Generation and metabolism of 12/15-LOX esterified products

12/15-LOX is suspected to be involved in inflammatory disorders such as atherosclerosis but the exact mechanism of its action is unclear. Novel esterified products called 12- and 15- H(p)ETE-PEs were recently identified in murine macrophages and human monocytes respectively, which may at least partially account for the effects of 12/15-LOX (Maskrey et al, 2007). In this thesis the generation and metabolism of 12- and 15-H(p)ETE-PEs was characterised in murine macrophages and human monocytes. 12- and 15-H(p)ETE-PEs increased by 15 minutes following cell activation and were metabolised by three hours. However, endogenous 12- and 15-HETE-PEs increased then remained stable. Novel lipids believed to be precursors of 15-HETE-PEs, called 15-KETE-PEs, were identified in human monocytes. 15-KETE-PEs increased by 15 minutes and were metabolised by three hours and may therefore account for the temporal generation pattern of 15-H(p)ETE-PEs. 14C radiolabeled products of 15-LOX were generated and characterised in human monocytes. Detection of the 14C radiolabel in activated monocytes suggested that products of 15-LOX may form Michael adducts with proteins by three hours post activation. This may account for the 15-KETE-PE decrease in time-course samples. A method was developed to synthesise 15-H(p)ETE-PE standards, to use in a new assay for their direct quantification following MS analysis. The 15-HETE-PE standard was also used to investigate its pro- versus anti-inflammatory effect on LPS-stimulated human monocytes. 15- HETE-PE down-regulated IL-ip, TNF-a, IL-6 and G-CSF generation, suggesting that products of 15-LOX are anti-inflammatory in human monocytes. 12-H(p)ETE-PEs were externalised in activated WT macrophages, which may be important for cell-cell interactions and re-structuring the cell membrane. EM analysis indicated that 12/15-LOX"7" macrophages have a lysosomal storage disease, symptoms included the presence of lysosomal storage bodies, vacuoles and damaged mitochondria. This may be caused by detected raised levels of PE, PS, PG and PI phospholipids and cholesterol esters in 12/15-LOX"7" macrophages. The data described herein provides a platform to further investigate the function of esterified products generated by 15- and 12/15-LOX.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Generation and metabolism of 12/15-LOX esterified products

12/15-LOX is suspected to be involved in inflammatory disorders such as atherosclerosis but the exact mechanism of its action is unclear. Novel esterified products called 12- and 15- H(p)ETE-PEs were recently identified in murine macrophages and human monocytes respectively, which may at least partially account for the effects of 12/15-LOX (Maskrey et al, 2007). In this thesis the generation and metabolism of 12- and 15-H(p)ETE-PEs was characterised in murine macrophages and human monocytes. 12- and 15-H(p)ETE-PEs increased by 15 minutes following cell activation and were metabolised by three hours. However, endogenous 12- and 15-HETE-PEs increased then remained stable. Novel lipids believed to be precursors of 15-HETE-PEs, called 15-KETE-PEs, were identified in human monocytes. 15-KETE-PEs increased by 15 minutes and were metabolised by three hours and may therefore account for the temporal generation pattern of 15-H(p)ETE-PEs. 14C radiolabeled products of 15-LOX were generated and characterised in human monocytes. Detection of the 14C radiolabel in activated monocytes suggested that products of 15-LOX may form Michael adducts with proteins by three hours post activation. This may account for the 15-KETE-PE decrease in time-course samples. A method was developed to synthesise 15-H(p)ETE-PE standards, to use in a new assay for their direct quantification following MS analysis. The 15-HETE-PE standard was also used to investigate its pro- versus anti-inflammatory effect on LPS-stimulated human monocytes. 15- HETE-PE down-regulated IL-ip, TNF-a, IL-6 and G-CSF generation, suggesting that products of 15-LOX are anti-inflammatory in human monocytes. 12-H(p)ETE-PEs were externalised in activated WT macrophages, which may be important for cell-cell interactions and re-structuring the cell membrane. EM analysis indicated that 12/15-LOX"7" macrophages have a lysosomal storage disease, symptoms included the presence of lysosomal storage bodies, vacuoles and damaged mitochondria. This may be caused by detected raised levels of PE, PS, PG and PI phospholipids and cholesterol esters in 12/15-LOX"7" macrophages. The data described herein provides a platform to further investigate the function of esterified products generated by 15- and 12/15-LOX

Unacylated-Ghrelin Impairs Hippocampal Neurogenesis and Memory in Mice and Is Altered in Parkinson’s Dementia in Humans

Blood-borne factors regulate adult hippocampal neurogenesis and cognition in mammals. We report that elevating circulating unacylated-ghrelin (UAG), using both pharmacological and genetic methods, reduced hippocampal neurogenesis and plasticity in mice. Spatial memory impairments observed in ghrelin-O-acyl transferase-null (GOAT/) mice that lack acyl-ghrelin (AG) but have high levels of UAG were rescued by acyl-ghrelin. Acyl-ghrelin-mediated neurogenesis in vitro was dependent on non-cell-autonomous BDNF signaling that was inhibited by UAG. These findings suggest that post-translational acylation of ghrelin is important to neurogenesis and memory in mice. To determine relevance in humans, we analyzed circulating AG:UAG in Parkinson disease (PD) patients diagnosed with dementia (PDD), cognitively intact PD patients, and controls. Notably, plasma AG:UAG was only reduced in PDD. Hippocampal ghrelin-receptor expression remained unchanged; however, GOAT+ cell number was reduced in PDD. We identify UAG as a regulator of hippocampal-dependent plasticity and spatial memory and AG:UAG as a putative circulating diagnostic biomarker of dementia

Calorie restriction activates new adult born olfactory‐bulb neurones in a ghrelin‐dependent manner but acyl‐ghrelin does not enhance subventricular zone neurogenesis

The ageing and degenerating brain show deficits in neural stem/progenitor cell (NSPC) plasticity that are accompanied by impairments in olfactory discrimination. Emerging evidence suggests that the gut hormone ghrelin plays an important role in protecting neurones, promoting synaptic plasticity and increasing hippocampal neurogenesis in the adult brain. In the present study, we investigated the role of ghrelin with respect to modulating adult subventricular zone (SVZ) NSPCs that give rise to new olfactory bulb (OB) neurones. We characterised the expression of the ghrelin receptor, growth hormone secretagogue receptor (GHSR), using an immunohistochemical approach in GHSR‐eGFP reporter mice to show that GHSR is expressed in several regions, including the OB but not in the SVZ of the lateral ventricle. These data suggest that acyl‐ghrelin does not mediate a direct effect on NSPC in the SVZ. Consistent with these findings, treatment with acyl‐ghrelin or genetic silencing of GHSR did not alter NSPC proliferation within the SVZ. Similarly, using a bromodeoxyuridine pulse‐chase approach, we show that peripheral treatment of adult rats with acyl‐ghrelin did not increase the number of new adult‐born neurones in the granule cell layer of the OB. These data demonstrate that acyl‐ghrelin does not increase adult OB neurogenesis. Finally, we investigated whether elevating ghrelin indirectly, via calorie restriction (CR), regulated the activity of new adult‐born cells in the OB. Overnight CR induced c‐Fos expression in new adult‐born OB cells but not in developmentally born cells, whereas neuronal activity was absent following re‐feeding. These effects were not present in ghrelin−/− mice, suggesting that adult‐born cells are uniquely sensitive to changes in ghrelin mediated by fasting and re‐feeding. In summary, ghrelin does not promote neurogenesis in the SVZ and OB; however, new adult‐born OB cells are activated by CR in a ghrelin‐dependent manner

Acyl-ghrelin attenuates neurochemical and motor deficits in the 6-OHDA model of Parkinson’s Disease

The feeding-related hormone, acyl-ghrelin, protects dopamine neurones in murine 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-based models of experimental Parkinson’s disease (PD). However, the potential protective effect of acyl-ghrelin on substantia nigra pars compacta (SNpc) dopaminergic neurones and consequent behavioural correlates in the more widely used 6-hydroxydopamine (6-OHDA) rat medial forebrain bundle (MFB) lesion model of PD are unknown. To address this question, acyl-ghrelin levels were raised directly by mini-pump infusion for 7 days prior to unilateral injection of 6-OHDA into the MFB with assessment of amphetamine-induced rotations on days 27 and 35, and immunohistochemical analysis of dopaminergic neurone survival. Whilst acyl-ghrelin treatment was insufficient to elevate food intake or body weight, it attenuated amphetamine-induced circling behaviour and SNpc dopamine neurone loss induced by 6-OHDA. These data support the notion that elevating circulating acyl-ghrelin may be a valuable approach to slow or impair progression of neurone loss in PD

Current trends in oxysterol research

In this short review we provide a synopsis of recent developments in oxysterol research highlighting topics of current interest to the community. These include the involvement of oxysterols in neuronal development and survival, their participation in the immune system, particularly with respect to bacterial and viral infection and to Th17-cell development, and the role of oxysterols in breast cancer. We also discuss the value of oxysterol analysis in the diagnosis of disease

Cholesterolomics: An update

Cholesterolomics can be regarded as the identification and quantification of cholesterol, its precursors post squalene, and metabolites of cholesterol and of its precursors, in a biological sample. These molecules include 1,25-dihydroxyvitamin D3, steroid hormones and bile acids and intermediates in their respective biosynthetic pathways. In this short article we will concentrate our attention on intermediates in bile acid biosynthesis pathways, in particular oxysterols and cholestenoic acids. These molecular classes are implicated in the aetiology of a diverse array of diseases including autoimmune disease, Parkinson's disease, motor neuron disease, breast cancer, the lysosomal storage disease Niemann-Pick type C and the autosomal recessive disorder Smith-Lemli-Opitz syndrome. Mass spectrometry (MS) is the dominant technology for sterol analysis including both gas-chromatography (GC)-MS and liquid chromatography (LC)-MS and more recently matrix-assisted laser desorption/ionisation (MALDI)-MS for tissue imaging studies. Here we will discuss exciting biological findings and recent analytical improvements