京都大学 / 新制・課程博士 / 博士(医学) / 甲第23413号 / 医博第4758号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 小柳 義夫, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A major challenge in developing disease altering therapeutics for the treatment of Parkinson’s disease (PD) has been the delivery of compounds across the blood-brain barrier (BBB) to the central nervous system (CNS). While direct surgical infusion has been utilized to deliver compounds to the brain that don’t cross the BBB, issues of poor biodistribution in the CNS due in part to properties of the molecules being delivered and/or infusion device protocols have limited the widespread success of this invasive approach. To avoid the issues of surgically delivering compounds to the CNS, numerous studies have examined the use of intranasal administration as a non-invasive delivery method. The data presented in this dissertation examines intranasal administration of dopamine neuron stimulating peptide-11 (DNSP-11), a small, amidated peptide with neuroprotective and restorative properties, and its effects on the nigrostriatal system in animal models of PD. Here we demonstrate that severely lesioned 6-hydroxydopamine (6-OHDA) F344 rats repeatedly administered DNSP-11 intranasally exhibited a decrease in damphetamine- induced rotation, dopamine (DA) turnover, and an increase in tyrosine hydroxylase positive neuronal sparing. Additionally, tracer studies indicated rapid distributed throughout the CNS and CSF following a one-time bilateral intranasal dose of 125I-labeled DNSP-11. These results demonstrate that DNSP-11 can be delivered to the CNS intranasally, and maintains its neuroactive properties on the nigrostriatal system in a rat model of PD. In a dose escalation study of DNSP-11, we evaluated the efficacy of repeated intranasal administration in awake, vertically chaired trained, 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) hemiparkinsonian rhesus macaques using an atomizer system over a 10-week period. Here we report that animals did not exhibit observable adverse effects at the DNSP-11 concentrations examined, bilateral increases in fine motor performance of the upper limbs, and changes in tissue levels of DA and its metabolites. Finally, tracer studies indicated signal present throughout the CNS and CSF following a one-time bilateral intranasal dose of 125I-labeled DNSP-11. These studies support the efficacy of the repeated intranasal administration of DNSP-11 in awake Rhesus macaques over 10-weeks, while also enhancing motor performance and striatal neurochemistry in a non-human primate model of PD.

La coqueluche est une pathologie due à la bactérie Bordetella pertussis qui touche les voies respiratoires des patients infectés causant toux, leucocytose, fièvre, et dont les symptômes peuvent aller jusqu’au décès chez les individus les plus à risque (nouveau-nés et enfants immunodéprimés en particulier). Ciblée par différents programmes vaccinaux depuis de nombreuses années, cette pathologie sévit à nouveau dans de nombreux pays développés où le nombre de cas augmente fortement depuis la fin des années 2000. Cette résurgence montre la nécessité de développer de nouvelles stratégies afin de comprendre les mécanismes de l’infection par B. pertussis. Dans ce contexte, la recherche préclinique apparaît comme essentielle pour comprendre la physiopathologie de la coqueluche. De nombreux modèles animaux ont été décrits pour l’étude de la coqueluche mais aucun de ces modèles ne permet de reproduire l’ensemble du spectre des symptômes cliniques de la pathologie, notamment la toux. Cependant, au cours des dernières années un modèle d’infection par Bordetella pertussis chez le jeune babouin a été développé aux Etats-Unis et permet de reproduire la pathologie observée chez l’homme, notamment concernant la toux et la transmission. Ce modèle semble ainsi très prometteur pour l’étude de la physiopathologie de la coqueluche.Cependant, de nombreuses inconnues subsistent dans ce modèle, notamment concernant la colonisation bactérienne et les interactions entre la bactérie et l’hôte. Nous avons ainsi cherché dans cette étude à évaluer d’une part l’impact de différents facteurs comme l’âge des animaux, la dose d’infection ainsi que la voie d’exposition sur la pathologie déclarée par les babouins suite à l’infection par la souche B1917 de B. pertussis afin de pouvoir proposer un parallèle avec les données cliniques disponibles. Nous avons également développé l’utilisation de techniques d’imagerie in vivo comme l’endomicroscopie confocale couplée à la bronchoscopie afin d’étudier la localisation et la cinétique de colonisation et certaines interactions du pathogène dans le tractus respiratoire inférieur au cours de la pathologie. Cette étude nous a ainsi permis d’approfondir les connaissances de physiopathologie de la coqueluche dans ce modèle babouin et consolidera cet outil précieux pour l’évaluation des futures stratégies de prévention contre cette pathologie. / Whooping cough, or pertussis, is a respiratory disease caused by Bordetella pertussis bacterial colonization of human airways. Main symptoms are cough, leukocytosis, fever and may even be lethal for some patients (e.g. newborn infants and immuno-deficient patients). Despite a good vaccination coverage worldwide against pertussis, whooping cough cases have been re-increasing in several developed countries in the past twenty years. This resurgence points out the crucial need to develop new control strategies and to better understand pertussis pathophysiology, notably using appropriate animal models. Numerous preclinical models including mice, rats, rabbits and swine have been described for B. pertussis infection studies. However, none of these models reproduce the full spectrum of clinical pertussis symptoms, especially cough. The recent baboon model of whooping cough described in the last few years in the US appears to be a very relevant model for pertussis pathophysiology studies as these animals reproduced all clinical symptoms as observed in humans including cough.However, many aspects of bacterial colonization and interactions with the host have yet to be described in this model.We have then evaluated diverse parameters such as animal age, the inoculum dose and the exposition route on the pathology symptoms and immune responses developed by baboons following B. pertussis B1917 strain inoculation in order to draw a parallel with human clinical data. We also developed in this model in vivo imaging techniques like confocal endomicroscopy coupled with bronchoscopy in order to evaluate bacterial colonization kinetics, localization and some interactions in the lower respiratory tract of infected baboons. Then, this study brought additional data on whooping cough physiopathology in this baboon model, which will be crucial for evaluating future prevention strategies against pertussis disease

Excerpt: The title and conclusions of the study recently published by Jones et al. (1) concluded that monkeys were protected from dying from a form of simian-human immunodeficiency virus (SHIV) by an psoralen- and ultraviolet-inactivated vaccinia virus in a multi-envelope DNA-VV-protein (DVP). However, the findings in the study are more equivocal than indicated by the title because the effectiveness of the modified vaccinia virus was not decisively demonstrated.

The Lateral Prefrontal Cortex (LPFC) and the Dorsal Premotor cortex (PMd) are two cortical structures that are involved in cognitive processes such as motor planning and decision-making. The LPFC is extensively connected to sensory, somatosensory, and motor cortices that help it control several cognitive functions [for review, see: (Tanji & Hoshi, 2008)]. Similarly, the PMd can integrate information from the prefrontal and motor cortex, acting as a link, in action planning and decision making [for review, see: (Hoshi & Tanji, 2007)]. Therefore, it is important to study the cortical pathways between these areas because of their common role in processing and selecting relevant information in tasks requiring decision-making. Using neural tract-tracing, immunolabeling and microscopy in rhesus monkeys (M. mulatta), we assessed the distribution and layer-specific organization of projection neurons from LPFC area 46 and PMd area 6 directed to the LPFC area 9. Our study revealed that projection neurons to area 9 were found originating from upper (L2-3) and deep (L5-6) layers of both areas, but with a slight upper layer bias. We found that the LPFC area 46 had a higher density of projection neurons directed to LPFC area 9 compared to the PMd area 6. Additionally, our data also revealed laminar differences in the perisomatic parvalbumin (PV) inhibitory inputs onto area 9 projection neurons, which were dependent on area of origin. Within ventral LPFC area 46, perisomatic PV+ inhibitory inputs onto upper layer projection neurons to area 9 was greater than those onto deep layer projection neurons. The opposite pattern was found for PMd area 6DR, where perisomatic PV+ inhibition onto deep layer projection neurons to area 9 was greater than those onto upper layer neurons. These findings provide additional insights into the layer-specific organization of prefrontal and premotor pathways that play an important role in action planning and decision-making.

While the success of combination antiretroviral therapy (ART) has extended the lifespan of people with human immunodeficiency virus (HIV)(PWH), approximately half of PWH on suppressive ART will experience HIV-associated neurological dysfunction. While ART has decreased the incidence of severe neurological disease and dementia in PWH, the incidence of milder neurological and cognitive complications remains stable. Despite the frequency of HIV neurological disease, contributing factors and inflammatory pathogenesis are difficult to observe in PWH over time. Extracellular vesicles (EVs) constitute an understudied method of intercellular communication and molecule delivery in viral infections. EVs carry inflammatory mediators to areas of the periphery during ART suppression but are understudied in the brain. In this dissertation, we use a biologically relevant simian-human immunodeficiency virus (SHIV)-infected non-human primate (NHP) model of HIV persistence in the central nervous system (CNS) to investigate the formation of a myeloid viral reservoir, inflammation during ART-mediated viral suppression, and the roles of myeloid EVs in persistent SHIV neuropathogenesis.In Chapter 2, we characterized viral and immune persistence in the CNS using SHIV.D, a novel model of HIV-1 in rhesus macaques (RM). Here, we demonstrate viral replication in the brain and neuropathogenesis after ART in RM using novel macrophage-tropic transmitted/founder (TF) SHIV.D.191859. Using quantitative immunohistochemistry (IHC) and DNA/RNAscope, we demonstrated myeloid-mediated neuroinflammation, viral replication, and proviral DNA in the brain in all animals. These findings were replicated in a second cohort of RM necropsied after 6 months of suppressive ART. We concluded that TF SHIV.D models HIV-1 CNS replication, pathogenesis, and persistence on ART in rhesus macaques, and is a biologically relevant model to study HIV neuropathogenesis. In Chapter 3, we investigated EVs in a SHIV.D/RM model of HIV. To determine the potential roles of different cell-derived EV populations in SHIV/HIV neuropathogenesis, we developed a method to investigate changes in the cellular origin of EVs in vivo in RM. EVs that are released by neural and glial cells into the blood circulatory system can serve as biomarkers for injury and illness as well as give insight into CNS dysfunction and other disease processes in a non-invasive manner. Here, we present a bead-free multiparameter conventional flow cytometry method to phenotype, characterize, and determine cellular origin of plasma extracellular vesicles. Using RM plasma and two four-parameter panels, we identified the following subsets of plasma EVs: tetraspanin CD81+, CD11b+ macrophage-derived, CD14+ monocyte-derived, TMEM119+ microglia-derived, CD171+ neuron-derived, CD3+ T cell-derived, and CD31+ endothelium-derived EVs. EVs were isolated from RM plasma before infection with SHIV.D, during acute viremia, and after ART suppression. EV flow cytometry on these samples revealed a significant increase in TMEM119+ microglial EVs and CD171+ neuronal EVs in RM plasma during viremia and ART suppression. In Chapter 4, we investigate myeloid-specific EVs in an in vitro SHIV.D/RM model. Using primary RM monocyte-derived macrophages (MDM), we determined that MDMs increased EV production after SHIV.D infection. Whole proteomic analysis was conducted on EVs from SHIV-infected and uninfected MDM. Gene ontology pathway analysis and gene set enrichment analysis reveal pathways associated with overrepresented proteins in myeloid EVs. Finally, differential abundance analysis demonstrated that myeloid EVs isolated from SHIV.D-infected MDMs carried significantly increased levels of neuropathogenic and inflammatory proteins. Altogether, these studies improve our understanding of SHIV.D viral persistence and persistent neuropathogenesis in the RM brain as a model for HIV-1 chronic neuropathogenesis and describe the contribution of myeloid EVs to neurological disease during SHIV/HIV infection. / Biomedical Sciences

Behaviour is guided by accumulated experience, valuation and comparison. While many aspects associated with these functions are mediated by the frontal lobes, the precise contribution from particular regions remains debated. This thesis will deal with how an organism comes to select an option and will specifically focus on the role of the orbitofrontal cortex (OFC) in two mechanisms in this process: learning of outcome specificities and selecting between multiple options based on their expected values. Despite evidence emphasizing anatomical and connective heterogeneity within this structure, the OFC is often regarded as a uniform region. This thesis aims to resolve some of this uncertainty by assuming that the medial and lateral regions of the OFC contribute differentially to learning and decision-making. Two distinct methodologies were used in these investigations. First, the contribution of the medial OFC to social and emotional processing was examined. The findings from this study disprove previously held beliefs that the medial regions of the OFC guide social and emotional behaviours, but indicted a role for this region in value-guided decision-making. The second study examined functional differences between the lateral and medial OFC by making circumscribed lesions to either region in macaque monkeys. The animals performed a number of 3-armed bandit tasks which were designed to investigate different aspects of value assignment and comparison. The results show that while lateral OFC was required for "credit assignment" – the correct assignment of values to visual cues – medial OFC was critical for comparison of the cues' values during decision-making. In unchanging probabilistic environments, mOFC lesions induced decision-making impairments when value comparison was difficult without affecting credit assignment and associative learning. By contrast, lateral OFC lesions caused the opposite pattern of impairment. The final study used human-neuroimaging techniques to investigate the differential representation of outcome-specific contingency learning and found not only that the expectation of a unique outcome facilitated learning and memory recall but that this was supported by a neural network which included the lateral regions of the OFC and the anterior cingulate cortex. Activity in the mOFC did not correlate with outcome-specific contingency learning but instead reflected both the value associated with the receipt and expectation of a reward. Taken together, the results from this thesis suggest that specific parts of the OFC make markedly different contributions to these very different cognitive functions.

Introduction : La dystonie est définie comme un syndrome de cocontractions musculaires soutenues aboutissant à des mouvements répétitifs et des postures anormales. Cependant la physiopathologie des dystonies reste mal comprise. Les études menées chez l’homme soulignent le rôle crucial des ganglions de la base dans la physiopathologie des dystonies. Des données récentes obtenues chez le rongeur suggèrent l’implication d’un désordre de la transmission cholinergique striatale mais es modèles qu’ils soient génétiques ou pharmacologiques n’aboutissent pas toujours à un phénotype de dystonie. C’est pourquoi il était important de proposer une étude chez le primate non humain, visant à vérifier notre hypothèse de travail, à savoir : est-ce qu’une augmentation de la transmission cholinergique dans le putamen est capable d’induire un phénotype clinique de dystonie similaire à celui rencontré chez l’homme.Méthodes : Nous avons réalisé des infusions chroniques d’un agoniste muscarinique non sélectif (Oxotremorine) au sein du territoire sensori-moteur du striatum chez le primate non-humain. Les symptômes cliniques induits par ce produit ont été évalués à l’aide de l’échelle de Burke-Fahn-Marsden (BFM) adaptée à l’animal. Nous avons également utilisé une approche électromyographique pour caractériser l’activité musculaire en lien avec la clinique ainsi que des enregistrements de l’activité Multi-Unitaire et Unitaire au sein des ganglions de la base afin d’établir des corrélations électro-cliniques.Résultats : Les infusions d’Oxotremorine nous ont permis d’observer : (i) des postures et des mouvements anormaux similaires aux mouvements dystoniques rencontrés en pathologie humaine ; (ii) une fréquence de décharge neuronale anormalement basse dans le GPi (13,5Hz) et un pattern de décharge de type « bursty » principalement lorsque les symptômes sont sévères ; (iii) une activité oscillatoire (28-30Hz) au sein du putamen, du GPe et du GPi; (iv) l’absence de cohérence de l’activité oscillatoire entre ces structures ; (v) que le GPi est la seule structure à présenter une cohérence de l’activité oscillatoire.Conclusion : Nos travaux démontrent pour la première fois qu’un modèle de dystonie chronique peut être obtenu chez le primate non humain par augmentation du tonus cholinergique dans le putamen. Ce travail valide l’hypothèse de l’implication des interneurones cholinergiques dans la physiopathologie des dystonies. Ils confortent l’idée qu’une augmentation du tonus cholinergique peu à elle seule induire un phénotype de dystonie. / Introduction: Dystonia is defined as a syndrome of sustained muscular cocontractions leading to repetitive movements and abnormal postures. However, the pathophysiology of dystonia remains poorly understood. Studies in humans emphasize the crucial role of basal ganglia in the pathophysiology of dystonia. Recent data in rodents suggest the involvement of a disorder in the striatal cholinergic transmission. But these genetic or pharmacological rodent models do not always express the phenotype of dystonia. Therefore, it was important to propose a primate study to test whether an increase of cholinergic transmission within the putamen is able to induce a clinical phenotype of dystonia similar to that seen in humans.Methods: To verify our hypothesis, we chronically infused non-selective muscarinic agonist (Oxotremorine) in the sensory-motor striatum in non-human primates. Dystonic clinical symptoms induced by this drug were assessed using the Burke-Fahn-Marsden (BFM) scale adapted to animals. We used electromyographic approach to characterize muscular activity linked to clinical symptoms, and we recorded Multi-Unit and Single-Unit neuronal activity in basal ganglia to establish electro-clinical correlations.Results: The infusions of Oxotremorine allowed us to observe: (i) abnormal postures and movements similar to the dystonic movements encountered in human pathology; (ii) an abnormally low neuronal firing frequency in the GPi (13.5Hz) and a bursty firing pattern mainly when the symptoms where severe; (iii) oscillatory activity (28-30Hz) within the putamen, GPe and GPi; (iv) the lack of coherence of the oscillatory activity between these structures; (v) that the GPi is the only structure to present a coherence of the oscillatory activity.Conclusion: We have demonstrated for the first time that a model of chronic dystonia can be obtained in non-human primates by increasing cholinergic tone in the putamen. This work validates the hypothesis of an involvement of cholinergic interneurons and striatal acetylcholine levels in the pathophysiology of dystonia.

La maladie de Parkinson (MP) est la seconde maladie neurodégénérative la plus répandue. Les symptômes moteurs répondent initialement bien aux médicaments dopaminergiques. Toutefois, des complications motrices de ces médicaments finissent par survenir. Certains patients se voient alors proposer un traitement neurochirurgical par stimulation cérébrale profonde (SCP) et chronique du Noyau Subthalamique (NST). Plusieurs études suggèrent que la modulation du faisceau cortico-subthalamique moteur explique au moins en partie l’efficacité de la SCP du NST. Approfondir la compréhension du mécanisme d’action de la SCP du NST pour le traitement de la MP devrait permettre d’optimiser le rapport innocuité/efficacité de cette procédure qui s’adresse à des dizaines de milliers de patients dans le monde. L’objectif primaire de cette thèse est de caractériser le faisceau cortico-subthalamique moteur sur les plans anatomique et fonctionnel chez le primate non humain. Les objectifs secondaires sont : Permettre la visualisation directe et en conditions stéréotaxiques du NST chez le primate non humain et le patient parkinsonien - Mettre au point une méthodologie permettant de caractériser sur les plans anatomique et fonctionnel tout faisceau de fibres entre deux régions cérébrales anatomiquement distinctes, chez le primate non humain - Elaborer une loi de commande pour la stimulation du NST par optogénétique et en boucle fermée, afin de détruire sélectivement les oscillations béta et tester l’hypothèse de leur rôle dans l’émergence des symptômes moteurs de la MP... / Parkinson’s Disease (PD) is the second most widespread neurodegenerative disease. Motor symptoms initially respond well to dopaminergic medecines ; however, motor complications will eventually occur. Some patients are then proposed a neurosurgical treatment by chronical electrical deep brain stimulation (DBS) of the subthalamic nucleus (STN). Several studies suggested that the modulation of the motor cortico-subthalamic bundle might explain the efficacy of STN DBS, at least to a certain extent. A better understanding of the mechanism of action of DBS of the STN in order to treat PD should help to optimize the safety/efficacy of this surgical procedure from which tens of thousands of patients could benefit all over the world.The main purpose of the present thesis is to characterize, both anatomically and functionally, the motor cortico-subthalamic bundle in non-human primates. Secundary objectives are : (1) to allow the direct visualization, under stereotactic conditions, of the STN of non-human primates and of patients with PD ; (2) to develop a methodology enabling to characterize, both anatomically and functionally, any fiber bundle between two anatomically distinct cerebral areas, in non-human primates ; (3) to establish a command law for closed-loop stimulation of STN by optogenetics, in order to selectively destroy beta-oscillations and assess the hypothesis of their role in the occurrence of motor symptoms in PD...

Dopamine deficiency in the striatum is a central feature of Parkinson’s disease (PD). Symptomatic therapy with L-3,4-dihydroxyphenylalanine (L-DOPA) aims at restoring physiological dopaminergic neurotransmission within the brain. Unfortunately, current treatment paradigms fail to achieve this goal, which leads to the emergence of motor complications, secondary to long term L-DOPA administration, including dyskinesia and wearing-OFF, and non-motor symptoms related to disease progression, including neuropsychiatric symptoms such as psychosis. However, degenerative changes in PD are not limited to the dopaminergic system, but also affect the serotonergic system. There is increasing evidence suggesting an involvement of the serotonergic system in the pathophysiology of both motor and non-motor complications of PD. The work presented in this Thesis has investigated the serotonergic and dopaminergic systems in PD, by performing post mortem studies in the brains of PD patients and of parkinsonian non-human primates (NHPs), and by performing behavioural studies in the parkinsonian rat and NHP models of PD. The main conclusions presented are that: 1) serotonergic type 1A (5-HT1A) and 2A (5-HT2A) levels are altered in the brains of dyskinetic parkinsonian NHPs, suggesting abnormal 5-HT1A- and 5-HT2A-mediated neurotransmission in dyskinesia; 2) 5-HT2A receptor levels are altered in the brains of PD patients with visual hallucinations (VH), suggesting abnormal 5-HT2A-mediated neurotransmission in VH; 3) some of the anti-dyskinetic actions attributed to stimulating 5-HT1A or antagonising 5-HT2A receptors might in fact be due to an antagonist action at D4 receptors, as antagonising D4 receptors significantly alleviates L-DOPA-induced dyskinesia in rat and NHP models of PD; 4) concurrent inhibition of the serotonin and dopamine transporters (SERT and DAT, respectively) enhances duration of L-DOPA-induced ON-time in the parkinsonian NHP. However, the ratio of SERT/ DAT inhibition appears crucial in determining the quality of this extra ON-time; SERT > DAT inhibition exacerbates the severity of L-DOPA-induced dyskinesia, whereas SERT = DAT and DAT > SERT inhibition do not worsen the severity of L-DOPA-induced dyskinesia. Together these data extend our knowledge of the interaction between serotonin and dopamine, specifically as they relate to symptoms and side effects of dopamine replacement therapy in PD and highlight potential novel therapeutic approaches to PD.