Bioactive lipids have emerged as prominent regulators of neural stem and progenitor cell (NPC) function under both physiological and pathological conditions. However, how lipid metabolism is regulated, and its role in modulation of NPC function remains unknown. In this regard, my study defines a novel epigenetic pathway that regulates lipid metabolism to determine NPC proliferation versus differentiation. Specifically, I show that activation of an atypical protein kinase C (aPKC)-mediated Ser436 phosphorylation of CREB binding protein (CBP) by aging, metformin stimulation and continued passaging in vitro, represses expression of monoacylglycerol lipase (Mgll) to promote neuronal differentiation of adult NPCs. Mgll, a lipase that hydrolyzes the endocannabinoid 2-arachidonoyl glycerol (2-AG) to produce arachidonic acid (ARA), is thus a key regulator of two critical bioactive lipid signaling pathways in the brain and a potential modulator of NPC function. I observed elevated Mgll levels, concomitant with neuronal differentiation deficits in both the lateral ventricle sub-ventricular zone (SVZ) and the hippocampal subgranular zone (SGZ) NPCs of phospho-null CBPS436A mice, that lack a functional aPKC-CBP pathway. Genetic knockdown of Mgll or inhibition of Mgll activity rescued these neuronal differentiation deficits. In addition, I found that CBPS436A SVZ NPCs exhibit enhanced proliferation at the expense of differentiation as an outcome of increased Mgll levels in culture. Interestingly, I also observed that SVZ NPCs from an Alzheimer’s disease (AD) model, the 3xTg mice, closely resemble CBPS436A NPC behaviour in culture. 3xTg NPCs exhibit attenuation of the aPKC-CBP pathway, which is associated with elevated Mgll expression and increased NPC proliferation at the expense of neuronal differentiation. Reactivation of the aPKC-CBP mediated-Mgll repression in 3xTg AD NPCs mitigates their differentiation deficits. These findings implicate Mgll as a critical switch that regulates NPC function by altering bioactive lipid signaling (2-AG versus ARA). They demonstrate that the aPKC-CBP mediated Mgll repression is essential for normal NPC function, and that when perturbed in AD, it causes impaired NPC function to generate fewer neurons, contributing to AD predisposition.

There have been dramatic advances in melanoma therapy in the last 10 years, yet there is still a demand for effective and affordable therapies. To identify novel therapeutic pathways a transcriptome analysis was performed on zebrafish melanoma models representing the different stages of melanoma progression. Transcriptomic differences between pre-malignant and malignant conditions highlighted lipid metabolism as a potential mediator of progression. A mass spectrometry analysis confirmed multiple changes in lipid composition between wild type fish, pre-malignant and advanced melanoma models. To better investigate metabolism a positron emission tomography (PET) technique was developed in zebrafish. Tumours in the zebrafish were successfully scanned with FDG used to detect human tumours. A novel tracer of unconjugated FA was then developed and, consistent with inferences from the transcriptome and mass spectrometry, was shown to be incorporated into tumours. Demonstrating the feasibility of PET in zebrafish now opens the way to systematic use of this organism in tracer development with potential time-saving and cost benefits. One of the most significantly up-regulated genes exclusive to the malignant state encodes lipoprotein lipase (LPL). LPL is involved in the release and uptake of FA from circulating triglyceride. LPL was found to increase the rate of tumour appearance and tumour growth in a zebrafish tumour assay. LPL was expressed in human tumours and expression correlated with progression. Melanoma cell lines expressed LPL and knocking-down LPL resulted in reduced cell numbers. The effect was most dramatic in WM852 cells. A novel role for LPL in autophagy was identified. WM852 cells treated with LPL siRNA showed a stabilisation of p62/SQSTM and induction of LC3B II. Electron microscopy revealed large autolysosomal vacuoles in the cytoplasm. Additionally many cells showed damaged mitochondria with absent cristae. The dependency of cells on LPL seemed to be modified by the co-expression of fatty acid synthase (FASN) required for de novo FA synthesis, as the magnitude of the effect of LPL-knockdown was dependent on the levels of FASN expressed in melanoma cell lines. Moreover, combining LPL and FASN inhibitors synergised to kill cells previously less sensitive to LPL inhibitor. FASN and LPL co-inhibition could provide a unique combinatorial therapeutic strategy.

G-protein coupled receptor 50 (GPR50) is a genetic risk factor for psychiatric illness. It is a member of the melatonin receptor family, which includes the well characterised melatonin receptors 1 and 2 (MT1 and MT2). However, the ligand for GPR50 remains elusive and little is known about GPR50 signalling pathways. Despite this, GPR50 is known to enhance neurite outgrowth and inhibit the actions of the neurite outgrowth inhibitor NOGO-A. Existing evidence also indicates a role in lipid metabolism; GPR50 knockout mice displayed abnormalities in energy homeostasis and weight control, whilst sequence variants are associated with altered lipid levels in humans. Further, a yeast-2-hybrid screen identified SREBF2 and ABCA2, regulators of lipid homeostasis, as GPR50 interactors. This thesis explores the role of GPR50 in neuronal development and lipid metabolism. The work presented in this thesis shows that GPR50 promotes neuronal differentiation. Overexpression significantly increased the number of neurites per cell in SH-SY5Y cells. Further, dendritic branching was enhanced by GPR50 transfection in hippocampal and cortical neurons (DIV 14). In hippocampal neurons, GPR50 transfection also lead to a shift towards spine maturity although it had no effect on spine morphology, suggesting GPR50 enhances spine development but may not alter synaptic strength. The effect of GPR50 on neuronal morphology may be driven by actin remodelling. Immunocytochemistry showed an enrichment of GPR50 in highly dynamic regions of the membrane, i.e. the lamellipodia and dendritic spines. Overexpression in SH-SY5Y cells also resulted in an increase in WAVE-2 and phosphorylated RAC1/CDC42, key modulators of actin dynamics. Additionally, GPR50 transfection altered the protein level and localisation of α- catenin, another regulator of actin organisation, in HEK293 and SH-SY5Y cells respectively. An involvement of GPR50 in lipid metabolism has also been demonstrated in this thesis. Verification of the Y2H study suggested GPR50 does not physically interact with SREBF2 or ABCA2. However, ABCA2 appears to induce the intracellular localisation of GPR50 in several cell lines. In SH-SY5Y cells, this was mimicked by the inhibition of cholesterol trafficking, suggesting the translocation of GPR50 to the plasma membrane is dependent on cholesterol transport. Further, the depletion of lipoproteins resulted in the downregulation of GPR50, indicating a responsiveness to lipid levels. Finally, GPR50 increased lipid metabolism, as seen by a decrease in intracellular lipid droplets upon GPR50 overexpression. The data presented here extends previous work indicating a role of GPR50 in neurodevelopment. It also highlights a potential mechanism by which GPR50 regulates neuronal morphology, i.e. via actin remodelling. Reports that GPR50 is involved in energy homeostasis is also supported in this thesis, further, results presented here suggest GPR50 is specifically involved in lipid metabolism. These processes are often disrupted in mental illness, thus this work may provide a functional link between GPR50 and psychiatric disorders.

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Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The present view of the lipid metabolism of tumors appears to be, as stated by Gore and Popjak (32) and Gore (11), that tumors in general lack the capacity to utilize acetate for the biosynthesis of lipids. The results of Medes et al. (12), Busch (13), and Busch and Baltrush (14), lend support to the view expressed by Gore and Popjak. However, evidence to the contrary, namely that the lipid metabolism in tumors is not impaired, also exists. In this regard the results of Trew and Begg (16), Jablonski and Olson (17), Olson et al. (54), and Haven (19,20) are of special significance. The lack of agreement in the results of various investigators was attributed by Henderson and LePage (15) to differences between the tumors studied, but it was felt that the discrepancies in the results could also be attributed to differences in the conditions of incubation, the importance of which was emphasized by Busch (27) [TRUNCATED]. / 2031-01-01

The aged T cell is characterized by decreased responsiveness to stimulation. Aging is associated with reduced membrane glycerophospholipid (GPL) to cholesterol ratios so it is interesting that deletion of mitochondrial glycerol-3-phosphate acyltransferase-1 which catalyzes the first step in de novo GPL synthesis induces an aged T cell phenotype in otherwise healthy mice. GPAT-1 could regulate T cell function through three possible mechanisms: maintenance of membrane GPL ratios and membrane based signaling, providing a specific substrate for downstream signaling, or direct regulation of cellular metabolism. Therefore, the goal of this project was to determine whether these mechanisms contribute to the dysfunctional T cell phenotype observed with decreased GPAT-1 activity. T cell stimulation requires significant upregulation of metabolic processes to drive clonal expansion and cytokine production. T cell dysfunction in GPAT-1 knockout mice may be partially explained by altered metabolic function. We found that GPAT-1 KO T cells have significantly reduced basal respiration rates and spare respiratory capacity which is not compensated for by increased glycolytic metabolism suggesting an inherent metabolic defect in GPAT-1 KO T cells. To better understand mechanistically how GPAT-1 regulates T cell function we moved into the Jurkat T cell line and found that shRNA mediated knockdown of the human isoform of GPAT-1 (GPAM) recapitulated key aspects of the dysfunctional T cell phenotype we observed in the mouse including highly significant reductions in IL-2 production and altered membrane GPL to cholesterol ratios. Phosphatidic acid addition was not capable of rescuing these deficiencies suggesting that GPAT-1/GPAM activity is required for proper T cell function. This was the first time that GPAT-1 activity has been shown to be important for T cell function in a non-murine model system and strongly suggests that GPAT-1/GPAM deficiency regulates T cell function at the cellular level. We further demonstrate that phosphorylation of ZAP-70 a proximal effector of T cell activation is significantly reduced in GPAM knock down Jurkat T cells, suggesting that membrane based signaling is dysfunctional. Taken together these data suggest that GPAT-1 is necessary for regulating cellular energy demands in T cells and essential for optimal T cell activation following stimulation. / text

A brief review was provided of lipid metabolism, mechanisms of lipid lowering and coronary heart disease risk factors (including the role of antioxidants). In addition, studies of lipid-lowering and other anti-atherosclerotic effects of garlic materials were also reviewed. Evaluation and development of techniques required to perform studies of the anti-atherosclerotic effects of garlic were implemented, including evaluation of methods for measuring total antioxidant capacity and the development of methods for the rapid isolation of low density lipoprotein (LDL). The main objective of this project was to investigate the effects of garlic upon plasma lipids in rats and healthy human subjects and the effects upon antioxidant status in healthy human subjects. In a long-term study with old male rats, a low dose of garlic oil (c. O.5mg/Kg body weight/day) for 12.5 weeks provided in a high fat diet produced no significant reduction in plasma lipids or glucose. Upward trends in plasma lipids early in the treatment period were similar to those reported from several previous studies in which eventual reductions in plasma lipids were observed suggesting the possibility of a small effect of garlic oil upon lipid metabolism. Two randomised, double-blind, placebo-controlled human studies were carried out to investigate the effects of capsules containing garlic oil dissolved in vegetable oil upon coronary heart disease risk factors in normal subjects. In the first study, 8.2 mg/day of garlic oil (or placebo) capsules were given to 51 subjects over an I 1- week period. 95% confidence intervals were calculated for differences between garlic oil and placebo groups of changes in plasma lipids, glucose and total antioxidant capacity between baseline and end-oftreatment. No significant effects were observed, but it was found that plasma lipids and glucose trends were more favourable with garlic oil treatment. Effects of garlic oil over placebo for cholesterol-related variables and glucose were in opposite directions for males and females and this interaction between genders reached significance for high density lipoprotein cholesterol, total cholesterol/high density lipoprotein cholesterol ratio, calculated low density lipoprotein cholesterol and glucose of plasma. The strong gender effect of garlic oil evidenced for plasma lipids and glucose implies potent effects of garlic oil and suggests future garlic studies should include gender analyses. In the second human study, 12 mg/day garlic oil (or placebo) capsules were given to 27 trained young male athletes for 16 weeks. 95% confidence intervals were calculated for differences between garlic oil and placebo groups of changes in plasma lipids, total antioxidant capacity and LDL composition between baseline and end-of-treatment. It was found that the reduction in plasma triglycerides (p=O.09) of the garlic oil group in comparison with the placebo group was accompanied by increases in density, a significant reduction in triglycerides / protein and a trend towards reduction of cholesterol/protein of the LDL fraction. A trend towards reduced total antioxidant capacity / protein of LDL appear to reflect garlic oil effects upon lipid composition of LDL rather than directly upon antioxidants. Non-significant trends towards small improvements in maximal oxygen intake test-duration and less plasma glucose depletion with garlic oil were observed. Overall, the results from the human studies provide some encouragement to the view that garlic oil could reduce coronary heart disease risk in normal persons and that this possibility deserves further investigation.

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