京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18874号 / 医博第3985号 / 新制||医||1008(附属図書館) / 31825 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 山田 亮, 教授 福山 秀直 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In translational research, disease models in preclinical studies are used as media for discovery of drugs or novel therapeutics. Development of in vitro models for various neurological diseases that enable efficient pharmacological or toxicological screening has been ongoing but challenging. Recognizing the potential benefit of in vitro disease models, dysfunctions in the cortical neuronal networks were induced to mimic the functional pathology of neurological symptoms using microelectrode arrays. Two different disease states – tinnitusand excitotoxicity – were investigated and discussed. In this model, pentylenetetrazol-induced increase in spontaneous firing rate and synchrony in the auditory cortical networks was used as correlate of tinnitus. Potential tinnitus treatment drugs from several different classes – including the novel class of potassium channel openers – were screened and quantified. The potentialtherapeutic values of these drugs were also discussed as the basis for drug repurposing. Functional excitotoxicity was induced by cisplatin (a cancer drug that causes neurological sideeffects) and glutamate (the major excitatory neurotransmitter). As proof-of-principle that the model may contribute to expediting the development of therapeutics, cisplatin excitotoxicity wasprevented by the antioxidant D-methionine, while glutamate excitotoxicity was prevented by ceftriaxone (a modulator of a glutamate reuptake transporter). In the latter part of the study, with results linking two of the screened drugs L-carnitine and D-methionine to GABAA receptor activation, it was demonstrated that this model not only served as an efficient drug-screening platform, but can be utilized to functionally investigate the underlying mechanism of drugs. Inaddition, several practical or conceptual directions for future studies to improve on this in vitro disease model are suggested.

No description available.

Neuronal homeostasis is an essential process to protect neurons from over/under-stimulation driven from systematic changes such as synapsis plasticity or tissue damage. Functional stability in neurons relays on the homeostatic plasticity that its disturbance causes irreversible injuries. Hence, a large body of studies elaborated to investigate the underlying mechanism for changes in synaptic connectivity and neuronal function. HIV-1 Tat (Transactivation of transcription), is a well-established neurotoxic protein, released by HIV-1 infected cells in the brain and disturbs neuronal homeostasis. The effects of Tat have been addressed in numerous studies investigating the molecular events associated with neuronal cell survival and death. The emergence of lncRNAs as critical players in disease etiology placed them in the spotlight to study pathogenesis of human diseases. Due to its capacity to modulate host transcriptome, HIV-1 Tat protein has been subjected to increasing genome-wide examinations. This study showed that exposing primary rat neurons to Tat resulted in the up-regulation of an uncharacterized long-non-coding RNA (lncRNA), LOC102549805 (lncRNA-U1). Evidence exists that increased expression of lncRNA-U1 in neurons disrupts bioenergetic pathways by dysregulating homeostasis of Ca2+, mitigating mitochondrial oxygen reduction, and decreasing ATP production leading to mitochondrial impairment in neurons. These changes were associated with imbalances in autophagy and apoptosis pathways via the Tat-mediated lncRNA-U1 induction. Additionally, this study showed the ability of Tat to modulate the expression of the neuropeptide B/W receptor 1 (NPBWR1) gene via the up-regulation of lncRNA-U1. Collectively, my results identified the Tat-mediated lncRNA-U1 elevation disturbs neuronal homeostasis. Our observations of lncRNA-U1 knock-down experiments indicated the novel lncRNA LOC102549805 (U1) as a viable therapeutic target to prevent HIV-1 Tat neurotoxicity. / Biology

Tesi realitzada a l'Equip de Neurociència de Sistemes - IDIBAPS / Since the discovery of the place cells in 1971 by John O’Keefe and colleagues an extensive work over the hippocampus has been developed as the mammal model of spatial navigation. Place cells are rodents’ hippocampal neurons whose firing is associated to certain locations of the environment. A majority of studies have focused on how the place fields (the area where the firing of a neuron is restricted) are generated in relation to the static cues of the environment (O'Keefe and Conway, 1978; Muller et al., 1987; Gothard et al., 1996). The present work assessed a similar question but regarding the dynamic cues surrounding the subject, and with the hypothesis that the hippocampus is also representing the position of other moving objects. In order to demonstrate if that was the case, we developed a behavioural protocol in which rats learnt to discriminate the movements of a robot in order to obtain reward, an Operant Position Discrimination Task (OPDT). Once the protocol was validated, the subjects were chronically implanted with tetrodes in the CA1 region of the hippocampus. In this way the activity of single hippocampal cells could be isolated off-line and the LFP of the area stored during the recordings. Using this method, the relationship between the firing of the cells and the field activity with the spatial parameters of the robot could be evaluated. The results showed a modulation by the dynamic cue of the theta oscillation. While the locomotor activity of the subjects is directly related to the power of theta in natural conditions (Vanderwolf, 1969), during the movement of the robot such relationship was disrupted and the band power between 4-12 Hz showed a trough at this time. The analysis of the single cells’ activity showed neurons locked to several spatial features of the dynamic cue. First, the position of the rat and the robot where analysed by information content parameters. Skaggs Index and Positional information (Markus et al., 1994; Olypher et al., 2003) showed neurons locked to the position of the subject as expected in CA1 and also other neurons locked to the positions of the robot. Second, moving from the spatial analysis to the temporal one, we found responses to the movement of the robot like OFF/ON variations of the basal activity of the neurons. Such changes in the firing patterns where quantified by the Mutual Information index (Nelken and Chechik, 2007) demonstrating that a large fraction of the neurons have a significant differential pattern of activity during the movement of the robot towards one side or the other. The use of the same index, MI, for the evaluation of the static or dynamic condition of the robot, also resulted in a set of neurons spiking with significant disparity during such epochs. In conclusion, the present work has demonstrated the existence of neural correlates locked to a dynamic cue in the hippocampus. Both the field activity at the theta range, LFP between 4 and 12 Hz, and the activity of the hippocampal neurons were found to reflect and/or encode the spatial features of a dynamic cue. The present work has in this way enlarged the limited evidence present in the prior literature about the role of the hippocampus in the tracking and localization of dynamic cues with the use of a behavioural protocol where both the spatial and temporal dynamics could be assessed. / La correcta localización y seguimiento de las pistas dinámicas que se encuentran en el ambiente es una tarea crucial para el individuo. Comportamientos fundamentales como la caza, el apareamiento o el escape necesitan una correcta identificación de la posición de presas, congéneres y depredadores para su correcta realización. El sistema cerebral encargado de localizar al propio sujeto en el ambiente se sabe que se encuentra en la formación hipocampal después de que diversos estudios hayan demostrado la necesidad del mismo para una correcta orientación (Morris et al., 1982) y, aún más importante, tras el descubrimiento en roedores de neuronas que disparan únicamente en espacios restringidos del entorno, las células de lugar (O'Keefe and Dostrovsky, 1971). Si bien se conoce que estos procesos están fundados en una correcta representación de la posición de las pistas estáticas del ambiente (O'Keefe and Conway, 1978; Muller et al., 1987; Gothard et al., 1996), que sirven de referencia para la propia localización, poco se sabe acerca de cómo se integra la información relativa a los objetos y/o sujetos móviles que se encuentran en el mismo ambiente. Este trabajo tiene como objetivo principal intentar responder a esta pregunta, es decir, ¿en qué modo el hipocampo procesa la información relativa a las pistas dinámicas? Para el desarrollo del estudio, primero, se diseñó una tarea comportamental que asegurara el hecho de que la pista dinámica resultase relevante para los sujetos de forma que los mismos prestaran atención a sus movimientos. Con este fin elegimos utilizar un robot cuyos desplazamientos pueden ser finamente controlados y asociar una recompensa a determinados patrones de navegación del robot. Después de probar con diferentes tareas de discriminación se llegó a una configuración (Operant Position Discrimination Task, OPDT) que permitía a los animales seguir los movimientos del robot desde un espacio separado en el cual recibían la recompensa en caso de discernir correctamente los desplazamientos de la pista. Una vez validada la tarea comportamental, a los sujetos que alcanzaron altas tasas de rendimiento se les implantaron tetrodos en la zona CA1 del hipocampo, lugar en el que se encuentran las células de lugar más estables. Una vez hecho el implante se procedió a registrar la actividad cerebral durante la ejecución de la tarea. Por una parte se aislaron los potenciales de acción pertenecientes a neuronas únicas y el potencial de campo de la zona, LFP. Respecto a la actividad de campo, LFP, se observó una disminución significativa de la potencia en la banda theta, 4-12 Hz, relacionada generalmente con la actividad locomotora del sujeto (Vanderwolf, 1969) durante el movimiento del robot. Durante el resto del registro la relación entre velocidad y potencia de theta se mantuvo y sólo en el periodo de discriminación del movimiento del robot esta relación se vio alterada con un mínimo de potencia observado en diferentes sujetos y registros. La actividad de las neuronas se analizó en función de los parámetros espaciales y dinámicos de la rata y el robot. Mirando la especificidad espacial del disparo de las neuronas a través de los parámetros Skaggs Index y Positional information (Markus et al., 1994; Olypher et al., 2003) se encontraron células significativamente ligadas en su actividad a la posición del sujeto o del robot. La actividad de las neuronas también se analizó de forma temporal, tomando como referencia el inicio de los estímulos, es decir el movimiento del robot hacia un lado u otro. Utilizando como índice la Mutual Information (Nelken and Chechik, 2007) se encontró que una larga proporción de las neuronas tienen respuestas diferenciales durante el movimiento del robot hacia uno de los lados. A su vez, el mismo análisis, pero en esta ocasión comparando los periodos en los que la pista se encuentra inmóvil con los que está en movimiento, determinó que otra fracción de las neuronas tiene patrones de disparo diferenciales según sea la condición dinámica de la pista. El conjunto de los resultados obtenidos indica claramente que el hipocampo se encuentra involucrado activamente en la localización y el seguimiento de las pistas dinámicas, siendo esto reflejado tanto en la actividad de sus neuronas como en la actividad de campo global. Los parámetros espaciales de la pista que resultaron modulados durante la tarea fueron su posición, la dirección de su movimiento y el hecho en sí de permanecer inmóvil o en desplazamiento.

La presente tesis doctoral presenta varios resultados fundamentales para la ampliación del conocimiento actual de procesos importantes en la generación de los circuitos neuronales, como son la migración y la ramificación de células neurales. En primer lugar, se ha determinado la expresión de la semaforina transmembranal 4F en cerebro de ratón en desarrollo y adulto. Así, se ha visto que se expresa en diversas áreas del cerebro, y se ha encontrado expresión de esta proteína en precursores neuronales y en neuronas maduras, principalmente en dendritas, y en células del linaje oligodendroglial. Para profundizar más en este aspecto se llevaron a cabo varios marcajes dobles de Sema4F con proteínas marcadoras de oligodendrocitos, observándose marca en el nervio óptico y otras regiones cerebrales, incluídas la materia blanca y vías de migración de oligodendrocitos. La localización de esta semaforina en el nervio óptico a edades embrionarias y su expresión en células precursoras de oligodendrocitos (OPCs), comprobada in vitro, nos llevó a sugerir que Sema4F funciona controlando la migración de OPCs. Una serie de experimentos con explantes de nervio óptico tratados con medio control o medio condicionado 4F nos permitió determinar que Sema4F actúa inhibiendo la migración de OPCs, sin afectar a su proliferación. Además, Sema4F induce la diferenciación de OPCs a oligodendrocitos maduros. Todos estos datos sugieren un posible papel de Sema4F en procesos de remielinización. Los efectos negativos de Sema4F sobre la migración de OPCs deben cursar con cambios en el citoesqueleto celular. La kinasa de adhesión focal (FAK) es un importante mediador de señales extracelulares (como factores tróficos, interacción de integrinas con proteínas de matriz extracelular, etc…) y el interior de las células. Actúa sobre el citoesqueleto de actina y de tubulina, influyendo en la generación de filopodios, lamelipodios y fibras de estrés. Tiene un papel crucial en migración, de modo que dedicimos estudiar si Sema4F ejerce sus efectos en OPCs a través FAK. Hemos visto que Sema4F es capaz de inducir la fosforilación en varios residuos tirosina de FAK en pocos minutos, y que ambas proteínas por separado ejercen efectos opuestos en la migración de oligodendrocitos. La vía de señalización de 4F, de la que se desconoce incluso el receptor, podría cursar mediante la modulación del estado de activación de FAK, aunque faltan experimentos definitivos. FAK presenta varias isoformas específicas del sistema nervioso central, originadas mediante procesos de splicing alternativo. En la presente tesis hemos determinado con gran especificidad la forma mayoritaria expresada en varias áreas cerebrales y en el desarrollo embrionario o el adulto, tanto en neuronas como en células de la glía. FAK responde a neurotrofinas y participa en procesos de ramificación neuronal, si bien su efecto final es controvertido. Otra proteína que responde a neurotrofinas, y actúa promoviendo la ramificación axonal, es la kinasa dependiente de cdc-42 activada 1 (Ack1). En esta tesis hemos determinado que ambas proteínas interaccionan en cerebro específicamente, de manera independiente de la isoforma de FAK presente. Mediante el uso de inhibidores hemos visto que la activación de FAK es necesaria para la fosforilación de Ack1 y viceversa. FAK es la responsable de la atracción ejercida por netrina-1, y hemos determinado que la ausencia de Ack1 elimina el efecto de esta molécula de señalización. Con técnicas de Espetrometría de Masas hemos identificado algunos posibles interactores de ambas proteínas. Además, hemos observado cambios en el estado de fosforilación de varios residuos de FAK y Ack1 en función del estado de desarrollo (ratones P5 Vs. Adultos) y del estado general de activación del cerebro (ratones inyectados con la droga epileptogénica PTZ Vs. Control). / This thesis presents several results related to important processes regarding neural circuit formation, i.e. migration and ramification of Central Nervous System (CNS) cells. First, we have determined the expression of transmembrane semaphorin 4F (Sema4F) in developing and adult mice brain. Expression of this protein is high in neuronal and oligodendrocyte precursor cells (OPCs), and in different areas including optic nerve (ON) and different migratory pathways. In vitro experiments confirmed Sema4F expression in OPCs. We investigated the role of this protein in functions important for OPC physiology, and found that Sema4F inhibits OPC migration from ON explants and induces their differentiation into mature progenitors. Negative effects of Sema4F in migration must involve cytoskeleton changes. Focal adhesion kinase (FAK) is an important integrator of different extracellular signals and modulates cytoskeleton dynamics to control generation of lamellipodia, fillopodia and stress fibers. In the present project we found that Sema4F is able to phosphorylate FAK, and that FAK enhances OPC migration. The exact implications of Sema4F-FAK relationship remain to be elucidated. FAK exists in different spliced isoforms, expressed preferentially in brain. In this project, we characterised the exact isoform expressed in different areas of the brain and by different cell types. Finally, FAK response to neurotrophins is well characterised. FAK also participates in ramification processes, with controversial final effects in neurons. Ack1 is a crucial transducer of neurotrophin-induced ramification. In this thesis we show that both proteins interact specifically in neurons. We have also found that the activation of FAK is necessary for Ack1 phosphorylation upon stimulation, and viceversa. FAK mediates netrin-1 attraction, and here we have determined that knocking-down Ack1 avoids netrin-1 effects in hippocampal explants. By Mass Spectrometry (MS) techniques, we have observed changes in the phosphorylation state of both proteins depending on the developmental stage of the brain (P5 mice) or its activation state (epileptic mice).

Master of Science / Department of Diagnostic Medicine/Pathobiology / Shafiqul I. Chowdhury / Bovine herpesvirus type 1 (BHV-1) is an important pathogen of cattle that can cause severe respiratory tract infection known as infectious bovine rhinotracheitis (IBR), abortion in pregnant cows, and is an important component of the Bovine Respiratory Disease Complex (BRDC, “Shipping fever”). The ability of BHV-1 to transport anterogradely from neuron cell bodies in trigeminal ganglia to axon termini in the nasal and ocular epithelia of infected cattle complicates the control of the disease in both vaccinated and infected cattle populations. In calves and rabbits, Us9 deleted viruses have defective anterograde neuronal spread from cell bodies in the trigeminal ganglia to nerve termini in the nose and eye but retrograde spread remains unaffected. To characterize the neuronal spread of BHV-1, we developed primary neuronal cultures using the dorsal root ganglia (DRG) of rabbit embryos. We successfully used microfluidic chamber devices to isolate DRG in the somal compartment and allowed for efficient growth of axons into the axonal compartment. This enabled us to study axon mediated neuronal spread of infection as well as viral transport in axons. Thus, rabbit DRG neuronal culture was susceptible to BHV-1 mutant and wild-type infection, and the method allowed visualization of viral spread in chamber cultures using live cell imaging and fluorescent microscopy. Lastly, using the microfluidic chamber compartmentalized neuron culture system we showed that Us9 acidic domain-deleted and Us9 null mutant BHV-1 viruses had defective anterograde neuronal transport relative to BHV-1 wild type and/or Us9 rescued viruses.

A first-of-its-kind, proof-of-concept drug screen with implications for two unmet medical needs. / The actin-bundling protein fascin is a key mediator of tumor invasion and metastasis and its activity drives filopodia formation, cell-shape changes and cell migration. Small-molecule inhibitors of fascin block tumor metastasis in animal models. Conversely, fascin deficiency might underlie the pathogenesis of some developmental brain disorders. To identify fascin-pathway modulators we devised a cell-based assay for fascin function and used it in a bidirectional drug screen. The screen utilized cultured fascin-deficient mutant Drosophila neurons, whose neurite arbors manifest the 'filagree' phenotype. Taking a repurposing approach, we screened a library of 1040 known compounds, many of them FDA-approved drugs, for filagree modifiers. Based on scaffold distribution, molecular-fingerprint similarities, and chemical-space distribution, this library has high structural diversity, supporting its utility as a screening tool. We identified 34 fascin-pathway blockers (with potential anti-metastasis activity) and 48 fascin-pathway enhancers (with potential cognitive-enhancer activity). The structural diversity of the active compounds suggests multiple molecular targets. Comparisons of active and inactive compounds provided preliminary structure-activity relationship information. The screen also revealed diverse neurotoxic effects of other drugs, notably the 'beads-on-a-string' defect, which is induced solely by statins. Statin-induced neurotoxicity is enhanced by fascin deficiency. In summary, we provide evidence that primary neuron culture using a genetic model organism can be valuable for early-stage drug discovery and developmental neurotoxicity testing. Furthermore, we propose that, given an appropriate assay for target-pathway function, bidirectional screening for brain-development disorders and invasive cancers represents an efficient, multipurpose strategy for drug discovery.

Damage to the adult mammalian central nervous system (CNS), either by traumatic injury or disease, usually results in permanent sensory and/or motor deficits. Regeneration of neural circuits is limited both by the lack of growthpromoting molecules and by the presence of growth-inhibitory molecules in the mature brain and spinal cord. The research described here examines the therapeutic potential of viral vectors and neuronal transplants to reconstruct damaged neural pathways in the CNS. Experimental neural transplantation techniques often fall short of expectations because of limited transplant survival and insufficient neurite outgrowth to repair connections and induce behavioral recovery. These shortcomings are addressed in the current studies by virus-mediated expression of cell-specific neurotrophic and guidance molecules in the host brain prior to cell transplantation. The initial proof-of-principle studies show that viral vectors can be used to create axon-guidance pathways in the adult mammalian brain. With such pathways in place, subsequent transplantation of neurons leads to longdistance, targeted outgrowth of neurites. Application of this technique to a rat model of Parkinsons disease demonstrates that circuit reconstruction leads to functional recovery. For this study, rats were lesioned on one side of their brain with 6-hydroxydopamine to produce a hemiparkinsonian state. The motor deficit was confirmed by amphetamine-induced rotation testing and spontaneous motor asymmetry testing. The rats were then divided into experimental groups to receive lentivirus injections along a path between the substantia nigra (SN) and the striatum to express glial cell-line derived neurotrophic factor (GDNF), GDNF family receptor alpha-1 (GFR1), netrin-1 or green fluorescent protein (GFP, control). One group received combination injections of lenti-GDNF and lenti-GFR1. One week after virus injections, animals received transplants of embryonic midbrain dopaminergic neurons into their SNs. They were tested for motor asymmetry every two weeks for a total of eight weeks and then brain tissue was harvested for immunohistochemical analysis. Results demonstrate that virus-induced expression of GDNF and GFR1 supports growth of dopaminergic fibers from cells transplanted into the SN all the way to the striatum, and these animals have a significant reduction in both drug-induced and spontaneous motor asymmetry.

Magnocellular neurones (MCNs) are capable of secreting vasopressin and oxytocin from the somato-dendritic compartment, which can occur independently to secretion from nerve terminals. One hypothesis of the mechanism that regulates this differential release is that dendrites utilise different vesicle pools compared to those found in terminals. Little is known for the function of neuronal dendrites, especially the mechanism for peptide release. One theory is that vesicles stored in dendrites are non-released vesicles ready for recycling or degradation. Immunofluorescent labelling was performed on hypothalamic slices of the transgenic rat where enhanced green fluorescent protein (eGFP) was tagged to vasopressin. Lysosomes were detected by the lysosome-associated membrane protein LAMP1. Correlation analysis of LAMP1 labelling and VP-eGFP had shown that localisation of lysosomes in dendrites is positively correlated to loci of high vasopressin expression. This suggests active degradation of vesicles in dendrites. It is not known whether preferential release of peptides occurs along the profile of dendrites. Experiments were carried out using a temperature block to block exit of vesicles from the Golgi apparatus. Release of the temperature block triggered release of a wave of newly synthesised vesicles from the Golgi apparatus. Measurement of the fluorescent intensity of VP-eGFP showed that preferential release of peptides does not occur along the profile of dendrites. I have also utilised confocal live cell imaging to study the dynamics of dendritic vasopressin release using VP-eGFP slice explants. Experiments using high potassium stimulation showed significant increase in the release of vasopressin after priming with thapsigargin (intracellular calcium mobiliser), in accordance to in vitro release and microdialysis studies. These results demonstrate that live cell imaging can be achieved in magnocellular neurons, providing a robust model system in the study of dendritic peptide release. Large dense core vesicles (LDCVs) in other cell types such as bovine adrenal chromaffin cells were shown to segregate according to vesicle age, suggesting that vesicle age is an important factor in the regulation of peptide release. Whether vesicles of different age groups exist in magnocellular dendrites is not known. Thus, biolistic transfection with exogenous fluorescent proteins for expression under temporal control was carried out. However, low transfection rate in magnocellular neurones and the high background fluorescence caused by scattered gold particles used as bullets for transfection deemed this method inappropriate for the purpose of imaging vesicles. Hence, development of an adenoviral transduction system was employed. By using an inducible adenovirus gene construct coupled with a fluorescent reporter gene, it is possible to visualise vesicle pool segregation under different experimental conditions. Subcloning of a red fluorescent construct tagged to ppANF was tested on PC12 cells to show targeting of fluorescence expression to LDCVs. Successful production of an inducible adenoviral DNA with the red fluorescent construct insert was confirmed by PCR and DNA sequencing. Whilst the generation of viral particles is still to be achieved, successful production of the virus will be an invaluable system for inducible gene expression in neurones.