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.

Acylation, a Conductor of Ghrelin Function in Brain Health and Disease.

Acyl-ghrelin (AG) is an orexigenic hormone that has a unique octanoyl modification on its third serine residue. It is often referred to as the "hunger hormone" due to its involvement in stimulating food intake and regulating energy homeostasis. The discovery of the enzyme ghrelin-O-acyltransferase (GOAT), which catalyses ghrelin acylation, provided further insights into the relevance of this lipidation process for the activation of the growth hormone secretagogue receptor (GHS-R) by acyl-ghrelin. Although acyl-ghrelin is predominantly linked with octanoic acid, a range of saturated fatty acids can also bind to ghrelin possibly leading to specific functions. Sources of ghrelin acylation include beta-oxidation of longer chain fatty acids, with contributions from fatty acid synthesis, the diet, and the microbiome. In addition, both acyl-ghrelin and unacyl-ghrelin (UAG) have feedback effects on lipid metabolism which in turn modulate their levels. Recently we showed that whilst acyl-ghrelin promotes adult hippocampal neurogenesis and enhances memory function, UAG inhibits these processes. As a result, we postulated that the circulating acyl-ghrelin:unacyl-ghrelin (AG:UAG) ratio might be an important regulator of neurogenesis and cognition. In this review, we discuss emerging evidence behind the relevance of ghrelin acylation in the context of brain physiology and pathology, as well as the current challenges of identifying the provenance of the acyl moiety.

Ghrelin Acylation-A Post-Translational Tuning Mechanism Regulating Adult Hippocampal Neurogenesis.

Adult hippocampal neurogenesis-the generation of new functional neurones in the adult brain-is impaired in aging and many neurodegenerative disorders. We recently showed that the acylated version of the gut hormone ghrelin (acyl-ghrelin) stimulates adult hippocampal neurogenesis while the unacylated form of ghrelin inhibits it, thus demonstrating a previously unknown function of unacyl-ghrelin in modulating hippocampal plasticity. Analysis of plasma samples from Parkinson's disease patients with dementia demonstrated a reduced acyl-ghrelin:unacyl-ghrelin ratio compared to both healthy controls and cognitively intact Parkinson's disease patients. These data, from mouse and human studies, suggest that restoring acyl-ghrelin signalling may promote the activation of pathways to support memory function. In this short review, we discuss the evidence for ghrelin's role in regulating adult hippocampal neurogenesis and the enzymes involved in ghrelin acylation and de-acylation as targets to treat mood-related disorders and dementia.

Analysis of target mRNAs in the fixed-frozen human brain using a modified BaseScope-ISH Assay protocol.

We describe a modified BaseScope™ Assay protocol (ACDBio) for RNA in situ hybridization on fixed-frozen human brain tissue. The original protocol caused tissue detachment due to harsh tissue pre-treatment. We therefore optimized it to improve tissue stability while providing high stain quality in fragile post-mortem tissue from aged donors with advanced neurodegeneration. The main changes include two additional fixation steps and modifications to the pre-treatment protocol. We also describe tissue imaging and stain quantification using the open-source QuPath software. For complete details on the use and execution of this protocol, please refer to Hornsby et al. (2020).

Automated Quantification of Mitochondrial Fragmentation in an In Vitro Parkinson's Disease Model.

Neuronal mitochondrial fragmentation is a phenotype exhibited in models of neurodegeneration such as Parkinson's disease. Delineating the dysfunction in mitochondrial dynamics found in diseased states can aid our understanding of underlying mechanisms of disease progression and possibly identify novel therapeutic approaches. Advances in microscopy and the availability of intuitive open-access software have accelerated the rate of image acquisition and analysis, respectively. These developments allow routine biology researchers to rapidly turn hypotheses into results. In this protocol, we describe the utilization of cell culture techniques, high-content imaging (HCI), and the subsequent open-source image analysis pipeline for the quantification of mitochondrial fragmentation in the context of a rotenone-based in vitro Parkinson's disease model. © 2020 The Authors. Basic Protocol 1: SN4741 neuron culture and treatment in a rotenone-based model of Parkinson's disease Basic Protocol 2: Identification of cell nuclei, measurement of mitochondrial membrane potential, and measurement of mitochondrial fragmentation in mouse-derived midbrain dopaminergic neurons.

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.

Ghrelin-Mediated Hippocampal Neurogenesis: Implications for Health and Disease.

There is a close relationship between cognition and nutritional status, however, the mechanisms underlying this relationship require elucidation. The stomach hormone, ghrelin, which is released during food restriction, provides a link between circulating energy state and adaptive brain function. The maintenance of such homeostatic systems is essential for an organism to thrive and survive, and accumulating evidence points to ghrelin being key in promoting adult hippocampal neurogenesis and memory. Aberrant neurogenesis is linked to cognitive decline in ageing and neurodegeneration. Therefore, identifying endogenous metabolic factors that regulate new adult-born neurone formation is an important objective in understanding the link between nutritional status and central nervous system (CNS) function. Here, we review current developments in our understanding of ghrelin's role in regulating neurogenesis and memory function.

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.

Ghrelin mediated neuroprotection - A possible therapy for Parkinson's disease?

Parkinson's disease is a common age-related neurodegenerative disorder affecting 10 million people worldwide, but the mechanisms underlying its pathogenesis are still unclear. The disease is characterised by dopamine nerve cell loss in the mid-brain and intra-cellular accumulation of α-synuclein that results in motor and non-motor dysfunction. In this review, we discuss the neuroprotective effects of the stomach hormone, ghrelin, in models of Parkinson's disease. Recent findings suggest that it may modulate mitochondrial function and autophagic clearance of impaired organelle in response to changes in cellular energy balance. We consider the putative cellular mechanisms underlying ghrelin-action and the possible role of ghrelin mimetics in slowing or preventing Parkinson's disease progression. This article is part of the Special Issue entitled 'Metabolic Impairment as Risk Factors for Neurodegenerative Disorders.'

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.

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.

A novel role for 12/15-lipoxygenase in regulating autophagy.

12/15-Lipoxygenase (LOX) enzymatically generates oxidized phospholipids in monocytes and macrophages. Herein, we show that cells deficient in 12/15-LOX contain defective mitochondria and numerous cytoplasmic vacuoles containing electron dense material, indicating defects in autophagy or membrane processing, However, both LC3 expression and lipidation were normal both basally and on chloroquine treatment. A LOX-derived oxidized phospholipid, 12-hydroxyeicosatetraenoic acid-phosphatidylethanolamine (12-HETE-PE) was found to be a preferred substrate for yeast Atg8 lipidation, versus native PE, while both native and oxidized PE were effective substrates for LC3 lipidation. Last, phospholipidomics demonstrated altered levels of several phospholipid classes. Thus, we show that oxidized phospholipids generated by 12/15-LOX can act as substrates for key proteins required for effective autophagy and that cells deficient in this enzyme show evidence of autophagic dysfunction. The data functionally link phospholipid oxidation with autophagy for the first time.

Novel keto-phospholipids are generated by monocytes and macrophages, detected in cystic fibrosis, and activate peroxisome proliferator-activated receptor-γ.

12/15-Lipoxygenases (LOXs) in monocytes and macrophages generate novel phospholipid-esterified eicosanoids. Here, we report the generation of two additional families of related lipids comprising 15-ketoeicosatetraenoic acid (KETE) attached to four phosphatidylethanolamines (PEs). The lipids are generated basally by 15-LOX in IL-4-stimulated monocytes, are elevated on calcium mobilization, and are detected at increased levels in bronchoalveolar lavage fluid from cystic fibrosis patients (3.6 ng/ml of lavage). Murine peritoneal macrophages generate 12-KETE-PEs, which are absent in 12/15-LOX-deficient mice. Inhibition of 15-prostaglandin dehydrogenase prevents their formation from exogenous 15-hydroxyeicosatetraenoic acid-PE in human monocytes. Both human and murine cells also generated analogous hydroperoxyeicosatetraenoic acid-PEs. The electrophilic reactivity of KETE-PEs is shown by their Michael addition to glutathione and cysteine. Lastly, both 15-hydroxyeicosatetraenoic acid-PE and 15-KETE-PE activated peroxisome proliferator-activated receptor-γ reporter activity in macrophages in a dose-dependent manner. In summary, we demonstrate novel peroxisome proliferator-activated receptor-γ-activating oxidized phospholipids generated enzymatically by LOX and 15-prostaglandin dehydrogenase in primary monocytic cells and in a human Th2-related lung disease. The lipids are a new family of bioactive mediators from the 12/15-LOX pathway that may contribute to its known anti-inflammatory actions in vivo.

Quantitative assays for esterified oxylipins generated by immune cells.

Phospholipid-esterified oxylipins include newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several esterified oxylipins--in particular hydro(pero)xyeicosatetraenoic acids, hydroxyoctadecadienoic acids, hydroxydocosahexaenoic acids and keto-eicosatetraenoic acids--attached to either phosphatidylethanolamine or phosphatidylcholine. Lipids are isolated from cells or tissue using a liquid-phase organic extraction, then analyzed by HPLC-tandem mass spectrometry (LC/MS/MS) in multiple reaction-monitoring mode. The protocol can simultaneously monitor up to 23 species. Generation of standards takes ∼2 d. Following this, extraction of 30 samples takes ∼3 h, with LC/MS/MS run time of 50 min per sample.

Phosphatidylethanolamine-esterified eicosanoids in the mouse: tissue localization and inflammation-dependent formation in Th-2 disease.

In this study, murine peritoneal macrophages from naïve lavage were found to generate four phospholipids that contain 12-hydroxyeicosatetraenoic acid (12-HETE). They comprise three plasmalogen and one diacyl phosphatidylethanolamines (PEs) (16:0p, 18:1p, 18:0p, and 18:0a at sn-1) and are absent in macrophages from 12/15-lipoxygenase (12/15-LOX)-deficient mice. They are generated acutely in response to calcium mobilization, are primarily cell-associated, and are detected on the outside of the plasma membrane. Levels of 12-HETE-PEs in naïve lavage are in a similar range to those of free 12-HETE (5.5 +/- 0.2 ng or 18.5 +/- 1.03 ng/lavage for esterified versus free, respectively). In healthy mice, 12/15-LOX-derived 12-HETE-PEs are found in the peritoneal cavity, peritoneal membrane, lymph node, and intestine, with a similar distribution to 12/15-LOX-derived 12-HETE. In vivo generation of 12-HETE-PEs occurs in a Th2-dependent model of murine lung inflammation associated with interleukin-4/interleukin-13 expression. In contrast, in Toll receptor-dependent peritonitis mediated either by live bacteria or bacterial products, 12-HETE-PEs are rapidly cleared during the acute phase then reappear during resolution. The human homolog, 18:0a/15-HETE-PE inhibited human monocyte generation of cytokines in response to lipopolysaccharide. In summary, a new family of lipid mediators generated by murine macrophages during Th2 inflammation are identified and structurally characterized. The studies suggest a new paradigm for lipids generated by 12/15-LOX in inflammation involving formation of esterified eicosanoids.

Activated platelets and monocytes generate four hydroxyphosphatidylethanolamines via lipoxygenase.

12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H](-)) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H (18)(2)O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.