Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

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

Dual Action Modulators of the Glycinergic Synapse

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

Kynurenine Pathway in Skin Cells: Implications for UV-Induced Skin Damage

The kynurenine pathway (KP) is the principle route of catabolism of the essential amino acid tryptophan, leading to the production of several neuroactive and immunoregulatory metabolites. Alterations in the KP have been implicated in various neuropsychiatric and neurodegenerative diseases, immunological disorders, and many other diseased states. Although the role of the KP in the skin has been evaluated in small niche fields, limited studies are available regarding the effect of acute ultra violet exposure and the induction of the KP in human skin-derived fibroblasts and keratinocytes. Since UV exposure can illicit an inflammatory component in skin cells, it is highly likely that the KP may be induced in these cells in response to UV exposure. It is also possible that some KP metabolites may act as pro-inflammatory and anti-inflammatory mediators, since the KP is important in immunomodulation

Characterisation of the kynurenine pathway in skin-derived fibroblasts and keratinocytes

Acute UVB exposure triggers inflammation leading to the induction of indoleamine 2,3 dioxygenase (IDO1), one of the first enzymes in the kynurenine pathway (KP) for tryptophan degradation. However, limited studies have been undertaken to determine the catabolism of tryptophan within the skin. The aim of this study was two fold: (1) to establish if the administration of the proinflammatory cytokine interferon-gamma (IFN-γ) and/or UVB radiation elicits differential KP expression patterns in human fibroblast and keratinocytes; and (2) to evaluate the effect of KP metabolites on intracellular nicotinamide adenine dinucleotide (NAD⁺) levels, and cell viability. Primary cultures of human fibroblasts and keratinocytes were used to examine expression of the KP at the mRNA level using qPCR, and at the protein level using immunocytochemistry. Cellular responses to KP metabolites were assessed by examining extracellular lactate dehydrogenase (LDH) activity and intracellular NAD⁺ levels. Major downstream KP metabolites were analyzed using GC/MS and HPLC. Our data shows that the KP is fully expressed both in human fibroblasts and keratinocytes. Exposure to UVB radiation and/or IFN-γ causes significant changes in the expression pattern of downstream KP metabolites and enzymes. Exposure to various concentrations of KP metabolites showed marked differences in cell viability and intracellular NAD⁺ production, providing support for involvement of the KP in the de novo synthesis of NAD⁺ in the skin. This new information will have a significant impact on our understanding of the pathogenesis of UV related skin damage and the diagnosis of KP related disease states.20 page(s

Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function:A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

Low-density lipoprotein (LDL) is the major source of lipid within atherosclerotic lesions. Myeloperoxidase (MPO) is present in lesions and forms the reactive oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). These oxidants modify LDL and have been strongly linked with the development of atherosclerosis. In this study, we examined the effect of HOCl, HOSCN and LDL pre-treated with these oxidants on the function of lysosomal enzymes responsible for protein catabolism and lipid hydrolysis in murine macrophage-like J774A.1 cells. In each case, the cells were exposed to HOCl or HOSCN or LDL pre-treated with these oxidants. Lysosomal cathepsin (B, L and D) and acid lipase activities were quantified, with cathepsin and LAMP-1 protein levels determined by Western blotting. Exposure of J774A.1 cells to HOCl or HOSCN resulted in a significant decrease in the activity of the Cys-dependent cathepsins B and L, but not the Asp-dependent cathepsin D. Cathepsins B and L were also inhibited in macrophages exposed to HOSCN-modified, and to a lesser extent, HOCl-modified LDL. No change was seen in cathepsin D activity or the expression of the cathepsin proteins or lysosomal marker protein LAMP-1. The activity of lysosomal acid lipase was also decreased on treatment of macrophages with each modified LDL. Taken together, these results suggest that HOCl, HOSCN and LDL modified by these oxidants could contribute to lysosomal dysfunction and thus perturb the cellular processing of LDL, which could be important during the development of atherosclerosis

Inhibition of cathepsin B and L activity after exposure of intact J774A.1 cells to HOSCN- and HOCl-modified LDL.

<p>LDL (1 mg protein mL^-1) was exposed to 0–500 μM HOSCN (A and B) or HOCl (C and D) for 24 h at 37°C respectively, prior to addition of each modified LDL (0.1 mg protein mL^-1) to J774A.1 cells for 4 (white bars) or 24 h (black bars), and determination of cathepsin B (A, C) or cathepsin L (B, D) activity, which is expressed relative to the no LDL control. * and # represent a significant decrease (p < 0.05) in cathepsin B or L activity compared with cells exposed to the incubation control LDL or no LDL. “a” represents a significant (p < 0.05) difference between cathepsin activity between cells incubated with LDL for 4 or 24 h.</p

Inhibition of cathepsin D activity after exposure of J774A.1 cell lysates to HOSCN- and HOCl-modified LDL.

<p>LDL (1 mg protein mL^-1) was exposed to 0–500 μM HOSCN (A) or HOCl (B) for 30 min (white) and 24 h (black) at 22°C and 37°C, respectively, prior to addition of each modified LDL (0.1 mg protein mL^-1) to J774A.1 lysates for 15 min at 22°C, followed by determination of cathepsin D activity expressed as activity/mg protein. * represents a significant decrease (p < 0.05) in cathepsin D activity compared with cells exposed to the incubation control LDL. There was no significant difference in enzyme inhibition between 30 min and 24 h LDL oxidant, as determined by 2-way ANOVA.</p

Inhibition of lysosomal acid lipase (LAL) activity after exposure of J774A.1 lysates and cells to modified LDL.

<p>LDL (1 mg protein mL^-1) was exposed to (A) HOSCN (0–500 μM) or (B) HOCl (0–500 μM) for 30 min (white) and 24 h (black) at 22°C and 37°C, respectively, prior to addition of each modified LDL (0.1 mg protein mL^-1) to J774A.1 lysates for 15 min at 22°C, and determination of LAL activity, which is expressed relative to the no LDL control. Graph (C) shows LAL activity after exposure of J774A.1 cells for 4 (white) or 24 h (black) at 37°C to LDL modified by HOSCN (250 μM), HOCl (250 μM), or OCN^- (2500 μM) for 24 h at 37°C. * and # represent a significant decrease (p < 0.05) in LAL activity compared with cells exposed to the incubation control LDL or no LDL.</p

Cathepsin B and L protein levels are unchanged in J774A.1 cells after exposure to HOSCN- and HOCl-modified LDL.

<p>LDL (1 mg protein mL^-1) was exposed to 0–500 μM HOSCN (A) or HOCl (B) for 24 h at 37°C, respectively, prior to addition of each modified LDL (0.1 mg protein mL^-1) to J774A.1 cells, followed by determination of cathepsin B (white) and L (black) protein expression. Cathepsin levels were normalised to β-actin levels, and then calculated as the fold change from the no LDL condition. Representative blots of the cathepsin B band at 25 kDa, the cathepsin L band at 25 kDa, or the β-actin band at 43 kDa, are displayed, of n = 3–4 separate experiments. There was no significant effect of oxidant treatment as determined by 1-way ANOVA on either cathepsin protein levels.</p

Inhibition of cathepsin B and L activity in J774A.1 cells after treatment with HOCl and HOSCN.

<p>(A) Cathepsin B and (B) cathepsin L activity in J774A.1 cells (1 × 10⁶ cells mL^-1) was determined after incubation with HOSCN (80–160 μM, white bars) or HOCl (80–160 μM, black bars) for 15 min at 22°C. (C) Cathepsin B and (D) cathepsin L activity in J774A.1 cell lysates (1 × 10⁶ cells mL^-1) after incubation with HOSCN (5–20 μM) for 15 min, followed by further incubation in the absence (white bars) or presence (black bars) of DTT (100 μM) for 15 min. Results are expressed as a percentage of the PBS-treated control cells. * and # represent a significant (p < 0.05) change in cathepsin B/L activity compared with control lysates or the presence / absence of DTT, respectively.</p