Characterising the role of GPR50 in neurodevelopment and lipid metabolism

Abstract

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.