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Autism spectrum disorder (ASD) encompasses a group of heterogeneous disorders that affect approximately 1% of children worldwide. ASD is characterized by two core symptoms, the first being deficits in social communication and interaction, and the second being restrictive and repetitive behaviours. Although environmental and genetic factors are known to contribute to the development of ASD, the etiology remains unknown. Genetic sequencing studies have implicated over 1000 genes with risk variants that are ASD-associated. Recent sequencing studies have highlighted that SCN2A, a gene that encodes the Voltage-Gated Sodium Channel Type II Alpha Subunit habours a large proportion of genetic risk variants for ASD. An emphasis was put on this gene because many of the top genes regulate transcription and cytoskeletal dynamics and not sodium flux aiding in regulating neuron excitability. Initial investigations of complete loss of Scn2a in mice led to perinatal lethality where heterozygous loss exhibited many behavioural phenotypes associated with ASD. Through our collaboration with Dr. Stephen Scherer (Hospital for Sick Children, Toronto) we identified two de novo truncating point variants in SCN2A. In our study, we focused on using human iPSC-derived neurons for disease modelling. We found these two variants caused a reduction in synapses suggesting that neuronal communication may be altered. Furthermore, electrophysiological characterization of the neurons harbouring the differing SCN2A variants showcased that loss-of-function (LoF) variants can produce differential phenotypes based on their location. Beyond the initial ion channel characterization, we wanted to probe whether cellular pathways were altered directly or indirectly by atypical neuronal activity. Proteomics of neurons expressing the more severe variant, p.R607*, found differentially expressed proteins (DEP)s that were upregulated and downregulated. Moreover, these DEPs were enriched and clustered into cellular pathways that were altered, with one of these clusters representing mitochondrial function. We functionally validated these findings in the same neurons and found corroboration between the molecular and cellular data of impaired mitochondria. Lastly, we used Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing to generate an isogenic model to validate our findings of the less severe p.G1744* variant. Together, this will aid in the discovery of new variant categorizations and targeted treatments for rescues of atypical neural connectivity or pathways that are altered downstream. / Thesis / Doctor of Philosophy (PhD)

<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>