The formation of neural connections in the mammalian nervous system is a complex process. During development, axons are initially overproduced and compete for limited quantities of target-derived growth factors. Axons which participate in functional circuits and secure appropriate amounts of growth factors are stabilized, while those axons that are either inappropriately connected or do not obtain sufficient concentrations of growth factors are eliminated in a process termed ‘axon pruning’. In this thesis, I examined the mechanisms that regulate pruning of peripheral, NGF-dependent sympathetic neurons that project to the eye. I determined that pruning of these projections in vivo requires the p75 neurotrophin receptor (p75NTR) and synthesis of brain-derived neurotrophic factor (BDNF) from the activity-dependent exon IV promoter. Furthermore, analysis of an in vitro model of axon competition, which is regulated by the interplay between nerve growth factor (NGF) and neuronal activity, revealed that p75NTR and BDNF are also essential for axon competition in culture. In this model, in the presence of NGF, neural activity confers a competitive growth advantage to stimulated, active axons by enhancing downstream TrkA (NGF receptor) signaling locally in axons. More interestingly, the unstimulated, inactive axons deriving from the same and neighboring neurons acquire a "growth disadvantage" due to secreted BDNF acting through p75NTR, which induces axon degeneration by suppressing TrkA signaling that is essential for axonal integrity. These data support a model where, during developmental axon competition, successful axons secrete BDNF in an activity-dependent fashion which activates p75NTR on unsuccessful neighboring axons, suppressing TrkA signaling, and ultimately promoting pruning by a degenerative mechanism.

During nervous system development, growing axons navigate towards their targets using signals from their environment. These signals may be biochemical or mechanical in nature; however, the role of mechanical cues in axon pathfinding in vivo, and the spatiotemporal dynamics of embryonic brain mechanics, are still largely uncharacterised. Here, I have identified a role for tissue mechanics in embryonic axon guidance in vivo, using retinal ganglion cell (RGC) axon outgrowth in the developing Xenopus laevis optic tract (OT) as a model system. Using atomic force microscopy (AFM) to map brain stiffness in vivo, I found that embryonic Xenopus brain tissue was mechanically heterogeneous at both early and later stages of OT outgrowth, i.e. just before RGC axons make a stereotypical turn in the mid-diencephalon, and when they reach their target, respectively. The final path of RGC axon turning followed a clear mechanical gradient: by the later stage, tissue rostral to the OT had become stiffer than tissue caudal to it. This mid-diencephalic stiffness gradient was an intrinsic property of the underlying brain tissue, and correlated with local cell body densities (with higher density rostral to the OT and lower density caudal to it). Crucially, inhibiting cell proliferation in vivo during OT outgrowth abolished the stiffness gradient and reduced OT turning at the later stage. Next, I developed a time-lapse AFM technique to track tissue stiffness and RGC axon behaviour simultaneously in vivo. Using this approach, I followed the evolution of the mid-diencephalic stiffness gradient during intermediate developmental stages, around the time when the OT’s caudal turn is initiated. The stiffness gradient was shallow pre-turn, but increased in magnitude during axon turning (mostly due to an increase in tissue stiffness rostral to the OT). This increase in stiffness gradient preceded the rise in OT turning angle, suggesting that the stiffness gradient is not caused by the invading axons. The observed rise in stiffness gradient correlated with stage-specific increases in local cell density, and was attenuated by blocking mitosis in vivo during time-lapse AFM measurements (which also reduced OT turning). As final confirmation that brain stiffness contributes to RGC axon pathfinding, I disrupted mechanical gradients by artificially stiffening brain tissue in vivo. Importantly, global stiffening via application of transglutaminase eliminated the mid-diencephalic stiffness gradient by increasing tissue stiffness caudal of the OT, and reduced the OT turning angle. Sustained mechanical compression of small areas using an AFM probe stiffened brain locally and repelled RGC axons, consistent with the way they turned away from rapidly stiffening tissue regions during time-lapse AFM experiments. Taken together, these results are consistent with a function for tissue mechanics in axon pathfinding in vivo.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19142号 / 生博第325号 / 新制||生||43 / 32093 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 豊島 文子, 教授 千坂 修 / 学位規則第4条第1項該当

La formation des neurites constitue une étape cruciale dans le processus de différenciation neuronale. Cependant, les mécanismes qui permettent de déterminer comment et à quelle position les neurites émergent sont toujours largement méconnus. Nous avons postulé qu'une marque moléculaire pouvait préfigurer la différenciation morphologique. Au cours de ma thèse, j'ai cherché à identifier de nouvelles molécules capables de s'accumuler aux sites d'initiation des neurites et d'en contrôler la protrusion. De manière intéressante, chez la levure, la marque moléculaire qui contrôle le site de protrusion du bourgeon a été caractérisée. Parmi les centaines de protéines contrôlant le site d'initiation chez la levure, les Septines constituent une famille de protéines bien conservée chez les vertébrés. Ces GTPases forment des filaments qui agissent comme barrière de diffusion ou « échafaudage » moléculaire. Au cours de ma thèse, je me suis donc intéressée au rôle des Septines lors de l'initiation axonale dans le modèle des neurones sensoriels de DRG chez l'embryon de poulet. Nous avons pu démontrer qu'aux stades précoces de leur développement, ces neurones formaient deux axones, un au pôle ventral et l'autre au pôle dorsal, indiquant que le nombre et la position des sites d'initiation des axones sont bien contrôlés dans ces neurones. Nous avons, ensuite, démontré que les Septines étaient bien exprimées dans les DRG aux stades précoces du développement. Mes analyses en vidéo-microscopie de la localisation de la septine 7 au cours de la différentiation des neurones de DRG montrent que les Septines s'accumulent au site d'émergence de l'axone, juste avant ou lors de sa formation. L'inhibition des Septines induite par une construction dominant-négative (DN) ou par ajout d'un inhibiteur pharmacologique bloque la formation des axones. De plus, cette inhibition entraine une modification précoce de la morphologie, qui se traduit par l'apparition de cellules multipolaires complexes et de cellules rondes sans prolongement suggérant que, conformément à notre hypothèse, les Septines sont impliquées dans l'initiation des neurites. L'ensemble de ces résultats montre que les Septines régulent la différenciation morphologique précoce des neurones sensoriels / Neurite formation is a crucial step of neuronal differentiation. However, the mechanisms that determine how and at which position neurites emerge in the soma are still poorly understood. We postulated that a molecular polarity could prefigure the morphological differentiation, with some molecules that could accumulate at the future site of axon initiation. Interestingly, such molecular polarity has been evidenced in the contest of yeast budding, with bud forming at specific position relatively to the previous bud site. Genome-wide screen identified hundreds of proteins that control bud site location. Among the vertebrate molecules homologous to those involved in budding site selection, we selected the Septins as promising candidates. These GTP-ases form filaments that act as diffusion barriers and molecular scaffolds. We investigated the contribution of Septins to axon initiation using the chick dorsal root ganglion (DRG) neurons as a model. Monitoring of cell morphology in nascent ganglia indicates that DRG neurons form a single axon at the ventral pole and a second one at the dorsal pole and that these axons seem to emerge directly after their last division. This suggests that two initiation sites are selected at opposite pole of the soma.We found that Septins homologous with those controlling budding are expressed in the early DRG developmental stages. My analyses by time-lapse video-microscopy showed that Septin7 accumulate at the site of axon emergence, just before or during its formation.We observed that a pharmacological inhibitor and a dominant-negative construct block axon formation both in vitro and in vivo respectively. Furthermore, blocking Septin function leads to the appearance of uncommon round or sea urchin-like neurons. Thus, Septins appear to regulate early step of morphological differentiation of DRG neurons, possibly by controlling axon initiation site selection

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22639号 / 医博第4622号 / 新制||医||1044(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 林 康紀, 教授 伊佐 正, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Proper synaptic connectivity is critical for communication between cells and information processing in the brain. Neurons are highly interconnected, forming synapses with multiple partners, and these connections are often refined during the course of development. While decades of research have elucidated many molecular players that regulate these processes, understanding their specific roles can be difficult due to the large number of synapses and complex circuitry in the brain. In this thesis, I investigate mechanisms that establish neural circuits in the simple organism C. elegans, allowing us to address this important problem with single cell resolution in vivo. First, I investigate remodeling of excitatory synapses during development. I show that the immunoglobulin domain protein OIG-1 alters the timing of remodeling, demonstrating that OIG-1 stabilizes synapses in early development but is less critical for the formation of mature synapses. Second, I explore how presynaptic excitatory neurons instruct inhibitory synaptic connectivity. My work shows that disruption of cholinergic neurons alters the pattern of connectivity in partnering GABAergic neurons, and defines a time window during development in which cholinergic signaling appears critical. Lastly, I define novel postsynaptic specializations in GABAergic neurons that bear striking similarity to dendritic spines, and show that presynaptic nrx-1/neurexin is required for the development of spiny synapses. In contrast, cholinergic connectivity with their other postsynaptic partners, muscle cells, does not require nrx-1/neurexin. Thus, distinct molecular signals govern connectivity with these two cell types. Altogether, my findings identify fundamental principles governing synapse development in both the developing and mature nervous system.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19142号 / 生博第325号 / 新制||生||43(附属図書館) / 32093 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 豊島 文子, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The neural cell adhesion molecule L1 is a member of the immunoglobulin superfamily and it mediates many adhesive interactions during brain development. Mutations in the L1 gene are associated with a spectrum of X-linked neurological disorders known as CRASH or L1 syndrome. The objective of this thesis was to use the zebrafish model to investigate the molecular mechanisms of L1 functions and the pathological effects of its mutations. Zebrafish has two L1 homologs, L1.1 and L1.2. Inhibition of L1.1 expression by antisense morpholino oligonucleotides resulted in phenotypes that showed resemblances to L1 patients. However, knockdown of L1.2 expression did not result in notable neural defects. Furthermore, analysis of the expression pattern of L1.1 has led to the discovery of a novel soluble L1.1 isoform, L1.1s. L1.1s is an alternatively spliced form of L1.1, consisting of the first four Ig-like domains and thus a soluble secreted protein. L1.1 morphants exhibited disorganized brain structures with many having an enlarged fourth/hindbrain ventricle. Further characterization revealed aberrations in ventricular polarity, cell patterning and proliferation and helped differentiate the functions of L1.1 and L1.1s. While L1.1 plays a pivotal role in axonal outgrowth and guidance, L1.1s is crucial to brain ventricle formation. Significantly, L1.1s mRNA rescued many anomalies in the morphant brain, but not the trunk phenotypes. Receptor analysis confirmed that L1.1 undergoes heterophilic interactions with neuropilin-1a (Nrp1a). Peptide inhibition studies demonstrated further the involvement of L1.1s in neuroepithelial cell migration during ventricle formation. In the spinal cord, spinal primary motoneurons expressed exclusively the full-length L1.1, and abnormalities in axonal projections of morphants could be rescued only by L1.1 mRNA. Further studies showed that a novel interaction between the Ig3 domain of L1.1 and Unplugged, the zebrafish muscle specific kinase (MuSK), is crucial to motor axonal growth. Together, these results demonstrate that the different parts of L1.1 contribute to the diverse functions of L1.1 in neural development.

Description of the cell cycle features of neural progenitors during late stages of neurogenesis in a gyrencephalic mammal, the ferret.

This study examined the effects of bilateral and unilateral deafness by measuring cortical auditory evoked potential (CAEP) responses in children at initial stages of bilateral cochlear implant (CI) use. We recorded cortical responses evoked by right and left CI stimulation in 127 children with early onset (< 12 months) deafness, with 72 children receiving the two devices in the same surgery (simultaneously implanted) and 55 children receiving the devices in separate procedures (sequentially implanted). Three different types of responses were identified in children with bilateral CIs. No significant effects of duration of deafness, age at implantation, or duration of unilateral CI use were found on response latencies and amplitudes within each type of cortical response, but there were clear differences in responses types between groups and ears. In the context of these findings, the effects of bilateral and unilateral deafness to the auditory pathways were discussed.