Intellectual disability (ID) is categorised by a significant reduction in cognitive function and adaptive abilities that begin in childhood. ID is part of a heterogeneous group of neurodevelopmental conditions associated with impairment in developmental domains and a cause of particularly adverse socioeconomic impact worldwide. There have been many recent advances in identifying causative genetic mutations in previously unexplained ID cases. With these advances comes an increasing demand for understanding mechanisms underpinning these pathogenic pathways. In this PhD thesis, I have studied rare monogenic novel neurodevelopmental disorders associated with ID. The objective of the thesis was to model a subset of mutations associated with novel neurodevelopmental disorders in mice to demonstrate a causal link between mutation and phenotype and to further understand the mechanisms by which these mutations result in human neurodevelopmental disorders. In order to achieve this, I adopted a multi-phase approach. Firstly, I designed a phenotyping platform, by combining behavioural and cognitive tests with morphometric brain analysis and genome-wide transcriptional analysis. I then used this approach to study KPTN-related syndrome, a novel developmental disorder that to date has not been characterised in mice, successfully recapitulating the main phenotypes described in the patients. Moreover, I gained further insight into the underlying pathogenic mechanisms associated with the disorder, opening the possibility of a therapy that could treat some aspects of cognitive and morphological impairments identified in the patients with KPTN-related syndrome. Lastly, I determined whether such an approach could be scaled-up to study multiple novel neurodevelopmental disorders, each with a mutation associated with a haploinsufficient novel neurodevelopmental disorder. I identified specific phenotypes for each of the four mouse lines under investigation, providing a platform for comparison between several developmental disorders. These refinements contributed to a larger five-year project starting at the Sanger Institute, aimed at characterising a wider diversity of human neurodevelopmental disorders.

The recent history of autoimmune neurology is marked by the discovery of many central nervous system (CNS) antibody-mediated diseases. These disorders are caused by antibodies that target important proteins expressed in the neuronal surface, which are believed to be directly pathogenic. These antibodies are immunoglobulin G (IgG) isotype and, as such, have the potential to cross the placenta during gestation. Foetal exposure to CNS-targeting antibodies could alter developing neuronal circuits, leading to disease. However, the consequences of exposure to these antibodies during neurodevelopment has hardly been considered. To study the relationship between maternal antibodies towards neuronal surface proteins and neurodevelopmental disorders in the foetus a dual approach was undertaken. First, pregnancy serum samples from mothers of children later diagnosed with a neurodevelopmental disorder and from mothers of children with typical development were screened for the presence of neuronal surface antibodies. Next, the effects of pathogenic neuronal surface antibodies in the offspring were assessed in a maternal-to-foetal transfer mouse model. Antibodies to neuronal surface proteins in the gestational serum, particularly CASPR2 antibodies, were found to associate with an increased risk of mental retardation and disorders of psychological development in the progeny. The animal model showed that mice exposed in utero to CASPR2 antibodies have long term behavioural sequelae and histological findings suggestive of abnormalities in brain development. These findings support a model in which maternal antibodies towards foetal neuronal proteins cause long-term behavioural deficits and permanent abnormalities at the cellular and synaptic level in a subset of children with neurodevelopmental disorders.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22690号 / 医博第4634号 / 新制||医||1045(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 古川 壽亮, 教授 滝田 順子, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Deformed epidermal autoregulatory factor 1 (DEAF1) encodes a transcription factor essential in early embryonic and neuronal development. In humans, mutations in the DNA binding domain of DEAF1 cause intellectual disability together with clinical characteristics collectively termed DEAF1-associated neurodevelopmental disorders (DAND). The objective of this study is to 1) assess the pathogenicity of newly identified variants using established functional assays, and 2) confirm and map the interaction domain of DEAF1 with HDAC1 and evaluate the importance of DEAF1-HDAC1 interaction on DEAF1-mediated transcriptional repression. Exome sequencing analysis identified six de novo DEAF1 mutations (p.D200Y, p.S201R, p.K250E, p.D251N, p.K253E, and p.F297S). Promoter activity experiments indicate DEAF1 transcriptional repression activity was altered by p.K250E, p.K253E, and p.F297S. Transcriptional activation activity was altered by p.K250E, p.K253E, p.F297S, and p.D251N. Combined with clinical phenotype of the patients, this work establishes the pathogenicity of new DEAF1 variants. Previous studies identified a potential protein interaction between DEAF1 and several proteins of the nucleosome remodeling and deacetylating (NuRD) complex including Histone Deacetylase 1 (HDAC1), Retinoblastoma Binding Protein 4 (RBBP4), Methyl CpG Binding Domain Protein 3 (MBD3). GST pull-down and co-immunoprecipitation (CoIP) assays confirmed and mapped the interaction with HDAC1 between amino acids 113 – 176 of DEAF1. To determine whether DEAF1-mediated repression requires HDAC1 activity, HEK293t wild type or CRISPR/Cas9-mediated DEAF1 knockout cells were treated with the HDAC inhibitor Trichostatin A (TSA). Interestingly, this study demonstrates that the requirement of HDAC1 activity on DEAF1-mediated transcriptional repression activity is target gene specific and expands our understanding of DEAF1 mediated transcriptional repression.

Thesis advisor: Laura Anne Lowery / Thesis advisor: Sarah McMenamin / Development of the central nervous system (CNS) is a complex process that requires the proper function of many genes in order for neurons to proliferate and divide, differentiate, and subsequently migrate long distances to form connections with one another. Abnormalities in any one of these cellular processes can lead to detrimental developmental defects. Growing evidence suggests that genetic mutations caused by rare copy number variants (CNVs) are associated with neurodevelopmental disorders including intellectual disabilities (ID), Autism spectrum disorder (ASD), and schizophrenia. Additionally, these pathogenic CNVs are characterized by extensive phenotypic heterogeneity, as affected individuals often present with microcephaly, craniofacial and heart defects, growth retardation, and seizures. Despite their strong association as risk factors towards neurodevelopmental disorders, the developmental role of individual CNV-affected genes and disrupted cellular mechanisms underlying these mutations remains poorly understood. Moreover, it is unclear as to how the affected genes both individually and combinatorially contribute to the phenotypes associated with pathogenic CNVs. Thus, in this thesis, we explore the functional basis of phenotypic variability of pathogenic CNVs linked to neurodevelopmental disorders. In particular, we focus on the 3q29 deletion and 16p12.1 deletion, to provide insight towards the convergent cellular, molecular, and developmental mechanisms associated with decreased dosage of the affected gene homologs using two complementary model systems, Xenopus laevis and Drosophila melanogaster. First, we examine the role of individual homologs of several CNV-affected genes at chromosome 3q29 and their interactions towards cellular processes underlying the deletion. We find that multiple 3q29-affected genes, including NCBP2, DLG1, FBXO45, PIGZ, and BDH1, contribute to disruptions in apoptosis and cell cycle pathways, leading to neuronal and developmental defects. We then expand further upon this work by discerning the individual contribution of four CNV-affected genes at chromosome 16p12.1, POLR3E, MOSMO, UQCRC2, and CDR2, towards neurodevelopment and craniofacial morphogenesis. We demonstrate that several of these genes affect multiple phenotypic domains during neurodevelopment leading to brain size alterations, abnormal neuronal morphology, and cellular proliferation defects. We then explore their functions during vertebrate craniofacial morphogenesis and demonstrate that some 16p12.1-affected genes are enriched in migratory neural crest, and contribute to early craniofacial patterning and formation of cartilaginous tissue structures. Together, these data are the first to suggest that signature neurodevelopmental phenotypes demonstrated in the 3q29 deletion and 16p12.1 deletion may stem from convergent cellular mechanisms including aberrations in neuronal proliferation, apoptosis and cell cycle regulation, and neural crest cell development. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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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.

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)

The factors contributing to the severity of the neurodevelopmental disorders autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) are largely unknown. Previous studies have indicated immune abnormalities in these disorders, such as increased inflammation and altered immune cell numbers. We, in collaboration with the Province of Ontario Neurodevelopmental Disorder (POND) Network, analyzed markers of intestinal permeability and inflammation in children diagnosed with ASD or ADHD, as well as typically developing controls. Plasma from these participants was used to investigate levels of soluble inflammation, denoted by circulating acute phase proteins, as well as circulating levels of markers of intestinal epithelial damage and bacterial translocation. Peripheral blood mononuclear cells were isolated from these participants and used to construct an immunophenotype of ASD and ADHD, focusing on monocytes and monocyte activation and maturation. These data were then compared with scores of behaviour severity to identify associations between inflammation and behaviour in these disorders. We identified increased soluble inflammation in ASD, indicated by increased circulating C-reactive protein. We associated this inflammation with intestinal permeability, indicated by increased circulating LPS. Classical monocyte frequency was significantly lower in ASD and these monocytes displayed an altered migratory phenotype, indicated by a reduction in CCR2 expression. Furthermore, we have identified potential maladaptive monocyte responses to soluble inflammation in both ASD and ADHD, with altered monocyte phenotypes in response to inflammatory mediators compared to typically developing controls. Finally, we identified that changes in monocyte phenotype are associated with more severe behaviours in both ASD and ADHD. These findings imply that inflammation and immune abnormalities contribute to the severity of neurodevelopmental disorders. / Thesis / Master of Science (MSc)

Assessment of executive functioning in neurodevelopmental disorders (e.g., autism) is a crucial aspect of neuropsychological evaluations. The executive functions are accomplished by the supervisory attentional system (SAS) and include such processes as inhibition, switching, and planning. Autism spectrum disorder (ASD) tends to present similarly to other neurodevelopmental disorders (e.g., ADHD). For example, ASD and ADHD may share similar etiological underpinnings in the frontostriatal system of the frontal lobe. Research on executive functioning in ASD has been mixed, thus the precise nature of executive functioning deficits in ASD remains equivocal. In recent years, simulation technologies have emerged as an avenue to assess neurocognitive functioning in individuals with neurodevelopmental disorders impacting frontostriatal function. Simulation technology enables neuropsychologists to assess neurocognitive functioning within a testing environment that replicates environments in which the subject is likely to be in everyday life, as well as present controlled, real-world distractions, which may be better able to tap “hot” executive functions. A Virtual Classroom Continuous Performance Test (CPT) has been used successfully to assess attention in individuals with neurodevelopmental disorders impacting frontostriatal function. The current study aimed to investigate executive functioning in individuals with high functioning ASD using a new construct driven Stroop assessment embedded into the Virtual Classroom. Group differences were found in the Virtual Classroom with distractions condition, indicating individuals with ASD may be more vulnerable to external interference control than neurotypical individuals.