L’intégrité fonctionnelle de la barrière hémato-encéphalique (BHE) est altérée dans de nombreuses pathologies neurologiques et métaboliques. Les vaisseaux cérébraux sont des tissus cibles des hormones stéroïdes sexuelles mais la contribution respective de ces effecteurs endocriniens et de leurs récepteurs dans l’intégrité de la BHE reste encore à être précisée. Les effets des hormones gonadiques sur l’unité neurovasculaire chez la souris ont été étudiés, en se concentrant sur l’aire préoptique médiane de l’hypothalamus, une région cérébrale hormono-sensible. L’augmentation de la perméabilité de la BHE chez des souris mâles et femelles gonadectomisés. Elle est associée chez le mâle à une désorganisation des jonctions serrées et une diminution de l’expression des protéines les constituant, à une activation des cellules gliales, et à une augmentation de l’expression de molécules inflammatoires, la supplémentation en testostérone permettant la restoration. Les récepteurs des androgènes et des estrogènes peuvent ainsi être impliqués dans la régulation hormono-dépendante du transport paracellulaire. La lignée de souris transgéniques sélectivement invalidées pour le récepteur neural des androgènes, a permis de mettre en évidence l’effet délétère de l’absence de ce récepteur sur l’intégrité de la BHE et des jonctions serrées. Pour compléter, un modèle ex vivo de tranches d’hypothalamus a permis d’appréhender les effets à court terme de la testostérone sur la BHE. Ces données soulèvent des questions sur les effets délétères potentiels des perturbateurs endo-criniens sur l'intégrité BBB et l'apparition de maladies neurologiques et métaboliques associées. / Functional integrity of the blood-brain barrier (BBB) is compromised in many neurological and metabolic pathologies. Cerebral blood vessels are target tissue for sex steroid hormones but the relative contribution of these endocrine effectors and their receptors in the BBB integrity are still unclear. Effects of gonadal hormones on the mouse neurovascular unit were studied, focusing on the hypothalamic medial preoptic area, a highly sensitive brain area to gonadal steroid hormones. BBB permeability increased in both gonactectomized male and female, is associated with tight junction disorganization and lower expression of tight junction proteins, glial activation, and up-regulation of inflammatory molecules in male. Testosterone supplementation restores the BBB impermeability, tight junction integrity, and almost completely abrogated the inflammatory features. Androgen and estrogen receptor may be involved in testosterone-induced regulation of the formation and maintenance of tight junction in males. Studying the involvement of these receptors using a trans-genic mice line selectively lacking neural AR in the CNS, highlighted the negative effect of this dele-tion on the BBB and TJ integrity. To complete, ex vivo slices of male mouse hypothalamus allowed to assess short-term molecular mechanisms of testosterone on the BBB structure and function.These data raise questions about the potential deleterious effects of endocrine disruptors on the BBB integrity and the occurrence of neurological and metabolic diseases.

La barrière hémato-encéphalique (BHE), localisée au niveau des capillaires cérébraux, contrôle les échanges entre le sang et le compartiment cérébral et assure ainsi le maintien de l'homéostasie du système nerveux central (SNC). La présence de la BHE est un atout lors du développement de médicament à visée périphérique. En effet, en limitant le passage de nombreuses molécules, la BHE protège le SNC des effets potentiellement neurotoxiques de ces molécules. Toutefois, l‟exposition des cellules endothéliales des capillaires cérébraux à des agents chimiques est susceptible d‟engendrer une augmentation transitoire de la perméabilité de la BHE. Cette augmentation peut perturber l‟homéostasie cérébrale et permettre l‟entrée massive de molécules potentiellement neurotoxiques dans le SNC. La prise en compte de la BHE en amont de l‟étude de la neurotoxicité d‟un médicament est donc un élément important. De plus, la majorité des médicaments sont utilisés de façon chronique et les effets secondaires indésirables résultant d‟une administration chronique sont fréquemment liés à une atteinte cérébrale. Afin de répondre à cette problématique, notre modèle in vitro de BHE, qui consiste en une co-culture de cellules endothéliales de capillaires cérébraux et de cellules gliales, a été adapté à l‟étude de la toxicité de molécules lors d‟un traitement prolongé. Les propriétés protectrices de la BHE deviennent une contrainte importante lors du développement de médicament à visée cérébrale. En effet, la présence de la BHE explique en partie les taux de succès très faibles des molécules lors du développement de médicaments à visée cérébrale. Afin de limiter les taux d‟échec, il est nécessaire de prédire efficacement la distribution cérébrale des composés en prenant en compte la BHE. Or, il est admis que l‟effet pharmacologique est lié à la concentration libre du médicament au niveau de sa cible. Ainsi, les nouvelles approches visent à prédire la concentration libre que la molécule atteindra dans le cerveau. Toutefois, les méthodes existantes pour prédire ce paramètre reposent sur une méthodologie in vivo et ne présentent pas un débit suffisant pour être utilisées lors des phases précoces du développement de médicaments. Une méthodologie in vitro pour obtenir le ratio de concentrations libres d‟une molécule entre le cerveau et le sang a été développée pour répondre à ce besoin. Le travail réalisé a permis de développer deux méthodologies in vitro. La première permet de prédire la toxicité chronique des molécules. En prédisant le ratio des concentrations libres entre le compartiment cérébral et sanguin des composés, la seconde facilite la sélection des médicaments candidats lors du développement de médicaments à visée cérébrale. Ces méthodologies pourront donc contribuer à diminuer les taux d‟échecs lors des phases précliniques et cliniques du développement de médicaments. / The blood-brain barrier (BBB), located at the level of brain capillaries, is responsible for brain homeostasis maintenance by tightly controlling blood-borne substances access to the brain. The presence of the BBB is an asset during peripheral drug development. Indeed, the BBB protects the central nervous system (CNS) against potential neurotoxic effects of compounds by strongly limiting their passage. However, exposure of brain capillaries endothelial cells to chemical agents is likely to cause a transient increase in BBB permeability. This increase can disrupt brain homeostasis and allow the massive entry of potentially neurotoxic molecules in the CNS. Hence, taking into account BBB toxicity in alternative neurotoxicity studies is important. In addition, the CNS side effects of several drugs used chronically could be at least partly attributed to their toxicity at the level of the BBB causing unwanted, indirect effect on brain cells. To address this issue, our in vitro BBB model, which consist of a co-culture of brain capillary endothelial cells and glial cells, has been adapted to the evaluation of repeated-dose toxicity at the BBB. The protective properties of the BBB become a major hurdle during CNS drug development. One way to reduce theimportant attrition rate, consists in predicting the CNS distribution of drug candidates early in CNS drug discovery programs. The use of unbound brain concentrations has been shown to provide the best correlations with pharmacological data. Hence, new approaches aim to predict the free brain concentration of compounds. However, the determination of free brain / free plasma ratios requires both in vitro and in vivo experiments that are both animal and time consuming. Consequently, we have explored the possibility to directly generate free brain / free plasma ratios under steady-state and non-steady state conditions in our in vitro BBB model, thereby greatly simplifying existing experimental procedures.. The work presented herein aimed to develop two in vitro methodologies. The first one allows the study of repeated-dose BBB toxicity. The second one allows free brain / free plasma ratios assessment using an in vitro model of the blood brain barrier, which can drive the selection of CNS drug candidates with the most favourable target engagement. The use of these two methodologies may help to reduce attrition rates in drug discovery and development by appreciating the eventual central toxicity of systemic drug associated with BBB dysfunction and by identifying centrally acting-compounds with a desirable in vivo response in the CNS early on in the drug discovery process.

Les entérovirus humains (EV) constituent un groupe de virus à ARN d'une grande diversité génétique. Ils sont responsables d'infections cérébrales graves mais rares et de méningites bénignes mais fréquentes. Les évènements conduisant à l'entrée des EVs dans le système nerveux central (SNC), l'importance de la charge virale dans le liquide céphalo rachidien (LCR) et sa corrélation éventuelle avec l'intensité du processus inflammatoire réactionnel restent peu explorés. Comme de nombreux génotypes d'EV sont associés à des manifestations neurologiques semblables, des processus pathologiques communs sont envisageables. Le premier volet de cette thèse avait pour objectif d'étudier prospectivement la charge virale EV dans le LCR de patients présentant une méningite à l'aide d'une technique de RT-QPCR. Nos résultats montrent que les différences significatives de charge virale retrouvées dans le LCR en fonction de l'âge, des leucocytes et de la protéinorachie sont reliées au génotype de l'EV responsable de l'infection. Le second volet de cette thèse avait pour but d'explorer l'hypothèse qu'une infection de la barrière hémato-Encéphalique (BHE), peut représenter une voie d'accès commune à une majorité d'EVs vers le SNC. La réplication et la translocation des EVs ont été évaluées avec un modèle in vitro de BHE basé sur la lignée cellulaire hCMEC/D3. Nous avons validé ce modèle cellulaire en montrant une permissivité différentielle à un large éventail d'EVs et montré les spécificités du franchissement de cette barrière par l'EV-A71. Ces données soulèvent la question de l'origine de l'ARN EV dans le LCR au cours des premiers stades d'une méningite. / Human enteroviruses (EV) are RNA viruses characterized by a large genetic variability. They are associated with severe but rare neurological infections and frequent but self-Limiting meningitis. The processes of entry into the central nervous system (CNS), the level of EV viral load in the cerebrospinal fluid (CSF) and its possible relation to the intensity of the associated inflammatory process remain poorly understood. As several EV genotypes are related to common neurological disorders, common pathological processes may be involved. In the first part of this PhD thesis, we have prospectively investigated the EV viral load in the CSF of patients with meningitis using a RT-QPCR assay. Our results showed that significant differences between viral load levels and the age groups, the leukocytes count, the protein levels in the CSF, and with the EV genotype involved in the infection. We also explored the hypothesis that an infection of the blood brain barrier (BBB) could be a common pathway used by EVs released in the bloodstream to gain access into the CNS. The EV replication and translocation were analyzed with an in vitro model of BBB based on the hCMEC/D3 cell line. We validated this cell model by showing different permissivity patterns among a large array of EV genotypes. In addition, we showed the specificities in how the EV-A71 crosses the endothelial barrier. The overall data raise the unresolved issue of the origin of viral RNA in the CSF and the sources of infection during the early acute stage of EV meningitis.

L'épilepsie du lobe temporal est fréquente et souvent pharmacorésistante. L'épileptogenèse est imputée à la mort neuronale, l'inflammation ou au déséquilibre de la neurotransmission. Récemment, la perméabilité vasculaire a été reconnue comme une cause de crises d'épilepsie. Dans un modèle d'épilepsie chronique, nous avons montré une angiogenèse associant vascularisation, surexpression de VEGF, perte de protéines des jonctions serrées et perméabilité de la BHE. L'observation des immunoglobulines G (IgGs) comme marqueurs de perméabilité vasculaire nous a permis de découvrir que les IgGs s'accumulent dans les neurones. Nous avons alors étudié le rôle de ces protéines dans l'épileptogenèse. Ensuite, afin de corréler la perméabilité de la BHE à l'épileptogenèse, nous avons étudié le kindling, un modèle dans lequel les crises sont induites mais pas spontanées. Nous n'avons observé aucun remaniement vasculaire, si ce n'est une dérégulation transitoire de deux protéines de jonctions serrées. La comparaison de ces deux modèles confirme la contribution de la dérégulation de la BHE dans la genèse des crises et la désigne comme une nouvelle cible thérapeutique. / Temporal lobe epilepsy is the most frequent form of pharmacoresistant epilepsies. Epileptogenesis is commonly imputed to neuronal loss, inflammation and an imbalance in neurotransmission. Now, vascular permeability was shown to participate in epileptic seizures generation. In a model of chronic epilepsy, we showed a neo-vascularisation associated with VEGF over expression, loss of tight junction proteins and BBB permeability. The use of immunoglobulins G (IgGs) as markers of permeability vascular allowed us to discover that the IgGs accumulates in neurones. We then studied the role of these proteins in epileptogenesis. Then, to correlate BBB permeability to epileptogenesis, we studied the kindling, a model in which seizures are induced but never spontaneous. We observed no vascular remodeling, except for a transient deregulation of tight junctions proteins. The comparison of these models confirms the contribution of BBB deregulation and points it as new therapeutic target.

Orientador: Maria Alice da Cruz-Höfling / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T16:36:47Z (GMT). No. of bitstreams: 1 Soares_EdileneSiqueira_M.pdf: 11448561 bytes, checksum: 00aace7be2d8fec579aea2fd4166f813 (MD5) Previous issue date: 2015 / Resumo: Neste trabalho investigamos a permeabilização da barreira hematoencefálica (BHE) pela peçonha da aranha Phoneutria nigriventer (PNV) através da via transcelular no cerebelo de ratos. As cavéolas foram analisadas nas células endoteliais pela expressão de proteínas associadas à sua formação (caveolina-1, Cav-1 e dinamina-2, Din2) e internalização (caveolina-1 fosforilada, pCav-1 e quinase da família Src, SKF), e nos astrócitos com avaliação da caveolina-3 (Cav-3) e da conexina-43 (Cx43) (formadora de junções comunicantes). A ação do PNV sobre o endotélio também foi avaliada pela ativação (acoplamento) ou inativação (desacoplamento) da enzima eNOS, produtora de óxido nítrico (NO). As estruturas que compõe a BHE foram avaliadas através de microscopia eletrônica de transmissão. Inicialmente, o estudo de Cav-1 contemplou sua localização, expressão gênica e proteica após o envenenamento em diferentes idades, ratos neonatos eram mais suceptíveis ao envenenamento do que ratos adultos. Após PNV, a imunomarcação para Cav-1 foi mais evidente na camada granular e molecular e em neurônios de Purkinje. A expressão Cav-1 e Din2 (foramdoras das vesículas e seu gargalo, respectivamente) aumentou em períodos de envenenamento agudo (1 h), de recuperação (5 h) e na ausência de sinais clínicos (24 h); em contrapartida SKF e pCav-1 envolvidas na internalização caveolar foram superexpressas em períodos opostos (às 2 h e 72 h). O PNV induziu aumento da metaloproteinase-9 da matriz (MMP9), importante mediadora de quebra da BHE e aumentou a formação e o tráfego de vesículas no endótelio após envenenamento. A análise de eNOS revelou desacoplamento (aumento de monômeros) nos períodos de envenenamento agudo (1-2 h) com progressivo retorno e super-expressão de dímeros (re-acoplamento) às 72 h; essas alterações foram relacionadas à ação do PNV sobre os níveis intracelulares de cálcio investigado pelo aumento na expressão de calmodulina e confimado pela localização de calbindina-D28. Os dados revelam a interferência do PNV sobre a homeostase endotelial e função vascular ao afetar o sistema eNOS/NO, importante controlador do tônus vascular. Nos astrócitos, as cavéolas são formadas por Cav-3 e sua superexpressão é associada a doenças neurológicas. O PNV aumentou significativamente os níveis basais de Cav-3 em astrócitos GFAP positivos (astrogliose reativa) em períodos de aumento de Cx43 (às 1, 5 e 24 h), e na vigência de edema citotóxico nos pés astrocitários e alterações nos contatos sinápticos axo-dendríticos e axo-somáticos. Em conjunto os resultados revelam que: (a) a quebra da via transcelular da BHE pelo PNV tem aumento da endocitose via cavéolas; (b) componentes da unidade neurovascular, como endotélio, astrócitos e neurônios estão intimamente envolvidos; (c) no endotélio, os efeitos são mediados pelo sistema eNOS/NO; (d) a SKF ativa o sistema endocítico e de transporte vesicular; (e) nos astrócitos, a dinamica expressão de Cx43 e Cav-3 e o retorno aos níveis basais em paralelo com a ausência de sinais de intoxicação nos animais (72 h) dá evidências de que ambas as proteínas interagem na resposta astrocitária. Os dados permitem sugerir que a presença de peptídeos neurotóxicos no veneno de Phoneutria nigriventer estão no centro dos efeitos aqui relatados / Abstract: In this work, we investigated the blood-brain barrier (BBB) permeabilization induced by Phoneutria nigriventer venom (PNV) in the transcellular route of rats¿ cerebellum. Caveolae was analyzed in endothelial cells accessing the expression of proteins involved in caveolae formation (caveolin-1, Cav-1 and dynamin-2, Dyn2) and internalization (phosphorylated Caveolin-1, pCav-1 and Src kinase family, SKF), in astrocytes caveolae role were evaluated with caveolin-3 (Cav-3) and connexin-43 (Cx43) (gap-junction main protein). PNV action on the endothelium was also investigated through activation (coupling) or inactivation (uncoupling) of eNOS enzyme, responsible for nitric oxide (NO) production. BBB components were evaluated using transmission electron microcopy. Initially, Cav-1 study addressed its localization along with Cav-1 protein and gene expression after envenoming in different age animals, neonate rats were more susceptible to envenoming than adult rats. After PNV, Cav-1 labeling was intense in granular and molecular layers and in Purkinje neurons. Cav-1 and Dyn2 (responsible for caveolae vesicles formation and scission, respectively) expression increased in periods of acute envenomation (1 h), recovery (5 h) and in the absence of clinical signals (24 h); in opposition SKF and pCav-1 involved in caveolae internalization were overexpress in opposite periods (at 2 h and 72 h). PNV induced increases in matrix metaloproteinases-9 (MMP9) an important BBB breakdown mediator, and increases in vesicles formation and traffic in the endothelium after envenoming. The study of eNOS activity revealed uncoupling (increasing in eNOS monomers) in acute periods after envenomation (1 h and 2 h) and progressive return followed by overexpression of dimers (re-coupling) at 72 h; those alterations were related to PNV action on calcium intracellular levels confirmed by Calmodulin increased expression and confirmed using Calbindin-D28 localization. Data revealed PNV interference on endothelial homeostasis and vascular function once affects the eNOS/NO system, an important vascular tonus controller. In astrocytes, caveolae are formed by Cav-3 and its overexpression is related to neurological disorders. PNV increased the basal levels of Cav-3 in GFAP-positive astrocytes (reactive astrogliosis) in the same periods as increased Cx43 (at 1, 5 e 24 h), during cytotoxic edema in astrocytes end-feet and alterations in axo-dendrites and axo-somatic synaptic contacts. Together, the results revealed that: (a) the BBB breakdown in transcellular route by PNV involves upregulation of caveolae endocytosis (b) the neurovascular unit components such as the endothelium, astrocytes and neurons are intimal involved (c) in the endothelium the effects are mediated by the eNOS/NO system and (d) SKF activates endocytic system and vesicular transport; (e) in the astrocytes, Cx43 and Cav-3 dynamic expression and their return to basal level in parallel with the absence of toxic signals in the animals (72 h) provides evidence that both protein interacts in astrocytes response. The data allows us to suggest that the neurotoxic peptides presented in Phoneutria nigriventer venom are in the center of the effects reported here / Mestrado / Biologia Tecidual / Mestra em Biologia Celular e Estrutural

[ES] El tratamiento de enfermedades neurológicas está muy limitado por la ineficiente penetración de los fármacos en el tejido cerebral dañado debido a la barrera hematoencefálica (BHE), lo que imposibilita mejorar la salud del paciente. La BHE es un mecanismo de protección natural para evitar la difusión de agentes potencialmente peligrosas para el sistema nervioso central. No obstante, la BHE se puede inhibir mediante ultrasonidos focalizados e inyección de microburbujas de forma segura, localizada y transitoria, una tecnología empleada mundialmente. La principal ventaja es su carácter no invasivo, siendo así muy atractiva y cómoda para el paciente. Normalmente, la zona cerebral enferma se trata en su parte central empleando un único foco. Sin embargo, enfermedades como el Alzheimer o el Parkinson requieren un tratamiento sobre estructuras de geometría compleja y tamaño elevado, situadas en ambos hemisferios cerebrales. Por tanto, la tecnología actual está muy limitada al no cumplir dichos requisitos. Esta tesis doctoral tiene como objetivo el desarrollo de una técnica novedosa, basada en hologramas acústicos, para resolver las limitaciones presentes en los tratamientos neurológicos empleando ultrasonidos. Se estudian las lentes acústicas holográficas impresas en 3D, que acopladas a un transductor mono-elemento, permiten el control preciso del frente de onda ultrasónico tanto para (1) compensar las distorsiones que sufre el haz hasta alcanzar el cerebro, como (2) focalizarlo simultáneamente en regiones múltiples y de geometría compleja o formando de vórtices acústicos, proporcionando así efectividad en tiempo y coste. Por ello, la investigación desarrollada en esta tesis abre un camino prometedor en el campo de la biomedicina que permitirá mejorar los tratamientos neurológicos, además de aplicaciones en neuroestimulación o ablación térmica del tejido. / [CA] El tractament de malalties neurològiques està molt limitat per la ineficient penetració del fàrmac en el teixit cerebral danyat a causa de la barrera hematoencefàlica (BHE), i així no és possible una millora de salut del pacient. La BHE és un mecanisme de protecció natural per a evitar la difusió d'agents potencialment perillosos per al Sistema Nervios Central. No obstant això, aquesta barrera es pot inhibir mitjancant una tecnologia emprada mundialment basada en ultrasons focalitzats i injeccio de microbombolles. El principal avantatge és el seu caràcter no invasiu, sent així molt atractiva i còmoda per al pacient, i permet obrir la BHE de manera segura, localitzada i transitòria. Normalment, la zona cerebral malalta es tracta en la seua part central, emprant un unic focus. No obstant això, malalties com l'Alzheimer o el Parkinson requereixen un tractament al llarg d'estructures de geometria complexa i grandària elevada, situades en tots dos hemisferis cerebrals. Per tant, la tecnologia actual està fortament limitada al no complir amb aquests requeriments. Aquesta tesi doctoral està enfocada a investigar i desenvolupar una tècnica nova, basada en hologrames acústics, per a solucionar les limitacions presents en els tractaments neurològics. Una lent acústica holograca de baix cost impresa en 3D acoblada a un transductor d'element simple permet el control precs del front d'ona ultrasònic punt per a (1) compensar les distorsions que pateix el feix en el seu camí cap al cervell, i (2) focalització simultània del feix en regions multiples i de geometria complexa, proporcionant aix un tractament efectiu en temps i cost. Per això, la investigació desenvolupada en aquesta tesi demostra la possibilitat de realitzar qualsevol tractament neurològic, a més d'aplicacions en la neuroestimulació o l'ablació tèrmica dins del camp biomèdic. / [EN] Treatments for neurological diseases are strongly limited by the inefficient penetration of therapeutic drugs into the diseased brain due to the blood-brain barrier (BBB), and therefore no health improvement can be achieved. In fact, the BBB is a protection mechanism of the human body to avoid the diffusion of potentially dangerous agents into the central nervous system. Nevertheless, this barrier can be successfully inhibited by using a worldwide spread technology based on microbubble-enhanced focused ultrasound. Its main advantage is its non-invasive nature, thus defining a patient-friendly clinical procedure that allows to disrupt the BBB in a safe, local and transient manner. Conventionally, the diseased brain structure has been targeted in its center, with a single focus. However, Alzheimer's or Parkinson's Diseases do require that ultrasound is delivered to entire, complex-geometry and large-volume structures located at both hemispheres of the brain. Therefore, current technology presents several limitations as it does not fulfill these requirements. This doctoral thesis aims to develop a novel technique based on using focused ultrasound acoustic holograms to solve the existing limitations to treat neurological diseases. In this dissertation, we study 3D-printed holographic acoustic lenses coupled to a single-element transducer that allow to accurately control the acoustic wavefront to both (1) compensate distortions suffered by the beam in its path to the brain, and (2) simultaneous focusing in multiple and complex-geometry structures or acoustic vortex generation, providing a time- and cost- efficient procedure. Therefore, the research carried out throughout this thesis opens a promising path in the biomedical field to improve the treatment for neurological diseases, neurostimulation or tissue ablation applications. / Acknowledgments to the Spanish institution Generalitat Valenciana, which funding grant allowed me to develop this doctoral thesis, and as well funded my research stay at Columbia University. The development of the entire thesis was supported through grant Nª. ACIF/2017/045. Particularly, the research carried out in Chapter 3 and Chapter 4 was possible thanks to and supported through grant BEFPI/2019/075. Action co-financied by the Agència Valenciana de la Innovació through grant INNVAL10/19/016 and by the European Union through the Programa Operativo del Fondo Europeo de Desarrollo Regional (FEDER) of the Comunitat Valenciana 2014-2020 (IDIFEDER/2018/022). / Jiménez Gambín, S. (2021). Transcranial Ultrasound Holograms for the Blood-Brain Barrier Opening [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171373 / TESIS

Parmi les récepteurs Frizzled, Frizzled 4 est le seul à avoir un phénotype vasculaire fort. Parcriblage, nous avons identifié l’ubiquitine ligase PDZRN3 en tant que nouveau partenaire de la protéineadaptatrice Dvl3 qui agit en aval de Fzd4. En utilisant des modèles murins inductibles, nous montronsque la délétion de PDZRN3 induit une létalité embryonnaire suite à des défauts de vascularisation dusac amniotique ; et que PDZRN3 est requis pour une vascularisation normale de la rétine. De par sonactivité d’ubiquitine ligase, PDZRN3 induit la prise en charge du complexe Fzd4/ Dvl3 par les vésiculesd’endocytose ce qui permet la transduction du signal après fixation du ligand Wnt5a sur le récepteurFzd4. PDZRN3 régule également le maintien des jonctions des cellules endothéliales et l’intégrité de labarrière hémato-encéphalique. La délétion de PDZRN3 stabilise les microvaisseaux après ischémiecérébrale. PDZRN3 induit la disruption des jonctions serrées et la rupture de la barrièrehématoencéphalique en ubiquitinant la protéine d’échafaudage MUPP1. / Fzd4 is the only Frizzled receptor that is essential for angiogenesis. By using a yeast twohybrid screening, we have identified the ubiquitin ligase PDZRN3 as a potential partner of the adaptorprotein Dvl3 that acts downstream of Fzd4. By using inducible mouse models, we have shown that lossof PDZRN3 leads to early embryo lethality due to vascular defects in the yolk sac when deleted inutero, and is then required during post natal retinal vascularization. PDZRN3 would target the Fzd4/Dvl3 complex to endosome, leading to signal transduction upon binding of Wnt5a to Fzd4. PDZRN3also regulates integrity of the blood brain barrier by acting on tight junctions stability. Loss of PDZRN3stabilizes microvessels after cerebral ischemia. PDZRN3 would induce tight junction disruption andblood brain barrier leakage by ubiquitinylating the scaffolding protein MUPP1.

Les traumatismes graves sont une cause principale d’hospitalisations et peuvent induire des handicaps physiques et psychologiques. Dans ce travail, nous nous intéressons au choc hémorragique (CH), défini par une perte sanguine de plus de 30% menant à une ischémie systémique causant de la mort cellulaire et la libération de médiateurs circulants. Parmi ces médiateurs, notre groupe a déjà démontré l’augmentation en circulation de l’acide urique (AU) après le CH et son rôle dans l’atteinte d’organes ; un effet qui est prévenu par l’utilisation d’une uricase, qui métabolise l’AU circulant. Notre objectif actuel est de démontrer le rôle de l’AU dans l’altération du système nerveux central. Pour ce faire nous utilisons un modèle murin de CH reperfusé avec 3 groupes expérimentaux : SHAM (contrôle), CH et CH+U (uricase IP au moment de la reperfusion). Nos résultats démontrent que l’altération de la perméabilité de la barrière hématoencéphalique (augmentation significative de la perméabilité à la fluorescéine de sodium (NaF)) et de l’expression de ICAM-1 après le CH peut être prévenu par l’administration d’uricase. Les résultats sont les mêmes pour la mesure de la neuroinflammation (activité de la myéloperoxidase (neutrophiles) ainsi que astrocytes et microglie activés) et de l’apoptose/dégérescence neuronale (caspase-3, coloration TUNEL et fluorojade). En conséquence à l’atteinte neuroinflammatoire et apoptotiques, nous observons une augmentation significative des comportements anxieux après le CH, détectés par le test de nage forcée, le labyrinthe en croix surélevé et l’intéraction sociale, et qui sont prévenus par le traitement avec uricase. En conclusion, ce projet permet de confirmer que l’AU joue un rôle important dans l’atteinte cérébrale et l’altération des comportements, après le CH reperfusé. / Polytrauma is one of the main causes of hospitalisations and can lead to physical and psychological handicaps. This work focuses on hemorrhagic shock (HS), defined by a blood-loss of at least 30%, leading to systemic ischemia, cell death and the release of various mediators in circulation. The importance of one of these mediators, uric acid (UA), in multiple organ failure after HS and the improvement by the use of an uricase, which can destroy UA, was already demonstrated by our lab. Our objective is to illustrate the implication of UA in central nervous system alterations after HS. To reach this goal, we use a murine model which is assigned to one of our 3 experimental groups: SHAM (control), HS and HS+U (IP injection of uricase at reperfusion). Our results show an altered blood-brain barrier permeability (significant infiltration of NaF in the brain after HS), an increased expression of ICAM-1 after HS and a prevention of both these results by uricase treatment. The same results are observed for neuroinflammation (myeloperoxidase activity (neutrophils), astrocytes and microglia) and for neuronal apoptosis/degeneration (caspase-3, TUNEL staining and FluoroJade staining). Furthermore, anxiety is increased after HS compared to SHAM but prevented with uricase treatment. The tests used to reach this conclusion are the elevated plus maze, the forced swim test and social interaction. In conclusion, this project confirms the central role of UA in brain lesions and subsequent behavioral alterations after resuscitated HS.

<p>A multitude of neurologic diseases are increasing in patients that both diminish quality and quantity of life. My dissertation research focused on unraveling the blood-brain barrier’s alterations (BBB), primarily in lung cancer brain metastases, the most common brain metastasis in patients. We optimized a reliable and reproducible mouse model for creating brain metastases using patient derived brain seeking cells of non-small lung cancer (NSCLC) using ultrasound-guided intracardiac injection. I then evaluated brain tissue with qualitative and quantitative immunofluorescence for individual components of the BBB. Using this experimental method, I was able to identify the specific shift of each BBB component over time in NSCLC brain metastases. I then used human brain metastases specimens to demonstrate the clinical relevance of my findings. These results show distinct alterations in the BBB, which have the potential for targeting therapeutic delivery to extend patient survival. I was also able to characterize a novel epithelial-mesenchymal (EMT) phenotype in vertebral metastases of NSCLC in our model, with features similar to those seen in human patients. Most recently, I analyzed patterns of paracellular permeability associated with each BBB component of NSCLC brain metastases which may provide direct passageways for therapeutic delivery. Altogether, this research offered foundational evidence for the future development of targeted novel therapeutics, including nanoparticles. Outside of the brain metastases field, we used an experimental framework to successfully characterize the BBB alterations in a traumatic brain injury model (bTBI). These findings provided the first description of this unique pathology and the framework for developing therapeutics in other neurologic diseases. Although my research work has focused on animal models of disease, future directions based on my research work include the developing a novel 3D BBB-on-chip device to enable high throughput novel therapeutic delivery through the BBB. Long-term, identifying targetable alterations in the restrictive BBB using <i>in vitro</i> and <i>in vivo</i> models provides a potential conduit for effective prevention and treatment of a myriad of neurologic diseases to prolong patient survival and quality of life.</p>

<p>There has been a rapid shift in food environment of Western cultures that has increased consumption of diets high in fat and sugar, which have imparted negative effects on metabolic and neurocognitive health. There is also building evidence that the adverse effects of Western diet</p> <p>(WD) are different in males and females, such that males are impacted more at an earlier age and females are impacted later in life. The underlying biological mechanisms linking WD and neurocognitive health are often associated with energy dysregulation or neuroinflammation. WD</p> <p>disrupts glucose homeostasis and causes low grade inflammation in the body, and these can impact</p> <p>the brain by disrupting the blood-brain barrier (BBB). The BBB is the microvasculature found throughout the entire brain that tightly regulates what compounds get into the brain to ensure optimal neuronal function. WD disrupts the BBB, however, the effects of WD on BBB integrity</p> <p>in females and younger individuals remain largely unknown. Based on the metabolic and behavioral effects of WD, we hypothesized that the effects are age- and sex- specific. To test this, we gave male and female rats access to a WD for 8-10 weeks starting in juvenile period (post-natal</p> <p>day 21) or in adulthood (post-natal day 75), then measured body weight, behavior, glucose tolerance, the density of two different markers of BBB integrity. We also measured density of resident immune cells (microglia) to assess the relationship between inflammation and BBB integrity. First, we focused on the impact of hyperglycemia on the BBB since elevated glucose alters glucose transporter 1 (GLUT1). We found sex- and age- specific decreases in GLUT1 density in the prefrontal cortex and hippocampus—two brain regions commonly associated with neurocognitive impairments associated with WD. Correlational comparisons between WD and chow (CH) animals also found that the typically relationship between glucose tolerance and</p> <p>GLUT1 in the PFC and hippocampus were overall disrupted in WD animals. Second, we measured the leakage of albumin, a blood protein, since WD depletes the tight junctions that would typically prevent albumin from entering the brain and triggering a neuroinflammatory response. We did not find an increase in albumin density in WD animals, however, we found a main effect of age which</p> <p>offers insight to differential susceptibilities to BBB leakage. Third, we focused on inflammation and found that WD did not impact microglia density in our experiments, nor did it correlate with GLUT1, albumin, or behavior. Collectively, our findings support the hypothesis that the impact of</p> <p>WD on the BBB is sex- and age- specific, suggest that WD does not increase leakage of compounds such as albumin, and highlights the nuanced relationships between WD, metabolic disruption, behavioral deficits, and neuroinflammation.  </p>