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Reduced inhibitory glycinergic neurotransmission is implicated in a number of neurological conditions including chronic pain and hyperekplexia and restoring glycinergic signalling may be an effective method of treating these pathologies. Glycine transporters (GlyTs) control synaptic and extra-synaptic glycine concentrations. Slowing the reuptake of glycine using non-competitive GlyT inhibitors will increase extracellular glycine concentrations and increase glycine receptor (GlyR) activation. The general approach to drug development, of modulating a single target with high affinity and efficacy, has not produced useful novel treatments and therapeutic outcomes for sufferers of chronic pain. Modulation of multiple related targets to induce robust, systemic modulation is suggested as a potential way to improve therapeutic outcomes. I propose that a multi-target drug approach could be taken to modulate two key proteins in a restricted system, GlyT2 and GlyRα1, to improve glycinergic inhibitory signalling and provide good leads for the development of novel analgesics. Furthermore, hyperekplexia is a disease caused by mutations in glycinergic proteins and I proposed that multi-target drugs could also be investigated for use in treating this condition. To study the dual modulation of the glycinergic synapse, in Chapter 3 I characterise a system where GlyTs are co-expressed with GlyRs in Xenopus laevis oocytes and show that GlyTs can reduce the concentration of glycine sensed by GlyRs and alter the efficacy of receptor activation. I use two-electrode voltage-clamp electrophysiology and confocal microscopy to measure the impact of GlyTs on GlyRs and show that increases in GlyT density near GlyRs increasingly diminish receptor currents. Reductions in GlyR mediated currents are not observed when non-transportable GlyR agonists are applied or when Na+ is not available, validating changes in GlyR activation as related to glycine uptake by GlyTs. GlyTs create diffusion-limited, concentration gradients across different agonist concentration ranges. Due to these diffusion barriers, the actual glycine concentrations sensed at the membrane by GlyRs can be greatly reduced and these are estimated. Full receptor currents can be restored when GlyTs are blocked with selective inhibitors. Modulation of glycinergic neurotransmission is a novel way of increasing inhibitory signalling in the CNS, however, attempts to pharmacologically target this system have thus far failed in the clinical treatment of chronic pain. Glycine activates inhibitory GlyRs, however it is also a co-agonist at excitatory NMDA receptors. Full inhibition of GlyTs has also been shown to cause motor defects and lead to death. Targeted and potent inhibition of GlyTs to increase synaptic levels of glycine may therefore have unintended and opposite effects. Bioactive lipid compounds developed in our lab have actions as both GlyT inhibitors and GlyR positive allosteric modulators (PAMs). In Chapter 4, I assess the usefulness of these compounds in the co-expression system characterised in Chapter 3, exploring the feasibility of a multi-target approach to drug design at the glycinergic synapse and show that features of glycinergic modulators which are not apparent when studied at single targets can be elucidated. Hyperekplexia is a rare hereditary disease caused by mutations at various glycinergic proteins, however the majority of hyperekplexia cases are caused by mutations in the GlyRα1 gene. GlyRs are modulated by alcohols and volatile anaesthetics, and a serine at position 267 has been identified as an important residue that mediates their actions. The S267 residue has also been implicated in a hyperekplexic family carrying the dominant missense hyperekplexic mutation GlyRα1S267N, and heterologous expression of this mutant GlyR displays decreased sensitivity to glycine and to potentiation by ethanol. In Chapter 5, I co-express the GlyRα1S267N mutant with GlyT2 and show that GlyT2 does not modulate this mutant receptor. I then show that although dual modulation is an efficacious approach at WT GlyRα1/GlyT2, robust positive potentiation of GlyRα1S267N/GlyT2 currents is only achieved by a high efficacy GlyR PAM. These results highlight the need to understand how disease mechanisms change the interaction between target proteins of interest when designing multi-target drugs. Finally, in Chapter 6 I undertake a joint investigation into the modulation of GlyRs by glutamate. In contradiction to a previous report by Liu and colleagues in 2010, we show that glutamate does not allosterically modulate GlyRs. Reciprocal glutamatergic modulation of GlyRs in the central nervous system (CNS) would imply crosstalk between these two signalling systems, which would have fundamental implications for our understanding of nociceptive circuits and approaches to modulating them. However, GlyRs have multiple sites of allosteric modulation and are known to be modulated by large number of compounds, and we suggest the presence of a contaminating substance as the basis for this discrepancy. This study highlights the importance of independent assessment of novel neurobiological concepts. In this thesis, I have described a novel way of assessing dual action pharmacological modulation of the glycinergic synapse which can be adapted to model diseases states. This work will support the development of good leads into novel glycinergic modulators with better tolerability, efficacy and therapeutic outcomes.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.