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.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/30493 |
| Date | January 2023 |
| Creators | CHALIL, LEON |
| Contributors | SINGH, KARUN K, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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