Using a genetic disorder and patient samples, the work in this thesis provides novel insights into the underlying causes of brain and nerve disorders. Patients with this disorder are missing a large amount of genetic material, and can develop disorders such as seizures, autism spectrum disorders, and ADHD and may also fail to achieve general milestones in socialization, growth, learning, and motor development. Because it is dangerous and invasive to access patient brain and nerve samples directly, this project converted patient blood or skin samples into neurons which were then studied. This thesis aimed to achieve three broad objectives. The first was to characterize an excitatory neuron subtype from three different families to identify changes in shape, connectivity, and function. The second objective involved identifying how these neurons might express different gene profiles, and what this means for the mechanisms involved in disease development. The third objective was to investigate a possible mechanism at the molecular level, which might offer insights into future therapies. The totality of the work in this thesis provides new insights into the cellular and molecular bases for disease in the 15q13.3 microdeletion disorder and offers future perspectives on how this disorder and others like it might be investigated and treated in the future. / Dissertation / Doctor of Philosophy (PhD) / The 15q13.3 microdeletion disorder is a clinically delineated set of neuropsychiatric phenotypes associated with the loss of genetic material from the 15q13.3 BP4-5 locus. To functionally characterize cellular features of the 15q13.3 microdeletion disorder and identify genetic and molecular elements contributing to disease pathophysiology, we assayed excitatory glutamatergic pyramidal neurons derived by the expression of the neurogenin-2 transcription factor in induced pluripotent stem cells (iPSCs) of 15q13.3 microdeletion patients and family members. Day 28 (DIV28) neurons were first functionally and morphologically assayed, revealing family-specific changes to population-level activity, individual action potential changes, and dendritic complexity with axon projection being decreased in all families. We followed up these experiments with RNA sequencing at an earlier timepoint (DIV14), identifying early changes in gene expression and pathway enrichment which varied appreciably between two families, potentially due to underlying clinical variations. Finally, we treated a proband and control with a potent, selective GSK3 inhibitor and found that the proband was comparatively insensitive to its effects on action potential properties. Taken together, these findings underscore the multi-layered heterogeneity in this disorder at the clinical, cellular and molecular level, and offer new insights into disease pathobiology and potential mechanisms.

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Neurogenin 2 (Ngn2) is a basic Helix-Loop-Helix (bHLH) transcription factor regulating differentiation and cell cycle exit in the developing brain. By transcriptional upregulation of a cascade of other bHLH factors, neural progenitor cells exit the cell cycle and differentiate towards a neuronal fate. Xenopus laevis Ngn2 (xNgn2) is a short-lived protein, targeted for degradation by the 26S proteasome. I have investigated the stability of Ngn2 mediated by post-translational modifications and structural disorder. Firstly I will describe work focused on ubiquitylation of xNgn2, targeting it for proteasomal degradation. xNgn2 is ubiquitylated on lysines, the recognized site of modification. I will discuss the role of lysines in ubiquitylation and stability of xNgn2. In addition to canonical ubiquitylation on lysines, I describe ubiquitylation of xNgn2 on non-canonical sites, namely its amino-terminal amino group, and cysteine, serine and threonine residues. I show that the ubiquitylation of cysteines in particular exhibits cell cycle dependence and is also observed in mammalian cell lines, resulting in cell cycle-dependent regulation of stability. I will then discuss whether phosphorylation, a regulator of xNgn2 activity, also affects xNgn2 stability. I will provide evidence of cell cycle-dependent phosphorylation of cyclin dependent kinase (cdk) consensus sites affecting the stability of xNgn2. Finally I describe studies on the folding properties of Ngn2 to assess their role in protein stability. xNgn2 associates with DNA and its heterodimeric binding partner xE12 and may interact directly with the cyclin-dependent kinase inhibitor Xic1. I will discuss the role of these interaction partners in xNgn2 stability. xNeuroD, a downstream target of xNgn2, is a related bHLH transcription factor which is stable. Here I describe domain swapping experiments between these two proteins highlighting regions conferring instability on the chimeric protein. Finally I will provide nuclear magnetic resonance (NMR) data looking at the effect of phosphorylation on protein structure in mouse Ngn2 (mNgn2).

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13141号 / 論医博第2141号 / 新制||医||1026(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 川口 義弥, 教授 横出 正之, 教授 萩原 正敏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM