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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.

The feeding-related hormone, acyl-ghrelin, protects dopamine neurons 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. While 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.HighlightsAcyl-ghrelin attenuates SNpc dopamine cell loss in rat 6-OHDA-lesion model of PDAcyl-ghrelin attenuates motor deficits in rat 6-OHDA-lesion model of PD

Circadian rhythms are recurring near‐24 hour patterns driven by an endogenous circadian timekeeping system. The master pacemaker in this system is the hypothalamic suprachiasmatic nucleus (SCN). Recently interest has been drawn to how the SCN clock responds to immune system stimulation. A major signalling component in the immune system is nuclear factor (NF)‐κB. In the present study we examined the role of NF‐κB in SCN function. Whilst serum shocked fibroblasts showed rhythmic nuclear localisation of p65 and p65‐dependent transcription, there were no circadian changes in the SCN in expression of the NF‐κB components p65, c‐Rel, p‐IκB or p‐IKK. Chronic treatment with the NF‐κB inhibitor PDTC did not impact on circadian or diurnal rhythms. Phase‐shifting light pulses did not impact on SCN expression of p65, and PDTC treatment did not attenuate the behavioural or molecular response to light pulses. Peripheral treatment with lipopolysaccharide resulted in increased NF‐κB component expression in the SCN. In vitro experiments with SCN slice cultures showed that treatment with NF‐κB inhibitors did not markedly alter rhythmic changes in PER2::LUC expression. Further, SCN slices from nf‐κb::luc mice did not show any evidence for circadian rhythms in NF‐κB‐mediated transcription. Experiments utilising older mice (~16 months old) showed that SCN treatment in vitro with PDTC resulted in increased amplitude of rhythmic PER2::LUC expression, and LPS treatment resulted in altered PER2::LUC rhythm acrophase. Overall, we interpret our results as providing evidence for the involvement of NF‐κB in the suprachiasmatic circadian clock following immune stimulation, but not under basal conditions.

© 2019 British Society for Neuroendocrinology 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.

Isolation of good quality, suitably fragmented chromatin is the most important prerequisite for successful chromatin immunoprecipitation. Chromatin can be sheared enzymatically or by mechanical methods such as sonication. Enzymatic shearing can be advantageous where expensive sonication equipment is not available or where native chromatin is to be examined. Illustrated here is the effectiveness of enzymatic shearing in the fragmentation and isolation of high quality ChIP grade chromatin. We also highlight comparable data for enzymatic shearing versus sonication.

Circadian clocks serve to impose a near-24-h temporal architecture on an organism's physiology, metabolism, and behavior. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus functions as the master circadian pacemaker. There is growing evidence that immunomodulators, such as cytokines, may impinge on circadian timekeeping. We examined whether there is endogenous expression of the proinflammatory cytokine interleukin-1β (IL-1β) and its signaling receptor IL-1R1 in the SCN of young and older mice across the diurnal cycle. We found expression of both IL-1β and IL-1R1 in the young SCN, although only IL-1R1 displayed temporal regulation. In the older SCN, levels of IL-1β were expressed at lower levels than in the young SCN, and IL-1R1 did not vary across the 24 h. We also report age-related day-night variation of IL-1β and IL-1R1 in the paraventricular nucleus (PVN) of the hypothalamus. Further, we examined the effect of peripheral immune challenge on IL-1β and IL-1R1 in the SCN. We found that IL-1β immunoreactivity was not altered 6 or 24 h after a septic dose of lipopolysaccharide (LPS; 5 mg/kg), whereas IL-1R1 was significantly up-regulated in the SCN both 6 and 24 h after LPS. We also demonstrate cellular activation in the SCN 24 h following LPS treatment, as evidenced by increased c-Fos and p65-NF-κB (nuclear factor kappa B) expression. Our results indicate that IL-1β and its associated signaling system may play a role in mediating the response of the circadian timing system to immune challenge as well as potentially contributing to the basal functioning of the SCN clock.

Background/Aims: The master mammalian circadian pacemaker is the suprachiasmatic nuclei (SCN) of the hypothalamus. In this study we examined the expression of transforming growth factor (TGF)-β and the associated signaller phosphorylated SMAD3 (pSMAD3) in the SCN and the paraventricular nucleus (PVN) of the hypothalamus of the young and older mouse across the diurnal cycle, in order to ascertain whether there are time-of-day and age influences on their expression in the hypothalamus. Methods: Immunohistochemistry coupled to densitometric analysis has been used to quantitate the expression of TGF-β and pSMAD3 in the SCN and PVN. Results: We have demonstrated a diurnal pattern of expression of TGF-β in the SCN of young animals, a rhythm that is lost in older mice. The PVN also shows diurnal expression of TGF-β and older mice exhibit elevated levels. Finally, we have demonstrated a novel day/night difference in the expression of pSMAD3, a part of the TGF signalling pathway, in the SCN, a rhythm that appears to be lost with age. Conclusion: We conclude that TGF-β and pSMAD3 are expressed under basal conditions in the SCN and PVN, and this expression is modulated in both a diurnal and age-dependent manner.

Highlights • Peripheral injections of acyl-ghrelin increase adult hippocampal neurogenesis. • Peripheral injections of acyl-ghrelin enhance pattern separation dependent memory. • Systemic administration of physiological levels of acyl-ghrelin has long-lasting memory benefits., Summary An important link exists between intact metabolic processes and normal cognitive functioning; however, the underlying mechanisms remain unknown. There is accumulating evidence that the gut hormone ghrelin, an orexigenic peptide that is elevated during calorie restriction (CR) and known primarily for stimulating growth hormone release, has important extra-hypothalamic functions, such as enhancing synaptic plasticity and hippocampal neurogenesis. The present study was designed to evaluate the long-term effects of elevating acyl-ghrelin levels, albeit within the physiological range, on the number of new adult born neurons in the dentate gyrus (DG) and performance on the Spontaneous Location Recognition (SLR) task, previously shown to be DG-dependent and sensitive to manipulations of plasticity mechanisms and cell proliferation. The results revealed that peripheral treatment of rats with acyl-ghrelin enhanced both adult hippocampal neurogenesis and performance on SLR when measured 8–10 days after the end of acyl-ghrelin treatment. Our data show that systemic administration of physiological levels of acyl-ghrelin can produce long-lasting improvements in spatial memory that persist following the end of treatment. As ghrelin is potentially involved in regulating the relationship between metabolic and cognitive dysfunction in ageing and neurodegenerative disease, elucidating the underlying mechanisms holds promise for identifying novel therapeutic targets and modifiable lifestyle factors that may have beneficial effects on the brain.

Background Ghrelin is an orexigenic stomach hormone that acts centrally to increase mid-brain dopamine neurone activity, amplify dopamine signaling and protect against neurotoxin-induced dopamine cell death in the mouse substantia nigra pars compacta (SNpc). In addition, ghrelin inhibits the lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines from peripheral macrophages, T-cells and from LPS stimulated microglia. Here we sought to determine whether ghrelin attenuates pro-inflammatory cytokine release from dopaminergic neurones. Findings The dopaminergic SN4741 cell-line, which derives from the mouse substantia nigra (SN) and expresses the ghrelin-receptor (growth hormone secretagogue receptor (GHS-R)) and the ghrelin-O-acyl transferase (GOAT) enzyme, was used to determine the neuro-immunomodulatory action of ghrelin. We induced innate immune activation via LPS challenge (1 μg/ml) of SN4741 neurones that had been pre-cultured in the presence or absence of ghrelin (1, 10, 100 nM) for 4 h. After 24 h supernatants were collected for detection of IL-1 beta (IL-1β ), TNF alpha (TNF-α) and IL-6 cytokines via enzyme linked immunosorbent assay (ELISA) analysis. Nuclear translocation of the transcription factor nuclear factor kappa B (NF-κB) was analyzed by Western blotting, and to determine viability of treatments a cell viability assay and caspase-3 immunohistochemistry were performed. We provide evidence that while IL-1β and TNF-α were not detectable under any conditions, SN4741 neurones constitutively released IL-6 under basal conditions and treatment with LPS significantly increased IL-6 secretion. Pre-treatment of neurones with ghrelin attenuated LPS-mediated IL-6 release at 24 h, an affect that was inhibited by the GHS-R antagonist [D-Lys3]-GHRP-6. However, while ghrelin pre-treatment attenuated the LPS-mediated increase in NF-κB, there was no alteration in its nuclear translocation. Cell viability assay and caspase-3 immunocytochemistry demonstrated that the results were independent from activation of cytotoxic and/or apoptotic mechanisms in the neuronal population, respectively. Conclusion Our results provide evidence that the gut-hormone, ghrelin, attenuates IL-6 secretion to LPS challenge in mid-brain dopaminergic neurones. These data suggest that ghrelin may protect against dopaminergic SN nerve cell damage or death via modulation of the innate immune response.

Chromatrap® 96 (C96) is a high-throughput analysis platform that profiles up to 96 transcription factors and epigenetic modifications simultaneously in less than 1 d. It enables sensitive, selective and reproducible target amplification with excellent signal-to-noise ratios, even from samples as small as 0.1 μg. Compatible with automated handling, C96 allows simultaneous investigation of parallel epigenetic landscapes, offering unprecedented assay flexibility and speed.

Background Ovarian cancer has a specific unmet clinical need, with a persistently poor 5-year survival rate observed in women with advanced stage disease warranting continued efforts to develop new treatment options. The amplification of BRD4 in a significant subset of high-grade serous ovarian carcinomas (HGSC) has led to the development of BET inhibitors (BETi) as promising antitumour agents that have subsequently been evaluated in phase I/II clinical trials. Here, we describe the molecular effects and ex vivo preclinical activities of i-BET858, a bivalent pan-BET inhibitor with proven in vivo BRD inhibitory activity. Results i-BET858 demonstrates enhanced cytotoxic activity compared with earlier generation BETis both in cell lines and primary cells derived from clinical samples of HGSC. At molecular level, i-BET858 triggered a bipartite transcriptional response, comprised of a ‘core’ network of genes commonly associated with BET inhibition in solid tumours, together with a unique i-BET858 gene signature. Mechanistically, i-BET858 elicited enhanced DNA damage, cell cycle arrest and apoptotic cell death compared to its predecessor i-BET151. Conclusions Overall, our ex vivo and in vitro studies indicate that i-BET858 represents an optimal candidate to pursue further clinical validation for the treatment of HGSC.

Background Epigenomic dysregulation has been linked to solid tumour malignancies, including ovarian cancers. Profiling of re-programmed enhancer locations associated with disease has the potential to improve stratification and thus therapeutic choices. Ovarian cancers are subdivided into histological subtypes that have significant molecular and clinical differences, with high-grade serous carcinoma representing the most common and aggressive subtype. Methods We interrogated the enhancer landscape(s) of normal ovary and subtype-specific ovarian cancer states using publicly available data. With an initial focus on H3K27ac histone mark, we developed a computational pipeline to predict drug compound activity based on epigenomic stratification. Lastly, we substantiated our predictions in vitro using patient-derived clinical samples and cell lines. Results Using our in silico approach, we highlighted recurrent and privative enhancer landscapes and identified the differential enrichment of a total of 164 transcription factors involved in 201 protein complexes across the subtypes. We pinpointed SNS-032 and EHMT2 inhibitors BIX-01294 and UNC0646 as therapeutic candidates in high-grade serous carcinoma, as well as probed the efficacy of specific inhibitors in vitro. Conclusion Here, we report the first attempt to exploit ovarian cancer epigenomic landscapes for drug discovery. This computational pipeline holds enormous potential for translating epigenomic profiling into therapeutic leads.