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.

Neurodevelopmental disorders (NDDs) are a heterogeneous set of disorders that are characterized by early disruptions to brain development and include autism spectrum disorder (ASD), attention deficit/hyperactivity disorder (ADHD), developmental delay (DD), intellectual disability (ID), epilepsy and schizophrenia (SZ). Although thousands of genetic risk variants have been identified, there is a lack of understanding of how they impact cellular and molecular mechanisms that underlie the clinical presentation and heterogeneity of NDDs. To investigate this, we used a combination of cellular, molecular, bioinformatic and omics methods to study NDD-associated molecular pathways in distinct neuronal populations. First, we studied the interaction between the high-confidence SZ risk genes DISC1 and NRG1-ErbB4 in cortical inhibitory neurons and found that NRG1-ErbB4 functions through DISC1 to regulate dendrite growth and excitatory synapses onto inhibitory neurons. Next, we studied the 15q13.3 microdeletion, a recurrent copy number variation (CNV) that is associated with multiple NDDs. Using a heterozygous mouse model [Df(h15q13)/+] and human sequencing data we identified OTUD7A (encoding a deubiquitinase) as an important gene driving neurodevelopmental phenotypes in the 15q13.3 microdeletion syndrome. Due to the paucity of literature on the function of OTUD7A in the brain, we used a proximity-labeling approach (BioID2) to elucidate the OTUD7A protein interaction network (PIN) in cortical neurons, and to examine how patient mutations affect the OTUD7A PIN. We found that the OTUD7A PIN was enriched for postsynaptic and axon initial segment proteins, and that distinct patient mutations have shared and distinct effects on the OTUD7A PIN. Further, we identified the interaction of OTUD7A with a high-confidence bipolar risk gene ANK3, which encodes AnkyrinG. We identified decreased levels of AnkyrinG in Df(h15q13)/+ neurons, and synaptic phenotypes were rescued by increasing AnkyrinG levels or targeting the Wnt pathway. Future investigation should include examination of the role of OTUD7A deubiquitinase activity in neural development. / Dissertation / Doctor of Philosophy (PhD) / Neurodevelopmental disorders result from disruptions to early brain development and include autism spectrum disorder (ASD), developmental delay (DD), epilepsy, and schizophrenia (SZ). These disorders affect more than 3% of children worldwide and can have a significant impact on an individual’s quality of life, including an increased risk of death in some cases. There is currently a lack of understanding of how these disorders develop and how to effectively treat them. Neurodevelopmental disorders are thought to arise from alterations in the connections between brain cells (neurons) and one of the major risk factors for these disorders is having certain variations in regions of the genome (DNA sequences), with more than 1000 of these risk variants having been identified so far. In this thesis, we analyzed how genetic risk factors interact in neurons to regulate neural connectivity. We discovered that risk variants found in individuals with different disorders actually work together to regulate similar processes important for neural connectivity, which suggests that distinct disorders may share a common underlying cause. Additionally, we established the importance of a new ASD risk gene and discovered that it interacts with other known risk genes to regulate neural connectivity. This thesis provides new insights into the processes in the brain that lead to neurodevelopmental disorders and has implications for future development of effective therapies for individuals affected by these disorders.