<p dir="ltr">The <i>SCN2A</i><i> </i>gene encodes for the neuronal sodium channel Na_V1.2, which mediates action potential initiation and propagation (Sanders et al., 2018). This protein is expressed mainly in the proximal axonal initial segment (AIS) and soma of glutamatergic excitatory cortical neurons (Kruth, Grisolano, Ahern, & Williams, 2020). <i>SCN2A</i> pathogenic variants have been associated with epilepsy. An example is the recurrent Nav1.2-L1342P variant, a heterozygous missense variant (Begemann et al., 2019) identified in five patients worldwide presenting an early-onset severe seizure phenotype that remains hard to treat with current medications (Que et al., 2021). Additionally, it is one of the few rare <i>SCN2A</i> variants that can impact brain structure (Miao et al., 2020).</p><p dir="ltr">Given that no disease-modifying treatment exists, there is an urgent need to generate novel tools to probe at variant-specific disease mechanisms, evaluate therapeutic interventions, and study interactions with other cell types. Previously, we demonstrated that hiPSC-derived 2D neuronal monolayers carrying the CRISPR/Cas9-edited Nav1.2-L1342P variant display a distinct hyperexcitability phenotype (Que et al., 2021). Despite these findings, questions persist regarding the Nav1.2-L1342P variant's influence on neurodevelopment in more physiologically relevant 3D models, such as organoids.</p><p dir="ltr">To address this, in Chapter 2 of this study, we generated human-induced pluripotent stem cell-derived cortical organoids carrying the epilepsy-related Nav1.2-L1342P variant to study its effect on neuronal hyperexcitability, neurodevelopment and other disease phenotypes. Our data suggests that Nav1.2-L1342P cortical organoid neurons display<b> </b>enhanced repetitive action potential firings, intrinsic excitability, enhanced calcium signaling, increased network neuronal firing, and excitatory postsynaptic currents (EPSCs), suggesting a marked hyperexcitability phenotype and enhanced excitatory neurotransmission. Moreover, cortical organoids with the Nav1.2-L1342P variant display significant changes in synaptic, glutamatergic, and development-related pathways. We also observed that Nav1.2-L1342P variant impacts cortical organoid synaptic and neuronal content.</p><p dir="ltr">The impact of the Nav1.2-L1342P variant was also demonstrated in the 2D-cortical neuron monolayer model, presenting a noticeable reduction in neuronal complexity, thus offering intriguing insights into their effect on neuronal morphology and developmental processes. Our findings recapitulate the hyperexcitable phenotype trends previously observed in the 2D-cortical neuron monolayer platform (Que et al., 2021) and provide evidence of non-autonomous cell development changes due to the Nav1.2-L1342P variant.</p><p dir="ltr">Chapter 3 of this dissertation established a co-culture of hiPSC-derived neurons and microglia, the brain's resident immune cells. Microglia originate from a different lineage (yolk sac) and are not naturally present in hiPSC-derived neuronal cultures. Therefore, they must be added to neuronal cultures to yield a heterogeneous environment. Microglia are also one of the few cell types able to respond to neuronal hypo and hyperexcitability changes. This unique capability prompted us to study how microglia responded to human neurons carrying a disease-causing variant and influenced neuronal excitability.</p><p dir="ltr">We found that microglia display increased branch length and enhanced process-specific calcium signal when co-cultured with the Nav1.2-L1342P neurons, recapitulating phenomena previously observed in rodent seizure models (Eyo et al., 2014; Nebeling et al., 2023). Moreover, the presence of microglia significantly lowered the repetitive action potential firing and current density of sodium channels in neurons carrying the variant, demonstrating the microglial capacity to influence and ameliorate the neuronal activity of the Nav1.2-L1342P mutant neurons. We hypothesized that this effect could be attributed to the increased release of glutamate or small molecules by the Nav1.2-L1342P mutant neurons, which could likely be triggering microglial responses. Additionally, we showed that co-culturing with microglia reduced sodium channel expression within the axon initial segment (AIS) of Nav1.2-L1342P neurons, explaining, in part, the mechanism behind the reduction of sodium current density.</p><p dir="ltr">Taken together, our observations with 2D cortical neurons and 3D cortical organoids revealed marked hyperexcitability and developmental changes associated with the Nav1.2-L1342P variant. Our work also reveals the critical role of human iPSCs-derived microglia in sensing and dampening hyperexcitability mediated by an epilepsy-causing SCN2A variant.</p>

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22700号 / 医科博第115号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 近藤 玄, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La surdité neurosensorielle est définie par une atteinte de l’oreille interne, il résulte principalement d’une perte de cellules ciliées (CC). Chez les mammifères, ce processus est malheureusement irréversible. Le développement de la thérapie cellulaire a fait naître de nouveaux espoirs pour le traitement des surdités neurosensorielles. Les cellules souches d’origine embryonnaire ou adulte seraient capables de se différencier in vitro en progéniteurs otiques et de restaurer partiellement les fonctions auditives in vivo après transplantation. Cependant, les protocoles de différenciation in vitro des CC à partir de cellules souches sont insatisfaisants, et les signaux qui contrôlent ce phénomène restent mal connus. Ainsi, l’objectif de ce travail de thèse était d’étudier in vitro la différenciation des CC à partir de cellules souches pluripotentes induites humaines (hiPSC). Nous nous sommes intéressés à deux voies de signalisation majeures impliquées dans le développement de l’oreille interne in vivo, la voie Notch et la voie Wnt. Dans une première partie, nous avons montré que l’inhibition tardive de la voie Notch favorise la différenciation des hiPSC en CC. Dans une seconde partie, nous avons étudié le rôle de la voie Wnt dans la différenciation des hiPSC en cellules otiques. Nos résultats indiquent que l'inhibition de la voie Wnt durant la première phase d’induction favorise l'expression des marqueurs de la placode otique et initie la spécification des CC.Les travaux présentés dans cette thèse améliorent ainsi les protocoles de différenciation des hiPSC et suggèrent que ce type de cellules serait parfaitement adapté pour traiter les surdités neurosensorielles. / Neurosensory hearing loss is associated to inner ear disorders and degeneration of hair cells (HCs). Unfortunately, this process is irreversible in mammals. Currently, no curative treatment allows these cells to regenerate. For this reason, the development of cell therapy arose new hopes for the treatment of neurosensory hearing loss. Stem cells, either of embryonic or adult origin, seem able to differentiate in vitro into otic progenitors and to partially restore auditory functions in vivo. However, current protocols for in vitro differentiation of stem cells into HCs are unsatisfactory, and the signals that control this phenomenon remain poorly understood. Thus, the objective of this thesis was to study in vitro HC differentiation from human induced pluripotent stem cells (hiPSCs). We were particularly interested in two major signaling pathways involved in vivo in inner ear development, the Notch and Wnt signaling pathways.In a first part, we demonstrated that Notch inhibition during late otic differentiation enhances hiPSC differentiation into hair cell-like cells. In a second part, we studied the role of the Wnt signaling pathway during otic induction and HC specification. Our results indicate that Wnt inhibition during early otic induction promotes the expression of otic placode markers and initiate HC specification. The work presented here thus propose improved protocols to obtain HCs from hiPSCs, and suggest that this cell type is perfectly adapted for the treatment of neurosensory hearing loss.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25197号 / 医博第5083号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 齋藤 潤, 教授 斎藤 通紀, 教授 長船 健二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22379号 / 医科博第109号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 武田 俊一, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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