Tesi sul tema "Ependymin"

"Alzheimer’s disease (AD) is a neurodegenerative disorder that leads to dementia in adults. The mechanism of neurodegeneration is thought to involve the extracellular production of a highly toxic A-beta peptide that engages cell surface receptors to induce cellular oxidative stress and apoptosis, but the signal transduction pathways that lead to A-beta induced cell death are unknown. We previously showed that a human ependymin neurotrophic peptide mimetic (hEPN-1) can promote cell survival in an in vitro AD model system. This initial observation was extended in this thesis by investigating the mechanism of A-beta induced apoptosis and hEPN-1 induced survival. Immunoblots were used to assay the total cellular levels of specific caspase proteins. The results show that A-beta induced apoptosis uses an extrinsic caspase pathway involving caspases-2 and -3, and that hEPN-1 treatment can reduce those caspase levels. A caspase activity assay showed that A-beta increased caspase-3/7 activity, while hEPN-1 treatment lowered it. Moreover, in vivo studies with AD transgenic mice showed that hEPN-1 treatment increased antioxidative superoxide dismutase levels in brain. Thus, hEPN-1 holds potential as a therapeutic to treat the underlying neurodegenerative cause of AD, not merely its symptoms as with other currently approved AD drugs."

"Alzheimer’s disease (AD) is the most widespread neurodegenerative disorder, affecting approximately 20 million people worldwide. AD pathology is primarily characterized by the formation of extracellular amyloid plaques resulting from the aggregation of insoluble amyloid-beta 1-42 (A-beta), and neurofibrillary tangles (NFT’s) resulting from intracellular aggregation of hyperphosphorylated tau protein. The current FDA-approved AD treatments do not stop or reverse neurodegeneration, but only treat the symptoms by increasing acetylcholine neurotransmitter. Our laboratory is attempting to provide an additional therapeutic approach by using neurotrophic factors to block apoptosis or to restore neurons. We previously demonstrated that, in an in vitro model for AD, hEPN-1 neurotrophic factor mimetics can block synthetic A-beta-induced neuronal cell death when added to cultures, presumably by blocking caspase activation. In this thesis, we extended these findings to study the effect of A-beta and hEPN-1 on tau hyperphosphorylation (as measured by immunoblots with phospho-specific antibodies) and nuclear DNA fragmentation (as measured by TUNEL staining), both in vitro and in vivo in AD transgenic mice. We found that A-beta induces the hyperphosphorylation of tau in both mouse N2a and human SHSY neuronal cells, and that hEPN-1 may lower this phosphorylation in N2a cells. Furthermore, we discovered that hEPN-1 can reduce nuclear DNA fragmentation when added both simultaneously to A-beta and 3 and 6 hours post A-beta addition. Finally, in vivo hEPN-1 may lower both tau hyperphosphorylation and caspase-7 related protein (C7RP) in AD transgenic (Tg) mice. The overall results validate our in vitro AD model, show the efficacy of hEPN-1 at blocking A-beta-induced DNA fragmentation even when added post-insult, and show that hEPN-1 may work in an AD mouse model. However, more studies must be conducted to confirm these findings. "

Densely ciliated ependymal cells line the ventricular surface of the brain, cerebral aqueduct, and the central canal of the spinal cord and beat continuously at up to 40Hz. Methodologies for the measurement and the containment of ependymal cilia were developed to allow their study in an ex-vivo setting. Three methods were used to measure ciliary beat frequency, the photomultiplier, photodiode and high speed imaging were established and compared. The method used, to hold ciliated ependymal strips affected ependymal ciliary beat frequency. Tissue held in a microscope cover glass system, or under direct observation of a water immersion lens, showed a beat frequency of half that of cilia observed in an incubation chamber using an inverted microscope. The incubation system, using an inverted microscope, and high speed video system were chosen for the studies in this thesis. Thes results obtained using this system may be summarised as follows:; there was no change in ciliary length or beat frequency between ependyma from infant and adult Wistar rats. However, ependymal cilia beat twice as fast as respiratory cilia. a pH of less than 7 was associated with dramatic fall in ciliary beat frequency which was shown to be directly related to changes in intracellular pH. increasing viscosity decreased ciliary beat frequency, though cilia demonstrated a capability to adapt to an increasing viscous load. pentobarbitone only slowed cilia at levels which were incompatible with life. Halothane, a volatile anaesthetic, caused significant, but reversible ciliary slowing at concentrations which are used in a clinical setting. adrenergic agents and forskolin had no effect on ependymal ciliary function. ethanol and acetaldehyde had no effect on ependymal ciliary beat frequency. low concentrations of the toxin MAM, thought to be implicated in the development of Parkinsonism, caused ciliary slowing.

OBJECTIVES: To retrospectively review the patient demographics, disease profile and treatment outcomes of paediatric patients treated for ependymoma at our institution. STUDY DESIGN AND METHODS: 51 eligible patients were treated between 1980 and 2013. The median age at presentation was 6 years. The majority of patients were male (66,7%), had infratentorial tumours (62,7%) and had low-grade tumours (70,6%). Gross total resection (GTR) was achieved in 22 patients (43,1%). Thirtyeight patients received adjuvant radiotherapy (76,5%) and 10 (19,6%) received adjuvant chemotherapy. RESULTS: The 5-year overall survival (OS) was 63,3 % (median follow up of 46 months). The 5 year progression free survival (PFS) was 50,70%. Seventeen (33,3%) patients experienced treatment failure, of which 13 (76,5%) represented local failure. The median time to first relapse was 20 months. The 5 year PFS for children > 3 was 50,0 % and 27,7% for children ≤ 3 years of age (p = 0.0356). GTR had a superior 5- year OS of 73,9% over subtotal resection with a value of 56,7% (p = 0.0016). Similarly an improved 5-year PFS of 70,3% versus 29,1% was observed with GTR over subtotal resection (p = <0.0001). Patients who received adjuvant radiotherapy (RT) had significantly better outcomes than those in whom RT was not given (p = <0.0001, 5 year OS of 69,7% versus 37,5%). CONCLUSION: This review confirms the finding that GTR is associated with improved outcomes and that adjuvant radiation therapy positively impacts survival. The worse outcomes in the younger age group requires further evaluation and possible change in treatment protocol for this group of patients.

This thesis describes the work that I have carried out in the Laboratory of Neurophysiolgy at the Université Libre de Bruxelles, under the supervision of Prof. Serge Schiffmann, in collaboration with Prof. Stéphane Schurmans of Université of Liège.The work is divided in two distinct but related projects and the results section is thus divided into two main chapters. The results described are presented in the form of two manuscripts, the first chapter is named “Ciliary phosphoinositides regulation by INPP5E controls Shh signaling by allowing trafficking of Gpr161 in neural stem cells primary cilium”.The second is named “Regulation of phosphoinositides ciliary levels controls trafficking and ciliogenesis in ependymal cells”.Since both manuscripts are comprehensive regarding the results, and methods, these are inserted as such into the thesis.An expanded introduction to the field, placing the results into context, precedes these two chapters. An extended discussion section follows each chapter; it presents some elements of discussion not included in the manuscripts, the implications of the results and the scope for further research.
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

The discovery of neural stem cells within the adult CNS has indicated an enormous potential in facilitating neuronal regeneration after injury. Studies from our laboratory have suggested that manipulation of the Rb/E2f pathway can directly impact embryonic and adult neurogenesis, thereby having tremendous potential for neuronal regeneration therapies. Recently, two new members of the Rb/E2f pathway have been discovered, the atypical E2fs: E2f7 and E2f8. Initial studies have suggested that atypical E2fs regulate diverse processes such as cell proliferation, DNA-stress response, apoptosis, however, their importance in the brain are unknown. To analyze their function during brain development, we crossed Nestin-Cre mice with mice bearing a conditional E2f7/E2f8 allele to delete both E2f7 and E2f8 in neural precursor cells. Whereas cortical development was largely unaffected by E2f7/E2f8 deficiency, we observed an enlargement of the lateral ventricles occurring postnatally, a brain condition named ventriculomegaly. We then looked at the ependymal cells, which are the cells lining the wall of the lateral ventricles, to determine if these cells were affected by the absence of atypical E2fs. We found progressive denaturation of the ependymal cell layer, distortion of the ependymal motile cilia and reactive astrocytes within the layer. We identified Gli3, a component of the Sonic hedgehog pathway (Shh), as a target for E2fs, including atypical E2fs. We unravelled a novel mechanism by which atypical E2fs regulate the expression of Gli3, leading to up-regulation of Numb/NumbL, which in consequence destabilizes cadherins organization within the ependymal cell layer. In conclusion, our work suggests that E2f7 and E2f8 transcription factors play an essential role in maintaining the ependymal cell barrier.

O maior nicho neurogênico no encéfalo adulto está ao redor dos ventrículos laterais, mas a identificação das células que iniciam tal formação é controversa. Há uma inervação do peptídeo CART que pode abrir perspectivas para o entendimento de seu papel na modulação da neurogênese. Propormos estudar a citoarquitetura ultra-estrutural das células proliferativas na região periventricular e descrever a organização dessa região e sua inervação por axonios imunorreativos ao CART. Utilizamos ratos e camundongos adultos, preparados para análise ultraestrutural e neuroquímica em microscópios eletrônicos de transmissão e varredura de alta-resolução, de luz e laser confocal. O estudo da proliferação e inervação ocorreu com a administração do marcador de fase S BrdU e anticorpos anti-BrdU, anti-CART, anti-DCX, anti-GFAP e anti-GFP. Cada tipo celular do nicho neurogênico apresentou uma densidade própria de ir-BrdU. Identificamos células de revestimento ventricular inervadas por axônios. A maior densidade de inervação ir-CART ocorre ao longo do trajeto dos neurônios em formação.
The major neurogenic niche in adult brains surrounds the lateral ventricles, but the identity of the cell that initiates this process in controversial. There is an innervation made by the CART peptide that may lead to perspectives for understanding its role in modulation of neurogenesis. We propose to study the ultrastructural cytoarchitecture of proliferative cells in this region and its innervation by CART immunoreactive axons. We used adult rats and mice, prepared for ultrastructural and neurochemical analysis by transmission and high-resolution scanning electron, light and laser confocal microscopes. The proliferation and innervation studies occured with the S-phase marker BrdU and anti-Brdu, anti-CART, anti-DCX, anti-GFAP, anti-GFP antibodies. Each sort of cells in neurogenic niche presented a proper density of BrdU staining. We identified the cells lining the ventricle being innervated by axons. The major density of CART innervation occurs along the pathway of neurons in maturation process.

Platelet-derived growth factor-C (PDGF-C) and its tyrosine kinase receptor PDGFRɑ have been shown to contribute to several key processes during central nervous system (CNS) development, including normal vascularization and formation of cerebral ventricles and basal membrane of the meninges. Due to redundancy between PDGF-C and PDGF-A, PDGF-C specific roles are sometimes masked and difficult to determine. Using the double mutant    Pdgfc-/-;PdgfraGFP/+ mouse (Mus musculus) strain we were able to detect and examine a new, undescribed phenotype of PDGF-C impaired mice, namely cerebellar malformations. These mutant mice displayed an upwards rotation of the cerebellar vermis with a severe posterior vermis hypoplasia and an enlarged fourth ventricle, suggesting PDGF-C/PDGFRɑ signaling as a novel candidate to induce Dandy-Walker malformation (DWM). Due to suspected cerebellar vascular malformation a quantification of diameter, density and number of vessels were performed. A significant increase (P < 0.05) of the number and density of vascular bed in the cerebellar nuclei was detected, however the vessel diameter was not significantly different (P > 0.05) in Pdgfc-/-;PdgfraGFP/+ mice in comparison with the control. Through immunofluorescence staining we detected discontinuation of the ependyma in the acute angle of the ventricular zone adjacent to the rhombic lip, interfacing the fourth ventricle and cerebellar anlagen. We further noted ectopic expression of rhombic lip derived cells in the ventricular zone, suggesting a misguided migration due to ablation of PDGF-C. We conclude that PDGF-C is an essential player in normal cerebellar development.

The developing mammalian spinal cord can generate all required cell types; in the adult, there exists a subpopulation of progenitors able to generate multiple cell types. The ependymal cell (EC) layer of the central canal (CC) of the adult spinal cord is a neural stem cell niche possessing the potential to self-renew and differentiate into astrocytes and oligodendrocytes in vivo and in vitro, and neurons in vitro. However, the differences between a developmental EC and an adult EC are not well established. Further, it is unknown whether ECs have equivalent stem/progenitor abilities, and whether certain subpopulations may be better targets for regeneration. Multiple Sclerosis (MS) is a debilitating disease characterized by areas of chronically demyelinated CNS, and an attractive target for regenerative therapy. Little is known about why endogenous regenerative programmes in MS are unsuccessful. Using in-situ hybridization (ISH) data from the Allen Spinal Cord Atlas and immunofluorescent detection of antigen in mice, we revealed temporal and spatial segmentation of ECs expressing distinct groups of genes. The ages analyzed (postnatal day 4 and 56) are relevant to the mechanisms of neurogenesis and gliogenesis, as the spinal cord is capable of regaining greater functionality following injury at P4 than at P56. Gene ontology analysis suggests different regions of the CC may contribute in different ways to spinal cord repair. Furthermore, we found the EC niche experiences altered levels of proliferation after two different types of insult, SCI and demyelinating injury in mice. We then used MS spinal cord to test what extent such repair from demyelination takes place. We found that remyelination occurred, but was inadequate and highly variable both between and within patients. Abnormal myelinating cells of a unique phenotype were found in the CNS and PNS; the extent and prevalence of peripheral involvement has not been previously reported. Further work is needed to determine whether the human EC layer could be targeted to enhance remyelination. Overall, these results reveal novel cell phenotypes engaged in repair in mouse and human spinal cord that may serve a basis for future therapeutic intervention.
Medicine, Faculty of
Graduate

Les cellules épendymaires sont des cellules multiciliées qui tapissent les parois de toutes les cavités du cerveau. Une fois différenciées, ces cellules ne se divisent plus au cours de la vie. Le battement de ces multiples cils motiles joue un rôle important pour maintenir un flux constant de liquide cérébrospinal à travers toutes les cavités cérébrales. Les cellules épendymaires assurent également des fonctions critiques d’échanges moléculaires avec le liquide cérébrospinal. Dans son ensemble, l’implication des cellules épendymaires et de leurs cils motiles s’avère d’une importance majeure dans le maintien des circuits neuraux ainsi que dans le fonctionnement plus global du cerveau. Récemment, une nouvelle caractéristique des cellules épendymaires a été identifiée ; elles font partie d’un microenvironnement appelé une « niche » centrée autour de cellules souches neurales dans le cerveau du rongeur adulte. Ces cellules souches neurales adultes sont capables de produire de nouveaux neurones qui migreront vers le bulbe olfactif des rongeurs adultes. Concernant leur origine, il a été montré que les cellules épendymaires multiciliées dérivent des cellules souches neurales durant les stades tardifs embryonnaires. Ces mêmes cellules souches peuvent d’ailleurs donner naissance à la plupart des différents types de cellules du cerveau. Cependant, les mécanismes par lesquels les cellules souches décident de leur destin cellulaire restent largement méconnus. Dans ce projet, nous étudions quel type de division donne naissance à des cellules épendymaires et nous nous intéressons également au lignage épendymaire. Nos données suggèrent que les cellules épendymaires ne migrent pas après leur dernière division et qu’elles restent à proximité de l’endroit où elles ont été produites. Chose particulièrement intéressante, nous montrons que les cellules épendymaires peuvent être générées par division symétrique ou asymétrique. Nos résultats révèlent aussi que les cellules souches neurales embryonnaires se divisent de manière asymétrique pour donner naissance à la fois à une celluleépendymaire et à une cellule souche neurale adulte. Ces données viennent s’ajouter à la connaissance actuelle que nous avons du développement du cerveau. De plus, elles pourraient contribuer à ouvrir de nouvelles perspectives et stratégies thérapeutiques pour soigner les maladies neurodégénératives à beaucoup plus long terme
Ependymal cells are multiciliated cells lining the walls of all brain cavities. Once they are mature, they do not divide during life. Their motile ciliary beating endorses a crucial role in maintaining a proper flow of cerebrospinal fluid throughout all brain cavities. Ependymal cells also ensure critical molecular exchanges of the cerebrospinal fluid. On the whole, the involvement of ependymal cells and their multiple motile cilia in the maintenance of the neural circuits and more globally in the well-functioning of the entire brain have proven paramount. More recently, a new characteristic of ependymal cells has been brought to light. Namely, they are part of a microenvironment so called a “niche” surrounding adult neural stem cells in the adult rodent brain. Noteworthy, these adult neuralstem cells are capable of producing new neurons that will migrate to the olfactory bulb of rodents. In terms of their origin, it was shown that multiciliated ependymal cells derive from neural stem cells during late embryonic stages. Besides, the same stem cells can give rise to most cell types of the brain. However, little is known about how fate-decision is made in neural stem cells. In this project, we tackle more particularly how multiciliated ependymal cells arise from the neural stem cells. Most specifically, we address the type of celldivision and the ependymal cell lineage. We find that ependymal cells are not migrating subsequent to their last division, but rather stay where they were first produced. Most interestingly, they can be generated through both symmetric and asymmetric cell division. We also show that embryonic neural stem cells divide asymmetrically to give rise to both an ependymal cell and an adult stem cell. We are confident that these data bring major new insights in the current understanding of neural development. Additionally, these findingscould contribute in opening new therapeutic perspectives and strategies to cure neurodegenerative diseases in a much longer term

Ischaemic stroke is a major cause of mortality and chronic disability for which there is no effective treatment. The subventricular zone (SVZ) is an adult neurogenic niche which mediates limited endogenous repair following stroke. To harness this phenomenon for therapy, it is important to understand how the SVZ niche is altered in stroke, and the processes that recruit neural precursors to the site of injury, which becomes a de facto neurogenic niche. Galectin-3 (Gal-3) is a β-galactoside binding protein involved in cellular adhesion, inflammation and tumour metastasis. Gal-3 is specifically expressed in the SVZ and maintains neuroblast migration to the olfactory bulb, although its role in post-stroke neurogenesis is not well-understood. Therefore, this project aimed to (1) characterise the cytoarchitecture of the SVZ in response to stroke, and (2) examine the role of Gal-3 in stroke outcome and tissue remodelling, and test the hypothesis that Gal-3 is required for neuroblast ectopic migration into the ischaemic striatum. Using the intraluminal filament model of middle cerebral artery occlusion (MCAO) in mice, and whole mounts of the lateral ventricular wall, significant SVZ reactive astrocytosis and increased vascular branching were observed, thereby disrupting the neuroblast migratory scaffold. Stroke increased SVZ cell proliferation without increase in cell death. Post-stroke ependymal cells were enlarged and non-proliferative, and assumed a reactive astroglial phenotype, expressing de novo high levels of glial fibrillary acidic protein. This was associated with focal planar cell polarity misalignment, and turbulent and decreased rate of cerebrospinal fluid flow. These findings demonstrate significant changes in multiple SVZ cell types which are positioned to influence post-stroke neurogenesis and regulation of the neural stem cell niche Gal-3 was up-regulated in the ischaemic brain and ipsilateral SVZ. To elucidate the role of Gal-3 after stroke, MCAO was performed in wildtype and Gal-3 null (Gal-3^-/-) mice, and parameters of stroke outcome and post-stroke neurogenesis compared. The deletion of Gal-3 did not affect infarct volumes or neurological outcomes, although neuroblast migration into the ischaemic striatum was increased in Gal-3^-/- brains. Gal-3^-/- mice failed to mount an angiogenic response in the ischaemic striatum, and this was associated with lower levels of vascular endothelial growth factor (VEGF) and increased anti-angiogenic protein levels. Loss of Gal-3 further disrupted the pro-proliferative neural-vascular interaction at the basement membrane. The current data indicate that Gal-3 is a pleiotropic molecule which has distinct roles in both the SVZ and the post-stroke striatum as niches of adult neurogenesis.

Les lésions médullaires traumatiques (LMT) conduisent à une atteinte des voies nerveuses sensitives et motrices. Leur taux de mortalité reste très élevé, d'où la nécessité de trouver de nouveaux traitements. Les Cellules Gliales Olfactives (CGOs) représentent un candidat intéressant de par leur fonction au sein du système olfactif primaire. La découverte d'une population de cellule souche neurale bordant le canal central de la moelle spinale (MS) adulte, appelées cellules épendymaires, suscite un nouvel espoir dans le domaine des biothérapies. Ce travail de thèse a permis d'étudier l'effet d'une transplantation de CGOs sue le comportement des cellules résidentes de la moelle spinale et notamment les cellules souches épendymaires qui, en association avec les astrocytes et les péricytes, participent aux mécanismes de guérison des LMT. L'utilisation du modèle murin hFoxJ1-CreERT2::YFP (permettant le suivi spécifique des cellules épendymaires et de leur progénie), a montré que les CGOs augmentaient in vitro le potentiel d'auto-renouvellement des cellules souches de la MS et modifiaient leur voie de différenciation vers un type neural. In vivo, la transplantation de CGOs augmente la prolifération des cellules épendymaires ainsi que leur différenciation en astrocytes hypo-réactifs conduisant à la formation d'un environnement post-lésionnel bénéfique à la survie neuronal et l'établissement d'une neurogenèse. Nos travaux ont montré pour la première fois que la transplantation de CGOs après LMT permettait la génération de nouveaux neurones. Ceci constitue un nouvel espoir dans l'établissement de stratégies thérapeutiques pour le traitement des LMT chez l'Homme
The spinal cord injuries (SCI) lead to the damages of the spinal cord or nerves and often cause permanent changes in body functions leading to the death. Cell therapies have raised great hope for regenerative medicine. Clinical data showed that the olfactory ensheathing cells (OECs) enhanced functional recovery after SCI and could be a very attractive therapeutic approach. Moreover, the discovery of a new endogenous resident stem cell population, lining the central canal of the spinal cord, named ependymal stem cells, represents a new hope for the therapy. This thesis analyzed the role of OECs transplantation, on the behaviour of ependymal stem cells since these cells, together with astrocytes and pericytes significantly contribute to the recovery of SCI. The use of the mouse model hFoxJ1-CreERT2::YFP (allowing to specifically follow the ependymal stem cells ant their progeny) showed that OECs increased in vitro the self-renewal potential of spinal cord stem cells and modified their differentiation pathway towards a neural type. In vivo, OECs transplantation significantly increases the proliferation of ependymal cells and their differenciation into hypo-reactive astrocytes leading to the formation of a beneficial environment to neuronal survival and the neurogenesis establishment. Our results also showed for the first time that OECs transplantation after SCI allows the generation of new neurons by non-ependymal cell-derived progenitors. These results represent a new hope in the establishment of therapeutic strategies for the treatment of SCI in humans

by Tong Yuen Kwan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1998.
Includes bibliographical references (leaves 124-141).
Abstract also in Chinese.
acknowledgements --- p.i
Abstract (English/Chinese) --- p.ii
contents --- p.vi
list of tables --- p.viii
ost of figures --- p.x
Chapter I. --- introduction --- p.1
Chapter I.1. --- Tumors of the Central Nervous System --- p.1
Chapter I.2. --- Histopathological Classification of Human Glial Tumors --- p.3
Chapter I.2.1. --- Histopathology of Astrocytic Gliomas --- p.3
Chapter I.2.1.1. --- Diffuse Astrocytomas --- p.3
Chapter I.2.1.2. --- Others --- p.6
Chapter I.2.2. --- Histopathology of Non-Astrocytic Gliomas --- p.6
Chapter I.2.2.1. --- Oligodendroglial Tumors --- p.6
Chapter I.2.2.2. --- Ependymal Tumors --- p.9
Chapter I.3. --- Tumor Suppressor Genes --- p.14
Chapter I.3.1. --- p53 --- p.14
Chapter I.3.1.1. --- Historical Perspectives --- p.14
Chapter I.3.1.2. --- Structure of p53 Gene and Protein --- p.15
Chapter I.3.1.3. --- Functions of Wild-Type p53 Protein --- p.18
Chapter I.3.1.4. --- Regulation and Modulation of the Functions of p53 --- p.21
Chapter I.3.1.5. --- Mechnism of p53 Inactivation --- p.23
Chapter I.3.1.6. --- p53 Mutation Profiles in Human Tumors --- p.25
Chapter I.3.2. --- Novel Genes --- p.28
Chapter I.3.2.1. --- PTEN/MMAC1 --- p.28
Chapter I.3.2.2. --- DMBT1 --- p.31
Chapter I.4. --- Cytogenetic and Molecular Genetic Studies in Gliomas --- p.34
Chapter I.4.1. --- Astrocytic Gliomas --- p.34
Chapter I.4.2. --- Non-Astrocytic Gliomas --- p.39
Chapter I.4.2.1. --- Oligodendroglial Tumors --- p.39
Chapter I.4.2.2. --- Ependymal Tumors --- p.46
Chapter II. --- objectives of study --- p.49
Chapter III. --- materials and methods --- p.52
Chapter III.l. --- Patients and Materials --- p.52
Chapter III.2. --- Collection of Samples --- p.57
Chapter III.3. --- DNA Extraction --- p.58
Chapter III.3.1. --- Extraction of Genomic DNA from Formalin-Fixed Paraffin Embedded Tissues --- p.58
Chapter III.3.2. --- Extraction of Genomic DNA from Blood --- p.60
Chapter III.4. --- Loss of Heterozygosity (LOH) Analysis on Chromosome 10q --- p.61
Chapter III.4.1. --- Microsatellite Markers --- p.62
Chapter III.4.2. --- Amplification of Target Sequences by PCR --- p.63
Chapter III.4.3. --- Denaturing Polyaerylamide Gel Electrophoresis --- p.64
Chapter III.4.4. --- Detection of Loss of Heterozygosity (LOH) --- p.64
Chapter III.5. --- Mutational Analysis of p53 and PTEN/MMAC1 --- p.66
Chapter III.5.1. --- Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) Analysis --- p.66
Chapter III.5.1.1. --- PCR Primers --- p.66
Chapter III.5.1.2. --- PCR Amplification of Target Sequences --- p.68
Chapter III.5.1.3. --- Non-denaturing Polyacrylamide Gel Electrophoresis --- p.71
Chapter III.5.2. --- Direct DNA Sequencing Analysis --- p.72
Chapter III.5.2.1. --- Cycle Sequencing --- p.72
Chapter III.5.2.2. --- Denaturing Gel Electrophoresis --- p.73
Chapter III.6. --- Differential PCR for Detection of MDM2 Amplification --- p.74
Chapter III.6.1. --- DNA Amplification by PCR --- p.74
Chapter III.6.2. --- Polyacrylamide Gel Electrophoresis --- p.75
Chapter III.6.3. --- Detection of Gene Amplification --- p.75
Chapter IV. --- Results --- p.77
Chapter IV.1. --- LOH Analysis of Chromosome l0q --- p.77
Chapter IV.2. --- Mutational Analysis ofp53 and PTEN/MMAC1 --- p.92
Chapter IV.3. --- Differential PCR Analysis of MDM2 Amplification --- p.103
Chapter V. --- discussion --- p.109
Chapter V.l. --- p53 Gene Inactivation Studies --- p.110
Chapter V.2. --- Molecular Genetic Studies on Chromosome l0q --- p.113
Chapter V.3. --- Microsatellite Instability in Non-Astrocytic Gliomas --- p.117
Chapter V.4. --- Significance of This Study --- p.118
Chapter V.5. --- Limitations of This Study --- p.119
Chapter V.6. --- Future Studies --- p.122
Chapter VI. --- REFERENCES --- p.124

Basierend auf die vorangegangene Studie der Abteilung für Neuropathologie des Pathologischen Instituts der Universität Würzburg, in der genetische Aberrationen auf dem Chromosom 9 in Ependymome beschrieben wurden, sollte eine Screeninguntersuchung des gesamten Chromosoms sowie eine verfeinerte Analyse der zuvor auffälligen Regionen erfolgen. Ziel der vorliegenden Arbeit war es, mögliche Tumorsuppressorgene oder Onkogene, die mit der Entstehung oder Progression von Ependymome verbunden sind, zu definieren. Weiterhin sollte eine Korrelation zu den klinischen Daten erfolgen. Dabei konnte bei erwachsenen Patienten gezeigt werden, dass Aberrationen auf Chromosom 9 die Prognose verbessern. Speziell der Marker D9S1872 bei DCR1 (DBCCR1) zeigte einen Trend zu einem Überlebensvorteil bei Erwachsenen. Deshalb könnte er in Zukunft zur prognostischen Einteilung von Ependymompatienten in der Klinik beitragen. Des Weiteren konnten bei Kindern in supratentoriellen Tumoren signifikant mehr Aberrationen gefunden wurden als in infratentoriellen. Dies deutet darauf hin, dass Ependymome zumindest bei Kindern abhängig von der Lokalisation unterschiedliche Entstehungswege haben. Es konnten zwei Regionen identifiziert werden, in denen sich häufig Aberrationen fanden: 9p21.1-22.3 und 9q31.3-33.3. Weiterhin konnten Lokalisation und Resektionsausmaß als unabhängige Prognosefaktoren bestätigt werden
Based on a former study of the departmentof neuropathology of the pathologic instisute of the university of Wuerzburg, in which aberrations on chromosome 9 in ependymomas had been described, the whole chromosome should be screened and a finer analysis of previously aberrated regions should be carried out. The aim of the thesis was defining possible tumor suppressor genes or oncogenes being involved in the development and progression of ependymomas. Moreover there should be a correlation to the clinical data. Adults with ependymomas harboring aberrations on chromosome 9 showed significantly longer overall survival than patients of the same group without this aberration, irrespective of the extent of resection in multivariate analysis. Aberrations of chromosome 9, and particularly of DCR1 (DBCCR1), may play a role in the prognostic evaluation for ependymomas in adults in the future. In pediatric patients, genetic aberrations were found significantly more often in supratentorial tumors than in tumors with infratentorial location. Two common regions of deletions were identified (9p21.1 approximately p22.3 and 9q31.3 approximately q33.2). Localization and extend of resection could be confirmed as prognostic factors

Zur Identifizierung geeigneter Routinemarker für die Prognose von Ependymompatienten führten wir immunhistochemische Untersuchungen und statistische Auswertungen an Ependymomen und Daten von 32 Erwachsenen und 23 pädiatrischen Patienten durch. Davon wurden bei drei Tumoren auch Rezidive untersucht, so dass insgesamt 59 Ependymome in die Untersuchung eingeschlossen wurden. Im Einzelnen handelte es sich um 11 myxopapilläre Ependymome, 6 Subependymome, 19 Ependymome und 23 anaplastische Ependymome. Die größten Fallgruppen bildeten pädiatrische Patienten unter drei Jahren und Erwachsene zwischen 50 und 70 Jahren. Bei Kindern war mit 45,8% die infratentorielle, bei Erwachsenen mit 65% die spinale Tumorlokalisation am häufigsten. Die untersuchten spinalen Ependymome entsprachen zu gleichen Teilen myxopapillären Ependymomen WHO Grad I und Ependymomen WHO Grad II. In supratentorieller Lage fanden sich mit 67% überwiegend anaplastische Ependymome WHO Grad III. Auch bei den infratentoriell gelegenen Ependymomen waren mit 63% die Mehrzahl anaplastische Ependymome, daneben fanden sich 29,6% Ependymome WHO Grad II. Beim Vergleich des von uns definierten und bestimmten Ki67-Scores als Zeichen für die Ependymomproliferation und der immunhistochemischen Positivität für HCK fiel nach Anwendung des Chi-Quadrat-Tests mit p=0,067 ein deutlicher Trend zu schwächerer punktförmiger Positivität bei höherem Ki67-Score auf. Dieser Trend setzte sich in der Erwachsenengruppe separat fort, während er in der Kindergruppe allein nicht nachweisbar war. In der Erwachsenengruppe war mit 28% ein deutlicher Anteil myxopapillärer Ependymome vorhanden, welche bei den Kindern nur 8% ausmachten.Möglicherweise spielt die veränderte HCK-Expression in der Subgruppe der myxopapillären Ependymome eine Rolle. Unsere Untersuchungen zeigten außerdem mit p=0,057 einen deutlichen Trend zu längerem Überleben bei immunohistochemischer DBC1-Negativität. Die Multivarianzanalyse mittels Cox-Regression wies eine Positivität für DBC1 als unabhängigen Risikofaktor für eine kürzere Überlebenszeit nach. Des Weiteren konnte eine mit p=0,013 signifikante Korrelation zwischen immunhistochemischer Positivität für DBC1 und höherem Ki67-Score gezeigt werden. Auch mit höherem WHO-Grad korrelierte die DBC1-Positivität mit p=0,009. Besonders infratentoriell gelegene Ependymome zeigten DBC1-Reaktivität. Hier treten bekannterweise häufiger anaplastische Ependymome mit höherem Proliferationsindex auf. Unsere Ergebnisse legen somit die Eignung des Markers DBC1 als immunhistochemische Routineuntersuchung für die Beurteilung der vom Resektionsstatus unabhängigen Prognose und Überlebenszeit von Ependymompatienten nahe
Immunohistochemical markers for prognosis and proliferation in ependymomas of children and adults

Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017
As lesões da espinal medula afectam actualmente milhões de pessoas em todo o mundo, que se deparam com um agravamento radical da sua qualidade de vida que, na maioria dos casos, não poderá ser recuperada. A Organização Mundial de Saúde define “lesão da espinal medula” como a perda total ou parcial de função neural provocada por um trauma ou uma patologia da espinal medula, resultando na diminuição do controlo motor abaixo do local da lesão assim como na perda de sensibilidade e regulação do sistema nervoso autónomo. A resposta clínica padrão actual tem como objectivo apenas tentar impedir o alastramento da lesão e consiste na realização de cirurgias para estabilizar a estrutura da coluna vertebral e descomprimir o local da lesão, seguidas de tratamento com metilprednisolona. No entanto, dado que o ambiente da lesão em mamíferos é extremamente inibitório para a ocorrência de regeneração, novos estudos têm tentado obter soluções terapêuticas que actuem na promoção de um ambiente pró-regenerativo, assim como na protecção dos tecidos que permanecerem intactos e funcionais após a lesão. A maior parte destas novas terapêuticas tem como objectivo prevenir o aparecimento de fenómenos associados à chamada lesão secundária ou de mitigar o seu efeito, dado que estes são em grande parte responsáveis pelo grau da lesão a longo prazo. Ao contrário do que se considerou durante décadas, investigações dos últimos 20 anos têm demonstrado que existem determinadas áreas no sistema nervoso central em que ocorre a formação de novos neurónios durante a vida adulta. Verificou-se também que existem células com capacidades estaminais neurais nestas e noutras zonas do sistema nervoso central, o que sugere que algumas células no adulto poderão ser estimuladas a originar novos neurónios e glia após uma lesão. As células ciliadas que revestem o interior do canal central na espinal medula, designadas células ependimárias, são um exemplo disso. Vários estudos observaram que estas células ependimárias respondem a uma lesão na espinal medula através da proliferação e migração para o local da lesão, onde originam astrócitos e oligodendrócitos; no entanto, são também capazes de originar neurónios quando cultivadas in vitro. O seu perfil de expressão inclui vários marcadores associados a células estaminais neurais, apoiando observações que indicam estas células como as únicas a possuir capacidades multipotentes no nicho do canal central. Pelo facto de serem as únicas células multiciliadas no canal central, as células ependimárias de mamífero podem ser identificadas pela expressão do factor de transcrição Foxj1, conhecido pelo seu papel como regulador da ciliogénese. O peixe-zebra (Danio rerio) já é conhecido como modelo em biologia do desenvolvimento há mais de 30 anos, mas a sua utilização para o estudo da regeneração de tecidos e órgãos é bastante mais recente. As extraordinárias capacidades de regeneração deste organismo, aliadas à bateria de métodos genéticos e moleculares desenvolvidos e adaptados para este modelo, tornaram o peixe-zebra num aliado inestimável para entender processos regenerativos e compará-los com a situação dos mamíferos, cujas capacidades regenerativas são muito inferiores. As características biológicas do peixe-zebra são particularmente apelativas, por exemplo para geração de linhas transgénicas: são capazes de gerar um grande número de embriões – transparentes, que permitem a observação de fenótipos relativamente cedo – e atingem a maturidade aos 3 meses, acelerando o processo de geração da linha. Os métodos desenvolvidos para a geração de peixes transgénicos também têm demonstrado elevados níveis de sucesso, especialmente o sistema Tol2, que se baseia na injecção do DNA desejado juntamente com mRNA que codifica para uma transposase; esta reconhece sequências específicas que flanqueiam o DNA injectado e insere-o no genoma do embrião. Entre as várias estruturas que o peixe-zebra consegue regenerar encontra-se a espinal medula, que obtém uma recuperação funcional quase completa um mês após a lesão. Este resultado deve-se tanto ao crescimento de axónios seccionados como à formação de novos neurónios no local da lesão, mecanismos promovidos por um ambiente pró-regenerativo. Os novos neurónios e células da glia são produzidos por células que revestem o canal central da espinal medula, tendo por isso função de epêndima, mas que também apresentam outras características morfológicas reminiscentes das células da glia radial, progenitores neurais durante o desenvolvimento (tanto em peixe-zebra como em mamífero). A utilização de marcadores moleculares para identificação destas células tem sido controversa, mas existe um marcador que poderá ser utilizado de forma clara. Devido à sua função ependimária, estas células possuem cílios e expressam por isso o factor de transcrição Foxj1a, um dos ortólogos do Foxj1 de mamífero. Será por isso importante desenvolver ferramentas que permitam estudar o papel que as células foxj1a+ da espinal medula do peixe-zebra desempenham após uma lesão, que pode ser realizado através da identificação da sua descendência (experiência de lineage tracing) e da sua função real no contexto de uma lesão promovendo a sua ablação específica. Este trabalho apresentava assim como objectivo principal o estabelecimento de linhas transgénicas estáveis em peixe-zebra que permitissem efectuar de forma eficaz e separadamente: 1) a marcação permanente das células foxj1a+ e da sua descendência; 2) a ablação específica das células foxj1a+, em ambos os casos após uma lesão da espinal medula. Para a geração da linha para lineage tracing foram testados três constructs separadamente, mas apenas com um deles foi possível observar passagem evidente do transgene para embriões F1. Ainda assim, o sucesso desta transgénese foi inferior ao reportado para o método utilizado. Os outros dois constructs apresentaram percentagens muito baixas de embriões positivos e de níveis de expressão dos transgenes após injecção, mesmo depois de tentativas de optimização, e de todos os que cresceram até atingir maturidade não foram detectados peixes fundadores de forma inequívoca. Para a geração da linha para ablação das células foxj1a+ foram testados dois constructs com os quais não foi possível obter um número suficiente de embriões positivos para crescer. O primeiro foi abandonado quando se observou que várias características do plasmídeo em que estava inserido não seriam adequadas para manter um nível de expressão adequado à experiência, como a falta da sequência Kozak e do sinal de poliadenilação a flanquear a sequência codificante. O segundo foi desenhado de forma a optimizar todas as sequências para promover uma expressão eficiente do transgene, contendo sequências de reconhecimento da transposase e sequências regulatórias a 5’ e 3’ da sequência codificante vindas de um plasmídeo utilizado como controlo positivo para as injecções, assim como a sequência codificante de uma proteína fluorescente (como repórter) que apresentara bons níveis de expressão com o promotor foxj1a. Não obstante, este construct parece não ter sido integrado no genoma dos embriões em que foi injectado dado que praticamente toda a expressão observada às 24 horas-pós-fertilização tinha sido perdida 4 dias depois. As razões que levaram ao insucesso na obtenção de embriões positivos após a injecção são desconhecidas para a maioria dos constructs testados, mas poderão estar relacionadas com mutações não detectadas nas regiões regulatórias ou nas sequências de reconhecimento da transposase, que impediriam a correcta expressão do construct ou a sua inserção no genoma, respectivamente. A experiência de lineage tracing concebida baseia-se na utilização do sistema de recombinação CreERT2/LoxP, que utiliza uma versão modificada da enzima Cre a que foi acrescentado um receptor de estrogénio. Esta modificação adiciona um passo de controlo temporal à experiência visto que é necessário activar a CreERT2 com administração da droga 4-OHT para que a enzima possa efectuar a recombinação entre locais LoxP e assim promover a marcação de células foxj1a+. O segundo objectivo deste trabalho foi então a optimização do protocolo de activação da CreERT2, que foi conseguido pela primeira vez neste laboratório. Inicialmente foram utilizados embriões que expressavam a CreERT2 e a cassete repórter na maioria das células (expressão induzida pelo promotor de uma proteína heat shock), tendo-se verificado que a eficácia da recombinação foi directamente influenciada pela concentração de 4-OHT administrada. A remoção do córion dos embriões por adição de pronase também promoveu este efeito ao permitir um acesso mais rápido da droga aos tecidos. Embriões e larvas F1 dos fundadores obtidos com a injecção de um dos constructs para lineage tracing foram também utilizados para testar a activação da CreERT2, mas neste caso não se observou a ocorrência de recombinação em células foxj1a+, mesmo após a sua proliferação ter sido induzida através da realização de uma lesão na espinal medula. Devido ao insucesso em gerar linhas transgénicas funcionais neste trabalho, será necessário utilizar novos métodos e/ou novos constructs para obter ferramentas biológicas que permitam no futuro compreender a função das células foxj1a+ durante a regeneração da espinal medula no peixe-zebra.
Spinal cord injury (SCI) is a disabling condition affecting millions of people worldwide. In order to improve functional recovery, new therapies are being devised to counteract the non-regenerative environment of the mammalian spinal cord. Ependymal cells (EC) of the spinal cord central canal have been proven to hold neural stem cell properties in vitro, suggesting that a pro-neurogenic fate could potentially be promoted in vivo after a lesion. The zebrafish Danio rerio has proved a valuable tool for developmental studies and, in recent years, for regenerative processes due to its remarkable ability to regenerate several organs and tissues. After SCI new neurons and glia are generated by the cells lining the central canal, which hold a strong resemblance to mammalian EC. Both mammalian and zebrafish EC have in common the expression of the transcription factor responsible for cilia formation: Foxj1/Foxj1a. However, no study has been performed to discover the progeny of zebrafish foxj1a+ cells after a lesion or their functional role in regeneration. This work aimed at generating stable transgenic zebrafish lines that allowed the investigation of foxj1a+ ependymal cell progeny after SCI, using lineage tracing, and also their specific ablation with a suicide gene. A total of three constructs for lineage tracing lines and two constructs for cell ablation lines was injected with transposase mRNA into one-cell stage embryos but only one of the lineage tracing constructs resulted in clear germline transmission to F1 embryos. All the other four constructs proved very difficult to generate stable expression in injected embryos as well as an adequate number of positives, even after several optimization attempts. Reasons for this lack of transgenesis efficiency are not completely understood but may include inadequate coding sequence features, undetected problems in untranslated regions, or obstacles to transposition such as mutations in transposase recognition sequences. Since the lineage tracing is based on the CreERT2/LoxP technology, optimization of CreERT2 activation was also performed. It was found that recombination efficiency is directly influenced by 4-OHT concentration and enhanced by treatment with pronase. However, when using a lineage tracing line no recombination was detected in foxj1a+ cells even after induced proliferation, prompting the need to validate CreERT2 function in this line.

La neurogenèse persiste à l’âge adulte dans deux régions du système nerveux central (SNC) des mammifères : la zone sous-ventriculaire (SVZ) du cerveau antérieur et la zone sous-granulaire (SGZ) de l’hippocampe. Cette neurogenèse est possible grâce à la capacité de prolifération des cellules souches présentes dans les niches de la SVZ et la SGZ, mais en vieillissant, le cerveau subit une diminution dramatique du nombre de cellules souches neurales adultes (CSNa), une diminution de la prolifération cellulaire et une altération des niches de neurogenèse. Cependant, une importante question reste sans réponse : comment la perte tardive des CSNa est temporellement reliée aux changements de l’activité de prolifération et de la structure de la principale niche de neurogenèse (la SVZ)? Afin d’avoir un aperçu sur les événements initiaux, nous avons examiné les changements des CSNa et de leur niche dans la SVZ entre le jeune âge et l’âge moyen. La niche de la SVZ des souris d’âge moyen (12 mois) subit une réduction de l’expression des marqueurs de plusieurs sous-populations de précurseurs neuraux en comparaison avec les souris jeunes adultes (2 mois). Anatomiquement, cela est associé avec des anomalies cytologiques, incluant une atrophie générale de la SVZ, une perte de la couche de cellules sousépendymaires par endroit et l’accumulation de gouttelettes lipidiques de grande taille dans l’épendyme. Fonctionnellement, ces changements sont corrélés avec une diminution de l’activité de la SVZ et une réduction du nombre de nouveaux neurones arrivant aux bulbes olfactifs. Pour déterminer si les CSNa de la SVZ ont subi des changements visibles, nous avons évalué les paramètres clés des CSNa in vivo et in vitro. La culture cellulaire montre qu’un nombre équivalent de CSNa ayant la capacité de former des neurosphères peut être isolé du cerveau du jeune adulte et d’âge moyen. Cependant, à l’âge moyen, les précurseurs neuraux semblent moins sensibles aux facteurs de croissance durant leur différenciation in vitro. Les CSNa donnent des signes de latence in vivo puisque leur capacité d’incorporation et de rétention du BrdU diminue. Ensemble, ces données démontrent que, tôt dans le processus du vieillissement, les CSNa et leur niche dans la SVZ subissent des changements significatifs, et suggèrent que la perte de CSNa liée au vieillissement est secondaire à ces événements.
Neurogenesis persists throughout the adulthood in two regions of the mammalian central nervous system (SNC): the sub-ventricular zone (SVZ) of the forebrain and the sub-granular zone (SGZ) of the hippocampus. Neurogenesis is possible due to the proliferation capacity of stem cells present within both the SVZ and SGZ niches, but with aging, the forebrain undergoes a drastic reduction in its number of adult neural stem cells (aNSCs), a decrease of cell proliferation and an alteration of the neurogenic niches. However, a key unresolved question remains: how the onset of aNSC loss is temporally related to changes of proliferating activity and to structural alterations within the principal stem cell niche (the SVZ)? To gain insights into the initial events leading to aging-associated aNSC loss, we investigated the changes occurring to aNSCs and the SVZ niche between young adulthood and middle-age. The SVZ niche of middle-aged mice (12-months-old) was found to display reduced expression of markers for multiple neural precursor sub-populations when compared to young adult mice (2-months-old). Anatomically, this was associated with significant cytological aberrations, including an overall atrophy of the SVZ, loss of sub-ependymal cells, and accumulation of large lipid droplets within the ependyma. Functionally, these changes correlated with diminished SVZ activity and reduced number of newly born neurons reaching the principal target tissue: the olfactory bulbs. To determine whether changes were evident at the level of the SVZ stem cells, we evaluated key in vitro and in vivo parameters of aNSCs. Tissue culture experiments showed that equal numbers of neurosphere-forming aNSCs could be isolated from young adult and middle-aged forebrains. However, at middle-age, neural precursors seemed to be less sensitive to growth factors during their in vitro differentiation and displayed signs of increased quiescence in vivo. Collectively, these findings demonstrate that, with early aging, aNCS and their SVZ niche go through significant changes, and suggest that aging-associated aNSC loss is secondary to these events.