Cell death in the Schwann cell lineage and its regulation by neuregulin.

The development of Schwann cells, the myelin-forming glial cells of the vertebrate peripheral nervous system, involves a neonatal phase of proliferation in which cells migrate along and segregate newly formed axons. Withdrawal from the cell cycle, around postnatal days 2-4 in rodents, initiates terminal differentiation to the myelinating state. During this time, Schwann cell number is subject to stringent regulation such that within the first postnatal week, axons and myelinating Schwann cells attain the one-to-one relationship characteristic of the mature nerve. The mechanisms that underly this developmental control remain largely undefined. In this report, we examine the role of apoptosis in the determination of postnatal Schwann cell number. We find that Schwann cells isolated from postnatal day 3 rat sciatic nerve undergo apoptosis in vitro upon serum withdrawal and that Schwann cell death can be prevented by beta forms of neuregulin (NRG-beta) but not by fibroblast growth factor 2 or platelet-derived growth factors AA and BB. This NRG-beta-mediated Schwann cell survival is apparently transduced through an ErbB2/ErbB3 receptor heterodimer. We also provide evidence that postnatal Schwann cells undergo developmentally regulated apoptosis in vivo. Together with other recent findings, these results suggest that Schwann cell apoptosis may play an important role in peripheral nerve development and that Schwann cell survival may be regulated by access to axonally derived NRG.

The Neurofibromatosis 2 tumor suppressor merlin in cytoskeleton organization and cell cycle regulation

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder, which predisposes to multiple tumours of the nervous system, typically schwannomas and meningiomas. Biallelic inactivation of the NF2 gene occurs both in sporadic and NF2-related schwannomas and in most meningiomas. The NF2 gene product merlin (or schwannomin) is structurally related to the ERM proteins, ezrin, radixin and moesin, which act as molecular linkers between the actin cytoskeleton and the plasma membrane. Merlin is a tumor suppressor that participates in cell cycle regulation. Merlin s phosphorylation status appears to be associated with its tumour suppressor activity, i.e. non-phosphorylated merlin functions as a tumour suppressor, whereas protein phosphorylation results in loss of functional activity. This thesis study was initiated to investigate merlin s role as a tumor suppressor and growth inhibitor. These studies show, that like many other tumor suppressors, also merlin is targeted to the nucleus at some stages of the cell cycle. Merlin s nuclear localization is regulated by cell cycle phase, contact inhibition and adhesion. In addition, a potential nuclear binding partner for merlin was identified, Human Enhancer of Invasion 10 (HEI10), a cyclin B interacting protein. Many tumor suppressors interact with microtubules and this thesis work shows that also merlin colocalizes with microtubules in mitotic structures. Merlin binds microtubules directly, and increases their polymerization in vitro and in vivo. In addition, primary mouse Schwann cells lacking merlin displays disturbed microtubule cytoskeleton. Fourth part of this thesis work began from the notion that PKA phosphorylates an unidentified site from the merlin N-terminus. Our studies show that serine 10 is a target for PKA and modulation of this residue regulates cytoskeletal organization, lamellipodia formation and cell migration. In summary, this thesis work shows that merlin s role is much more versatile than previously thought. It has a yet unidentified role in the nucleus and it participates in the regulation of both microtubules and the actin cytoskeleton. These studies have led to a better understanding of this enigmatic tumor suppressor, which eventually will aid in the design of specific drugs for the NF2 disease.

Surface markers of regionalization in the vertebrate nervous system

In order to identify new molecules that might play a role in regional specification of the nervous system, we generated and characterized monoclonal antibodies (mAbs) that have positionally-restricted labeling patterns.

The FORSE-1 mAb was generated using a strategy designed to produce mAbs against neuronal cell surface antigens that might be regulated by regionally-restricted transcription factors in the developing central nervous system (CNS). FORSE-1 staining is enriched in the forebrain as compared to the rest of the CNS until E18. Between E11.5-E13.5, only certain areas of the forebrain are labeled. There is also a dorsoventrally-restricted region of labeling in the hindbrain and spinal cord. The mAb labels a large proteoglycan-like cell-surface antigen (>200 kD). The labeling pattern of FORSE-1 is conserved in various mammals and in chick.

To determine whether the FORSE-1 labeling pattern is similar to that of known transcription factors, the expression of BF-1 and Dlx-2 was compared with FORSE-1. There is a striking overlap between BF-1 and FORSE-1 in the telencephalon. In contrast, FORSE-1 and Dlx-2 have very different patterns of expression in the forebrain, suggesting that regulation by Dlx-2 alone cannot explain the distribution of FORSE-1. They do, however, share some sharp boundaries in the diencephalon. In addition, FORSE-1 identifies some previously unknown boundaries in the developing forebrain. Thus, FORSE-1 is a new cell surface marker that can be used to subdivide the embryonic forebrain into regions smaller than previously described, providing further complexity necessary for developmental patterning.

I also studied the expression of the cell surface protein CD9 in the developing and adult rat nervous system. CD9 is implicated in intercellular signaling and cell adhesion in the hematopoetic system. In the nervous system, CD9 may perform similar functions in early sympathetic ganglia, chromaffin cells, and motor neurons, all of which express the protein. The presence of CD9 on the surfaces of Schwann cells and axons at the appropriate time may allow the protein to participate in the cellular interactions involved in myelination.

Pio del Rio Hortega and the discovery of the oligodendrocytes

Rio del Rio Hortega (1882-1945) discovered microglia and oligodendrocytes (OLGs), and after Ramon y Cajal, was the most prominent figure of the Spanish school of neurology. He began his scientific career with Nicolas Achucarro from whom he learned the use of metallic impregnation techniques suitable to study non-neuronal cells. Later on, he joined Cajal's laboratory. and Subsequently, he created his own group, where he continued to develop other innovative modifications of silver staining methods that revolutionized the study of glial cells a century ago. He was also interested in neuropathology and became a leading authority on Central Nervous System (CNS) tumors. In parallel to this clinical activity, del Rio Hortega rendered the first systematic description of a major polymorphism present in a subtype of macroglial cells that he named as oligodendroglia and later OLGs. He established their ectodermal origin and suggested that they built the myelin sheath of CNS axons, just as Schwann cells did in the periphery. Notably, he also suggested the trophic role of OLGs for neuronal functionality, an idea that has been substantiated in the last few years. Del Rio Hortega became internationally recognized and established an important neurohistological school with outstanding pupils from Spain and abroad, which nearly disappeared after his exile due to the Spanish civil war. Yet, the difficulty of metal impregnation methods and their variability in results, delayed for some decades the confirmation of his great insights into oligodendrocyte biology until the development of electron microscopy and immunohistochemistry. This review aims at summarizing the pioneer and essential contributions of del Rio Hortega to the current knowledge of oligodendrocyte structure and function, and to provide a hint of the scientific personality of this extraordinary and insufficiently recognized man.

Hanseníase neural, aspectos diagnósticos da forma neural pura e mecanismos imunopatogênicos da lesão do nervo na doença. Participação de quimiocinas CCL2 e CXCL10 e metaloproteinases 2 e 9

O diagnóstico da hanseníase neural pura baseia-se em dados clínicos e laboratoriais do paciente, incluindo a histopatologia de espécimes de biópsia de nervo e detecção de DNA de Mycobacterium leprae (M. leprae) pelo PCR. Como o exame histopatológico e a técnica PCR podem não ser suficientes para confirmar o diagnóstico, a imunomarcação de lipoarabinomanana (LAM) e/ou Glicolipídio fenólico 1 (PGL1) - componentes de parede celular de M. leprae foi utilizada na primeira etapa deste estudo, na tentativa de detectar qualquer presença vestigial do M. leprae em amostras de nervo sem bacilos. Além disso, sabe-se que a lesão do nervo na hanseníase pode diretamente ser induzida pelo M. leprae nos estágios iniciais da infecção, no entanto, os mecanismos imunomediados adicionam severidade ao comprometimento da função neural em períodos sintomáticos da doença. Este estudo investigou também a expressão imuno-histoquímica de marcadores envolvidos nos mecanismos de patogenicidade do dano ao nervo na hanseníase. Os imunomarcadores selecionados foram: quimiocinas CXCL10, CCL2, CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, e metaloproteinases 2 e 9. O estudo foi desenvolvido em espécimes de biópsias congeladas de nervo coletados de pacientes com HNP (n=23 / 6 BAAR+ e 17 BAAR - PCR +) e pacientes diagnosticados com outras neuropatias (n=5) utilizados como controle. Todas as amostras foram criosseccionadas e submetidas à imunoperoxidase. Os resultados iniciais demonstraram que as 6 amostras de nervos BAAR+ são LAM+/PGL1+. Já entre as 17 amostras de nervos BAAR-, 8 são LAM+ e/ou PGL1+. Nas 17 amostras de nervos BAAR-PCR+, apenas 7 tiveram resultados LAM+ e/ou PGL1+. A detecção de imunorreatividade para LAM e PGL1 nas amostras de nervo do grupo HNP contribuiu para a maior eficiência diagnóstica na ausência recursos a diagnósticos moleculares. Os resultados da segunda parte deste estudo mostraram que foram encontradas imunoreatividade para CXCL10, CCL2, MMP2 e MMP9 nos nervos da hanseníase, mas não em amostras de nervos com outras neuropatias. Além disso, essa imunomarcação foi encontrada predominantemente em células de Schwann e em macrófagos da população celular inflamatória nos nervos HNP. Os outros marcadores de ativação imunológica foram encontrados em leucócitos (linfócitos T e macrófagos) do infiltrado inflamatório encontrados nos nervos. A expressão de todos os marcadores, exceto CXCL10, apresentou associação com a fibrose, no entanto, apenas a CCL2, independentemente dos outros imunomarcadores, estava associada a esse excessivo depósito de matriz extracelular. Nenhuma diferença na frequência da imunomarcação foi detectada entre os subgrupos BAAR+ e BAAR-, exceção feita apenas às células CD68+ e HLA-DR+, que apresentaram discreta diferença entre os grupos BAAR + e BAAR- com granuloma epitelioide. A expressão de MMP9 associada com fibrose é consistente com os resultados anteriores do grupo de pesquisa. Estes resultados indicam que as quimiocinas CCL2 e CXCL10 não são determinantes para o estabelecimento das lesões com ou sem bacilos nos em nervo em estágios avançados da doença, entretanto, a CCL2 está associada com o recrutamento de macrófagos e com o desenvolvimento da fibrose do nervo na lesão neural da hanseníase.

Alterações da expressão da proteína PHEX na Hanseníase

A hanseníase é uma doença infecciosa causada pelo Mycobacteruim leprae, um bacilo intracelular obrigatório, que prolifera principalmente na pele e nos nervos periféricos no interior de células como macrófagos e células de Schwann. A transmissão ocorre por meio das mucosas das vias respiratórias, provavelmente por aerossóis expelidos por indivíduos infectados. O homem é o seu hospedeiro natural sendo a multiplicação do bacilo muito lenta, com um período de geração estimado de 14 dias. Apesar da mínima variação no genoma do M. leprae, a doença é caracterizada por um espectro de formas clínicas bem definido, decorrente da capacidade de resposta imune do hospedeiro. Em pacientes classificados como multibacilares (MB) a doença é disseminada, com inúmeras lesões de pele e proliferação bacilar considerável. Nesses indivíduos ocorre hiporresponsividade celular ao M. leprae. Nas formas paucibacilares (PB), os pacientes apresentam uma ou poucas lesões, a carga bacilar é pequena e, às vezes não observada por meio da baciloscopia tradicional e ocorre resposta imune patógeno-específica. As incapacidades físicas nos pacientes decorrem da neuropatia e osteopatia e podem ser irreversíveis. Essas deformidades podem avançar mesmo após a diminuição da carga bacilar com o final do tratamento poliquimioterápico. A presente tese teve por objetivo estudar as implicações da proteína PHEX nas alterações fisiopatológicas da hanseníase, em especial as alterações ósseas. A proteína PHEX (Phosphate-regulating gene with Homologies to Endopeptidase on the X chromosome) é expressa em várias células humanas e, no primeiro artigo que compõe essa tese, demonstramos que o M.leprae leva à diminuição da expressão de PHEX em linhagens de células de Schwann e osteoblastos humanos. Este efeito foi igualmente causado por outras espécies de micobactérias. No segundo manuscrito ora submetido, observamos que em leucócitos sanguíneos de pacientes hansenianos também ocorreu modulação negativa de PHEX. Este efeito não se relacionou com a capacidade de produção de citocinas inflamatórias frente ao M. leprae in vitro ou com alterações bioquímicas. O efeito inibidor da mineralização ocasionado pela modulação negativa de PHEX talvez contribua para a doença óssea da hanseníase, auxiliando a explicar a capacidade do M. leprae de penetrar e sobreviver no osso.

Immobilization of cell-binding peptides on poly-ε-caprolactone film surface to biomimic the peripheral nervous system.

Cell-material interactions are crucial for cell adhesion and proliferation on biomaterial surfaces. Immobilization of biomolecules leads to the formation of biomimetic substrates, improving cell response. We introduced RGD (Arg-Gly-Asp) sequences on poly-ε-caprolactone (PCL) film surfaces using thiol chemistry to enhance Schwann cell (SC) response. XPS elemental analysis indicated an estimate of 2-3% peptide functionalization on the PCL surface, comparable with carbodiimide chemistry. Contact angle was not remarkably reduced; hence, cell response was only affected by chemical cues on the film surface. Adhesion and proliferation of Schwann cells were enhanced after PCL modification. Particularly, RGD immobilization increased cell attachment up to 40% after 6 h of culture. It was demonstrated that SC morphology changed from round to very elongated shape when surface modification was carried out, with an increase in the length of cellular processes up to 50% after 5 days of culture. Finally RGD immobilization triggered the formation of focal adhesion related to higher cell spreading. In summary, this study provides a method for immobilization of biomolecules on PCL films to be used in peripheral nerve repair, as demonstrated by the enhanced response of Schwann cells.

Extracellular matrix components in peripheral nerve repair: how to affect neural cellular response and nerve regeneration?

Peripheral nerve injury is a serious problem affecting significantly patients' life. Autografts are the "gold standard" used to repair the injury gap, however, only 50% of patients fully recover from the trauma. Artificial conduits are a valid alternative to repairing peripheral nerve. They aim at confining the nerve environment throughout the regeneration process, and providing guidance to axon outgrowth. Biocompatible materials have been carefully designed to reduce inflammation and scar tissue formation, but modifications of the inner lumen are still required in order to optimise the scaffolds. Biomicking the native neural tissue with extracellular matrix fillers or coatings showed great promises in repairing longer gaps and extending cell survival. In addition, extracellular matrix molecules provide a platform to further bind growth factors that can be released in the system over time. Alternatively, conduit fillers can be used for cell transplantation at the injury site, reducing the lag time required for endogenous Schwann cells to proliferate and take part in the regeneration process. This review provides an overview on the importance of extracellular matrix molecules in peripheral nerve repair.

Cellular schwannomas of the intracranial and intraspinal compartment: morphological and immunological characteristics compared with classical benign schwannomas.

The concept of cellular schwannoma as an unusual benign tumor is well established for peripheral nerves but has never been tested in neurosurgical series. In order to test the validity of this concept in cranial nerves and spinal roots we performed an analysis of the clinical and morphological characteristics of 12 cellular and 166 classical benign schwannomas. Immunohistochemical detection of antigen expression in Schwann cells including proliferating cell nuclear antigen (PCNA) was also performed. This study shows that cellular schwannomas in neurosurgical series manifest at a lower age than the classical benign variant and occur mainly in the spinal roots. Mitotic activity and sinusoidal vessels appear more frequently in cellular schwannomas and constitute with high cellularity, the most valuable criteria separating both entities. The postoperative course in both types of tumors was free of metastases or sarcomatous changes. Immunoexpression of S-100 protein, vimentin, epithelial membrane antigen and glial fibrillary acidic protein is not statistically different between the two variants. In contrast, PCNA is more highly expressed in cellular schwannomas. These These results confirm the concept that cellular schwannomas are a clinico-pathological variant of benign schwannomas and provide significant support for the introduction of this entity in neurosurgical oncology.

Plasticité présynaptique et gliale à long-terme en réponse à un changement chronique de l’activité synaptique,à la jonction neuromusculaire d’amphibien

La plasticité synaptique est une importante propriété du système nerveux, impliquée dans l’intégration de l’information. Cette plasticité a généralement été décrite par des changements aux niveaux pré et postsynaptiques. Notamment, l’efficacité présynaptique, soit la probabilité de libération de neurotransmetteurs associée au contenu quantique d’une synapse, peut être augmentée ou diminuée selon l’activité antérieure de la synapse. Malgré cette caractérisation, les mécanismes à l’origine de la détermination de l’efficacité présynaptique demeurent obscurs. Également, la plasticité synaptique reste encore mal définie au niveau glial, limitant, de ce fait, notre compréhension de l’intégration de l’information. Pourtant, la dernière décennie a mené à une redéfinition du rôle des cellules gliales. Autrefois reléguées à un rôle de support passif aux neurones, elles sont désormais reconnues comme étant impliquées dans la régulation de la neurotransmission. Notamment, à la jonction neuromusculaire (JNM), les cellules de Schwann périsynaptiques (CSPs) sont reconnues pour moduler l’efficacité présynaptique et les phénomènes de plasticité. Un tel rôle actif dans la modulation de la neurotransmission implique cependant que les CSPs soient en mesure de s’adapter aux besoins changeants des JNMs auxquelles elles sont associées. La plasticité synaptique devrait donc sous-tendre une forme de plasticité gliale. Nous savons, en effet, que la JNM est capable de modifications tant morphologiques que physiologiques en réponse à des altérations de l'activité synaptique. Par exemple, la stimulation chronique des terminaisons nerveuses entraîne une diminution persistante de l’efficacité présynaptique et une augmentation de la résistance à la dépression. À l’opposé, le blocage chronique des récepteurs nicotiniques entraîne une augmentation prolongée de l’efficacité présynaptique. Aussi, compte tenu que les CSPs détectent et répondent à la neurotransmission et qu’elles réagissent à certains stimuli environnementaux par des changements morphologiques, physiologiques et d’expression génique, nous proposons que le changement d'efficacité présynaptique imposé à la synapse, soit par une stimulation nerveuse chronique ou par blocage chronique des récepteurs nicotiniques, résulte en une adaptation des propriétés des CSPs. Cette thèse propose donc d’étudier, en parallèle, la plasticité présynaptique et gliale à long-terme, en réponse à un changement chronique de l’activité synaptique, à la JNM d’amphibien. Nos résultats démontrent les adaptations présynaptiques de l’efficacité présynaptique, des phénomènes de plasticité à court-terme, du contenu mitochondrial et de la signalisation calcique. De même, ils révèlent différentes adaptations gliales, notamment au niveau de la sensibilité des CSPs aux neurotransmetteurs et des propriétés de leur réponse calcique. Les adaptations présynaptiques et gliales sont discutées, en parallèle, en termes de mécanismes et de fonctions possibles dans la régulation de la neurotransmission. Nos travaux confirment donc la coïncidence de la plasticité présynaptique et gliale et, en ce sens, soulèvent l’importance des adaptations gliales pour le maintien de la fonction synaptique.

L’altération des interactions neurone-glie à la jonction neuromusculaire de souris âgées

Durant le vieillissement, l’ensemble des fonctions de l’organisme se détériore, que ce soit aussi bien au niveau moteur que cognitif. Le vieillissement s’accompagne d’une diminution de la force, ainsi que de la masse musculaire. Des études récentes tendent à montrer que cette perte de masse musculaire que l’on appelle sarcopénie aurait pour origine un dérèglement de la jonction neuromusculaire. Les changements au niveau du présynaptique et du post synaptiques lors du vieillissement normal font l’objet de plusieurs études, mais les changements relatifs aux cellules de Schwann périsynaptique sont très peu connus. Le but de cette étude est donc d’analyser les modifications des interactions neurone-glie à la jonction neuromusculaire. Dans cette étude, nous montrons que certaines fonctions des cellules gliales de la synapse âgée sont déréglées, en particulier, le type de récepteurs activés par une stimulation nerveuse à haute fréquence. D’autre part, nos résultats montrent que les mécanismes responsables de l’augmentation de la transmission synaptique suite à cette stimulation nerveuse à haute fréquence sont altérés à la synapse âgée. Enfin, outre les modifications de la terminaison axonale et de la fibre musculaire, les cellules gliales montrent des signes de réorganisation structurelle propre à une synapse en réparation. Ces résultats montrent que le fonctionnement de la jonction neuromusculaire et a fortiori les interactions neurones-glie sont altérées lors du vieillissement normal.

The role of Wnt signalling in the development of somites and neural crest

The Wnt family of secreted signalling molecules controls a wide range of developmental processes in all metazoans. In this investigation we concentrate on the role that members of this family play during the development of (1) the somites and (2) the neural crest. (3) We also isolate a novel component of the Wnt signalling pathway called Naked cuticle and investigate the role that this protein may play in both of the previously mentioned developmental processes. (1) In higher vertebrates the paraxial mesoderm undergoes a mesenchymal-to-epithelial transformation to form segmentally organised structures called somites. Experiments have shown that signals originating from the ectoderm overlying the somites or from midline structures are required for the formation of the somites, but their identity has yet to be determined. Wnt6 is a good candidate as a somite epithelialisation factor from the ectoderm since it is expressed in this tissue. In this study we show that injection of Wnt6-producing cells beneath the ectoderm at the level of the segmental plate or lateral to the segmental plate leads to the formation of numerous small epithelial somites. We show that Wnts are indeed responsible for the epithelialisation of somites by applying Wnt antagonists which result in the segmental plate being unable to form somites. These results show that Wnt6, the only member of this family to be localised to the chick paraxial ectoderm, is able to regulate the development of epithelial somites and that cellular organisation is pivotal in the execution of the differentiation programmes. (2) The neural crest is a population of multipotent progenitor cells that arise from the neural ectoderm in all vertebrate embryos and form a multitude of derivatives including the peripheral sensory neurons, the enteric nervous system, Schwann cells, pigment cells and parts of the craniofacial skeleton. The induction of the neural crest relies on an ectodermally derived signal, but the identity of the molecule performing this role in amniotes is not known. Here we show that Wnt6, a protein expressed in the ectoderm, induces neural crest production. (3) The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement (Pandur et al. 2002b). Recent work has identified the protein Naked cuticle as an intracellular switch promoting the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked cuticle-1 (cNkd1) and demonstrate that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly our study shows that the expression of cNkd1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

NEUREGULINS 1-ALPHA AND 1-BETA ON THE REGENERATION THE PERIPHERAL NERVES

Objective: To evaluate the effect of the neuregulins 1-alpha and 1-beta on the regeneration the sciatic nerves of male adult C57BL/6J mice, using the tubulization technique. Methods: Eighteen animals were used, divided into three groups. A polyethylene prosthesis was implanted in a 4.0 mm defect of the left sciatic nerve, as follows: group 1 containing only purified collagen (Vitrogen (R)); group 2, collagen with neuregulin 1-alpha; group 3, collagen with neuregulin 1-beta. The control group consisted of six segments of right sciatic nerves. After four weeks, the animals were sacrificed. A segment from the midpoint of the nerve regenerated inside the prostheses was extracted; histological sections were standardized, and slides were made up for histomorphometric analysis. Results: the results were statistically compared using the Tukey multiple comparisons test and The Student`s t test. The animals treated with neuregulins had greater numbers of myelinized axons, with a statistically significant difference in relation to the collagen-only group. There was no statistical difference between the neuregulin 1-alpha and 1-beta groups. Conclusion: The addition of neuregulins provided a significant increase in the number of myelinized fibers.

Pharmacological and local toxicity studies of a liposomal formulation for the novel local anaesthetic ropivacaine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Implante de tubo de silicone com e sem colágeno na regeneração de nervos em eqüinos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)