Terminally differentiated Schwann cells (SCs), the glial cells in the adult peripheral nerves, display a remarkable plasticity by adopting a de-differentiated phenotype following injury and becoming specialised to repair-type cells for promoting nerve regeneration. Adult SC plasticity is also hijacked by leprosy-causing Mycobacterium leprae during peripheral nerve infection, which make SCs susceptible to reprogramming and generation of progenitor/stem-like cells for bacterial advantage. Interestingly, de-differentiated SCs generated during nerve injury and infection reactivated stem cell transcription factor Sox2, which is essential for maintaining pluripotency in embryonic stem cells (ESCs). In this study we address what role Sox2 plays and how it is involved in adult SC plasticity. We identified that Sox2 binds to a network of gene targets in de-differentiated adult SCs across the mouse genome. This Sox2 target network is distinct from Sox2 target genes in core ESC pluripotency, and appears to be modulated by SC microenvironmental changes and pathological conditions, as nerve crush injury and infection-induced reprogramming expanded Sox2 binding to target genes. In vivo knockdown by shRNA of Sox2 in wild type adult nerves demonstrated reduction in SC de-differentiation. Mutant mice defective in natural nerve degeneration, de-differentiation and regeneration (Wallerian degeneration slow mice; Wlds) not only show impaired Sox2 binding to its target genes but also a delay in Sox2 and target gene expression after nerve crush injury. Together, these in vivo data reveal an impact of Sox2 and its target network on SC plasticity. Furthermore, altered expression of many of these target genes after Sox2 knockdown in wild type adult Schwann cells in vitro and in vivo as well as in injured Wlds nerves suggests a functional role of a Sox2 target network in nerve injury-repair processes. This includes Sox2 target genes such as Megf10, Btc, Atf3 and Nestin. By acting on these genes Sox2 may coordinate relevant gene functions ranging from phagocytosis/clearance, proliferation, transcription and cytoskeletal dynamics. Thus, this study proposes a novel concept of how reactivation of an embryonic stem cell regulator like Sox2 in adult tissues coordinates a gene network regulating Schwann cell plasticity and multiple biological functions facilitating the nerve injury-repair process. These findings may aid in developing strategies towards promoting nerve regeneration, or designing treatments for neuropathies in which deregulation of Schwann cell de-differentiation contributes to pathogenesis.

In the peripheral nervous system (PNS), Schwann cells ensheathe and myelinate axons to promote saltatory conduction of nerve impulses. Close interactions between Schwann cells and axons, and Schwann cells and the basal lamina are essential for the regulation of Schwann cell development and function. Myelinating Schwann cells are highly polarized radially and longitudinally for specifying distinct domains in the axon, which is required for fast action potential propagation. In addition, the Schwann cell cytoplasm is organized into discrete compartments, called Cajal bands, which contain different dystrophin-glycoprotein complexes that are believed to segregate the Schwann cell plasma membrane into appositions between the outer surface of the myelin sheath and the cytoplasmic face of the Schwann cell plasma membrane. Periaxin is expressed in myelinating Schwann cells, and homodimerizes at its PDZ domain to form a transmembrane complex with dystrophin-related protein 2 (DRP2) and dystroglycan. This PDG complex is concentrated at the appositions, and is essential for myelin sheath maintenance and stability in the mature PNS. In mice lacking Periaxin, an intact myelin sheath is formed but subsequently becomes unstable. Periaxin-null Schwann cells are also shorter, which has been proposed to result in a reduction in nerve conduction velocity. This thesis is a study of how Periaxin PDZ domain dimerization contributes to the regulation of PDG complex stability, apposition maintenance, Schwann cell internodal distance and myelin stability. I have studied the function of Periaxin by generating a conditional mutant mouse that lacks the PDZ domain, which is predicted to abrogate dimerization. In these mutants, DRP2 is severely depleted and appositions containing DRP2 fail to form. Mutant Schwann cells also have disrupted Cajal bands and shorter internodal lengths. In the mature peripheral nerves, mutant mice display a peripheral neuropathy characterized by hypermyelination with focally folded myelin. Nerve conduction velocity, motor coordination and sensory function were also studied in these mutant mice. Taken together, these data suggest that dimerization of the Periaxin PDZ domain is required for the stabilization of the PDG and appositions, and regulation of Schwann cell elongation and myelin maintenance. By analyzing a tamoxifen-inducible conditional mouse lacking Periaxin’s PDZ domain in mature myelinating Schwann cells, this work also shows that Periaxin dimerization is essential for maintaining Schwann cell compartmentalization and myelin stability in adult nerves. Finally, studies of single amino acid mutations of the Periaxin PDZ reveal that subtle changes in the structure of the PDZ domain can abrogate dimerization,and a possible mechanism for PDZ-PDZ homodimerization of Periaxin is proposed.

No description available.

Abstract The function of the vertebrate nervous system is based on the rapid and accurate transmission of electrical impulses. The myelin sheath is a lipid-rich membrane that envelops the axon, preventing the leakage of the nervous impulse to the environment. Myelin is formed when the plasma membrane of a myelinating glial cell differentiates and wraps around an axon. The compaction of myelin leads to the extrusion of most of the glial cell cytoplasm from the structure. Both the compact and noncompact regions of myelin carry distinct subsets of proteins. The myelin-associated glycoprotein (MAG) is present in noncompact myelin. It is a cell adhesion molecule expressed only by myelinating glial cells. Two isoforms of MAG, S- and L-MAG, exist, and these forms differ from each other only by their cytoplasmic domains. Until now, little information has been available on the differences between the MAG isoforms. This study was carried out in order to gain information on the cytoplasmic domains of S- and L-MAG. Significant differences were observed in the properties of the MAG cytoplasmic domains. An interaction between the L-MAG cytoplasmic domain and the S100b protein was characterised, and a role for this interaction was found in the regulation of L-MAG phosphorylation. Evidence was also obtained for the dimerisation of the L-MAG cytoplasmic domain. The S-MAG cytoplasmic domain bound zinc, which induced a change in the surface properties of the protein. The S-MAG cytoplasmic domain was also found to interact directly with tubulin, the core component of microtubules. In conclusion, this study has brought information on the functions of the MAG cytoplasmic domains. The results are complementary with ealier hypotheses on the roles of the MAG isoforms in myelinating glia. While the properties of L-MAG suggest a role as a signaling molecule, a dynamic structural role for S-MAG during myelin formation and maintenance can be envisaged.

The transplantation of primary Schwann cells (SC) has been shown to improve nerve regeneration. However, to monitor the survival of transplanted cells within the host, a stable labelling method is required. The in vitro characteristics of green fluorescent protein labelled SC (GFP SC) and their effects in an in vivo peripheral nerve injury model were investigated.   The GFP-SC were readily visualised ex vivo and stimulated significantly better axonal regeneration compared to controls. Clinical use of autologous SC for the treatment of nerve injuries is of limited use due to difficulty in obtaining clinically useful numbers. However, bone marrow mesenchymal stem cells (MSC) can trans-differentiate into SC like cells (dMSC). The in vitro and in vivo differentiation of MSC was explored, and the study extended to include the easily-accessible adipose stem cells (ASC).  In vitro, glial growth factor stimulated MSC express S100, a SC marker, and its expression is maintained following in vivo transplantation.  Similarly, untreated MSC transplanted in vivo also expressed S100, which indicates glial differentiation in response to local cytokines and growth factors. Using an in vitro model, comprising dMSC or dASC co-cultured with adult dorsal root ganglia (DRG) neurons, the capacity of the dMSC and SC like differentiated ASC (dASC) to promote axon myelination was verified: both cell types expressed transcripts for protein zero, peripheral myelin protein-22 and myelin basic protein. The potential of stem cells in nerve repair may be limited by innate cellular senescence or donor age affecting cell functionality thus it was essential to determine the effects of donor age on morphology and functionality of stem cells.  The proliferation rates, expression of senescence markers (p38 and p53) and the stimulation of neurite outgrowth from DRG neurons by stem cells isolated from neonatal, young or old rats were very similar. However, the distribution and ultrastructure of mitochondria in dMSC and dASC from young and old rats were quite different, and seem to indicate physiological senescence of the aged cells.  Given the wide-ranging influence of Notch signalling in cell differentiation, including the neural crest to a glial cell type switch, and self-renewal in mammals, its role in the differentiation of stem cells to SC was investigated. The mRNA for notch-1 and -2 receptors were expressed in the dASC, blockage of notch signaling did not affect the neurotrophic and myelination potential of dASC.  In conclusion, these findings show that GFP labelling has no deleterious effect on SC survival and function. MSC and ASC differentiated into glial-type cells acquire SC morphology, and express characteristic SC markers, and the differentiation process was independent of the Notch signaling pathway. Also, following transplantation into a nerve gap injury dMSC improve regeneration. This study established that following co-culture with DRG neurons, dMSC and dASC were able to express peripheral myelin proteins.  Also, the functional bioactivity of these cells is independent of the donor animal age. Finally, although the glial lineage differentiated aged cells characterized in this study expressed markers typical of senescence they retained the potential to support axon regeneration.

No description available.

Chez les vertébrés, la vitesse de la conduction nerveuse dépend du processus de myélinisation. Dans le système nerveux périphérique, ce sont les cellules de Schwann (CS) qui en s’enroulant autour de l’axone, constituent les gaines de myéline, séparés par des nœuds de Ranvier. La succession de ces gaines augmente la vitesse de conduction nerveuse car les potentiels d’action sont forcés de « sauter » d’un nœud de Ranvier à un autre, ce qui accélère leur vitesse de propagation. La géométrie (l’épaisseur et la longueur) de la gaine de myéline est donc un paramètre essentiel de la conduction de l’influx. Une publication à laquelle j’ai participé, a mis en évidence la polarisation cellulaire de la cellule de Schwann myélinisante. Notre hypothèse est que ce processus est capital pour la formation d’une gaine de myéline fonctionnelle. Comme trois complexes protéiques, conservés au cours de l’évolution, établissent et maintiennent la polarisation cellulaire (ces complexes sont: aPKC/Par3/Par6, Pals1/Patj/Crb3 et Dlg1/Lgl/Scrib chez les mammifères), mon travail consiste à étudier le rôle fonctionnel des protéines de la polarité Dlg1 et Crb3 lors de la myélinisation. Comme l’altération de la géométrie de la myéline est la cause d’un grand nombre de pathologies du système nerveux périphérique mais aussi central. Mon travail sur la mise en lumière des mécanismes qui préside à ce phénomène permet d’envisager de nouvelles voies thérapeutiques. / In the mammalian nervous system, the nerve conduction velocity depends on the myelin sheath. Myelin is produced by Schwann cells in the peripheral nervous system. The myelin sheath, together with the highly specialized nodes of Ranvier that are regulary arrayed along the myelinated fibers, is responsible for efficient and rapid propagation of action potentials along the nerve. Optimal conduction is obtained by adjusting the geometry (length and thickness) of the myelin sheath When I arrived in the laboratory, the team just showed the polarization of the myelinating Schwann cell ( mSC). We hypothesized then that cell polarity proteins are key players for the formation of the myelin sheath. Three complexes, well conserved among species, organize polarized cellular processes. In mammals, these complexes are aPKC/Par3/Par6, Pals1/Patj/Crb3 et Dlg1/Lgl/Scrib. Using an approch allowing the in vivo transduction of mSC, I investigate the relevance of Dlg1 and Crb3 in myelin formation. Changes in the myelin geometry is linked to several human neuropathies in the central and peripheral nervous system. This work highlights mechanisms which control correct myelin formation and allow designing strategies for their treatment.

Abstract The function of the nervous system is based on the targeted transmission of electrical impulses assuring the coordinated function of tissues and organs. Myelination of the neuronal axons allows the fast saltatory conduction by producing repetitive sites where sodium channels cluster on the axolemma. In the peripheral nervous system (PNS), myelin is formed by differentiation of the Schwann cell plasma membrane. The cells form myelin by first wrapping consecutive layers of the plasma membrane around the axons and then excluding almost all of the cytoplasm from the structure, forming compacted and non-compacted membrane compartments. The myelin-associated glycoprotein (MAG) is located in all of the non-compacted regions of the PNS myelin sheath. Its two isoforms, L-MAG and S-MAG, differ only by the carboxy-terminal tails of their respective cytoplasmic domains. Both of the MAG isoforms are found in PNS myelin. They are differentially expressed during development and, until now, it has been thought that L-MAG is not present in the mature PNS myelin sheaths of murines. This study shows that both MAG isoforms can be found in different non-compacted membrane compartments in the mature PNS myelin sheaths in dorsal root ganglia (DRG)/Schwann cell cocultures. Early myelin development and myelin maturation were analyzed by means of a study of the expression of two early myelin markers, MAG and galactosyl cerebrosides (Gal-CB), believed to play roles in both myelin formation and maintenance. In order to allow the exploitation of the full potential of the DRG/Schwann cell coculture model through the use of mouse mutants, a coculture method was developed in which mouse DRGs and Schwann cells are able, for the first time, to produce significant amounts of myelin. To further explore the role of MAG in myelin maintenance and stability, the stability of purified MAG was studied through extensive degradation experiments.

yes / Peripheral nerve injuries affect a large proportion of the global population, often causing significant morbidity and loss of function. Current treatment strategies include the use of implantable nerve guide conduits (NGC's) to direct regenerating axons between the proximal and distal ends of the nerve gap. However, NGC's are limited in their effectiveness at promoting regeneration Current NGCs are not suitable as substrates for supporting either neuronal or Schwann cell growth, as they lack an architecture similar to that of the native extracellular matrix (ECM) of the nerve. The aim of this study was to create an acellular porcine peripheral nerve using a novel decellularisation protocol, in order to eliminate the immunogenic cellular components of the tissue, while preserving the three-dimensional histoarchitecture and ECM components. Porcine peripheral nerve (sciatic branches were decellularised using a low concentration (0.1%; w/v) sodium dodecyl sulphate in conjunction with hypotonic buffers and protease inhibitors, and then sterilised using 0.1% (v/v) peracetic acid. Quantitative and qualitative analysis revealed a ≥95% (w/w) reduction in DNA content as well as preservation of the nerve fascicles and connective tissue. Acellular nerves were shown to have retained key ECM components such as collagen, laminin and fibronectin. Slow strain rate to failure testing demonstrated the biomechanical properties of acellular nerves to be comparable to fresh controls. In conclusion, we report the production of a biocompatible, biomechanically functional acellular scaffold, which may have use in peripheral nerve repair. / Engineering and Physical Sciences Research Council. Grant Number: EPSRC EP/F500513/1

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