouse MCT1, MCT2, and MCT4 are expressed in the PNS. While DRG neurons express MCT1, myelinating Schwann cells (SCs) coexpress MCT1 and MCT4 in a domain-specific fashion, mainly in regions of noncompact myelin. Interestingly, SC-specific downregulation of MCT1 expression in rat neuron/SC cocultures led to increased myelination, while its downregulation in neurons resulted in a decreased amount of neurofilament. Finally, pure rat SCs grown in the presence of lactate exhibited an increase in the level of expression of the main myelin regulator gene Krox20/Egr2 and the myelin gene P0. These data indicate that lactate homeostasis participates in the regulation of the SC myelination program and reveal that similar to CNS, PNS axon-glial metabolic interactions are most likely mediated by MCTs.

Missense mutations in TENM4, a regulator of axon guidance and central myelination, cause essential tremor.

Essential tremor (ET) is a common movement disorder with an estimated prevalence of 5% of the population aged over 65 years. In spite of intensive efforts, the genetic architecture of ET remains unknown. We used a combination of whole-exome sequencing and targeted resequencing in three ET families. In vitro and in vivo experiments in oligodendrocyte precursor cells and zebrafish were performed to test our findings. Whole-exome sequencing revealed a missense mutation in TENM4 segregating in an autosomal-dominant fashion in an ET family. Subsequent targeted resequencing of TENM4 led to the discovery of two novel missense mutations. Not only did these two mutations segregate with ET in two additional families, but we also observed significant over transmission of pathogenic TENM4 alleles across the three families. Consistent with a dominant mode of inheritance, in vitro analysis in oligodendrocyte precursor cells showed that mutant proteins mislocalize. Finally, expression of human mRNA harboring any of three patient mutations in zebrafish embryos induced defects in axon guidance, confirming a dominant-negative mode of action for these mutations. Our genetic and functional data, which is corroborated by the existence of a Tenm4 knockout mouse displaying an ET phenotype, implicates TENM4 in ET. Together with previous studies of TENM4 in model organisms, our studies intimate that processes regulating myelination in the central nervous system and axon guidance might be significant contributors to the genetic burden of this disorder.

Redox dysregulation in schizophrenia : effect on myelination of cortical structures and connectivity

Cette thèse traite du rôle qu'un facteur de risque génétique développé chez les patients souffrant de schizophrénie, à savoir un déficit de la synthèse du glutathion, peut jouer dans les anomalies de la connectivité cérébrale trouvées chez ces patients. L'essentiel du travail a été consacré à évaluer la structure de la substance blanche dans l'ensemble du cerveau chez un modèle animal par une méthode similaire à celle utilisée en recherche clinique avec l'imagerie par résonance magnétique (IRM). Cette approche de translation inverse chez la souris knock-out de glutamate-cystéine ligase modulateur sous-unité (Gclm KO), avait l'objectif d'étudier l'effet des défenses redox déficientes sur le développement des connexions cérébrales, tout en excluant celui des facteurs non liés au génotype. Après avoir établi le protocole de recherche, l'influence d'une manipulation environnementale a également été étudiée. Pour effectuer une analyse statistique fiable des données d'IRM obtenues, nous .avons d'abord créé un atlas du cerveau de la souris afin de l'utiliser comme modèle pour une segmentation précise des différentes régions du cerveau sur les images IRM obtenues in vivo. Les données provenant de chaque région d'intérêt ont ensuite été étudiées séparément. La qualité de cette méthode a été évaluée dans une expérience de simulation pour déduire la puissance statistique réalisable dans chaque région en fonction du nombre d'animaux utilisés. Ces outils d'analyse nous ont permis d'évaluer l'intégrité de la substance blanche dans le cerveau des souris durant le développement grâce à une expérience longitudinale, en utilisant l'imagerie du tenseur de diffusion (DTI). Nous avons ainsi observé des anomalies dans les paramètres dérivés du tenseur (diffusivité et anisotropie) dans la Commissure Antérieure et le Fimbria/Fornix des souris Gclm KO, par rapport aux animaux contrôles. Ces résultats suggèrent une substance blanche endommagée dans ces régions. Dans une expérience électrophysiologique, Pascal Steullet a montré que ces anomalies ont des conséquences fonctionnelles caractérisées par une réduction de la vitesse de conduction dans les fibres nerveuses. Ces données renforcent les conclusions des analyses d'imagerie. Le mécanisme par lequel une dérégulation redox affecte la structure de la substance blanche reste encore à définir, car une analyse immunohistochimique des protéines constituantes de la couche de myéline des fibres concernées n'a pas donné de résultats concluants. Nous avons également constaté un élargissement des ventricules dans les jeunes souris Gclm KO, mais pas chez les adultes et des anomalies neurochimiques déjà connues chez ces animaux (Duarte et al. 2011), à savoir une réduction du Glutathion et une augmentation de l'acide N-acétylaspartique, de l'Alanine et du ratio Glutamine/Glutamate. Nous avons ensuite testé l'effet d'un stress environnemental supplémentaire, l'élevage en isolement social, sur le phénotype. Ce stress n'a eu aucun effet sur la structure de la substance blanche évaluée par DTI, mais a réduit la concentration de myo-Inositol et augmenté le ratio de Glutamine/Glutamate dans le cortex frontal. Nous avons aussi reproduit dans ce groupe indépendant d'animaux les effets du génotype sur le profil neurochimique, sur la taille des ventricules et aussi sur les paramètres dérivés du tenseur de diffusion dans le Fimbria/Fornix, mais pas dans la Commissure Antérieure. Nos résultats montrent qu'une dérégulation redox d'origine génétique perturbe la structure et la fonction de la substance blanche dans des régions spécifiques, causant ainsi l'élargissement des ventricules. Ces phénotypes rassemblent certaines caractéristiques neuro-anatomiques de la schizophrénie, mais les mécanismes qui en sont responsables demeurent encore inconnus. L'isolement social n'a pas d'effet sur la structure de la substance blanche évaluée par DTI, alors qu'il est prouvé qu'il affecte la maturation des oligodendrocytes. La neurochimie corticale et en particulier le rapport Glutamine/Glutamate a été affecté par le dérèglement redox ainsi que par l'isolement social. En conséquence, ce ratio représente un indice prometteur dans la recherche sur l'interaction du stress environnemental avec le déséquilibre redox dans le domaine de la schizophrénie. -- The present doctoral thesis is concerned with the role that a genetic risk factor for the development of schizophrenia, namely a deficit in Glutathione synthesis, may play in the anomalies of brain connectivity found in patients. Most of the effort was devoted to perform a whole-brain assessment of white matter structure in the Glutamate-Cysteine ligase modulatory knockout mouse model (Gclm KO) using Magnetic Resonance Imaging (MRI) techniques similar to those used in state-of-the-art clinical research. Such reverse translational approach taking brain imaging from the bedside to the bench aimed to investigate the role that deficient redox defenses may play in the development of brain connections while excluding all influencing factors beside the genotype. After establishing the protocol, the influence of further environmental manipulations was also studied. Analysis of MRI images acquired in vivo was one of the main challenges of the project. Our strategy consisted in creating an atlas of the mouse brain to use as segmentation guide and then analyze the data from each region of interest separately. The quality of the method was assessed in a simulation experiment by calculating the statistical power achievable in each brain region at different sample sizes. This analysis tool enabled us to assess white matter integrity in the mouse brain along development in a longitudinal experiment using Diffusion Tensor Imaging (DTI). We discovered anomalies in diffusivity parameters derived from the tensor in the Anterior Commissure and Fimbria/Fornix of Gclm KO mice when compared to wild-type animals, which suggest that the structure of these tracts is compromised in the KO mice. In an elegant electrophysiological experiment, Pascal Steullet has provided evidence that these anomalies have functional consequences in form of reduced conduction velocity in the concerned tracts, thus supporting the DTI findings. The mechanism by which redox dysregulation affects WM structure remains unknown, for the immunohistochemical analysis of myelin constituent proteins in the concerned tracts produced inconclusive results. Our experiments also detected an enlargement of the lateral ventricles in young but not adult Gclm KO mice and confirmed neurochemical anomalies already known to affect this animals (Duarte et al. 2011), namely a reduction in Glutathione and an increase in Glutamine/Glutamate ratio, N-acetylaspartate and Alanine. Using the same methods, we tested the effect of an additional environmental stress on the observed phenotype: rearing in social isolation had no effect on white matter structure as assessed by DTI, but it reduced the concentration of myo-Inositol and increased the Glutamine/Glutamate ratio in the frontal cortex. We could also replicate in this separate group of animals the effects of genotype on the frontal neurochemical profile, ventricular size and diffusivity parameters in the Fimbria/Fornix but not in the Anterior Commissure. Our data show that a redox dysregulation of genetic origin may disrupt white matter structure and function in specific tracts and cause a ventricular enlargement, phenotypes that resemble some neuroanatomical features of schizophrenia. The mechanism responsible remains however unknown. We have also demonstrated that environmental stress in form of social isolation does not affect white matter structure as assessed by DTI even though it is known to affect oligodendrocyte maturation. Cortical neurochemistry, and specifically the Glutamine to Glutamate balance was affected both by redox dysregulation and social isolation, and is thus a good target for further research on the interaction of redox imbalance and environmental stress in schizophrenia.

Endocytic trafficking and oligodendroglial exosome secretion in axon-glia communication and myelination

Oligodendrocytes form specialized plasma membrane extensions which spirally enwrap axons, thereby building up the myelin sheath. During myelination, oligodendrocytes produce large amounts of membrane components. Oligodendrocytes can be seen as a complex polarized cell type with two distinct membrane domains, the plasma membrane surrounding the cell body and the myelin membrane. SNARE proteins mediate the fusion of vesicular cargoes with their target membrane. We propose a model in which the major myelin protein PLP is transported by two different pathways. VAMP3 mediates the non-polarized transport of newly synthesized PLP via recycling endosomes to the plasma membrane, while transport of PLP from late endosomes/lysosomes to myelin is controlled by VAMP7. In the second part of the thesis, the role of exosome secretion in glia to axon signaling was studied. Further studies are required to clarify whether VAMP7 also controls exosome secretion. The thesis further focused on putative metabolic effects in the target neurons. Oligodendroglial exosomes showed no obvious influences on neuronal metabolic activity. Analysis of the phosphorylation levels of the neurofilament heavy subunit revealed a decrease in presence of oligodendrocytes, indicating effects of oligodendroglial exosomes on the neuronal cytoskeleton. Finally, candidates for kinases which are possibly activated upon influence of oligodendroglial exosomes and could influence neuronal survival were identified.

Fyn kinase targets in oligodendroglial physiology and myelination

In the central nervous system (CNS), oligodendrocytes form the multilamellar and compacted myelin sheath by spirally wrapping around defined axons with their specialised plasma membrane. Myelin is crucial for the rapid saltatory conduction of nerve impulses and for the preservation of axonal integrity. The absence of the major myelin component Myelin Basic Protein (MBP) results in an almost complete failure to form compact myelin in the CNS. The mRNA of MBP is sorted to cytoplasmic RNA granules and transported to the distal processes of oligodendrocytes in a translationally silent state. A main mediator of MBP mRNA localisation is the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 which binds to the cis-acting A2 response element (A2RE) in the 3’UTR of MBP mRNA. A signalling cascade had been identified that triggers local translation of MBP at the axon-glial contact site, involving the neuronal cell adhesion molecule (CAM) L1, the oligodendroglial plasma membrane-tethered Fyn kinase and Fyn-dependent phosphorylation of hnRNP A2. This model was confirmed here, showing that L1 stimulates Fyn-dependent phosphorylation of hnRNP A2 and a remodelling of A2-dependent RNA granule structures. Furthermore, the RNA helicase DDX5 was confirmed here acting together with hnRNP A2 in cytoplasmic RNA granules and is possibly involved in MBP mRNA granule dynamics.rnLack of non-receptor tyrosine kinase Fyn activity leads to reduced levels of MBP and hypomyelination in the forebrain. The multiadaptor protein p130Cas and the RNA-binding protein hnRNP F were verified here as additional targets of Fyn in oligodendrocytes. The findings point at roles of p130Cas in the regulation of Fyn-dependent process outgrowth and signalling cascades ensuring cell survival. HnRNP F was identified here as a novel constituent of oligodendroglial cytoplasmic RNA granules containing hnRNP A2 and MBP mRNA. Moreover, it was found that hnRNP F plays a role in the post-transcriptional regulation of MBP mRNA and that defined levels of hnRNP F are required to facilitate efficient synthesis of MBP. HnRNP F appears to be directly phosphorylated by Fyn kinase what presumably contributes to the initiation of translation of MBP mRNA at the plasma membrane.rnFyn kinase signalling thus affects many aspects of oligodendroglial physiology contributing to myelination. Post-transcriptional control of the synthesis of the essential myelin protein MBP by Fyn targets is particularly important. Deregulation of these Fyn-dependent pathways could thus negatively influence disorders involving the white matter of the nervous system.rnrn

Myelination of the right parahippocampal cingulum is associated with physical activity in young healthy adults.

Recent evidence suggests that individual differences in physical activity (PA) may be associated with individual differences in white matter microstructure and with grey matter volume of the hippocampus. Therefore, this study investigated the association between PA and white matter microstructure of pathways connecting to the hippocampus. A total of 33 young, healthy adults underwent magnetic resonance imaging (MRI). High angular resolution diffusion-weighted imaging and multi-component relaxometry MRI scans (multi-component driven equilibrium pulse observation of T1 and T2) were acquired for each participant. Activity levels (AL) of participants were calculated from 72-h actigraphy recordings. Tractography using the damped Richardson Lucy algorithm was used to reconstruct the fornix and bilateral parahippocampal cinguli (PHC). The mean fractional anisotropy (FA) and the myelin water fraction (MWF), a putative marker of myelination, were determined for each pathway. A positive correlation between both AL and FA and between AL and MWF were hypothesized for the three pathways. There was a selective positive correlation between AL and MWF in the right PHC (r = 0.482, p = 0.007). Thus, our results provide initial in vivo evidence for an association between myelination of the right PHC and PA in young healthy adults. Our results suggest that MWF may not only be more specific, but also more sensitive than FA to detect white matter microstructural alterations. If PA was to induce structural plasticity of the right PHC this may contribute to reverse structural alterations of the right PHC in neuropsychiatric disorder with hippocampal pathologies.

MYELINATION IN UNDERNOURISHED RAT BRAIN

Several interactive parameters of protein-calorie malnutrition imposed during postnatal ontogeny on the myelination of rat brain wre investigated. Postnatal starvation depresses the rate of myelin protein synthesis to approximately the same extent in all major brain regions examined (cerebral cortex, cerebellum, striatum, hippocampus, hypothalamus, midbrain and medulla), indicating a relatively uniform reduction in myelination throughout the brain. Early starvation from birth through 8 days, as well as starvation occurring late, from 14 to 30 days, produced no lasting deficit in myelin accumulation. Starvation from birth through 14 days or from birth through 20 days produces lasting, significant myelin deficits in all brain regions when examined following ad libitum feeding to 60 days of age. These data, in combination with the metabolic studies of myelin synthesis, show that severe starvation occurring during the 2nd and 3rd weeks of postnatal life produces an immediate reduction in myelin synthesis, and that the subsequent deficit in myelin accumulation is irreversible by nutritional rehabilitation. With respect to the relative severity of nutritional restriction occurring during this "critical" interval of brain ontogeny, additional studies showed that mild undernourishment (producing less than 20 percent growth lag) produces no myelin deficit. There appears to be a threshold effect such that undernutrition producing a growth lag of between 20 to 30 percent first produces a measurable deficit. Increasingly severe regimens of nutritional restriction which produce approximately 30, 40 and 50 percent body weight lags result in initial myelin deficits of 25, 55 and 60 percent, respectively. Initial myelin deficits do not recover following nutritional rehabilitation, although myelin continues to increase in both normal and all undernourished populations. At the cellular level, severe postnatal nutritional restriction appears to depress both the initial synthesis of myelin precursor proteins (as demonstrated for proteolipid protein) as well as their subsequent assembly into myelin membrane. All of the findings of the present studies are consistent with a hypothetical model of undernutrition-induced brain hypomyelination in which the primary defect consists of a failure of oligodendroglia to myelinate a substantial percentage of axons, resulting in a greatly decreased ratio of myelinated to unmyelinated axons. ^

Adult brain retains the potential to generate oligodendroglial progenitors with extensive myelination capacity

Remyelination of focal areas of the central nervous system (CNS) in animals can be achieved by transplantation of glial cells, yet the source of these cells in humans to similarly treat myelin disorders is limited at present to fetal tissue. Multipotent precursor cells are present in the CNS of adult as well as embryonic and neonatal animals and can differentiate into lineage-restricted progenitors such as oligodendroglial progenitors (OPs). The OPs present in adults have a different phenotype from those seen in earlier life, and their potential role in CNS repair remains unknown. To gain insights into the potential to manipulate the myelinating capacity of these precursor and/or progenitor cells, we generated a homogenous culture of OPs from neural precursor cells isolated from adult rat subependymal tissues. Phenotypic characterization indicated that these OPs resembled neonatal rather than adult OPs and produced robust myelin after transplantation. The ability to generate such cells from the adult brain therefore opens an avenue to explore the potential of these cells for repairing myelin disorders in adulthood.

Neurotransplantation of magnetically labeled oligodendrocyte progenitors: Magnetic resonance tracking of cell migration and myelination

Demyelination is a common pathological finding in human neurological diseases and frequently persists as a result of failure of endogenous repair. Transplanted oligodendrocytes and their precursor cells can (re)myelinate axons, raising the possibility of therapeutic intervention. The migratory capacity of transplanted cells is of key importance in determining the extent of (re)myelination and can, at present, be evaluated only by using invasive and irreversible procedures. We have exploited the transferrin receptor as an efficient intracellular delivery device for magnetic nanoparticles, and transplanted tagged oligodendrocyte progenitor cells into the spinal cord of myelin-deficient rats. Cell migration could be easily detected by using three-dimensional magnetic resonance microscopy, with a close correlation between the areas of contrast enhancement and the achieved extent of myelination. The present results demonstrate that magnetic resonance tracking of transplanted oligodendrocyte progenitors is feasible; this technique has the potential to be easily extended to other neurotransplantation studies involving different precursor cell types.

Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia

Neuropathological and brain imaging studies suggest that schizophrenia may result from neurodevelopmental defects. Cytoarchitectural studies indicate cellular abnormalities suggestive of a disruption in neuronal connectivity in schizophrenia, particularly in the dorsolateral prefrontal cortex. Yet, the molecular mechanisms underlying these findings remain unclear. To identify molecular substrates associated with schizophrenia, DNA microarray analysis was used to assay gene expression levels in postmortem dorsolateral prefrontal cortex of schizophrenic and control patients. Genes determined to have altered expression levels in schizophrenics relative to controls are involved in a number of biological processes, including synaptic plasticity, neuronal development, neurotransmission, and signal transduction. Most notable was the differential expression of myelination-related genes suggesting a disruption in oligodendrocyte function in schizophrenia.

Induction of myelination in the central nervous system by electrical activity.

The oligodendrocyte is the myelin-forming cell in the central nervous system. Despite the close interaction between axons and oligodendrocytes, there is little evidence that neurons influence myelinogenesis. On the contrary, newly differentiated oligodendrocytes, which mature in culture in the total absence of neurons, synthesize the myelin-specific constituents of oligodendrocytes differentiated in vivo and even form myelin-like figures. Neuronal electrical activity may be required, however, for the appropriate formation of the myelin sheath. To investigate the role of electrical activity on myelin formation, we have used highly specific neurotoxins, which can either block (tetrodotoxin) or increase (alpha-scorpion toxin) the firing of neurons. We show that myelination can be inhibited by blocking the action potential of neighboring axons or enhanced by increasing their electrical activity, clearly linking neuronal electrical activity to myelinogenesis.

Mesenchymal Stem Cells Engrafted In A Fibrin Scaffold Stimulate Schwann Cell Reactivity And Axonal Regeneration Following Sciatic Nerve Tubulization.

The present study investigated the effectiveness of mesenchymal stem cells (MSCs) associated with a fibrin scaffold (FS) for the peripheral regenerative process after nerve tubulization. Adult female Lewis rats received a unilateral sciatic nerve transection followed by repair with a polycaprolactone (PCL)-based tubular prosthesis. Sixty days after injury, the regenerated nerves were studied by immunohistochemistry. Anti-p75NTR immunostaining was used to investigate the reactivity of the MSCs. Basal labeling, which was upregulated during the regenerative process, was detected in uninjured nerves and was significantly greater in the MSC-treated group. The presence of GFP-positive MSCs was detected in the nerves, indicating the long term survival of such cells. Moreover, there was co-localization between MSCs and BNDF immunoreactivity, showing a possible mechanism by which MSCs improve the reactivity of SCs. Myelinated axon counting and morphometric analyses showed that MSC engrafting led to a higher degree of fiber compaction combined with a trend of increased myelin sheath thickness, when compared with other groups. The functional result of MSC engrafting was that the animals showed higher motor function recovery at the seventh and eighth week after lesion. The findings herein show that MSC+FS therapy improves the nerve regeneration process by positively modulating the reactivity of SCs.

Effectiveness of iron repletion in the diet for the optic nerve development of anaemic rats

Purpose To compare the process of myelination in the developing optic nerve (ON) of anaemic rats with the subsequent recovery after being fed an iron-recovery diet. Methods In this study, the morphometrical parameters in the ON were assessed by electron microscopy in Wistar rats that were on an iron-deficient diet for 32 days or for 21 days followed by 10 days on an iron-recovery diet. Qualitative and quantitative analyses were performed using representative electron ultramicrographs. Data were analysed by one-way analysis of variance (ANOVA). When differences were detected, comparisons were made using Tukey`s post hoc test (P<0.05 was considered to be significant). Results Qualitative analysis of the ONs in anaemic and recovered animals showed a higher rate of deformed axons and increased lamellar separation in the myelin sheath when compared with the respective control group. The ON of the anaemic group showed a reduced mean density of myelinated fibres when compared with the control group. The fibre area ratio, axon area ratio, and myelin area ratio of large axons/small axons in the ONs of the control group showed the highest values for the myelin areas, axon areas, and total fibre areas. The control group showed a significantly higher myelin sheath thickness when compared with the anaemic and recovered groups. Conclusions Our data indicate that iron is necessary for maintenance of the ON cell structure, and that morphological damage from iron deficiency is not easily reverted by iron repletion. Eye (2010) 24, 901-908; doi:10.1038/eye.2009.205; published online 14 August 2009