Molecular genetics of the imprinted gene - SPROUTY3 (SPRY3)

Sprouty proteins are key regulators of cell growth and branching morphogenesis during development. Human SPRY3 which maps to the pseudoautosomal region 2, undergoes random X-inactivation in females and preferential Y-inactivation in males, behaving as though genetically X-linked. Spry3 is widely expressed in neuronal tissues, being found at high levels in the cerebellum and particularly in the Purkinje cells which, notably, are deficient in the autistic brain. Spry3 is also highly expressed in other ganglia in adults including retinal ganglion cells, dorsal root ganglion and superior cervical ganglion. SPRY3 enhancer can drive SPRY3 expression in the lung airway, which is consistent with a role in branching morphogenesis and the function of the original Drosophila Spry gene, which is critical for lung morphogenesis, providing a possible explanation for an observed anatomic abnormality in the autistic lung airway. In the human and mouse, the SPRY3 core promoter contains an AG-rich repeat and we found evidence of coexpression, promoter binding and regulation of SPRY3 expression by transcription factors EGR1, ZNF263 and PAX6. Spry3 over-expression in mouse superior cervical ganglion cells inhibits axon branching and Spry3 knockdown in those cells increases axon branching, consistent with known functions of other Sprouty proteins. Novel SPRY3 upstream transcripts that I characterised originate from three start sites in the X-linked F8A3 – TMLHE gene region, which is recently implicated in autism causation. Arising from these findings, I propose that the lung airway abnormality and low levels of blood carnitine found in autism suggest that deregulation of SPRY3 may underpin a subset of autism cases.

Fractionation and compositional studies of rabbit skeletal muscle membranes

The observations of Hooke (1665), Schleiden & Schwann (1839) and Virchow (1855) led to the identification of the cell as the basic structural unit of living material. In the intervening years, it has been firmly established that the chemical processes which underlie the proper functioning, development and reproduction of the organism are cellular activities. The development of the electron microscope has enabled cell structure to be studied in detail. A picture of the cell as an entity with a complex and highly organised internal structure has emerged from the work of Palade, Porter, Fernandez-Moran and many others. Although cells from different tissues and organisms differ in aspects of their structure and consequently in function, they have several features in common. A retentive membrane encloses a number of cell constituents, which include membrane-enclosed subcellular structures known as organelles. The cells of most tissues also contain a reticulum or system of branching tubules. The interplay of the biochemical activities of these structures enables the cell to function. Almost thirty years ago, Claude, Palade, Schneider, Hogeboom, de Duve and others set out to analytically fractionate the subcellular components obtained after the fragmentation of liver cells. This approach has become known as subcellular fractionation, and signalled a major conceptual breakthrough in biochemistry (reviewed by de Duve, 1964, 1967, 1971). The significance of this breakthrough has been underlined by the award of the 1974 Nobel Prize in Medicine to de Duve, Palade and Claude. This thesis is concerned with the application of subcellular fractionation techniques to the separation and characterisation of the membrane systems of the rabbit skeletal muscle cell.

Investigations into the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy and maternal immune activation

The amygdala is a limbic structure that is involved in many of our emotions and processing of these emotions such as fear, anger and pleasure. Conditions such as anxiety, autism, and also epilepsy, have been linked to abnormal functioning of the amygdala, owing to improper neurodevelopment or damage. This thesis investigated the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy (TLE) and maternal immune activation (MIA). The kainic acid (KA) model of temporal lobe epilepsy (TLE) was used to induce Ammon’s-horn sclerosis (AHS) and to investigate behavioural and cytoarchitectural changes that occur in the amygdala related to Neuropeptide Y1 receptor expression. Results showed that KA-injected animals showed increased anxiety-like behaviours and displayed histopathological hallmarks of AHS including CA1 ablation, granule cell dispersion, volume reduction and astrogliosis. Amygdalar volume and neuronal loss was observed in the ipsilateral nuclei which was accompanied by astrogliosis. In addition, a decrease in Y1 receptor expressing cells in the ipsilateral CA1 and CA3 sectors of the hippocampus, ipsi- and contralateral granule cell layer of the dentate gyrus and ipsilateral central nucleus of the amygdala was found, consistent with a reduction in Y1 receptor protein levels. The results suggest that plastic changes in hippocampal and/or amygdalar Y1 receptor expression may negatively impact anxiety levels. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain and tight regulation and appropriate control of GABA is vital for neurochemical homeostasis. GABA transporter-1 (GAT-1) is abundantly expressed by neurones and astrocytes and plays a key role in GABA reuptake and regulation. Imbalance in GABA homeostasis has been implicated in epilepsy with GAT-1 being an attractive pharmacological target. Electron microscopy was used to examine the distribution, expression and morphology of GAT-1 expressing structures in the amygdala of the TLE model. Results suggest that GAT-1 was preferentially expressed on putative axon terminals over astrocytic processes in this TLE model. Myelin integrity was examined and results suggested that in the TLE model myelinated fibres were damaged in comparison to controls. Synaptic morphology was studied and results suggested that asymmetric (excitatory) synapses occurred more frequently than symmetric (inhibitory) synapses in the TLE model in comparison to controls. This study illustrated that the amygdala undergoes ultrastructural alterations in this TLE model. Maternal immune activation (MIA) is a risk factor for neurodevelopmental disorders such as autism, schizophrenia and also epilepsy. MIA was induced at a critical window of amygdalar development at E12 using bacterial mimetic lipopolysaccharide (LPS). Results showed that MIA activates cytokine, toll-like receptor and chemokine expression in the fetal brain that is prolonged in the postnatal amygdala. Inflammation elicited by MIA may prime the fetal brain for alterations seen in the glial environment and this in turn have deleterious effects on neuronal populations as seen in the amygdala at P14. These findings may suggest that MIA induced during amygdalar development may predispose offspring to amygdalar related disorders such as heightened anxiety, fear impairment and also neurodevelopmental disorders.