Morphological analysis of the Axon Initial Segment in novel monogenic rat models of Autism Spectrum Disorder and Intellectual Disability

Monogenic mutations in synaptic proteins are known to cause severe and low functioning cases of autism spectrum disorder and intellectual disability (ASD/ID) (Wright et al., 2018). One of the most prevalent monogenic causes of moderate to severe non-syndromic and syndromic ID with a high co-morbidity of seizures and altered sensory processing is SYNGAP1 haploinsufficiency (Hamdan et al., 2009; Hoischen et al., 2014). Resulting from de novo truncating or frameshift mutations in the SYNGAP1 gene on human chromosome 6, SYNGAP1 haploinsufficiency leads to a 50% reduction of the encoded synaptic GTP-ase activating protein (SynGAP) (Hamdan et al., 2009). Pre-clinical models of Syngap1 haploinsufficiency have provided an invaluable tool to understand the underlying pathophysiology resulting from a reduction of SynGAP. The heterozygous mouse model of Syngap1 haploinsufficiency (Syngap1+/-) demonstrates a range of cellular, physiological and behavioural abnormalities from early development to adulthood (Chen et al., 1998; Kim et al., 1998; Komiyama et al., 2002; Rumbaugh et al., 2006; Muhia et al., 2010). Key among these phenotypes is brain region and cell-type specific hyper- and hypo-excitability, causing an excitation/inhibition imbalance which manifests as disrupted circuit function (Clement et al., 2012, 2013; Ozkan et al., 2014; Aceti et al., 2015; Berryer et al., 2016; Katsanevaki, 2017). Neuronal excitability is defined as the probability of action potential (AP) generation in response to a given current. The site of AP generation is a specialised cellular compartment located between the somatodendritic and axonal compartments of a neuron, called the axon initial segment (AIS). The AIS is a dynamic structure, shown to alter its length and position along the axon in response to altered physiological and pathological conditions (for review see Rasband (2010)). Prolonged increase in neuronal activity causes shortening and a distal shift of the AIS (Evans et al., 2015; Grubb and Burrone, 2010) while extended periods of decreased neuronal activity result in lengthening and proximal shift of the AIS (Kuba et al., 2006; Kuba, 2012). The functional consequence of this morphological plasticity is altered intrinsic excitability such that neurons with distally located, shorter AISs exhibit increased AP threshold with subsequent production of fewer APs in response to a given current while neurons with longer, proximal AISs exhibit decreased AP threshold and increased frequency of AP firing (Kuba et al., 2006; Grubb and Burrone, 2010; Kuba et al., 2010; Kuba, 2012; Evans et al., 2015). Given this ability of the AIS to alter cellular excitability and the evidence of altered excitability in pre-clinical models of SYNGAP1 haploinsufficiency, this thesis tests the hypothesis that alterations in AIS length will underlie some the altered cellular excitability observed upon reduction of SynGAP. In order to test this hypothesis, first a characterisation and comparison of AIS lengths between Syngap1+/- and control juvenile rats was undertaken across six brain regions previously known to exhibit altered cellular excitability or be involved in behaviours that are altered in Syngap1 haploinsufficiency. These brain regions include the prelimbic medial prefrontal cortex (mPFC-PL), somatosensory cortex – barrel fields (S1BF), Cornu Ammonis 1 and 3 (CA1, CA3) sub-fields of the dorsal hippocampus, the lateral (LA) and basal (BA) nuclei of the amygdala and the primary visual cortex 1 (V1). Juvenile animals were chosen to reflect the developmental underpinnings of this disorder as Syngap1+/- mice exhibit alterations in synaptic physiology at this age (Barnes et al., 2015) and it coincides with the developmental critical period for some of the brain regions analysed, including the V1 and BLA. Further, the AIS has been shown undergo developmentally regulated alternations in its morphology during this time-period. Statistical analysis of results and post-hoc interactions of brain-region and genotype were analysed using a linear mixed modelling (LMM) approach to account for the non-normal distribution of the data-sets. Subsequently, the effects of Syngap1 haploinsufficiency on morphological plasticity of the AIS was tested. This included analysis and comparison of genotype specific changes in AIS lengths over development and following a cued fear conditioning associative-learning paradigm. Lastly, as cell-type specific differences in cellular excitability have been noted in the Syngap1+/- mouse, AIS lengths were compared across genotypes in cells of the mPFC and BLA with specific projection targets. In addition to studying the difference of AIS length in rat models of SYNGAP1 haploinsufficiency, a similar study was undertaken in this thesis in rat models of Fragile-X syndrome, Cowden’s syndrome, NLGN3-associated non-syndromic ID, NRXN1 and CNTNAP2 associated ASD/ID. This work was undertaken to study the hypothesis that diverse genetic causes of ASD/ID converge onto common cellular pathways, and, as preclinical models of these disorders all exhibit evidence of altered cellular excitability, alterations in AIS length might underlie some of these changes. The work documented in this thesis provides the first characterisation of AIS lengths across pre-clinical models of Syngap1 haploinsufficiency as well as across multiple monogenic rat models of ASD/ID. The results indicate that in almost all the brain-regions and models studied, AIS morphology remained unaltered between heterozygous/homozygous animals and their wild-type littermate controls, causing a rejection of the hypothesis that alterations in AIS morphology is a common cellular pathway underlying changes in cellular excitability across these models. However, provided here is the first evidence of AIS morphology in the mPFC-PL being differentially altered in Syngap1 haploinsufficiency compared to control animals when subjected to a cued fear conditioning paradigm as well as alterations in AIS morphology in the mPFC, S1 and BA in a rat model of Fragile-X syndrome

A sex difference in the response of the rodent postsynaptic density to synGAP haploinsufficiency

SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-95. We previously reported that heterozygous deletion of Syngap1 in mice is correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was increased in young Syngap1+/- mice. Here we show that only females and not males show a highly significant correlation between an increase in TARP and a decrease in synGAP in the PSDs of Syngap1+/- rodents. The data reveal a sex difference in the adaptation of the PSD scaffold to synGAP haploinsufficiency

A sex difference in the composition of the rodent postsynaptic density

SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-95. We previously reported that heterozygous deletion of synGAP in mice is correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was increased in young synGAP+/- mice. Here we show that a highly significant increase in TARP in the PSDs of young synGAP+/- rodents is present only in females and not in males. The data reveal a sex difference in the adaptation of the PSD scaffold to synGAP heterozygosity

A sex difference in the composition of the rodent postsynaptic density

SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-95. We previously reported that heterozygous deletion of synGAP in mice is correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was increased in young synGAP+/- mice. Here we show that a highly significant increase in TARP in the PSDs of young synGAP+/- rodents is present only in females and not in males. The data reveal a sex difference in the adaptation of the PSD scaffold to synGAP heterozygosity

Common variants in CLDN2 and MORC4 genes confer disease susceptibility in patients with chronic pancreatitis

A recent Genome-wide Association Study (GWAS) identified association with variants in X-linked CLDN2 and MORC4 and PRSS1-PRSS2 loci with Chronic Pancreatitis (CP) in North American patients of European ancestry. We selected 9 variants from the reported GWAS and replicated the association with CP in Indian patients by genotyping 1807 unrelated Indians of Indo-European ethnicity, including 519 patients with CP and 1288 controls. The etiology of CP was idiopathic in 83.62% and alcoholic in 16.38% of 519 patients. Our study confirmed a significant association of 2 variants in CLDN2 gene (rs4409525—OR 1.71, P = 1.38 x 10-09; rs12008279—OR 1.56, P = 1.53 x 10-04) and 2 variants in MORC4 gene (rs12688220—OR 1.72, P = 9.20 x 10-09; rs6622126—OR 1.75, P = 4.04x10-05) in Indian patients with CP. We also found significant association at PRSS1-PRSS2 locus (OR 0.60; P = 9.92 x 10-06) and SAMD12-TNFRSF11B (OR 0.49, 95% CI [0.31–0.78], P = 0.0027). A variant in the gene MORC4 (rs12688220) showed significant interaction with alcohol (OR for homozygous and heterozygous risk allele -14.62 and 1.51 respectively, P = 0.0068) suggesting gene-environment interaction. A combined analysis of the genes CLDN2 and MORC4 based on an effective risk allele score revealed a higher percentage of individuals homozygous for the risk allele in CP cases with 5.09 fold enhanced risk in individuals with 7 or more effective risk alleles compared with individuals with 3 or less risk alleles (P = 1.88 x 10-14). Genetic variants in CLDN2 and MORC4 genes were associated with CP in Indian patients

Identification of Gli3 target genes during corpus callosum development

The corpus callosum (CC) is the largest white matter fibre tract in the mammalian telencephalon responsible for the transfer and coordination of information between the two cerebral hemispheres. The development of this structure is tightly regulated by a complex interactions between signalling molecules and transcription factors. Gli3 is a zinc finger transcription factor best known for its effects in mediating sonic hedgehog (Shh) signalling. It has previously been shown that conditional inactivation of Gli3 from cortical progenitor cells leads to patterning defects resulting in an agenesis of corpus callosum (AgCC) phenotype. However, the exact molecular mechanisms by which Gli3 controls this patterning In this project I take two approaches to identify downstream targets of Gli3 through which it controls CC development using the Gli3 conditional knockout mouse Emx1Cre;Gli3fl/fl. First, I look at the interaction between Gli3 and the Hippo pathway, with the hypothesis that Gli3 is an upstream regulator of the pathway. In order to do this I analyse the Hippo pathway target Yap and compare the protein levels of its phosphorylated and non-phosphorylated state using quantitative western blot analyses at E14.5 and E16.5. However, no significant difference is observed in the protein levels between mutant and control embryos at either age. The second approach uses data from a previously performed RNA-Seq experiments to identify potential directly regulated targets of Gli3. In situ hybridisation was used to analyse the expression profiles of eight genes identified from the RNA-seq. From this expression analysis two genes were chosen for further study: Foxb1 and Pappa. Following the expression analysis, promoter sequences and predicted enhancer regions for Foxb1 and Pappa were subjected to bioinformatics analysis to identify potential Gli3 binding sites. This analysis revealed two enhancer regions with evolutionarily conserved Gli3 candidate binding sites for Pappa while one conserved binding site was identified in the Foxb1 promoter. The in vitro and in vivo functionality of these sites were then tested using a protein-DNA binding assay and in utero electroporation respectively. These analyses showed that one of the putative Pappa enhancers is significantly repressed by Gli3 in the septum

Liver X Receptor Inverse Agonist GAC0001E5 Impedes Glutaminolysis and Disrupts Redox Homeostasis in Breast Cancer Cells

Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors which regulate the expression of lipid and cholesterol metabolism genes. Moreover, LXRs and their ligands have been shown to inhibit tumor growth in a variety of cancers. We have previously identified the small molecule compound GAC0001E5 (1E5) as an LXR inverse agonist and a potent inhibitor of pancreatic cancer cells. Transcriptomic and metabolomic studies showed that 1E5 disrupts glutamine metabolism, an essential metabolic pathway commonly reprogrammed during malignant transformation, including in breast cancers. To determine the role of LXRs and potential application of 1E5 in breast cancer, we examined LXR expression in publicly available clinical samples, and found that LXR expression is elevated in breast tumors as compared to normal tissues. In luminal A, endocrine therapy-resistant, and triple-negative breast cancer cells, 1E5 exhibited LXR inverse agonist and “degrader” activity and strongly inhibited cell proliferation and colony formation. Treatments with 1E5 downregulated the transcription of key glutaminolysis genes, and, correspondingly, biochemical assays indicated that 1E5 lowered intracellular glutamate and glutathione levels and increased reactive oxygen species. These results indicate that novel LXR ligand 1E5 is an inhibitor of glutamine metabolism and redox homeostasis in breast cancers and suggest that modulating LXR activity and expression in tumor cells is a promising strategy for targeting metabolic reprogramming in breast cancer therapeutics