This body of work details the exploitation of an incredibly powerful neural culture system, which enables the in vitrostudy of events involved in peripheral nervous system (PNS) development. Using a myelinating dorsal root ganglion (DRG) explant culture system, node of Ranvier formation and maintenance and the associated generation and maturation of myelin segments was examined. In addition, Schwann cell (SC) development, dynamics, and migration were extensively studied. First, in characterizing these cultures, the discrete axonal localization of specific ankyrin isoforms was revealed. Ankyrins are peripheral membrane proteins that immobilize classes of integral membrane proteins to the spectrin based-membrane skeleton. Ankyrins interact with proteins such as the voltage-dependent/gated sodium channel (vgsc) and members of the L1 family of cell adhesion molecules. These interactions are physiologically relevant to the formation of membrane specializations involved in axon guidance and the initiation and propagation of action potentials. We examined ankyrinB and ankyrinG expression in cultured DRG explants, which allowed visualization of individual axons. AnkyrinB and ankyrinG exhibited differential localizations to specific axonal populations. This was evident as early as one day in vitro and persisted over time. In mature pre-myelinated cultures, axons having an apparent diameter of less than 1 µm predominantly expressed ankyrinB, whereas axons having a diameter greater than or equal to 1 µm predominantly expressed ankyrinG (based on immunocytochemical reactivity). When myelination was induced, ankyrinGwas appropriately localized to sites of nodal development flanked by myelinating glial processes in the large caliber axons. These observations suggest that axons destined for myelination may express a distinct complement of peripheral, and perhaps integral, membrane proteins as compared to those observed in non-myelinated axons. These distinguishing features may play a role in the selection of axons for myelination. This work was followed with defining the role axo-glial interactions play in organizing domains along the axon being myelinated. Nodes of Ranvier are specialized, highly polarized axonal domains crucial to the propagation of saltatory action potentials. In the PNS, axon-glial cell contacts have been implicated in SC differentiation and the formation of nodes of Ranvier. SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes. However, a role for these contacts in node formation remains controversial. Using the myelinating explant culture system, we observed that SCs reorganize and polarize microvillar components, such as the ezrin-binding phosphoprotein 50kDa (EBP50)/regulatory co-factor of the sodium-hydrogen exchanger isoform 3 (NHERF-1), actin, and the activated ezrin, radixin, and moesin (ERM) family of proteins, concomitant with myelination in response to inductive signals. These components were targeted to the SC distal tips where live cell imaging revealed novel, dynamic growth cone-like behavior. Further, localized activation of the Rho signaling pathway at SC tips gave rise to these microvillar component-enriched “caps” and influenced the efficiency of node formation. Extending these findings, a more profound examination of SC dynamics was undertaken. This was a particularly important experimental transition, as SC motility is crucial in the development and regeneration of the PNS. The seemingly equivalent bipolarity of mature SCs represents a conundrum in terms of directed motility. Fluorescence time-lapse microscopy of transfected SCs within the myelinating DRG explants revealed a novel cycling of these cells between static, bipolar and motile, unipolar morphologies via asymmetric process retraction and extension. Concentrations of PIP2 (phosphatidylinositol (4,5)-bisphosphate), activated ERMs, and EBP50 delineated the transitory asymmetry associated with the generation and neuron-like migration of the unipolar cell. EBP50 over-expression enhanced unipolar SC migration, suggesting a new role for this adaptor protein in cell motility. Further, the ERMs themselves were found to be essential to both motility and process dynamics with ERM disruption yielding a dysfunctional, multipolar SC phenotype. We propose this novel form of motility may be associated with the correct alignment and spacing of SCs along axons prior to elaboration of the myelin sheath. These compiled studies present significant advances in understanding and examining axo-glial interactions in the PNS. This work establishes the foundation for further, novel exploration of normal PNS development and the regeneration and repair mechanisms involved in PNS injury and disease states.

La phénylcétonurie (PCU est une maladie métabolique génétique causée par une déficience d'activité phénylalanine hydroxylase (PAH). Une hypomyélinisation du cerveau a été documentée chez les patients non traités, mais sa pathophysiologie reste floue. Nous avons investigué l'influence de la phénylalanine (Phe), phénylpyruvate (PP) et phénylacétate (PA) sur les oligodendrocytes. Nous avons premièrement montré dans un modèle murin de PCU que le nombre d'oligodendrocytes n'était pas différent dans le corps calleux entre animaux PCU et sains. Ensuite, en utilisant la technique des co-cultures myélinisantes nous avons pu déterminer que Phe, PP et PA n'ont pas d'effet direct sur la synthèse des gaines de myéline. Ces données indiquent que ces trois composés n'exercent probablement pas de rôle direct dans l'hypomyélinisation du système nerveux central constatée dans le cadre de la PCU. Ces données suggèrent donc des mécanismes d'action indirects. De plus, nous avons investigué la faisabilité d'un modèle de thérapie génique pour la PCU. Celui-ci implique la transduction ex vivo d'hépatocytes ou cellules souches mésenchymateuses par un vecteur lentiviral puis leur implantation dans le foie de l'organisme receveur. Phenylketonuria (PKU) is a metabolic genetic disease characterized by deficient phenylalanine hydroxylase (PAH) enzymatic activity. Brain hypomyelination has been reported in untreated patients, but its mechanism remains unclear. We therefore investigated the influence of phenylalanine (Phe), phenylpyruvate (PP), and phenylacetate (PA) on oligodendrocytes. We fisrt showed in a mouse model of PKU that the number of oligodendrocytes is not different in corpus callosum sections from adult mutants or from control brains. Then, using enriched oligodendroglial cultures, we detected no cytotoxic effect of high concentrations of Phe, PP, or PA. Finally, we analyzed the impact of Phe, PP, and PA on the myelination process in myelinating cocultures using both an in vitro index of myelination, based on activation of the myelin basic protein (MBP) promoter, and the direct quantification of myelin sheaths by both optical measurement and a bioinformatics method. None of these parameters was affected by the increased levels of Phe or its derivatives. Taken together, our data demonstrate that high levels of Phe, such as in PKU, are unlikely to directly induce brain hypomyelination, suggesting involvement of alternative mechanisms in this myelination defect. Moreover, we investigated the feasibility of a gene therapy for phenylketonuria. This project involved the ex vivo transduction of hepatocytes and mesenchymal stem cells with lentivirus vector and the engraftment of these cells in the liver's recipient.

For the past 45 years, QKI has been studied for its role in the processes of development and central nervous system myelination using the qkv mouse. The presence of a single KH domain and the recent identification of a high-affinity binding site in mRNAs, suggests that it can bind to and regulate mRNAs through processes such as stability, splicing and transport. As a member of the STAR RNA binding family of proteins the QKI isoforms may also be involved in cell signaling pathways. / QKI's involvement in all of these processes, lead us to examine both the protein partners and the mRNA targets of the QKI complex in order to identify potentially new pathways regulated by QKI. In doing so, we identified a novel direct protein-protein interaction with PABP and for the first time described the relocalization of QKI to cytoplasmic granules following oxidative stress. In addition, in vivo mRNA interaction studies were performed and allowed the identification of approximately 100 new mRNA targets in human glioblastoma cells. One of the targets identified was VEGF mRNA. / Another QKI target mRNA is MBP, a major protein component of the myelin sheath and the candidate auto-antigen in multiple sclerosis (MS). In vivo MBP is symmetrically dimethylated on a single arginine residue. To further establish the role of the methylation of MBP in myelination, a methyl-specific antibody and an adenovirus expressing a recombinant protein arginine methyltransferase 5 (PRMT5) was generated. We show that methylated MBP is found in areas of mature myelin and that overexpression of the PRTM5 blocked the differentiation of oligodendrocytes. / Taken together these datas implicate QKI for the first time in the process of human cancer angiogenesis and could explain the vascularization defects observed in some of the qkI mutant mice. In addition, arginine methylation of MBP may prove to have an important role in the process of myelination and in the pathogenesis of demyelination and the autoimmune reaction in diseases such as MS.

Healthy white matter in the brain and spinal cord is composed primarily of myelinated axons and glial cells. Myelinated axons transfer information between the peripheral nervous system and the central nervous system (CNS) as well as between centres within the CNS. Demyelination, a hallmark of neurodegenerative autoimmune diseases such as multiple sclerosis (MS), can cause nerve damage and degrade signal propagation. Magnetic resonance imaging (MRI) methods thought to assess myelin integrity and the structural integrity of axons are improving both the diagnosis and understanding of white matter diseases such as MS. Current methods, however, are sensitive to many different pathologies, making the interpretation of individual MRI results difficult. For this dissertation, several quantitative MRI methods were developed and compared, including single component T1 and T2 relaxometry, multicomponent T2 relaxometry, diffusion tensor imaging (DTI), and quantitative magnetization transfer imaging (qMTI). These methods were tested on agarose gels, fixed rat spinal cords, healthy control mice, and the cuprizone mouse model of demyelination. Quantitative MRI measurements were correlated to ultrastructural measurements of white matter to determine the influence myelin content and axonal structure have on different MRI methods. Cellular distributions measured in electron micrographs of the corpus callosum correlated strongly to several different quantitative MRI metrics. The largest Spearman correlation coefficient varied depending on cellular type: longitudinal relaxation rates (RA/T1) vs. the myelinated axon fraction ( r = 0.90/-0.90), the qMTI-derived bound pool fraction (f) vs. the myelin sheath fraction ( r = 0.93), and the DTI-derived axial diffusivity vs. the non-myelinated cell fraction (r = 0.92). Using Pearson’s correlation coefficient, f was strongly correlated to the myelin sheath fraction (r = 0.98) with a linear equation predicting myelin content (5.37f −0.25). Of the calculated MRI metrics, f was the strongest indicator of myelin content while longitudinal relaxation rates and diffusivity measurements were the strongest indicators of changes in tissue structure. Multiparametric MRI measurements of relaxation, diffusion, and magnetization transfer give a more complete picture of white matter integrity.

No description available.

Healthy white matter in the brain and spinal cord is composed primarily of myelinated axons and glial cells. Myelinated axons transfer information between the peripheral nervous system and the central nervous system (CNS) as well as between centres within the CNS. Demyelination, a hallmark of neurodegenerative autoimmune diseases such as multiple sclerosis (MS), can cause nerve damage and degrade signal propagation. Magnetic resonance imaging (MRI) methods thought to assess myelin integrity and the structural integrity of axons are improving both the diagnosis and understanding of white matter diseases such as MS. Current methods, however, are sensitive to many different pathologies, making the interpretation of individual MRI results difficult. For this dissertation, several quantitative MRI methods were developed and compared, including single component T1 and T2 relaxometry, multicomponent T2 relaxometry, diffusion tensor imaging (DTI), and quantitative magnetization transfer imaging (qMTI). These methods were tested on agarose gels, fixed rat spinal cords, healthy control mice, and the cuprizone mouse model of demyelination. Quantitative MRI measurements were correlated to ultrastructural measurements of white matter to determine the influence myelin content and axonal structure have on different MRI methods. Cellular distributions measured in electron micrographs of the corpus callosum correlated strongly to several different quantitative MRI metrics. The largest Spearman correlation coefficient varied depending on cellular type: longitudinal relaxation rates (RA/T1) vs. the myelinated axon fraction ( r = 0.90/-0.90), the qMTI-derived bound pool fraction (f) vs. the myelin sheath fraction ( r = 0.93), and the DTI-derived axial diffusivity vs. the non-myelinated cell fraction (r = 0.92). Using Pearson’s correlation coefficient, f was strongly correlated to the myelin sheath fraction (r = 0.98) with a linear equation predicting myelin content (5.37f −0.25). Of the calculated MRI metrics, f was the strongest indicator of myelin content while longitudinal relaxation rates and diffusivity measurements were the strongest indicators of changes in tissue structure. Multiparametric MRI measurements of relaxation, diffusion, and magnetization transfer give a more complete picture of white matter integrity.

Downstream purification and analysis of a model biopharmaceutical protein (recombinant human myelin basic protein) is described. The recombinant protein was expressed in the milk of transgenic cows and was found exclusively associated with the casein micellar phase. Binding of milk calcium to the active sites of a cation exchanger resin was used beneficially in this study in order to gently disrupt the casein micelles and liberate the recombinant protein. This approach was found superior to the conventional micelle disruption procedures with respect to product recovery, resin fouling due to milk components and column hydrodynamic properties. Further purification was carried out using Ni2+ affinity chromatography and resulted in purity more than 90% and a total recovery of 78%. A capillary electrophoresis total protein assay employing large volume sample stacking and a microsphere-based, sandwich-type immunoassay were developed and validated. Both methods were successfully integrated with the downstream purification protocol in order to evaluate various quality attributes of the recombinant protein. A onestep capillary isoelectric focusing protocol was developed in order to monitor the recombinant protein in milk samples. The results showed extra protein bands in the transgenic milk that had isoelectric points significantly lower than the theoretically calculated one which indicated that the protein had been modified during expression. The association between the recombinant protein and bovine milk caseins was explored at the molecular level using the surface plasmon resonance technique. Results showed a calciummediated interaction between the recombinant protein and the phosphorylated caseins. This selective interaction was not noted between the human myelin basic protein and milk caseins which indicated mammary gland-related posttranslational modifications, most likely phosphorylation. The co-expression of the recombinant protein and caseins in the mammary gland, along with the ability of the recombinant protein to form calcium bridges with caseins explained its association with the casein micellar phase in the transgenic milk. Despite this and owing to the low expression levels of the recombinant protein in milk, light scattering investigations using diffusing wave spectroscopy showed no significant differences between the transgenic and the non-transgenic milk samples with respect to the average micelle size and the micelle surface charges.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

Multiple sclerosis (MS) is a chronic neurological disease characterized by the destruction of central nervous system (CNS) myelin. Although the neurotrophin, brain derived neurotrophic factor (BDNF) has a beneficial role in re-myelination and/or myelin repair, these effects are hampered by the over-expression of a transcriptional repressor isoform of methyl CpG binding protein 2 (MeCP2) called MeCP2E1. We hypothesize that following experimental autoimmune encephalomyelitis (EAE) -induced myelin damage, the immune system induction of the pathogenic MeCP2E1 isoform hampers the re-myelination and/or myelin repair process by repressing BDNF expression. Our research identified the temporal gene and protein expression changes of MeCP2E1, MeCP2E2 and BDNF in an EAE mouse model of MS, and correlated them with the changes in the neurological disability scores (NDS). Our results indicated MeCP2E1 mRNA levels are elevated in EAE animals which is responsible for the repressed BDNF production in the spinal cord that prevents re-myelination and/or myelin repair. / February 2016

No description available.