Le vieillissement et la dégénérescence du cerveau, associés à des déficits cognitifs, comportementaux et neurologiques, représentent aujourd'hui un problème majeur de santé publique. L'une des principales maladies liées à l'âge est la maladie d'Alzheimer (MA). L'une des caractéristiques de la MA est l'apparition de plaques amyloïdes, résultant de l'agrégation du peptide ßA4. Physiologiquement, le précurseur de la protéine amyloïde (APP) est clivé par une alpha-sécrétase qui génère un fragment soluble de l'APP (sAPP), important pour la formation de nouvelles cellules nerveuses (neurogenèse). Ce clivage en prévient deux autres, par les béta- et gamma-sécrétases, impliqués dans la MA, et conduisant à la formation du ßA4 toxique. Une analyse du plexus choroïde (PCh) a mis en évidence la forte expression de l’APP par cette structure cérébrale. Le PCh est une structure facilement accessible et produisant le liquide cérébro-spinal : son impact peut donc être répercuté à l’ensemble du cerveau. Il pourrait être une source cérébrale importante d’APP, et contribuer fortement à la pathologie. Mon projet de thèse s'inscrivait dans la possibilité de réguler génétiquement l'expression des formes sauvages et mutées de l'APP au niveau de cette source, et suivre les conséquences sur la neurogenèse adulte et la formation des plaques amyloïdes, marqueur histopathologique de la MA. Par l’utilisation de la thérapie génique pour moduler l’expression de l’APP dans les PCh, nous avons confirmé l’importance de l’APP soluble provenant des PCh dans la neurogenèse adulte. Les PCh semble être une source importante d’APP dans le cerveau, et pourraient avoir un rôle clé dans la maladie d’Alzheimer. / Aging and degeneration of the brain with cognitive decline and neurologic symptoms are major individual and societal problems. The major age-related brain degeneration disease is Alzheimer’s disease (AD) with about 40 million people affected in 2015.Physiologically, the Amyloid Precursor Protein (APP) is cleaved by an alpha-secretase, releasing soluble APP (sAPP) an important regulator of adult neurogenesis. This cleavage prevents two others in positions beta and gamma that generate the ßA4 toxic peptide, a hallmark of Alzheimer Disease.Next generation RNA-sequencing has revealed that APP is the 16th most expressed genes in the choroid plexus (CP), suggesting that it may be a major source of sAPP and ßA4 in the cerebrospinal fluid (CSF). If so, adult neurogenesis in the SVZ and hippocampus may be regulated by the choroid plexus and impeded in mutations favoring ßA4 production. My thesis project fell under the possibility to regulate App expression in the CP, and follow consequences on adult neurogenesis and plaques formation in AD. Using viral vectors to modulate App expression in the CP, we confirmed the importance of sAPP coming from CP in adult neurogenesis. With so, CP seems to be an important source of APPin the brain, and could have a key role in AD.

Prognostic factors come in a variety of forms and may be patient, tumour or environmental related. This thesis examines the interaction of prognostic factors for a variety of tumour types. It particularly focuses on single nucleotide polymorphisms (SNPs) of the vascular endothelial growth factor (VEGF) gene. The first section on meningiomas describes the frequency of sex steroid receptors in meningiomas. In this study, absence of progesterone receptors is associated with high tumour grade and male gender. Tumours that are progesterone receptor negative have an odds ratio for recurrence of 5.Choroid plexus carcinomas are aggressive malignant tumours generally occurring in young children. Gross total surgical resection has been shown to be a highly significant factor in tumour recurrence and survival. This study describes a treatment paradigm of neoadjuvant ICE chemotherapy in these children which decreases the vascularity and increase the chance of a complete removal. The operative blood loss with this regimen is reduced to 0.22 blood volumes from 1.11 blood volumes without neoadjuvant chemotherapy. The VEGF gene is highly polymorphic and SNPs of the region have previously been shown to influence VEGF protein expression. This study looks at cohorts of both adult gliomas and a variety of paediatric brain tumours; comparing them to controls. There are several associations described between the development of certain tumours and specific SNP genotypes. In addition to this, certain genotypes and haplotypes have an influence on survival of adult grade 2 astrocytomas and paediatric medulloblastomas and ependymomas. There are consistent themes to the prognostic genotypes throughout both the adult and the paediatric tumours.Prognostic factors come in a variety forms as described in this thesis. It is vital to understand the complex interaction between factors to best utilise them for the benefit of patients.

Vitamin A is an essential nutrient; it is not synthesized by mammals and therefore must be obtained through the diet. During times of fasting or dietary vitamin A insufficiency, retinol, the alcohol form of the vitamin is released from the liver, its main storage tissue, for circulation in complex with retinol-binding protein 4 (RBP) to provide an adequate supply to peripheral tissues. Stimulated by Retinoic Acid 6 (STRA6), the transmembrane RBP receptor, mediates retinol uptake across blood-tissue barriers such as the retinal pigment epithelium of the eye, the placenta and the choroid plexus of the brain. Our understanding as to how this protein functions has been greatly enhanced by the high-resolution 3D structure of zebrafish STRA6 in complex with calmodulin (CaM) solved by single-particle cryogenic-electron microscopy. However, the nature of the interaction of STRA6 with retinol remains unclear. Here, I present the high-resolution structures of zebrafish and sheep STRA6 reconstituted in nanodisc lipid bilayers in the presence and absence of retinol. The nanodisc reconstitution system has allowed us to study this protein in a close to physiological environment and examine its interaction with the cell membrane and relationship with its ligand, retinol. We also present the structure of sheep STRA6 in complex with human RBP. The structure of the STRA6-RBP complex confirms predictions in the literature as to the nature of the protein-protein interaction needed for retinol transport. Calcium-bound CaM is bound to STRA6 in the RBP-STRA6 structure, consistent with a regulatory role of this calcium binding protein in STRA6-RBP interaction. The analysis of the three states of STRA6 – pre, post and during interaction with retinol – provide unique insights into the mechanism of STRA6-mediated cellular retinol uptake.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19568号 / 医博第4075号 / 新制||医||1013(附属図書館) / 32604 / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊佐 正, 教授 渡邉 大, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>  </p> <p>Pediatric hydrocephalus is a complex neurological condition associated with a pathological accumulation of cerebrospinal fluid (CSF), typically within the brain ventricular system. Pediatric hydrocephalus can be primary (due to genetic abnormalities or idiopathic causes), or secondary to injuries such as hemorrhage, trauma, or infection. The current permanent treatment paradigms for pediatric hydrocephalus are exclusively surgical and include the diversion of CSF via shunt or ventriculostomy. These surgical interventions are wrought with failures, burdening both the United States healthcare system and patients with repeat neurosurgical procedures. Thus, the development of nonsurgical interventions to treat hydrocephalus represents a clinically unmet need. To study hydrocephalus, we use a genetic rat model of primary neonatal hydrocephalus, the <em>Tmem67</em>P394L mutant. In several proof-of-concept studies, we identify antagonism of the transient receptor potential vanilloid 4 (TRPV4) channel and associated upstream regulatory kinase, serum-and-glucocorticoid-induced kinase 1 (SGK1) as therapeutics for the treatment of hydrocephalus. Using <em>in vitro</em> models of the choroid plexus epithelium, the tissue which produces CSF, we show compelling proof-of-mechanism for TRPV4 antagonism and SGK1 inhibition at preventing CSF production. Therefore, the studies in this dissertation provide substantive evidence on the role of TRPV4 in the choroid plexus in health and disease. </p>

Fundamental to mammalian brain development is the integration of cell intrinsic and extrinsic signals that direct the proliferation and differentiation of neural stem cells. Precise expression of transcription factors together with other intracellular components instruct progenitor cell fate, whereas interaction with extracellular signaling factors refines this process. We have elucidated the composition of the cerebrospinal fluid that is the source of multiple extrinsic cues during brain development. The choroid plexus, a highly vascularized tissue located in each ventricle of the brain, actively secretes cerebrospinal fluid. By RNA sequencing, we obtained transcriptome data on the choroid plexi from lateral and fourth ventricles of the mouse brain and discovered that they include transcripts unique to each tissue. Transcription factor expression in the macaque and human choroid plexi suggests that positional identities of these tissues are conserved in the primate brain. Based on transcriptional results, we defined the choroid plexus secretome, a prediction of secreted factors from the choroid plexus. By quantitative mass spectrometry, we detected proteins secreted by each choroid plexus, and comparison of these proteomic results with transcriptional profiling suggests that choroid plexus transcriptomes contribute to availability of regionalized cerebrospinal fluid factors during development. In the second part of my dissertation research, I studied the role of DNA repair mechanisms in regulating neural stem cells. These studies focused on DNA LigaseIV, an essential component of DNA double-stranded break repair, during cerebral cortical development. Deficiency of LigaseIV activity caused by a missense mutation leads to LigaseIV syndrome, in which a key clinical feature is microcephaly. Using the Lig4 R278H mouse mutant, we found increased cell death in the developing cortex, contributing to reduced cortical thickness and cellularity in the anterior cerebral cortex. These results indicate that DNA LigaseIV is essential for proper cortical development. Together, these findings illustrate the complexity of regulatory mechanisms that guide brain development, requiring the integration of mechanisms from within and outside the cell. We have investigated two such mechanisms, extrinsic cues from regionalized cerebrospinal fluid and DNA LigaseIV. These results should provide greater insight into mechanisms of normal brain development and neuropathological states. / 2017-11-02T00:00:00Z

No description available.

<p dir="ltr">A neurological disorder, hydrocephalus, has an estimated global pediatric prevalence of 380,000 new cases each year [<a href="#_ENREF_1" target="_blank">1</a>]. It is a family of diseases that can occur at any age when cerebrospinal fluid builds up within the ventricles of the brain. Thus, the only available treatments are surgical, invasive, and prone to complications. There is a global need for successful treatment strategies without brain surgery.</p><p><br></p><p dir="ltr">Choroid plexus epithelial cells (CPEC) are responsible for production of cerebrospinal fluid (CSF). Ependymal cells line the ventricles and play roles in CSF maintenance and waste clearance. Astrocytes perform various functions, one being blood-brain barrier (BBB) maintenance. Collectively these cells contribute to brain fluid/electrolyte regulation and barrier integrity. Increased glial fibrillary acidic protein (GFAP) fluorescence, a marker of activated astrocytes, appeared in hydrocephalic (<i>Tmem67</i>^-/-) animals by immunohistochemistry as early as postnatal day (P)10. The tight junction proteins expressed in choroid plexus (CP); claudin-1 (Cl-1) and zona occludin 1 (ZO-1) fluorescent intensity increased in P15 hydrocephalic animals compared to wildtype (<i>Tmem67</i>^+/+). These cells also contain aquaporins (AQP), aquaporin-1 (AQP1) and aquaporin-4 (AQP4), important in regulating CSF and interstitial fluid (ISF). Increased fluorescent intensity of AQP4 in the subventricular zone and increased AQP1 apical localization and protein amount in the CP was observed in hydrocephalic animals at postnatal day (P)15. Many of these may be targeted for the treatment of hydrocephalus. However, there is no consensus in pathological findings between models of hydrocephalus and these finding may not translate to common pharmacological targets.</p><p><br></p><p dir="ltr">A transient receptor potential cation channel, subfamily vanilloid, member 4 (TRPV4) antagonist (RN1734) ameliorates hydrocephalus in a rat model of congenital hydrocephalus (<i>Tmem67</i> model). It was hypothesized that targeting this mechanosensitive ion channel may slow production of CSF by targeting the CP. However, hydrocephalus pathology can have various effects on the brain. Astrocytes were visualized using fluorescent immunohistochemistry of glial fibrillary acidic protein (GFAP) and RN1734 did not seem to change immunoreactivity to wildtype untreated levels. Increased immunoreactivity of TRPV4 and AQP1 was observed in CP of untreated and RN1734 treated <i>Tmem67</i>^-/- rats. AQP4 and TRPV4 immunoreactivity increased in the subventricular zone and periventricular white matter (WM) of hydrocephalic rats. With RN1734, TRPV4 immunoreactivity, but not AQP4, had similar immunoreactivity to wildtype untreated. Increased GFAP and AQP immunoreactivity may indicate residual inflammation in the <i>Tmem67</i>^-/- rats. More experiments must be done to further elucidate TRPV4’s role in hydrocephalus pathology.</p><p><br></p><p dir="ltr">Serum and glucocorticoid-regulated kinase 1 (SGK1) is a kinase implicated in cell volume regulation and CSF production. SI113, an SGK1 inhibitor, ameliorates hydrocephalus in the <i>Tmem67</i> rodent model. The goal of this study was to determine if SI113 could be used with a new solvent other than dimethyl sulfoxide (DMSO), which can have possible toxic effects. 1-methyl-2-pyrrolidinone (NMP) has high solubility and ability to cross the BBB. These studies showed that NMP as a solvent did not have adverse effects on body weight, however thus far, it has not ameliorated hydrocephalus significantly at the concentration used in this study. There is a possibility that the concentration in NMP that we used was not efficacious enough. CSF and blood plasma samples from animals treated with SI113 24 hours and 30 minutes before euthanasia will be used to investigate the concentration of SI113 that remains in the circulation and the amount that crosses the BBB and blood-cerebrospinal fluid (BCSFB) barriers. We hope that the results will inform dosage for our future studies. Future studies may also examine SI113 mechanism of action in hydrocephalus.</p><p><br></p><p dir="ltr">This thesis addresses hydrocephalus cell and molecular pathology in the <i>Tmem67</i> model and examines potential treatment strategies. Future directions include comparing models of hydrocephalus to find common treatment strategies in the hope to find pharmaceutical strategies to better manage human hydrocephalus.</p>

La neurogenèse adulte permet la formation de nouveaux neurones dans les bulbes olfactifs de la souris. Les propriétés des cellules souches neurales situées dans la zone sous-ventriculaire (ZSV) et des précurseurs sont régulées par la niche contenant des cellules de support et une matrice extracellulaire (MEC). Des facteurs contenus dans le liquide cérébrospinal (LCS), produits par les plexus choroïdes (PC), contrôlent aussi la niche. L'homéoprotéine Otx2 est secrétée dans le LCS par les PC, et internalisée spécifiquement par certaines cellules du parenchyme cérébral. Otx2 est impliquée dans différentes étapes du développement du cerveau, dont celui des PC, et peut agir de manière non-cellulaire-autonome. Ma thèse vise à comprendre comment Otx2 régule les fonctions des PC et participe à la neurogenèse adulte. Grâce à des études génomiques d'un modèle murin knockdown (KD) d'Otx2 dans les PC adultes, nous avons montré que (i) les PC de différents ventricules présentent des profils d'expression différents (ii) le KD d'Otx2 modifie l'expression de gènes impliqués dans des fonctions importantes des PC (iii) la dérégulation de certains gènes après KD est spécifique d'un type de PC. Une étude protéomique suggère (iv) qu'Otx2 pourrait être impliquée à d'autres niveaux que la régulation transcriptionnelle. L'étude de la neurogenèse adulte dans des modèles murins KD d'Otx2 nous a permis de montrer que (i) l'expression d'Otx2 dans les PC régule la neurogenèse adulte (ii) Otx2 transfère dans les astrocytes de la ZSV (iii) le transfert d'Otx2 est suffisant pour réguler la neurogenèse (iv) le KD d'Otx2 dans les PC modifie l'expression de protéines de la MEC secrétées par les astrocytes. / Adult neurogenesis in mice involves neural stem cells in the subventricular zone (SVZ) whose progenitors integrate into the olfactory bulbs. The neurogenic niche, which contains supporting cells and extracellular matrix (ECM), regulates the properties (proliferation, migration and differentiation) of progenitor cells. This niche is influenced by factors from cerebrospinal fluid (CSF), which is produced by the choroid plexus (CP) in the brain ventricles. The Otx2 homeoprotein transcription factor is secreted into CSF by CP, and taken up by a specific subset of cells within the brain parenchyma. Otx2 is involved in various stages of brain development, including CP development, and has non-cell autonomous functions. The aim of my thesis is to understand how Otx2 regulates adult CP function and participates in adult SVZ neurogenesis. Through genomic studies, we investigated the consequence of Otx2 knockdown (KD) in adult CP and found: (i) adult CP from different ventricles exhibit different expression profiles; (ii) Otx2 KD alters the expression of genes with important CP functions; and (iii) deregulation of certain genes after Otx2 KD can be CP specific. Through proteomics studies, we found that (iv) adult Otx2 could be involved in functions beyond transcriptional regulation, such as RNA processing.To evaluate the role of Otx2 in SVZ neurogenesis, we also used Otx2 KD mouse models. We found that: (i) the expression of Otx2 in CP regulates adult neurogenesis; (ii) Otx2 transfers to astrocytes of the SVZ; (iii) Otx2 transfer is sufficient to regulate adult neurogenesis; and (iv) Otx2 KD in CP alters the expression of ECM proteins secreted by astrocytes in the neurogenic niche.

No description available.