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Pumilio 2 (PUM2) is a member of the Puf family of mRNA binding proteins and translational regulators which are involved in various processes including embryonic patterning and memory formation. Nevertheless, its functions in the outgrowth of neuronal axons have not been studied. This study shows endogenous expression of PUM2 in neurites of dorsal root ganglia (DRG) neurons and transport of PUM2 along retinal ganglion cell (RGC) axons and their growth cones. Overexpression of PUM2 in DRG neurons resulted in shorter axons when compared to control neurons. Expression of either dominant negative mutation (dnPUM2) or PUM2W349G displayed a reduction in axonal length. PUM2 downregulation with microRNA (miRNA) also caused a reduction in neurite length compared to control neurons. Finally, PUM2 silencing did not alter eye size at E4, which allows investigation of axonal outgrowth in RGC in vivo. These results suggest a novel role for PUM2 in axonal outgrowth.

Merrilactone A (1) was isolated in only 0.004% yield from the methanol extracts of the pericarps of Illicium merrillianum. Structural elucidation of Merrilactone A revealed a compact, cage-like pentacyclic architecture of high molecular complexity, featuring seven stereocentres, five of which as contiguous fully substituted carbon atoms, two γ-lactones and a central oxetane ring. Merrilactone A also exhibits an important neurotrophic activity, significantly promoting neurite outgrowth in the primary cultures of foetal rat cortical neurons at very low concentrations. Structurally, merrilactone A is related to anislactones A and B, a pair of epimeric sesquiterpene dilactones discovered ten years earlier by Kouno and co-workers from the related Illicium anisatum plant. Fukuyama has shown that anislactone B can be converted into merrilactone A using a simple three step sequence, suggesting that the anislactones may be biogenetic precursors to merrilactone A. Described in this thesis are our research efforts directed towards developing a conceptually novel synthetic route enabling regiodivergent total synthesis of both anislactone A / B and merrilactone A. Our synthetic route (around 22 steps) features several key reactions, which include a [2+2] photo-cycloaddition reaction, Tiffeneau-Demjanov ring expansion and titanium(III) mediated radical cyclization.

Eukaryotic Elongation Factor 1A (eEF1A) exists in two forms in vertebrates. The first form, eEF1A1, is expressed ubiquitously throughout development but is downregulated postdevelopmentally and replaced with eEF1A2, an isoform sharing 92% amino acid identity, in neurons and muscle. The primary function of eEF1A is to recruit amino-acylated tRNAs in a GTP-dependent manner to the A site of the ribosome during protein translation, but it also has non-canonical roles in the cell, some of which are isoform dependent. The reasons for the cell-type dependent switch from eEF1A1 to eEF1A2 are poorly understood. The first aim of this project was to examine the role played by eEF1A isoforms in neuritogenesis. To do this I used RNAi to significantly reduce expression of one or other isoform in neuronal cells and measure the effects this had on neurite outgrowth. Neurite outgrowth was significantly reduced in cells depleted of eEF1A1, but not eEF1A2. The complete loss of eEF1A2 is fatal, as has been demonstrated in the wasted mouse, an eEF1A2-null model characterised by muscle wastage, neurodegeneration and death at 4 weeks of age. Mice heterozygous for the wasted mutation have normal motor function. Recent work has found that heterozygous missense mutations in eEF1A2 can cause epilepsy and intellectual disability. It is not yet known whether the seven different de novo mutations identified to date confer loss or gain of function – a crucial piece of information required before possible treatments can be sought. The second aim of this project therefore was to investigate the role of eEF1A2 in epilepsy and intellectual disability. I achieved this by using CRISPR in two ways; firstly to model one of the mutations, D252H, in vitro in a neuronal cell line, and secondly to model another of the mutations, G70S, in vivo. No mice that recapitulated the human disease condition of EEF1A2G70S/+ were obtained however, due to the error-prone nature of the non-homologous end joining repair pathway activated by CRISPR-mediated DNA cleavage, 17 of the 35 mice born were found to be homozygous nulls at the Eef1a2 locus. Nine of these had fatal audiogenic seizures caused by sudden loud noises within the animal unit. Three mice were Eef1a2G70S/- and one Eef1a2G70S/G70S but these nonetheless showed a wasted phenotype, indicating that this mutant form of eEF1A2 has compromised function, at least in terms of translation elongation. Whether it has a toxic function ca not yet be known, but the severity of the phenotype in the G70S homozygous animal could suggest a gain of function. In in vitro experiments with exogenous eEF1A2 carrying the epilepsy-causing mutation R423C, protein expression of the mutant construct in immortalised cell lines was significantly higher when cotransfected with the wildtype construct, which mirrors the condition in humans, than when transfected alone, so the mutant protein could be stabilised in the presence of wildtype eEF1A2. I used CRISPR on LUHMES cells to make a mutant neuronal cell line containing the D252H mutation in eEF1A2. Due to time restraints no phenotypic differences between the wild type line and the D252H mutation line have yet been identified, but would form the focus of a future project.

Pumilio 2 (PUM2) is a member of the Puf family of mRNA binding proteins and translational regulators which are involved in various processes including embryonic patterning and memory formation. Nevertheless, its functions in the outgrowth of neuronal axons have not been studied. This study shows endogenous expression of PUM2 in neurites of dorsal root ganglia (DRG) neurons and transport of PUM2 along retinal ganglion cell (RGC) axons and their growth cones. Overexpression of PUM2 in DRG neurons resulted in shorter axons when compared to control neurons. Expression of either dominant negative mutation (dnPUM2) or PUM2W349G displayed a reduction in axonal length. PUM2 downregulation with microRNA (miRNA) also caused a reduction in neurite length compared to control neurons. Finally, PUM2 silencing did not alter eye size at E4, which allows investigation of axonal outgrowth in RGC in vivo. These results suggest a novel role for PUM2 in axonal outgrowth.

The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth.

The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth.

Master of Philosophy (Medicine) / The actin cytoskeleton is important in many cellular processes such as motility, and establishing and maintaining cell morphology. Members of the tropomyosin protein family associate with the actin cytoskeleton along the major groove of actin filaments (F-actin), stabilising them and regulating actin-filament dynamics. To date over 40 non-muscle tropomyosin isoforms have been identified, which are encoded by 4 different genes (α, β, γ, δ). Individual tropomyosin isoforms define functionally distinct F-actin populations. Previous studies have shown that tropomyosins sort to distinct subcellular compartments at different stages of development in polarised cells. Neuronal growth cones are highly dynamic polarised structures, dependent on a constant reorganisation of the actin cytoskeleton. By eliminating tropomyosins in a knockout (KO) mouse model, we investigated the role of two tropomyosin isoforms, Tm5NM1 and Tm5NM2 (γTm gene products) in growth cone dynamics and neurite outgrowth. Growth cone protrusion rates were significantly increased in one day old Tm5NM1/2 KO hippocampal neurons compared to WT controls. Neuritogenesis was significantly affected by the elimination of Tm5NM1/2, with a slight decrease in neurite length and an increase in neuronal branching in neurons cultured for four days. At the molecular level, the depletion of Tm5NM1/2 had no impact on the protein levels and activity of ADF/cofilin in hippocampal neurons while in cortical neurons a subtle but significant increase in ADF/cofilin activity was observed. The subtle phenotype in the early stages of neuritogenesis observed from eliminating Tm5NM1/2 may be explained with functional compensation by other tropomyosin isoforms. Functional compensation for the loss of Tm5NM1/2 may be provided by isoforms Tm5a/5b, TmBr2 and Tm4 as they localise to the growth cones, structures where Tm5NM1/2 are normally found. These results suggest that Tm5NM1/2 may not be required for early stages of neuritogenesis but may still play a fine-tuning role for this process.

This thesis describes a molecular-based study undertaken to analyse the expression of the RAMOSUS1 (RMS1) and RAMOSUS5 (RMS5) genes in pea (Pisum sativum). Both genes encode carotenoid cleavage dioxygenase (CCD) enzymes that are together proposed to control the synthesis of an inhibitor of bud outgrowth termed SMS (Shoot Multiplication Signal). SMS was recently identified as strigolactone. Expression analyses of RMS1 presented here have built upon earlier experiments which demonstrate it to be a highly regulated transcript. RMS1 mRNA levels are known to be rapidly decreased following removal of the shoot apex but are subsequently restored to that of intact plants by auxin (indole-3-acetic acid or IAA). This regulatory mechanism is retained in all five ramosus mutants tested to date. Together with physiological data, this indicates RMS1, and therefore SMS, are required in IAA-mediated suppression of bud outgrowth. Another significant aspect of RMS1 regulation identified in previous studies involves a graft-transmissible, long-distance feedback signal that moves from shoot to root. This feedback regulation is dependent on the RMS2 gene and enhances RMS1 expression levels. Prior to the cloning of RMS5 and its discovery as a second CCD enzyme in the RMS network, reciprocal grafting studies with the rms mutants indicated RMS5 may act in the same pathway as RMS1 to produce SMS. Multiple studies presented here demonstrate that these two CCD genes are expressed in similar tissues and are regulated by the same signals, specifically IAA and the RMS2-dependent feedback signal. Like RMS1, the RMS5 gene also retains its IAA response in the rms mutants. However, RMS5 is generally less responsive to changes in IAA and RMS2-dependent feedback, as it exhibits smaller fluctuations than RMS1 in its expression levels. Together these findings support a general view that RMS1 is more likely to control a rate-limiting step in SMS synthesis. A previous study indicated that RMS1 expression may be up-regulated by IAA through a posttranscriptional mechanism. This thesis sought to more closely examine the RMS1 and RMS5 IAA response by separately observing the effect of IAA on subsequent transcription. New transcripts, termed heterogenous nuclear RNAs (hnRNAs), were relatively quantified in parallel with existing mRNAs in the steady-state cytoplasmic pool. The experiments conducted here provide further evidence that IAA may act post-transcriptionally to stabilise RMS1 mRNA because the changes in hnRNA are not proportional to the changes in mRNA following IAA-modifying treatments. IAA may still function to induce transcription of RMS1, but this does not appear to be a significant mechanism by which IAA regulates RMS1 expression. In contrast, the IAA induction of RMS5 occurs predominantly via new transcription and RMS5 either lacks or is not as strongly subjected to the IAA-mediated mRNA stabilisation mechanism proposed for RMS1. Initial studies described in this thesis also suggest that IAA could act to regulate the expression of the Arabidopsis orthologues MORE AXILLARY BRANCHING (MAX) genes via a post-transcriptional mechanism. Analyses of MAX hnRNA and mRNA levels in Arabidopsis to date indicate it is the RMS5 orthologue MAX3 which exhibits an IAA response most like RMS1. Additional studies into the regulation of RMS1 and RMS5 presented in this thesis provide further insights into the molecular mechanisms controlling their expression levels. In vitro experiments with the translation inhibitor cycloheximide demonstrate that RMS5 expression levels are increased when protein synthesis is reduced, as previously shown for RMS1. Relative quantification of RMS1 and RMS5 hnRNA levels further demonstrate that the induction by cycloheximide is due primarily to an increase in new transcription, indicating that RMS1 and RMS5 are negatively regulated by a rapidly turned-over transcriptional repressor. Tissue specific effects on RMS1 expression were also observed which are consistent with a protein degradation function of the RMS4 F-box in the shoot. This thesis provides further evidence to suggest that SMS acts in concert with IAA to inhibit the sustained outgrowth of axillary buds. RMS1 and RMS5 expression levels are not regulated by a hypothetical fast decapitation signal which is proposed to cause the initial bud outgrowth occurring prior to decapitation-induced IAA depletion. RMS1, RMS5 and SMS are therefore unlikely to control the initial exit of buds from dormancy to an intermediate transition state. Studies here also suggest that enhanced shoot auxin transport and cytokinin biosynthesis are associated with axillary bud outgrowth because the rms mutants contain elevated shoot expression levels of a gene encoding the auxin efflux carrier PIN1 and two genes controlling cytokinin biosynthesis. Several approaches described in this study were used to characterise the RMS1 and RMS5 proteins. Anti-peptide antibodies were generated against both proteins and the results obtained show that although the antibodies are likely to recognise the full-length proteins, further work is required to effectively detect RMS1 and RMS5 in plant tissues via western blotting. Preliminary in situ immunolocalisation results indicate the RMS1 and RMS5 proteins are localised to the vasculature, consistent with gene expression analyses.

Master of Philosophy (Medicine) / The actin cytoskeleton is important in many cellular processes such as motility, and establishing and maintaining cell morphology. Members of the tropomyosin protein family associate with the actin cytoskeleton along the major groove of actin filaments (F-actin), stabilising them and regulating actin-filament dynamics. To date over 40 non-muscle tropomyosin isoforms have been identified, which are encoded by 4 different genes (α, β, γ, δ). Individual tropomyosin isoforms define functionally distinct F-actin populations. Previous studies have shown that tropomyosins sort to distinct subcellular compartments at different stages of development in polarised cells. Neuronal growth cones are highly dynamic polarised structures, dependent on a constant reorganisation of the actin cytoskeleton. By eliminating tropomyosins in a knockout (KO) mouse model, we investigated the role of two tropomyosin isoforms, Tm5NM1 and Tm5NM2 (γTm gene products) in growth cone dynamics and neurite outgrowth. Growth cone protrusion rates were significantly increased in one day old Tm5NM1/2 KO hippocampal neurons compared to WT controls. Neuritogenesis was significantly affected by the elimination of Tm5NM1/2, with a slight decrease in neurite length and an increase in neuronal branching in neurons cultured for four days. At the molecular level, the depletion of Tm5NM1/2 had no impact on the protein levels and activity of ADF/cofilin in hippocampal neurons while in cortical neurons a subtle but significant increase in ADF/cofilin activity was observed. The subtle phenotype in the early stages of neuritogenesis observed from eliminating Tm5NM1/2 may be explained with functional compensation by other tropomyosin isoforms. Functional compensation for the loss of Tm5NM1/2 may be provided by isoforms Tm5a/5b, TmBr2 and Tm4 as they localise to the growth cones, structures where Tm5NM1/2 are normally found. These results suggest that Tm5NM1/2 may not be required for early stages of neuritogenesis but may still play a fine-tuning role for this process.