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Bipolar disorder is a heritable psychiatric disorder, and several of the genes associated with bipolar disorder and related psychotic disorders are involved in the development and maintenance of white matter in the brain. Patients with bipolar disorder have an increased incidence of white matter hyper-intensities, and quantitative brain imaging studies collectively indicate subtle decreases in white matter density and integrity in bipolar patients. This suggests that genetic vulnerability to psychosis may manifest itself as reduced white matter integrity, and that white matter integrity is an endophenotype of bipolar disorder. This thesis comprises a series of studies designed to test the role of white matter in genetic risk to bipolar disorder by analysis of diffusion tensor imaging (DTI) data in the Bipolar Family Study. Various established analysis methods for DTI, including whole-brain voxel-based statistics, tract-based spatial statistics (TBSS) and probabilistic neighbourhood tractography, were applied with fractional anisotropy (FA) as the outcome measure. Widespread but subtle white matter integrity reductions were found in unaffected relatives of patients with bipolar disorder, whilst more localised reductions were associated with cyclothymic temperament. Next, the relation of white matter to four of the most prominent psychosis candidate genes, NRG1, ErbB4, DISC1 and ZNF804A, was investigated. A core haplotype in NRG1, and three of the four key single nucleotide polymorphisms (SNPs) within it, showed an association with FA in the anterior thalamic radiations and the uncinate fasciculi. For the three SNPs considered in ErbB4, results were inconclusive, but this was consistent with the background literature. Most notable however, was a clear association of a non-synonymous DISC1 SNP, Ser704Cys, with FA extending over most of the white matter in the TBSS and voxel-based analyses. Finally, FA was not associated with a genome-wide supported risk SNP in ZNF804A, a finding which could not be attributed to a lack of statistical power, and which contradicts a strong, but previously untested hypothesis. Whilst the above results need corroboration from independent studies, other studies are needed to address the cellular and molecular basis of these findings. Overall, this work provides strong support for the role of white matter integrity in genetic vulnerability to bipolar disorder and the wider psychosis spectrum and encourages its future use as an endophenotype.

Control of metabolic homeostasis is governed by the melanocortin system in the hypothalamus which includes the paraventricular nucleus of the hypothalamus (PVH). We previously identified that ErbB4 which is a risk gene for the development of obesity is expressed in the PVH yet the precise function of ErbB4 in the PVH remained unexplored. First, we characterized the function of ErbB4 in the PVH and its relationship to energy metabolism by specifically deleting ErbB4 in the PVH. Deletion of ErbB4 in the PVH increases mouse body weight and decreases energy expenditure in a food intake independent manner. Second, we identified that ErbB4 in the PVH is expressed in OXT neurons and deletion of ErbB4 in OXT cells decreases energy expenditure. Finally, we provide circuit level evidence that ErbB4-positive neurons of the PVH project to brainstem nuclei necessary for energy expenditure, the nucleus of the solitary tract (NTS).

The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease. KEY MESSAGES: Adenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting. An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons. A hybrid promoter allows preferential expression of transgenes in GABAergic neurons. Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.

ErbB4 is a receptor tyrosine kinase (RTK) of the epidermal growth factor receptor (EGFR) family that regulates numerous biological processes, from development to physiological function and degeneration. ErbB4 is important in many aspects of neurodevelopment including astrogenesis, neural migration, dendrite formation and synapse maturation. ErbB4 is also necessary for normal development of the heart, mammary glands, immune cells, and lungs. Furthermore, ErbB4 plays important roles in brain function and synaptic transmission in structures such as the hippocampus, amygdala, nucleus accumbens and substantia nigra. ErbB4, and its cognate ligand neuregulin 1 (NRG1) are both schizophrenia susceptibility genes. Mice lacking ErbB4 expression during development or in adulthood display ‘schizophrenia-like behavior,’ indicating that ErbB4 plays important roles in both development and maintenance of normal brain function. ErbB4, like all RTKs, signals through canonical signaling via phosphorylation-mediated signaling cascades. ErbB4 can also signal through a non-canonical mechanism whereby ErbB4 is cleaved and releases an intracellular domain (E4ICD) that shuttles to the nucleus and regulates gene transcription. Non-canonical signaling by ErbB4 requires an isoform-specific inclusion of a cleavage site in the extracellular juxtamembrane domain (EJD). Alternative splicing of the ERBB4 can generate two different ErbB4 isoforms: ErbB4-JMb, which is uncleavable, and ErbB4-JMa, which includes a cleavage site required to signal through the non-canonical mechanism. Non-canonical signaling by ErbB4-JMa has been implicated in the regulation of cortical, cardiac, mammary gland, pulmonary and immune cell development. While ErbB4-JMa was the first RTK identified to be capable of non-canonical signaling, it is now known that most RTKs can be cleaved and utilize direct ICD signaling. However, most studies of this non-canonical signaling by RTKs, including ErbB4, have been performed in vitro. Loss-of-function mouse models, in which non-canonical RTK signaling is abrogated, have not yet been developed to study non-canonical RTK signaling in vivo. The central aim of this dissertation is to create a novel mutant mouse in which non-canonical signaling by ErbB4-JMa is abolished and to phenotypically assess the roles of ErbB4 signaling in the intact organism. To accomplish this, I used germline CRISPR/Cas9 gene editing of mouse ERBB4 exon 16a to target ErbB4-JMa and create an isoform-specific knock out mouse, designated ErbB4-JMa-/-. In this mutant mouse expression of ErbB4-JMa, and hence non-canonical E4ICD signaling, is eliminated, while ErbB4-JMb canonical signaling is intact. I first confirmed that formation of E4ICD was abolished due to the absence of ErbB4-JMa transcription. I then used the ErbB4-JMa-/- mouse to showed that non-canonical signaling by ErbB4-JMa regulates the expression of GFAP during cortical development, consistent with previous results. Next, I used an unbiased gene-discovery approach to identify genes that are regulated by ErbB4 during cortical development. I found that the regulation of two genes, CRYM and DBi, is dependent on ErbB4-JMa. Finally, I used the ErbB4-JMa-/- mouse to assess whether non-canonical ErbB4 signaling plays a role in ErbB4-related developmental phenotypes. I found that ErbB4-JMa signaling is not required for the development of the heart, mammary glands, sensory ganglia or cortical interneurons, but may regulate the development of immune cells. Together these findings show that the ErbB4-JMa-/- mouse is a robust tool to study the role of non-canonical ErbB4-JMa signaling in vivo.

Poly (ADP-ribose) polymerase 1 (PARP1) is a ubiquitously expressed enzyme that post-translationally modifies proteins via poly (ADP-ribosylation) (PARylation). PARP1 serves various functions, including DNA damage repair, regulation of cell death pathways, chromatin modification, RNA processing, and transcriptional regulation. Accordingly, mutations in Parp1 or Adprhl2 (encoding the protein ADP-ribosylhydrolase 3, which removes PAR polymers) cause intellectual disability, ataxia, episodic psychosis, neurodegeneration, and developmental delay. Altered PARP1 expression is also associated with numerous neurodegenerative and neuroimmune disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, rheumatoid arthritis, major depressive disorder, and epilepsy. Despite ubiquitous expression and an apparent connection with brain disorders, PARP1's role in neurodevelopment has not been widely studied. Our lab has recently uncovered a novel interaction between PARP1 and the receptor tyrosine kinase ErbB4, which binds its ligand NRG1 to mediate numerous functions during neurodevelopment, including radial migration of excitatory neurons, tangential migration of inhibitory neurons, synaptogenesis, and differentiation. Additionally, ErbB4 has multiple splice forms that confer different signaling modalities. Specifically, the ErbB4-juxtamembrane (JM)-a isoform is cleavable via the enzymes tumor necrosis factor-alpha (TACE) and presenilin/gamma-secretase. Upon NRG1 binding and ErbB4-JMa cleavage, the ErbB4 intracellular domain (E4ICD) is released, which regulates transcription through direct promoter binding. Previous findings have shown that E4ICD complexes with co-factors to repress gliogenesis during early development. Due to PARP1's prominent roles in chromatin modification and transcriptional control, this begs the question as to whether PARP1 is likewise regulating glial gene expression via E4ICD. The aims of this dissertation are two-fold: 1) investigate the role of PARP1 in regulating astrocytic gene expression via E4ICD and 2) further characterize the effect of PARP1 loss on brain development. To explore the role of PARP1-E4ICD in the regulation of astrogenesis, I utilized mouse primary embryonic neural precursor cell (NPC) cultures and transgenic mice with a germline knockout of PARP1, ErbB4, or ErbB4-JMa. I found that NRG1-mediated repression of GFAP expression upon FGF removal from NPC cultures was dependent upon the presence of PARP1, ErbB4, and ErbB4-JMa. Additionally, I showed that PARP1 KO and ErbB4 KO mice overexpress GFAP at birth, indicating the importance of both proteins in vivo. To investigate the effect of PARP1 loss on neurodevelopment more broadly, I analyzed the brain and cortical size of PARP1 KO mice at birth, finding a reduction in brain weight relative to body size, which is associated with a thinner cortex and a reduced cortical surface area. Furthermore, I discovered that PARP1 loss alters early-born neuron migration and increases the density of deeper-layer neurons. To investigate changes in gene expression associated with these findings, I performed RNA-sequencing of the embryonic PARP1 KO cortex. I found that PARP1 loss increases the expression of genes involved in neuronal migration and adhesion, including Reln, which encodes the glycoprotein Reelin. Accordingly, my findings indicate that PARP1 loss increases the abundance of Reelin-expressing cells in the developing (E15.5) and adolescent (P5) mouse brain. I further demonstrated that PARP1 loss, inhibition, or acute knockdown increases Cajal-Retzius cell abundance in vitro, suggesting PARP1 regulates Cajal-Retzius cell development via a cell-autonomous mechanism. Finally, atomic force microscopy showed that NPCs isolated from the PARP1 KO cortex adhere more strongly to the cell adhesion molecule N-cadherin, likely due to excess Reelin. Overall, these findings demonstrate that PARP1 regulates astrogenesis, Cajal-Retzius cell development, and cell adhesion in the developing brain.