Düsseldorf, Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Düsseldorf, Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Düsseldorf, Universiẗat, Diss., 2003.

Düsseldorf, Universiẗat, Diss., 2001.

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.

Mutations in the gene for peripheral myelin protein 22 (PMP22) are associated with peripheral neuropathy in mice and humans. PMP22 is produced mainly in Schwann cells in the peripheral nervous system where it is localised to compact myelin. The function of PMP22 is unclear but its low abundance makes it unlikely to be a structural myelin protein. I have studied the peripheral nerves of two different mouse models with alterations in the pmp22 gene. (1) The Trembler-J (Tr^J) mouse which has a point mutation [L16P] in the first transmembrane domain of PMP22. (2) PMPP22 overexpressing transgenic mice which have 7 (C22), 4 (C61) and 2 (C2) copies of the human PMP22 gene in addition to the mouse pmp22 gene. In the nerves of adult Tr^J mice there was considerable evidence of abnormal Schwann cell-axon interactions. Abnormal features were reproduced in the early stages of regeneration following crush injury. This demonstrates that the abnormalities are a result of an intrinsic abnormality of Tr^J Schwann cells and not secondary changes related to demyelination. In the initial stages of postnatal development the number of axons that were singly ensheathed was the same in all the mutants examined, indicating that PMP22 does not function in the initial enclosure of groups of axons and subsequent separation of single axons. All strains examined had an increased proportion of fibres that were incompletely surrounded by Schwann cell cytoplasm indicating that this step is disrupted in PMP22 mutants. Increasing the number of copies of PMP22 resulted in an increasing severity of phenotype. In C22 (7 copy) animals myelin formation was delayed or non-existent in many fibres whereas in C61 animals myelination initially appeared normal with abnormality appearing later in a small population of fibres. The C2 strain appeared relatively unaffected. It is concluded that PMP22 functions in the initiation of myelination and most probably involves the ensheathment of the axon by the Schwann cell, and the extension of this cell along the axon. Abnormalities are most likely to result from defective interactions between the axon and the Schwann cell.

Orientador: Vânia Nieto Brito de Souza / Resumo: A infecção por Mycobacterium leprae (M. leprae) desencadeia alterações no status funcional das células de Schwann (SCs), responsáveis pela produção da bainha de mielina e homeostasia neural. Estudos prévios indicam que lesões nos nervos periféricos modulam a expressão de fatores-chave envolvidos na diferenciação e maturação das SCs. Na hanseníase, alterações funcionais nessas células podem estar envolvidas na patogênese do dano neural. O presente estudo buscou determinar a expressão dos fatores de transcrição KROX-20, SOX-10, JUN e do receptor p75NTR, envolvidos nos processos de diferenciação e maturação de SCs. Os dados foram obtidos a partir de cultura primária de SCs murinas expostas ao M. leprae durante sete e 14 dias, com diferentes multiplicidades de infecção (MOI 100:1, MOI 50:1). Os mesmos fatores foram avaliados in vivo, em nervos ciáticos de camundongos nude (NUFoxn1nu), após seis e nove meses de infecção. Comparando-se grupos experimentais em relação aos controles, nossos resultados em cultura de SCs demonstram redução significativa de KROX-20 e SOX-10, bem como elevação na frequência de células imunomarcadas por p75NTR. Paralelamente, nervos ciáticos de camundongos nude infectados por M. leprae apresentaram queda de KROX-20 e aumento de p75NTR, quando comparados aos animais não infectados. Em conjunto, nossos resultados corroboram achados prévios sobre a interferência de M. leprae na expressão de fatores-chave envolvidos na maturação celular, favorecendo a manuten... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mycobacterium leprae infection (M. leprae) triggers changes in the functional status of Schwann cells (SCs), which are responsible for the production of myelin sheath and neural homeostasis. Previous studies indicate that peripheral nerve injury modulates the expression of key factors involved in the differentiation and maturation of SCs. In leprosy, functional changes in these cells may be involved in the pathogenesis of neural damage. The present study sought to determine the expression of the transcription factors KROX-20, SOX-10, JUN and the neurotrophic receptor p75NTR, involved in the differentiation and maturation processes of SCs. Data were obtained from primary murine SCs culture exposed to M. leprae for seven and 14 days, with different multiplicities of infection (MOI 100:1, MOI 50:1). The same factors were evaluated in vivo on sciatic nerve of nude mice (NU-Foxn1nu), after six and nine months of infection. By comparing experimental groups versus controls, our SC culture results demonstrate significant reduction of KROX-20 and SOX-10, as well as elevation in the frequency of cells immunolabelled by p75NTR. In parallel, sciatic nerves of nude mice infected with M. leprae showed a decrease in KROX-20 and an increase of p75NTR when compared to uninfected animals. Our results corroborate previous findings on M. leprae interference in the expression of key factors involved in cell maturation, favoring the maintenance of a non-myelinating phenotype in SCs, with possible ... (Complete abstract click electronic access below) / Mestre

Submitted by Mariane Bertolucci Casalenovo null (mary_bc14@hotmail.com) on 2017-09-14T00:20:08Z No. of bitstreams: 1 DISSERTAÇÃO FINAL.pdf: 10749096 bytes, checksum: 4412c078d6e18d64f519d7ac0952d26d (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-09-15T13:49:22Z (GMT) No. of bitstreams: 1 casalenovo_mb_me_bot.pdf: 10749096 bytes, checksum: 4412c078d6e18d64f519d7ac0952d26d (MD5) / Made available in DSpace on 2017-09-15T13:49:22Z (GMT). No. of bitstreams: 1 casalenovo_mb_me_bot.pdf: 10749096 bytes, checksum: 4412c078d6e18d64f519d7ac0952d26d (MD5) Previous issue date: 2017-08-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A infecção por Mycobacterium leprae (M. leprae) desencadeia alterações no status funcional das células de Schwann (SCs), responsáveis pela produção da bainha de mielina e homeostasia neural. Estudos prévios indicam que lesões nos nervos periféricos modulam a expressão de fatores-chave envolvidos na diferenciação e maturação das SCs. Na hanseníase, alterações funcionais nessas células podem estar envolvidas na patogênese do dano neural. O presente estudo buscou determinar a expressão dos fatores de transcrição KROX-20, SOX-10, JUN e do receptor p75NTR, envolvidos nos processos de diferenciação e maturação de SCs. Os dados foram obtidos a partir de cultura primária de SCs murinas expostas ao M. leprae durante sete e 14 dias, com diferentes multiplicidades de infecção (MOI 100:1, MOI 50:1). Os mesmos fatores foram avaliados in vivo, em nervos ciáticos de camundongos nude (NUFoxn1nu), após seis e nove meses de infecção. Comparando-se grupos experimentais em relação aos controles, nossos resultados em cultura de SCs demonstram redução significativa de KROX-20 e SOX-10, bem como elevação na frequência de células imunomarcadas por p75NTR. Paralelamente, nervos ciáticos de camundongos nude infectados por M. leprae apresentaram queda de KROX-20 e aumento de p75NTR, quando comparados aos animais não infectados. Em conjunto, nossos resultados corroboram achados prévios sobre a interferência de M. leprae na expressão de fatores-chave envolvidos na maturação celular, favorecendo a manutenção de fenótipo não mielinizante nas SCs, com possíveis implicações na reparação de nervos periféricos adultos. / Mycobacterium leprae infection (M. leprae) triggers changes in the functional status of Schwann cells (SCs), which are responsible for the production of myelin sheath and neural homeostasis. Previous studies indicate that peripheral nerve injury modulates the expression of key factors involved in the differentiation and maturation of SCs. In leprosy, functional changes in these cells may be involved in the pathogenesis of neural damage. The present study sought to determine the expression of the transcription factors KROX-20, SOX-10, JUN and the neurotrophic receptor p75NTR, involved in the differentiation and maturation processes of SCs. Data were obtained from primary murine SCs culture exposed to M. leprae for seven and 14 days, with different multiplicities of infection (MOI 100:1, MOI 50:1). The same factors were evaluated in vivo on sciatic nerve of nude mice (NU-Foxn1nu), after six and nine months of infection. By comparing experimental groups versus controls, our SC culture results demonstrate significant reduction of KROX-20 and SOX-10, as well as elevation in the frequency of cells immunolabelled by p75NTR. In parallel, sciatic nerves of nude mice infected with M. leprae showed a decrease in KROX-20 and an increase of p75NTR when compared to uninfected animals. Our results corroborate previous findings on M. leprae interference in the expression of key factors involved in cell maturation, favoring the maintenance of a non-myelinating phenotype in SCs, with possible implications for the repair of adult peripheral nerves.

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.

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